Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques(Macaca mulatta, MMU)and crab-eating macaques(M. fascicularis, MFA) serve as...Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques(Macaca mulatta, MMU)and crab-eating macaques(M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from84 samples(41 MFA samples and 43 MMU samples)encompassing 14 common tissues. Our findings revealed a small fraction of genes(3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover,19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary,this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.展开更多
Rhesus monkey embryonic stem（rES） cells have similar characteristics to human ES cells,and might be useful as a substitute model for preclinical research.Notch signaling is involved in the formation of bile ducts,wh...Rhesus monkey embryonic stem（rES） cells have similar characteristics to human ES cells,and might be useful as a substitute model for preclinical research.Notch signaling is involved in the formation of bile ducts,which are composed of cholangiocytes.However,little is known about the role of Notch signaling in cholangiocytic commitment of ES cells.We analyzed the effect of Notch signaling on the induction of cholangiocyte-like cells from rES cells.About 80% of definitive endoderm（DE） cells were generated from rES cells after treatment with activin A.After treatment with BMP4 and FGF1 on matrigel coated wells in serum-free medium,rES-derived DE gave rise to cholangiocyte-like cells by expression of cholangiocytic specific proteins（CK7,CK18,CK19,CK20,and OV-6） and genes（GSTPi,IB4,and HNF1β）.At the same time,expression of Notch 1 and Notch 2 mRNA were detected during cell differentiation,as well as their downstream target genes such as Hes 1 and Hes 5.Inhibition of the Notch signal pathway by L-685458 resulted in decreased expression of Notch and their downstream genes.In addition,the proportion of cholangiocyte-like cells declined from ～90% to ～20%.These results suggest that Notch signaling may play a critical role in cholangiocytic development from ES cells.展开更多
The objective of this study was to set up a rhesus monkey model of polycystic ovary syndrome（PCOS）,which is globally prevalent among reproductive-aged human women,and to understand the reproductive traits of PCOS fe...The objective of this study was to set up a rhesus monkey model of polycystic ovary syndrome（PCOS）,which is globally prevalent among reproductive-aged human women,and to understand the reproductive traits of PCOS female monkeys.Six adult female rhesus monkeys aged 6-10 a,were divided into a PCOS group and a control group.The PCOS group were given two cycles of subcutaneous injections of propionic acid testosterone（PAT）,3.5 mg/kg body weight,on day 1,day 3,and day 5 of the menstrual cycle,respectively,and then given muscle injections of human chorionic gonadotropin（HCG）,350 IU/kg body wtight,on day 7,day 9,and day 11,respectively.Results showed that high levels of serum LH and T [（5.35±0.17） IU/L and（7.58±0.14） ng/mL,respectively],and a high ratio value of LH/FSH（5.35/1.30=4.12） were observed in the PCOS group.No significant differences were found in serum FSH,E2,and P in the PCOS group compared with those of the control.Polycystic ovaries in the PCOS monkeys were recorded by live ultrasound.The blastocysts rates of the PCOS vs.the control were 23.53% vs.66.67%,and there was a significant difference between the two groups.This study shows that PAT coupled with HCG can induce PCOS in rhesus monkeys in the short term.The reproductive features of PCOS monkeys were similar to those of PCOS patients.展开更多
In our previous study, five homologous feeder cell lines, Monkey ear skin fibroblasts （MESFs）, clonally derived fibroblasts from the MESFs （CMESFs）, monkey oviductal fibroblasts （MOFs）, monkey follicular granulo...In our previous study, five homologous feeder cell lines, Monkey ear skin fibroblasts （MESFs）, clonally derived fibroblasts from the MESFs （CMESFs）, monkey oviductal fibroblasts （MOFs）, monkey follicular granulosa fibroblast-like （MFGs） cells, monkey follicular granulosa epithelium-like （MFGEs） cells, were developed for the maintenance of rhesus embryonic stem cells （rESCs）. We found that MESFs, CMESFs, MOFs and MFGs, but not MFGEs, support the growth of rhesus embryonic stem cells. Moreover, we detected some genes that are upregulated in supportive feeder cell lines by semi-quantitative PCR. In the present study, we applied the GeneChip Rhesus Macaque Genome Array of Affymetrix Corporation to study the expression profiles of these five feeder cell lines, in purpose to find out which cytokines and signaling pathways were important in maintaining the rESCs, mRNAs of eight genes, including GREM2, bFGF, KITLG, DKK3, GREM1, AREG, SERPINF1 and LTBP1, were found to be upregulated in supportive feeder cell lines, but not in MFGE. The results indicate that many signaling pathways may play redundant roles in supporting the undifferentiated growth and maintenance of pluripotency in rESCs.展开更多
The purpose of this research was to study the pharmacokinetics and the bioavailability of recombinant human parathyroid hormone [rhPTH （1-34）] in Rhesus monkeys after single and multiple subcutaneous administration....The purpose of this research was to study the pharmacokinetics and the bioavailability of recombinant human parathyroid hormone [rhPTH （1-34）] in Rhesus monkeys after single and multiple subcutaneous administration. An immunoradiometric assay （IRMA） was used to determine the plasma drug concentration of rhFFH （1-34） after giving single dose of 10, 20 and 40 ug/kg and daily dose of 40 ug/kg for 7 d by subcutaneous administration, and intravenous injection of 20 ug/kg in Rhesus monkeys. The pharmacokinetic parameters were calculated by noncompartmental analysis. The drug plasma level quantitation range was from 0.027 to 2.22 ng/mL. The intra- and inter-assay precision （CV） of analysis were less than 15%, and the average recovery was about 93.0% ± 8.6% - 116.5% ± 14.0%. After subcutaneous administration of rhPTH（1-34） at dose of 10, 20 and 40 ug/kg, the average Tmax was 0.67, 0.5 and 0.83 h, Cmax were 1.85 ± 0.05, 3.23 ± 0.25 and 7.15 ± 1.19 ng/mL, the AUC（0-∞） were 3.4 ± 0.6, 10.7 ± 1.3 and 12.6 ± 1.5 ng/h/mL, and terminal-phase elimination T1/2 were 0.72 ± 0.10, 1.15 ± 0.10 and 1.03 ± 0.06 h, respectively. The absolute bioavailability of rhPTH （1-34） was 46.96% after subcutaneous administration of 20 ug/kg. There was no evidence of accumulation during systemic exposure of rhPTH （1-34） upon multiple dosing in Rhesus monkeys. The IRMA assay method provide reasonable sensitivity and specificity for the pharrnacokinetic study of rhPTH （1-34） after subcutaneous or intravenous administration in Rhesus monkeys. The pharmacokinetic characteristic of rhPTH （1-34） in monkeys shows linear relationship with the dose administered subcutaneously.展开更多
Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in respon...Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.展开更多
Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. Howe...Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. However, the outcomes are highly variable, which limits the accuracy of evaluations of ischemic stroke studies. In this study, eight healthy adult rhesus monkeys were randomized into two groups with four monkeys in each group： middle cerebral artery occlusion at origin segment （M1） and middle cerebral artery occlusion at M2 segment. The blood flow in the middle cerebral artery was blocked completely for 2 hours using the endovascular microcoil placement technique （1 mm × 10 cm） （undetachable）, to establish a model of cerebral ischemia. The microcoil was withdrawn and the middle cerebral artery blood flow was restored. A reversible middle cerebral artery occlusion model was identified by hematoxylin-eosin staining, digital subtraction angiography, magnetic resonance angiography, magnetic resonance imaging, and neurological evaluation. The results showed that the middle cerebral artery occlusion model was successfully established in eight adult healthy rhesus monkeys, and ischemic lesions were apparent in the brain tissue of rhesus monkeys at 24 hours after occlusion. The rhesus monkeys had symptoms of neurological deficits. Compared with the M1 occlusion group, the M2 occlusion group had lower infarction volume and higher neurological scores. These experimental findings indicate that reversible middle cerebral artery occlusion can be produced with the endovascular microcoil technique in rhesus monkeys. The M2 occluded model had less infarction and less neurological impairment, which offers the potential for application in the field of brain injury research.展开更多
Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technolo...Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non- human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca2~/calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates （NHPs）. These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.展开更多
To establish an in vitro system for isolating and culturing the mesenchymal stem cells （MSC） of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collect...To establish an in vitro system for isolating and culturing the mesenchymal stem cells （MSC） of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collected and separated by gradient centrifugation to discard most of the blood cells. The MSC contained in the monocyte centrifuging layer was obtained and cultured in Dulbecco＇s modified media （low glucose, L-DMEM） supplemented with 10% Fetal bovine serum （FBS） and 1 ng/ml basic fibroblast growth factor （bFGF）. The non-MSC was screened out by continuously renewing the medium. A passage culture was undertaken while the MSC monolayer formed. The spindle-shaped MSC formed a monolayer after 18 days of primary culturing, and the cells appeared in an oriented array with a swirling and irradiating growth trend. In the anaphase of passage culture, the cell proliferation rate was decreased and the morphology changed into triangular, polygon and flat appearance. These results suggested that mesenchymal stem cells （MSC） of the Rhesus monkey can be passaged in vitro with the established optimized culture system.展开更多
Background:Since December 2019,an outbreak of the Corona Virus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2)in Wuhan,China,has become a public health emergency of internatio...Background:Since December 2019,an outbreak of the Corona Virus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2)in Wuhan,China,has become a public health emergency of international concern.The high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models.Methods:Three 3-5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS-CoV-2,and then analyzed by clinical signs,viral replication,chest X-ray,histopathological changes and immune response.Results:Viral replication of nasopharyngeal swabs,anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge.Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema,notably,old monkeys exhibited diffuse severe interstitial pneumonia.Viral antigens were detected mainly in alveolar epithelial cells and macrophages.Conclusion:SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys.Rhesus macaque models infected with SARS-CoV-2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS-CoV-2 infection.展开更多
Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition. These allografts have low immunogenicity and are more readily available than autologous nerves for the repair o...Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition. These allografts have low immunogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects. In this study, we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve, and compared the outcome with that of autograft. The graft was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells. Pathomo- rphology, electromyogram and immunohistochemistry findings revealed the absence of palmar erosion or ulcers, and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively. There were no significant differences in the mean peak compound muscle action potential, the mean nerve conduction velocity, or the number of neurofilaments between the experimental and control groups. However, outcome was significantly better in the experimental group than in the blank group. These findings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey. The outcomes are similar to those obtained with autologous nerve graft.展开更多
Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infe...Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000single-nucleus transcriptomes of the lung, liver,kidney, and cerebral cortex in rhesus macaques(Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multiorgan dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019(COVID-19).Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway,which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy(an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection,which may facilitate the development of therapeutic interventions for COVID-19.展开更多
Objective To develop a model of SHIV-KB9/Chinese origin rhesus （Ch Rh） macaques for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh macaques with that reported in Indian rhesus （Ind Rh） macaq...Objective To develop a model of SHIV-KB9/Chinese origin rhesus （Ch Rh） macaques for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh macaques with that reported in Indian rhesus （Ind Rh） macaques. Methods Seven mamu-A＊01 negative Ch Rh macaques were inoculated intravenously with 1-10000 MID50 of SHIV-KB9. The monkeys were monitored for viral load, CD4, CDS, SHIV-specific antibody and virus genetic variation. The results were compared with those previously observed in Ind Rh macaques. Results As compared to that observed in Ind Rh macaques, SHIV-KB9 in Ch Rh macaques displayed three identical disease progression patterns. However, the primary pattern was not identical between the two subspecies. The level of plasma viremia differed in SHIV-KB9-infected Ch Rh macaques which exhibited different outcomes from those in Ind Rh macaques. Generally, the values of viral load and the maintenance of CD4^＋ T cells were associated with humoral responses. Otherwise, the viral genetic distances （divergence, diversity） were larger in animals （M419, M425） with their CD4^＋ T cells profoundly depleted. Conclusion The model of SHIV-KB9/Ch Rh macaques displays a relatively slow progression to AIDS compared with Ind Rh macaques, which may more accurately reflect the potential of candidate vaccines in humans.展开更多
Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. I...Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. In this study, we established a standardized experimental model of radial nerve defects in primates and evaluated the effect of repair on peripheral nerve injury. We repaired 2.5-cm lesions in the radial nerve of rhesus monkeys by transplantation of autografts, acellular allografts, or acellular allografts seeded with autologous bone marrow stem cells. Five months after surgery, regenerated nerve tissue was assessed for function, electrophysiology, and histomorphometry. Postoperative functional recovery was evaluated by the wrist-extension test. Compared with the simple autografts, the acellular allografts and allografts seeded with bone marrow stem cells facilitated remarkable recovery of the wrist-extension functions in the rhesus monkeys. This functional improvement was coupled with radial nerve distal axon growth, a higher percentage of neuron survival, increased nerve fiber density and diameter, increased myelin sheath thickness, and increased nerve conduction velocities and peak amplitudes of compound motor action potentials. Furthermore, the quality of nerve regeneration in the bone marrow stem cells-laden allografts group was comparable to that achieved with autografts. The wrist-extension test is a simple behavioral method for objective quantification of peripheral nerve regeneration.展开更多
In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and h...In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4+ T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.展开更多
Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previo...Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previous studies have verified the limit and validity of multiple ampli- fication of peripheral nerve regeneration using small gap sleeve bridging of small donor nerves to repair large receptor nerves in rodents. The present study sought to observe multiple ampli- fication of myelinated nerve fiber regeneration in the primate peripheral nerve. Rhesus monkey models of distal ulnar nerve defects were established and repaired using muscular branches of the right forearm pronator teres. Proximal muscular branches of the pronator teres were su- tured into the distal ulnar nerve using the small gap sleeve bridging method. At 6 months after suture, two-finger flexion and mild wrist flexion were restored in the ulnar-sided injured limbs of rhesus monkey. Neurophysiological examination showed that motor nerve conduction veloc- ity reached 22.63 _＋ 6.34 m/s on the affected side of rhesus monkey. Osmium tetroxide staining demonstrated that the number of myelinated nerve fibers was 1,657 ＋ 652 in the branches of pronator teres of donor, and 2,661 ~ 843 in the repaired ulnar nerve. The rate of multiple amplification of regenerating myelinated nerve fibers was 1.61. These data showed that when muscular branches of the pronator teres were used to repair ulnar nerve in primates, effective regeneration was observed in regenerating nerve fibers, and functions of the injured ulnar nerve were restored to a certain extent. Moreover, multiple amplification was subsequently detected in ulnar nerve axons.展开更多
基金supported by the National Natural Science Foundation of China (82021001 and 31825018 to Q.S., 32370658 to Y.M.,82001372 to X.Y.)National Key Research and Development Program of China (2022YFF0710901)+2 种基金National Science and Technology Innovation2030 Major Program (2021ZD0200900) to Q.S.Shanghai Pujiang Program (22PJ1407300)Shanghai Jiao Tong University 2030 Initiative (WH510363001-7) to Y.M。
文摘Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques(Macaca mulatta, MMU)and crab-eating macaques(M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from84 samples(41 MFA samples and 43 MMU samples)encompassing 14 common tissues. Our findings revealed a small fraction of genes(3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover,19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary,this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.
基金supported by research grants from Zhejiang Natural Sciences Foundation of China (Y2110911 Y2080996)the National Key Technologies R&D Program of China (2007CB947701)
文摘Rhesus monkey embryonic stem（rES） cells have similar characteristics to human ES cells,and might be useful as a substitute model for preclinical research.Notch signaling is involved in the formation of bile ducts,which are composed of cholangiocytes.However,little is known about the role of Notch signaling in cholangiocytic commitment of ES cells.We analyzed the effect of Notch signaling on the induction of cholangiocyte-like cells from rES cells.About 80% of definitive endoderm（DE） cells were generated from rES cells after treatment with activin A.After treatment with BMP4 and FGF1 on matrigel coated wells in serum-free medium,rES-derived DE gave rise to cholangiocyte-like cells by expression of cholangiocytic specific proteins（CK7,CK18,CK19,CK20,and OV-6） and genes（GSTPi,IB4,and HNF1β）.At the same time,expression of Notch 1 and Notch 2 mRNA were detected during cell differentiation,as well as their downstream target genes such as Hes 1 and Hes 5.Inhibition of the Notch signal pathway by L-685458 resulted in decreased expression of Notch and their downstream genes.In addition,the proportion of cholangiocyte-like cells declined from ～90% to ～20%.These results suggest that Notch signaling may play a critical role in cholangiocytic development from ES cells.
基金supported by Yunnan Key Laboratory of Animal Reproductive Biology(2010-03)
文摘The objective of this study was to set up a rhesus monkey model of polycystic ovary syndrome（PCOS）,which is globally prevalent among reproductive-aged human women,and to understand the reproductive traits of PCOS female monkeys.Six adult female rhesus monkeys aged 6-10 a,were divided into a PCOS group and a control group.The PCOS group were given two cycles of subcutaneous injections of propionic acid testosterone（PAT）,3.5 mg/kg body weight,on day 1,day 3,and day 5 of the menstrual cycle,respectively,and then given muscle injections of human chorionic gonadotropin（HCG）,350 IU/kg body wtight,on day 7,day 9,and day 11,respectively.Results showed that high levels of serum LH and T [（5.35±0.17） IU/L and（7.58±0.14） ng/mL,respectively],and a high ratio value of LH/FSH（5.35/1.30=4.12） were observed in the PCOS group.No significant differences were found in serum FSH,E2,and P in the PCOS group compared with those of the control.Polycystic ovaries in the PCOS monkeys were recorded by live ultrasound.The blastocysts rates of the PCOS vs.the control were 23.53% vs.66.67%,and there was a significant difference between the two groups.This study shows that PAT coupled with HCG can induce PCOS in rhesus monkeys in the short term.The reproductive features of PCOS monkeys were similar to those of PCOS patients.
文摘In our previous study, five homologous feeder cell lines, Monkey ear skin fibroblasts （MESFs）, clonally derived fibroblasts from the MESFs （CMESFs）, monkey oviductal fibroblasts （MOFs）, monkey follicular granulosa fibroblast-like （MFGs） cells, monkey follicular granulosa epithelium-like （MFGEs） cells, were developed for the maintenance of rhesus embryonic stem cells （rESCs）. We found that MESFs, CMESFs, MOFs and MFGs, but not MFGEs, support the growth of rhesus embryonic stem cells. Moreover, we detected some genes that are upregulated in supportive feeder cell lines by semi-quantitative PCR. In the present study, we applied the GeneChip Rhesus Macaque Genome Array of Affymetrix Corporation to study the expression profiles of these five feeder cell lines, in purpose to find out which cytokines and signaling pathways were important in maintaining the rESCs, mRNAs of eight genes, including GREM2, bFGF, KITLG, DKK3, GREM1, AREG, SERPINF1 and LTBP1, were found to be upregulated in supportive feeder cell lines, but not in MFGE. The results indicate that many signaling pathways may play redundant roles in supporting the undifferentiated growth and maintenance of pluripotency in rESCs.
文摘The purpose of this research was to study the pharmacokinetics and the bioavailability of recombinant human parathyroid hormone [rhPTH （1-34）] in Rhesus monkeys after single and multiple subcutaneous administration. An immunoradiometric assay （IRMA） was used to determine the plasma drug concentration of rhFFH （1-34） after giving single dose of 10, 20 and 40 ug/kg and daily dose of 40 ug/kg for 7 d by subcutaneous administration, and intravenous injection of 20 ug/kg in Rhesus monkeys. The pharmacokinetic parameters were calculated by noncompartmental analysis. The drug plasma level quantitation range was from 0.027 to 2.22 ng/mL. The intra- and inter-assay precision （CV） of analysis were less than 15%, and the average recovery was about 93.0% ± 8.6% - 116.5% ± 14.0%. After subcutaneous administration of rhPTH（1-34） at dose of 10, 20 and 40 ug/kg, the average Tmax was 0.67, 0.5 and 0.83 h, Cmax were 1.85 ± 0.05, 3.23 ± 0.25 and 7.15 ± 1.19 ng/mL, the AUC（0-∞） were 3.4 ± 0.6, 10.7 ± 1.3 and 12.6 ± 1.5 ng/h/mL, and terminal-phase elimination T1/2 were 0.72 ± 0.10, 1.15 ± 0.10 and 1.03 ± 0.06 h, respectively. The absolute bioavailability of rhPTH （1-34） was 46.96% after subcutaneous administration of 20 ug/kg. There was no evidence of accumulation during systemic exposure of rhPTH （1-34） upon multiple dosing in Rhesus monkeys. The IRMA assay method provide reasonable sensitivity and specificity for the pharrnacokinetic study of rhPTH （1-34） after subcutaneous or intravenous administration in Rhesus monkeys. The pharmacokinetic characteristic of rhPTH （1-34） in monkeys shows linear relationship with the dose administered subcutaneously.
基金Acknowledgment This study was supported by the National Natural Science Foundation of China （30230190）, the National Basic Science Research and Development Project （973） （G1999055901） and the Chinese Academy of Sciences （CAS） Knowledge Innovation Program （KSCX-2-SW-201）.
文摘Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.
基金supported by grants from the National Key Basic Research Program(973 Program)of China,No.2011CB707804Beijing Municipal Science and Technology Project,No.2121100005312016
文摘Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. However, the outcomes are highly variable, which limits the accuracy of evaluations of ischemic stroke studies. In this study, eight healthy adult rhesus monkeys were randomized into two groups with four monkeys in each group： middle cerebral artery occlusion at origin segment （M1） and middle cerebral artery occlusion at M2 segment. The blood flow in the middle cerebral artery was blocked completely for 2 hours using the endovascular microcoil placement technique （1 mm × 10 cm） （undetachable）, to establish a model of cerebral ischemia. The microcoil was withdrawn and the middle cerebral artery blood flow was restored. A reversible middle cerebral artery occlusion model was identified by hematoxylin-eosin staining, digital subtraction angiography, magnetic resonance angiography, magnetic resonance imaging, and neurological evaluation. The results showed that the middle cerebral artery occlusion model was successfully established in eight adult healthy rhesus monkeys, and ischemic lesions were apparent in the brain tissue of rhesus monkeys at 24 hours after occlusion. The rhesus monkeys had symptoms of neurological deficits. Compared with the M1 occlusion group, the M2 occlusion group had lower infarction volume and higher neurological scores. These experimental findings indicate that reversible middle cerebral artery occlusion can be produced with the endovascular microcoil technique in rhesus monkeys. The M2 occluded model had less infarction and less neurological impairment, which offers the potential for application in the field of brain injury research.
基金supported by the National Program on Key Basic Research Project(973 Programs 2015CB755605)the National Natural Science Foundation of China(81471312)
文摘Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non- human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca2~/calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates （NHPs）. These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.
文摘To establish an in vitro system for isolating and culturing the mesenchymal stem cells （MSC） of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collected and separated by gradient centrifugation to discard most of the blood cells. The MSC contained in the monocyte centrifuging layer was obtained and cultured in Dulbecco＇s modified media （low glucose, L-DMEM） supplemented with 10% Fetal bovine serum （FBS） and 1 ng/ml basic fibroblast growth factor （bFGF）. The non-MSC was screened out by continuously renewing the medium. A passage culture was undertaken while the MSC monolayer formed. The spindle-shaped MSC formed a monolayer after 18 days of primary culturing, and the cells appeared in an oriented array with a swirling and irradiating growth trend. In the anaphase of passage culture, the cell proliferation rate was decreased and the morphology changed into triangular, polygon and flat appearance. These results suggested that mesenchymal stem cells （MSC） of the Rhesus monkey can be passaged in vitro with the established optimized culture system.
基金This work was supported by the National Research and Development Project of China(Grant No.2020YFC0841100)Fundamental Research Funds for CAMS of China(Grant No.2020HY320001)+3 种基金National Key Research and Development Project of China(Grant No.2016YFD0500304)CAMS initiative for Innovative Medicine of China(Grant No.2016-I2M-2-006)National Mega projects of China for Major Infectious Diseases(Grant No.2017ZX10304402)and National Key Research and Development Programme of China(2016YFD0500301,2020YFC0840800,2020YFC0840900).
文摘Background:Since December 2019,an outbreak of the Corona Virus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2)in Wuhan,China,has become a public health emergency of international concern.The high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models.Methods:Three 3-5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS-CoV-2,and then analyzed by clinical signs,viral replication,chest X-ray,histopathological changes and immune response.Results:Viral replication of nasopharyngeal swabs,anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge.Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema,notably,old monkeys exhibited diffuse severe interstitial pneumonia.Viral antigens were detected mainly in alveolar epithelial cells and macrophages.Conclusion:SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys.Rhesus macaque models infected with SARS-CoV-2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS-CoV-2 infection.
基金supported by grants from the National Natural Science Foundation of China,No.30170962the Major Subject of Key Technology of Guangzhou City of China,No.2002Z1-E0031science and technology projects of Nanshan district,No.2014028
文摘Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition. These allografts have low immunogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects. In this study, we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve, and compared the outcome with that of autograft. The graft was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells. Pathomo- rphology, electromyogram and immunohistochemistry findings revealed the absence of palmar erosion or ulcers, and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively. There were no significant differences in the mean peak compound muscle action potential, the mean nerve conduction velocity, or the number of neurofilaments between the experimental and control groups. However, outcome was significantly better in the experimental group than in the blank group. These findings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey. The outcomes are similar to those obtained with autologous nerve graft.
基金supported by the National Basic Research Program of China(2020YFA0804000,2020YFC0842000,2020YFA0112200,2021YFC2301703)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB32010100)+6 种基金Special Associate Research Program of the Chinese Academy of Sciences(E1290601)National Natural Science Foundation of China(32122037,81891001,32192411,32100512,U1902215)Collaborative Research Fund of the Chinese Institute for Brain Research,Beijing(2020-NKX-PT-03)CAS Project for Young Scientists in Basic Research(YSBR-013)Young Elite Scientist Sponsorship Program by the China Association for Science and Technology(2020QNRC001)National Resource Center for Non-Human Primates。
文摘Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000single-nucleus transcriptomes of the lung, liver,kidney, and cerebral cortex in rhesus macaques(Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multiorgan dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019(COVID-19).Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway,which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy(an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection,which may facilitate the development of therapeutic interventions for COVID-19.
文摘Objective To develop a model of SHIV-KB9/Chinese origin rhesus （Ch Rh） macaques for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh macaques with that reported in Indian rhesus （Ind Rh） macaques. Methods Seven mamu-A＊01 negative Ch Rh macaques were inoculated intravenously with 1-10000 MID50 of SHIV-KB9. The monkeys were monitored for viral load, CD4, CDS, SHIV-specific antibody and virus genetic variation. The results were compared with those previously observed in Ind Rh macaques. Results As compared to that observed in Ind Rh macaques, SHIV-KB9 in Ch Rh macaques displayed three identical disease progression patterns. However, the primary pattern was not identical between the two subspecies. The level of plasma viremia differed in SHIV-KB9-infected Ch Rh macaques which exhibited different outcomes from those in Ind Rh macaques. Generally, the values of viral load and the maintenance of CD4^＋ T cells were associated with humoral responses. Otherwise, the viral genetic distances （divergence, diversity） were larger in animals （M419, M425） with their CD4^＋ T cells profoundly depleted. Conclusion The model of SHIV-KB9/Ch Rh macaques displays a relatively slow progression to AIDS compared with Ind Rh macaques, which may more accurately reflect the potential of candidate vaccines in humans.
基金supported by the National High-Technology Research and Development Program of China(863 Program),No.2006AA02A130the National Natural Science Foundation of China,No.81372041,31070869,30700847
文摘Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. In this study, we established a standardized experimental model of radial nerve defects in primates and evaluated the effect of repair on peripheral nerve injury. We repaired 2.5-cm lesions in the radial nerve of rhesus monkeys by transplantation of autografts, acellular allografts, or acellular allografts seeded with autologous bone marrow stem cells. Five months after surgery, regenerated nerve tissue was assessed for function, electrophysiology, and histomorphometry. Postoperative functional recovery was evaluated by the wrist-extension test. Compared with the simple autografts, the acellular allografts and allografts seeded with bone marrow stem cells facilitated remarkable recovery of the wrist-extension functions in the rhesus monkeys. This functional improvement was coupled with radial nerve distal axon growth, a higher percentage of neuron survival, increased nerve fiber density and diameter, increased myelin sheath thickness, and increased nerve conduction velocities and peak amplitudes of compound motor action potentials. Furthermore, the quality of nerve regeneration in the bone marrow stem cells-laden allografts group was comparable to that achieved with autografts. The wrist-extension test is a simple behavioral method for objective quantification of peripheral nerve regeneration.
基金Key Scientific and Technological projects of China (2004BA719A14) and Yunnan province (2004NG12, 2006PT08)National 973 project of China (2006CB504200, 2006CB504300)+3 种基金The Natural Science Foundation of China (30471605 30671960)The Knowledge Innovation Program (KSCX1-YW-R-15)"Western Light" Projects of Chinese Academy of Sciences.
文摘In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4+ T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.
基金supported by grants from the National Program on Key Basic Research Project of China(973 Program),No.2014CB542200the National Natural Science Foundation of China,No.31271284,81171146,31100860+1 种基金Program for Innovative Research Team in University of Ministry of Education of China,No.IRT1201the Natural Science Foundation of Beijing of China,No.7142164
文摘Multiple-bud regeneration, i.e., multiple amplification, has been shown to exist in peripheral nerve regeneration. Multiple buds grow towards the distal nerve stump during proximal nerve fiber regeneration. Our previous studies have verified the limit and validity of multiple ampli- fication of peripheral nerve regeneration using small gap sleeve bridging of small donor nerves to repair large receptor nerves in rodents. The present study sought to observe multiple ampli- fication of myelinated nerve fiber regeneration in the primate peripheral nerve. Rhesus monkey models of distal ulnar nerve defects were established and repaired using muscular branches of the right forearm pronator teres. Proximal muscular branches of the pronator teres were su- tured into the distal ulnar nerve using the small gap sleeve bridging method. At 6 months after suture, two-finger flexion and mild wrist flexion were restored in the ulnar-sided injured limbs of rhesus monkey. Neurophysiological examination showed that motor nerve conduction veloc- ity reached 22.63 _＋ 6.34 m/s on the affected side of rhesus monkey. Osmium tetroxide staining demonstrated that the number of myelinated nerve fibers was 1,657 ＋ 652 in the branches of pronator teres of donor, and 2,661 ~ 843 in the repaired ulnar nerve. The rate of multiple amplification of regenerating myelinated nerve fibers was 1.61. These data showed that when muscular branches of the pronator teres were used to repair ulnar nerve in primates, effective regeneration was observed in regenerating nerve fibers, and functions of the injured ulnar nerve were restored to a certain extent. Moreover, multiple amplification was subsequently detected in ulnar nerve axons.
