AIM To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4(AAV-Tβ4) on murine colitis via intracolonic administration.METHODS AAV-Tβ4 was prepared and intracolonically use...AIM To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4(AAV-Tβ4) on murine colitis via intracolonic administration.METHODS AAV-Tβ4 was prepared and intracolonically used to mediate the secretory expression of Tβ4 in mouse colons. Dextran sulfate sodium(DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid(TNBS) was used to establish a mouse colitis model resembling Crohn's disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tβ4 on colitis. The activities of myeloperoxidase(MPO) and superoxide dismutase(SOD) and the content of malondialdehyde(MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis andproliferation were detected by TUNEL assay and immunochemistry, respectively.RESULTS Recombinant AAVs efficiently delivered Lac Z and Tβ4 into the colonic tissues of the mice, and AAV-Tβ4 led to a strong expression of Tβ4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ4 significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tβ4 also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice.CONCLUSION Tβ4 exerts a protective effect on murine colitis, indicating that AAV-Tβ4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.展开更多
The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large a...The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large amount of RIFA venom allergen Sol i 4 for diagnosis of RIFA allergy and allergen-specific immunotherapy, the gene encoding this protein was amplified and cloned into the prokaryotic expression vector pET43, la. The recombinant plasmid was used to transform competent cells and the recombinant proteins were expressed in E. coll. SDS-PAGE and Western blotting analysis indicated that high-level expression of Sol i 4 protein was successfully achieved. Allergenic activity analysis of the recombinant allergen Sol i 4 was then performed on rabbit. The result showed that the recombinant protein obtained had significant allergenic activity. It indicated that the recombinant allergen Sol i 4 of RIFA venom was successfully expressed in E. coli, which provided foundation for further developing therapeutic and diagnosis reagents of RIFA allergy.展开更多
Background:It has been demonstrated that thymosinβ4(Tβ4)could inflect the severity of acute-on-chronic hepatitis B liver failure(ACHBLF),but the relationship between its methylation status and the prognosis of liver...Background:It has been demonstrated that thymosinβ4(Tβ4)could inflect the severity of acute-on-chronic hepatitis B liver failure(ACHBLF),but the relationship between its methylation status and the prognosis of liver failure is not clear.This study aimed to determine Tβ4 promoter methylation status in patients with ACHBLF and to evaluate its prognostic value.Methods:The study recruited 115 patients with ACHBLF,80 with acute-on-chronic hepatitis B pre-liver failure(pre-ACHBLF),and 86 with chronic hepatitis B(CHB).In addition,there were 36 healthy controls(HCs)from the Department of Hepatology,Qilu Hospital of Shandong University.The 115 patients with ACHBLF were divided into three subgroups:33 with early stage ACHBLF(E-ACHBLF),42 with mid-stage ACHBLF(M-ACHBLF),and 40 with advanced stage ACHBLF(A-ACHBLF).Tβ4 promoter methylation status in peripheral blood mononuclear cells(PBMCs)was measured by methylation-specific polymerase chain reaction,and mRNA was detected by quantitative real-time polymerase chain reaction.Results:Methylation frequency of Tβ4 was significantly higher in patients with ACHBLF than in those with pre-ACHBLF,CHB or HCs.However,expression of Tβ4 mRNA showed the opposite trend.In patients with ACHBLF,Tβ4 promoter methylation status correlated negatively with mRNA levels.The 3-month mortality of ACHBLF in the methylated group was significantly higher than that in the unmethylated group.Also,Tβ4 promoter methylation frequency was lower in survivors than in non-survivors.When used to predict the 1-,2-,and 3-month incidence of ACHBLF,Tβ4 methylation status was better than the model for end-stage liver disease(MELD)score.The predictive value of Tβ4 methylation was higher than that of MELD score for the mortality of patients with E-ACHBLF and M-ACHBLF,but not for A-ACHBLF.Conclusions:Tβ4 methylation might be an important early marker for predicting disease incidence and prognosis in patients with ACHBLF.展开更多
3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuro...3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.展开更多
Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic ...Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.展开更多
AIM:To investigate whether serum thymosinβ4 can provide diagnostic or prognostic information in liver failure patients caused by chronic hepatitis B virus(HBV) infection. METHODS:Serum thymosinβ4 levels were measure...AIM:To investigate whether serum thymosinβ4 can provide diagnostic or prognostic information in liver failure patients caused by chronic hepatitis B virus(HBV) infection. METHODS:Serum thymosinβ4 levels were measured in 30 patients with acute-on-chronic liver failure(ACLF), 31 patients with chronic liver failure(CLF),30 patients with compensated liver cirrhosis(CR)and 32 patients with chronic hepatitis B and 30 healthy controls.Serum thymosinβ4 levels were measured by enzyme-linked immunosorbent assay and Child-Pugh and model for end-stage liver disease(MELD)scores were calculated for each patient on admission.RESULTS:Compared with healthy controls,serum thymosinβ4 levels in ACLF,CLF,CR and chronic hepatitis B patients were significantly lower,6.5047 (4.7879-10.5314)μg/mL vs 0.4632(0.2759-0.8768) μg/mL,0.6981(0.5209-1.2008)μg/mL,1.8053 (0.8110-2.3397)μg/mL,3.7803(1.8570-6.4722)μg/mL, respectively(P<0.001).The levels of thymosinβ4 in liver failure(ACLF or CLF)patients were markedly lower than that in CR(P<0.001),and a difference was also found between CLF and ACLF patients(P=0.038).In patients with chronic liver disease,there was a positive relationship between thymosinβ4 levels and albumin, choline esterase,and platelet(P<0.001),and negative relationship with alanine aminotransferase(P=0.020), aspartate aminotransferase,total bilirubin,international normalized ratio of prothrombin time,and Child-Pugh and MELD scores(P<0.001).Of the 61 liver failure patients,the thymosinβ4 levels of non-survivors were significantly lower than that of survivors(P=0.007). Receiver operating characteristics analysis identified a thymosinβ4 cutoff level of 0.5708μg/mL for predicting poor prognosis in all liver failure patients.The serial thymosinβ4 values were observed in 13 liver failure inpatients.Lower initial values were observed in the death.While greater improvement in thymosinβ4 value was found in those who recovered from the disease. CONCLUSION:Serum thymosinβ4 can be used as an important potential predictor for liver failure caused by chronic HBV infection.展开更多
Thymosin β4 (Tβ4), a G-actin binding protein, has diverse biological functions. This study tested the effects of Tβ4 on oligodendrogenesis in a rat model of intracerebral hemorrhage (ICH). ICH was induced by stereo...Thymosin β4 (Tβ4), a G-actin binding protein, has diverse biological functions. This study tested the effects of Tβ4 on oligodendrogenesis in a rat model of intracerebral hemorrhage (ICH). ICH was induced by stereotactic injection of 100 μm of autologous blood into the striatum in 32 male Wistar rats. The rats were randomly divided into four groups: 1) saline control group (n = 8);2) 3 mg/kg Tβ4-treated group (n = 8);3) 6 mg/kg Tβ4-treated group (n = 8);and 4) 12 mg/kg Tβ4treated group (n = 8). Tβ4 or saline was administered intraperitoneally starting at 24 h post ICH and then every 3 days for 4 additional doses. The neurological functional outcome was evaluated by behavioral tests (i.e., modified Neurological Severity Score and corner turn test) at multiple time points after ICH. Animals were sacrificed at 28 days post ICH, and histological studies were completed. Tβ4 treatment improved neurological functional recovery significantly and increased actively proliferating oligodendrocytic progenitor cells and myelinating oligodendrocytes in the ICH-affected brain tissue, compared with the saline-treated group. The high-dose treatment of Tβ4 showed better restorative effects compared with the low-dose treatment. Tβ4 treatment enhanced ICH-induced oligodendrogenesis that may contribute to the enhanced functional recovery after ICH. Further investigation is warranted to determine the associated underlying mechanisms of Tβ4 treatment for ICH.展开更多
Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production ...Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production of antibodies and the consequent transfer of these to the piglets via colostrum to prevent diarrhea during the neonate period and after weaning. The objective of this study was to evaluate the immune response of the sows immunized with recombinant ETEC proteins (F4, F5, F6, F18 and F41). The immune response of the sows immunized with the recombinant proteins was compared with a commercial vaccine containing ETEC bacterins. The study was performed on a commercial farm and included nine pregnant sows divided into three groups: G1 was vaccinated with recombinant proteins (n = 3);G2 was vaccinated with the commercial vaccine (n = 3);and G3 was vaccinated with sterile buffered saline (PBS) (n = 3). All the sows were fed a balanced diet without antibiotics and water ad libitum. The recombinant fimbriae stimulated the specific humoral immune response of the immunized sows. There was a statistically significant increase in the levels of antibodies to the fimbriae F4 (K88), F5 (K99), F6 (987P) and F18 in the sows vaccinated with the recombinant proteins compared with the control group. The colostrum IgG titers for all fimbriae in all the immunized sows were significantly increased compared to the control group. Additionally, all the piglets exhibited significantly increased antibody levels relative to all fimbriae when compared with those in the unimmunized control group, demonstrating successful antibody transfer via colostrum of the sows to the piglets.展开更多
Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arter...Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arteries and internal iliac artery. Tβ4 was dissolved in sterile saline and intramuscularly injected into the centre and periphery of ligation area in the treatment group (n = 10) starting from the surgery day until 4 weeks after surgery, while control animals received saline injection only (n = 9). All animals were sacrificed at 6 weeks after surgery and used for immunohistochemistry studies. Results: Tβ4 stimulated angiogenesis was evidenced by increased vascular density based on CD31 immunostaining, which was sig- nifycantly increased in Tβ4 group (562.5 ± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2;p 0.05) groups. Tβ4 increased Pax3/7+ skeletal muscle progenitor cell density. Pax3/7+ cell density of Tβ4 group (13.7% ± 2%) was significantly higher than that of the control group (4.3% ± 1.6%, p < 0.05). However, the numbers of central nuclei fiber and central nuclei per fiber were insignificantly increased in Tβ4 group as compared to control group. The numbers of central nuclei fiber were 8.9 ± 2.1 and 9.5 ± 1.6, and the central nuclei per fiber were 0.25 ± 0.07 and 0.48 ± 0.09 for control and Tβ4 groups, respectively. Conclusions: This preliminary study suggests that localized delivery of Tβ4 increased angiogenesis and skeletal muscle progenitor cell density in ischemic skeletal muscle, but failed to promote skeletal muscle regeneration.展开更多
Thymosin β4（Tβ4） is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells（m ESCs...Thymosin β4（Tβ4） is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells（m ESCs） proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on m ESCs. Target genes during m ESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the m ESCs-derived cardiomyocytes. It was found that Tβ4 decreased m ESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, m ESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these m ESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant ？-catenin. Under m ESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of m ESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed m ESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.展开更多
基金Supported by National Foundation of Natural Sciences,China,No.81300293
文摘AIM To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4(AAV-Tβ4) on murine colitis via intracolonic administration.METHODS AAV-Tβ4 was prepared and intracolonically used to mediate the secretory expression of Tβ4 in mouse colons. Dextran sulfate sodium(DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid(TNBS) was used to establish a mouse colitis model resembling Crohn's disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tβ4 on colitis. The activities of myeloperoxidase(MPO) and superoxide dismutase(SOD) and the content of malondialdehyde(MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis andproliferation were detected by TUNEL assay and immunochemistry, respectively.RESULTS Recombinant AAVs efficiently delivered Lac Z and Tβ4 into the colonic tissues of the mice, and AAV-Tβ4 led to a strong expression of Tβ4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ4 significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tβ4 also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice.CONCLUSION Tβ4 exerts a protective effect on murine colitis, indicating that AAV-Tβ4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.
基金supported by the Key Pro-gram Foundation of General Administration of QualitySupervision, Inspection and Quarantine of China(2005IK169).
文摘The sting of red imported fire ant （RIFA） could cause serious allergic response in fraction of people. These allergic reactions are mainly caused by its venom, especially venom allergen Sol i 1-4. To produce large amount of RIFA venom allergen Sol i 4 for diagnosis of RIFA allergy and allergen-specific immunotherapy, the gene encoding this protein was amplified and cloned into the prokaryotic expression vector pET43, la. The recombinant plasmid was used to transform competent cells and the recombinant proteins were expressed in E. coll. SDS-PAGE and Western blotting analysis indicated that high-level expression of Sol i 4 protein was successfully achieved. Allergenic activity analysis of the recombinant allergen Sol i 4 was then performed on rabbit. The result showed that the recombinant protein obtained had significant allergenic activity. It indicated that the recombinant allergen Sol i 4 of RIFA venom was successfully expressed in E. coli, which provided foundation for further developing therapeutic and diagnosis reagents of RIFA allergy.
基金supported by grants from the Key Project of the Chinese Ministry of Science and Technology(2017ZX102022022)the National Natural Science Foundation of China(81970522)the Key Research and Development Project of Shandong Province(2019GSF108023).
文摘Background:It has been demonstrated that thymosinβ4(Tβ4)could inflect the severity of acute-on-chronic hepatitis B liver failure(ACHBLF),but the relationship between its methylation status and the prognosis of liver failure is not clear.This study aimed to determine Tβ4 promoter methylation status in patients with ACHBLF and to evaluate its prognostic value.Methods:The study recruited 115 patients with ACHBLF,80 with acute-on-chronic hepatitis B pre-liver failure(pre-ACHBLF),and 86 with chronic hepatitis B(CHB).In addition,there were 36 healthy controls(HCs)from the Department of Hepatology,Qilu Hospital of Shandong University.The 115 patients with ACHBLF were divided into three subgroups:33 with early stage ACHBLF(E-ACHBLF),42 with mid-stage ACHBLF(M-ACHBLF),and 40 with advanced stage ACHBLF(A-ACHBLF).Tβ4 promoter methylation status in peripheral blood mononuclear cells(PBMCs)was measured by methylation-specific polymerase chain reaction,and mRNA was detected by quantitative real-time polymerase chain reaction.Results:Methylation frequency of Tβ4 was significantly higher in patients with ACHBLF than in those with pre-ACHBLF,CHB or HCs.However,expression of Tβ4 mRNA showed the opposite trend.In patients with ACHBLF,Tβ4 promoter methylation status correlated negatively with mRNA levels.The 3-month mortality of ACHBLF in the methylated group was significantly higher than that in the unmethylated group.Also,Tβ4 promoter methylation frequency was lower in survivors than in non-survivors.When used to predict the 1-,2-,and 3-month incidence of ACHBLF,Tβ4 methylation status was better than the model for end-stage liver disease(MELD)score.The predictive value of Tβ4 methylation was higher than that of MELD score for the mortality of patients with E-ACHBLF and M-ACHBLF,but not for A-ACHBLF.Conclusions:Tβ4 methylation might be an important early marker for predicting disease incidence and prognosis in patients with ACHBLF.
基金financially supported by the National Natural Science Foundation of China(General Program),No.31271494Excellent Talent Support Program of Liaoning Province,No.LJQ2011004
文摘3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.
基金financially supported by National Natural Science Fund.Molecule mechanism of the resistance of HRD1 against diabetic retinopathy (NO.81271015)Molecule mechanism of the resistance of P58IPK against diabetic retinopathy (NO.30872823)
文摘Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.
基金Supported by The National Basic Research Program of China,No.2007CB512801the National 11th 5-year Plan for Hepatitis Research,No.2008ZX10002-005Tianjin Public Health Bureau Key Research Program,No.07KG9
文摘AIM:To investigate whether serum thymosinβ4 can provide diagnostic or prognostic information in liver failure patients caused by chronic hepatitis B virus(HBV) infection. METHODS:Serum thymosinβ4 levels were measured in 30 patients with acute-on-chronic liver failure(ACLF), 31 patients with chronic liver failure(CLF),30 patients with compensated liver cirrhosis(CR)and 32 patients with chronic hepatitis B and 30 healthy controls.Serum thymosinβ4 levels were measured by enzyme-linked immunosorbent assay and Child-Pugh and model for end-stage liver disease(MELD)scores were calculated for each patient on admission.RESULTS:Compared with healthy controls,serum thymosinβ4 levels in ACLF,CLF,CR and chronic hepatitis B patients were significantly lower,6.5047 (4.7879-10.5314)μg/mL vs 0.4632(0.2759-0.8768) μg/mL,0.6981(0.5209-1.2008)μg/mL,1.8053 (0.8110-2.3397)μg/mL,3.7803(1.8570-6.4722)μg/mL, respectively(P<0.001).The levels of thymosinβ4 in liver failure(ACLF or CLF)patients were markedly lower than that in CR(P<0.001),and a difference was also found between CLF and ACLF patients(P=0.038).In patients with chronic liver disease,there was a positive relationship between thymosinβ4 levels and albumin, choline esterase,and platelet(P<0.001),and negative relationship with alanine aminotransferase(P=0.020), aspartate aminotransferase,total bilirubin,international normalized ratio of prothrombin time,and Child-Pugh and MELD scores(P<0.001).Of the 61 liver failure patients,the thymosinβ4 levels of non-survivors were significantly lower than that of survivors(P=0.007). Receiver operating characteristics analysis identified a thymosinβ4 cutoff level of 0.5708μg/mL for predicting poor prognosis in all liver failure patients.The serial thymosinβ4 values were observed in 13 liver failure inpatients.Lower initial values were observed in the death.While greater improvement in thymosinβ4 value was found in those who recovered from the disease. CONCLUSION:Serum thymosinβ4 can be used as an important potential predictor for liver failure caused by chronic HBV infection.
文摘Thymosin β4 (Tβ4), a G-actin binding protein, has diverse biological functions. This study tested the effects of Tβ4 on oligodendrogenesis in a rat model of intracerebral hemorrhage (ICH). ICH was induced by stereotactic injection of 100 μm of autologous blood into the striatum in 32 male Wistar rats. The rats were randomly divided into four groups: 1) saline control group (n = 8);2) 3 mg/kg Tβ4-treated group (n = 8);3) 6 mg/kg Tβ4-treated group (n = 8);and 4) 12 mg/kg Tβ4treated group (n = 8). Tβ4 or saline was administered intraperitoneally starting at 24 h post ICH and then every 3 days for 4 additional doses. The neurological functional outcome was evaluated by behavioral tests (i.e., modified Neurological Severity Score and corner turn test) at multiple time points after ICH. Animals were sacrificed at 28 days post ICH, and histological studies were completed. Tβ4 treatment improved neurological functional recovery significantly and increased actively proliferating oligodendrocytic progenitor cells and myelinating oligodendrocytes in the ICH-affected brain tissue, compared with the saline-treated group. The high-dose treatment of Tβ4 showed better restorative effects compared with the low-dose treatment. Tβ4 treatment enhanced ICH-induced oligodendrogenesis that may contribute to the enhanced functional recovery after ICH. Further investigation is warranted to determine the associated underlying mechanisms of Tβ4 treatment for ICH.
文摘Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production of antibodies and the consequent transfer of these to the piglets via colostrum to prevent diarrhea during the neonate period and after weaning. The objective of this study was to evaluate the immune response of the sows immunized with recombinant ETEC proteins (F4, F5, F6, F18 and F41). The immune response of the sows immunized with the recombinant proteins was compared with a commercial vaccine containing ETEC bacterins. The study was performed on a commercial farm and included nine pregnant sows divided into three groups: G1 was vaccinated with recombinant proteins (n = 3);G2 was vaccinated with the commercial vaccine (n = 3);and G3 was vaccinated with sterile buffered saline (PBS) (n = 3). All the sows were fed a balanced diet without antibiotics and water ad libitum. The recombinant fimbriae stimulated the specific humoral immune response of the immunized sows. There was a statistically significant increase in the levels of antibodies to the fimbriae F4 (K88), F5 (K99), F6 (987P) and F18 in the sows vaccinated with the recombinant proteins compared with the control group. The colostrum IgG titers for all fimbriae in all the immunized sows were significantly increased compared to the control group. Additionally, all the piglets exhibited significantly increased antibody levels relative to all fimbriae when compared with those in the unimmunized control group, demonstrating successful antibody transfer via colostrum of the sows to the piglets.
文摘Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arteries and internal iliac artery. Tβ4 was dissolved in sterile saline and intramuscularly injected into the centre and periphery of ligation area in the treatment group (n = 10) starting from the surgery day until 4 weeks after surgery, while control animals received saline injection only (n = 9). All animals were sacrificed at 6 weeks after surgery and used for immunohistochemistry studies. Results: Tβ4 stimulated angiogenesis was evidenced by increased vascular density based on CD31 immunostaining, which was sig- nifycantly increased in Tβ4 group (562.5 ± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2;p 0.05) groups. Tβ4 increased Pax3/7+ skeletal muscle progenitor cell density. Pax3/7+ cell density of Tβ4 group (13.7% ± 2%) was significantly higher than that of the control group (4.3% ± 1.6%, p < 0.05). However, the numbers of central nuclei fiber and central nuclei per fiber were insignificantly increased in Tβ4 group as compared to control group. The numbers of central nuclei fiber were 8.9 ± 2.1 and 9.5 ± 1.6, and the central nuclei per fiber were 0.25 ± 0.07 and 0.48 ± 0.09 for control and Tβ4 groups, respectively. Conclusions: This preliminary study suggests that localized delivery of Tβ4 increased angiogenesis and skeletal muscle progenitor cell density in ischemic skeletal muscle, but failed to promote skeletal muscle regeneration.
基金supposed by grants from National Natural Science Foundation of China(No.81100818,No.31100828 and No.81070342)the Fundamental Research Funds for the Central Universities(HUST:No.2012TS036)
文摘Thymosin β4（Tβ4） is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells（m ESCs） proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on m ESCs. Target genes during m ESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the m ESCs-derived cardiomyocytes. It was found that Tβ4 decreased m ESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, m ESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these m ESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant ？-catenin. Under m ESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of m ESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed m ESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
