基于pH值敏感的荧光染料分析腺病毒诱导T淋巴细胞胞内体膜的裂解

【目的】探索基于pH值敏感的荧光染料分析腺病毒裂解T淋巴细胞胞内体膜的实验方法。【方法】本文以Jurkat细胞(T淋巴瘤细胞)为靶细胞,将pH值敏感的荧光染料pHrodo dextran与5型腺病毒(Ad5)共同孵育Jurkat细胞,对pHrodo dextran孵育的浓度与时间进行了优化,利用激光共聚焦显微镜分析胞内相对平均荧光强度百分比随时间的变化情况,反映Ad5诱导胞内体膜裂解情况。【结果】研究结果表明,在pHrodo dextran终浓度为80μg/m L,孵育时间为10 min条件下,在病毒感染后的30 min,相对平均荧光强度百分比出现显著下降;利用巴佛洛酶素A1抑制胞内体膜质子泵活性后,相对平均荧光强度百分比出现轻微下降。【结论】建立了基于pHrodo dextran分析腺病毒诱导T细胞胞内体膜裂解的新方法。

干扰素诱导跨膜蛋白抗甲型流感病毒的研究进展

干扰素诱导跨膜蛋白(interferon inducible transmembrane proteins,IFITM)是20世纪80年代人们通过小干扰RNA技术研究中发现的一种抗病毒蛋白,目前该蛋白的抗病毒作用及其机制已成为研究热点。迄今的研究发现,IFITM的抗甲型病毒感染作用是通过多种途径实现的。IFTIM能够有效作用于甲型流感病毒表面糖蛋白血凝素与宿主细胞表面受体结合之后、流感病毒包膜与内吞形成的胞内体膜融合之前的流感病毒感染的胞内体途径。在该作用过程中,IFITM下调网格蛋白的表达,扰乱液泡ATP酶(V型ATP酶,vacuolar ATPase,v-ATPase)的酸化功能,破坏胞内体胆固醇的平衡,从而抑制病毒包膜与胞内体膜融合,阻断病毒RNA释放到细胞质中。此外,IFITM也能够影响感染细胞与正常细胞之间的半融合,发挥抑制病毒感染的作用。近年来的研究提示,IFITM通过诱导记忆T细胞选择性高表达该蛋白,继而保护细胞免受甲型流感病毒感染。

TfR-Ubi融合蛋白稳定表达株的构建及功能初测

目的:通过构建转铁蛋白受体-泛素(transferrin receptor-ubiquitin,Tf R-Ubi)融合蛋白,研究胞内体蛋白分选机制。方法:首先扩增小鼠泛素和人源转铁蛋白受体基因,构建HA-Tf R-Ubi表达质粒,转染A431细胞后,采用Western blot验证融合蛋白的表达。在稳定表达Tf R-Ubi的A431和Hrs-KO MEF细胞株中,转铁蛋白刺激,免疫荧光法观察Tf R-Ubi融合蛋白在细胞内的位置。结果:成功构建HA-Tf R-Ubi表达质粒,免疫荧光结果提示Tf R-Ubi融合蛋白被分选到晚期内体(late endosome)/溶酶体,Hrs-KO MEF细胞中Tf R-Ubi融合蛋白分选受到影响。结论:Tf R-Ubi融合蛋白在胞内体被分选,证明了泛素修饰改变了转铁蛋白受体的降解方式,为胞内体分选转运装置(endosomal sorting complex required for transport,ESCRT)分选机制研究提供了实验工具。

Clinical Diagnosis Technique of Goat Pox Disease

[Objective] The aim was to provide theoretical basis for effective prevention of goat pox disease.[Method] 5 cases of infected goats were diagnosed for goat pox with microbiology examination.The poxes on their skin,rumen,reticulum,omasum,abomasum and submandibular lymph nodes,bronchial lymph nodes,lung and spleen were macroscopically and microscopically observed with pathanatomical and histopathological technique.[Result] Poxes on skin mainly showed ashen hemisphere state and gave prominence to the surface of skin; some cases had hemorrhage in the poxes and showed dark purplish red.Poxes on gastric mucosa showed ashen.Cytoplasmic inclusion body could be all observed in epithelial cells of the poxes and macrphages of lymph node,lung and spleen.[Conclusion] Poxes on skin,lung and the surface of gastric mucosa as well as cytoplasmic inclusion body in the epithelial cells of pox and the macrphages of lymphoid organs were the especial pathochanges of goat pox,which could be taken as the proof of goat pox＇s clinic diagnisis.

NPC蛋白研究进展

C型尼曼氏病 (Niem ann-Pick type C,N PC)是一种致命的神经退行性失调疾病 ,常见于儿童早期发作。其特征是患者细胞中胆固醇和鞘糖脂等脂类沉积在晚期胞内体 /溶酶体中。 NPC症状是由 N PC1或 NPC2的功能缺陷引起的。NPC1是一个跨膜蛋白 ,目前认为它参与内吞后的底物向高尔基复合体或胞内其它位点运输 ,在脂类物质循环和平衡控制机制中起重要作用。NPC2是一个可溶性溶酶体蛋白 ,可结合胆固醇。目前尚不清楚 NPC1和 N PC2如何调控溶酶体等细胞器的确切功能。

The BLOC Interactomes Form a Network in Endosomal Transport

With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.

Preparation of BCEC-Astrocyte Co-culturing Membrane Plate Insert

To prepare a hand-made micropore membrane culture plate insert forco-culture. Methods The plate insert was made using plastic centrifuge tube and micropore membrane.After seeding brain capillary endothelial cells (BCECs) on it (under the effect ofastrocyte-conditioned medium), the plate insert was assessed by analysis of trans-endothelialelectrical resistance (TEER). Results The plate insert has a stability of at least 15 d underculture condition. TEER increased significantly under co-culture condition from (66.1 +- 13.3)Ωcm^2 to (182.2 +- 6.7) Ωcm^2. Conclusion This micropore membrane culture plate insert can beeasily made, on which BCEC culture can be successfully performed. Moreover, it is adjustable andrecyclable. It follows that the plate insert is a useful tool for co-culture and the relatedresearch fields.

Possible Mechanism of Effects of Etimicin and Gentamicin on Intracellular Calcium Homeostasis

Aim Intracellular calcium ([Ca^(2+) ]_i) is mainly regulated by mitochondriaand endo-plasmic reticula. This study was carried out to ascertain whether the elementary mechanismof the effects of etimicin (EM) and gentamicin (GM) on [Ca^(2+) ]_i is related to their effects onmitochondrion Ca^(2+) -uptake and endoplasmic reticulum Ca^(2+) -uptake. Methods The effects of GMand EM on [Ca^(2+) ]_i in LLC-PK1 were determined with a fluorescent probe of Fura-2/AM. The effectsof EM and GM on mitochondrion Ca^(2+) -uptake and endoplasmic reticulum Ca^(2+) -uptake weredetermined by isotope indicator (^(45)Ca^(2+) ) . Results EM and GM at the concentration of 1mmol·L^(-1) had no significant effect on [Ca^(2+) ]_i(P. > 0.05) and at 10 mmol·L^(-1)significantly caused [Ca^(2+) ]_i to increase (P < 0.01). EM and GM at 1 mmol·L^(-1) causedmitochondrion Ca^(2+)-uptake to ascend dramatically (P < 0.05) and at 10 mmol·L^(-1) causedmitochondrion Ca^(2+) -uptake to descend significantly. EM and GM at more than 0.34 mrnol·L^(-1)significantly inhibited endoplasmic reticulum Ca^(2+) -uptake (P < 0.05 or 0.01). Conclusion Novariation of [Ca^(2+) ]_i caused by EM and GM at lower concentrations might relate to theequilibrium of their promotion of mitochondrion Ca^(2+) -uptake with their inhibition of endoplasmicreticulum Ca^(2+) -uptake. The elevation of [Ca^(2+) ]_i caused by EM and GM at higherconcentrations might correlate with their inhibition of mitochondrion Ca^(2+) -uptake andendoplasmic reticulum Ca^(2+) -uptake.

A Study of Rabbit Lens Epithelial Cells Survival and Growth on the Rabbit Capsular Bag in Vitro

Objective: To study the proliferation, migration and metaplasm of residual rabbit lens epithelial cells (LECs) after extracapsular cataract extraction(ECCE)based on the rabbit capsular bag model in vitro. Methods: Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression and aspiration of lens fibers, was performed on 20 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on petri dishes. The capsular bags were incubated with Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and monitored for 3 weeks by phase-contrast microscopy, after which light microscopy was performed on them.Results: After a latent period of 2-3 d, outgrowth was observed across the posterior capsule. Growth proceeded rapidly so that the posterior capsule was totally covered by a confluent monolayer of cell at 6-8 day. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also came.Conclusion: This model exhibits many of the in vito characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.

The Involvement of Hydrogen Peroxide in the Regulation of Cell Content Redistribution in the Excised Garlic Scape of Allium sativum

During long term storage at 25℃ in the dark,a large number of cell content were transferred from the senescing scape to the developing cloves,and the transfer from the basal part of scape was earlier than that from the apical part in the excised garlic scape ( Allium sativum var Taichang).Levels of H 2O 2 decreased in the cloves and significantly increased up to 10 folds and then declined quickly in the scape.Levels of H 2O 2 were enhanced early in the basal part of scape.In the treatment of GA 3 at the cloves,levels of H 2O 2 were strongly enhanced in the cloves and inhibited in the scape,coinciding with the distinct inhibition of cell content tansfer.The results indicated that H 2O 2 may be involved in cell content redistribution and its regulation.3-Amino-1,2,4-triazole (AT) is a specific inhibitor of catalase.Effects of AT on cell content redistribution and levels of H 2O 2 were almost similar to those of GA 3,It further proved the above concept.According to the changes of H 2O 2 leves and activities of peroxidase and catalase in the cloves and in the scape,we suggest that the accumulation of H 2O 2 in the scape was transducted from the cloves,and the decline of H 2O 2 level in the scape with GA 3 or AT at the cloves was mainly through the inhibition of H 2O 2 synthesis in the cloves.

Innate immunity in inflammatory bowel disease

The human intestinal tract is home to an enormous bacterial flora. The host defense against microorganisms can be divided into innate and adaptive immunity. The former is the most immediate line of response to immunologic challenges presented by bacteria, viruses, and fungi. The mucosal immune system has evolved to balance the need to respond to pathogens while co-existing with commensal bacteria and food antigens. In inflammatory bowel disease （IBD）, this hyporesponsiveness or tolerance breaks down and inflammation supervenes driven by the intestinal microbial flora. Bacteria contain compounds and are recognized by a variety of receptors, including Toll-like receptors （TLRs） and NODs （a family of intracellular bacterial sensors） and are potent stimuli of innate immune responses. Several mutations in these receptors have been associated with development of IBD.

The mitochondrial Na^＋/Ca^2＋ exchanger may reduce high glucose-induced oxidative stress and nucleotide-binding oligomerization domain receptor 3 inflammasome activation in endothelial cells

Background The mitochondrial Na^＋/Ca^2＋ exchanger, NCLX, plays an important role in the balance between Ca2. influx and efflux across the mitochondrial inner membrane in endothelial ceils. Mitochondrial metabolism is likely to be affected by the activity of NCLX because Ca^2＋ activates several enzymes of the Krebs cycle. It is currently believed that mitochondria are not only centers of energy produc- tion but are also important sites of reactive oxygen species （ROS） generation and nucleotide-binding oligomerization domain receptor 3 （NLRP3） inflammasome activation. Methods ＆ Results This study focused on NCLX function, in rat aortic endothelial cells （RAECs）, induced by glucose. First, we detected an increase in NCLX expression in the endothelia of rats with diabetes mellitus, which was induced by an injection of streptozotocin. Next, colocalization of NCLX expression and mitochondria was detected using confocal analysis. Suppression of NCLX expression, using an siRNA construct （siNCLX）, enhanced mitochondrial Ca^2＋ influx and blocked efflux induced by glucose. Unexpectedly, silencing of NCLX expression induced increased ROS generation and NLRP3 inflammasome activation. Conclusions These findings suggest that NCLX affects glucose-dependent mitochondrial Ca^2＋ signaling, thereby regulating ROS generation and NLRP3 in- flammasome activation in high glucose conditions. In the early stages of high glucose stimulation, NCLX expression increases to compensate in order to self-protect mitochondrial maintenance, stability, and function in endothelial cells.

miRNA studies in in vitro and in vivo activated hepatic stellate cells

AIM: To understand which and how different miRNAs are implicated in the process of hepatic stellate cell (HSC) activation. METHODS: We used microarrays to examine the differential expression of miRNAs during in vitro activation of primary HSCs (pHSCs). The transcriptome changes upon stable transfection of rno-miR-146a into an HSC cell line were studied using cDNA microarrays. Selected differentially regulated miRNAs were investigated by quantitative real-time polymerase chain reaction during in vivo HSC activation. The effect of miRNA mimics and inhibitor on the in vitro activation of pHSCs was also evaluated.RESULTS: We found that 16 miRNAs were upregulated and 26 were downregulated significantly in 10-d in vitro activated pHSCs in comparison to quiescent pHSCs. Overexpression of rno-miR-146a was characterized by marked upregulation of tissue inhibitor of metalloproteinase-3, which is implicated in the regulation of tumor necrosis factor-α activity. Differences in the regulation of selected miRNAs were observed comparing in vitro and in vivo HSC activation. Treatment with miR-26a and 29a mimics, and miR-214 inhibitor during in vitro activation of pHSCs induced significant downregulation of collagen type Ⅰ transcription. CONCLUSION: Our results emphasize the different regulation of miRNAs in in vitro and in vivo activated pHSCs. We also showed that miR-26a, 29a and 214 are involved in the regulation of collagen type I mRNA.

A novel gene delivery system targeting cells expressing VEGF receptors

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-β-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) andhuman malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with theGene delivery system targeting VEGF receptors relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

Objective To investigate the possible involvement of erythropoietin （EPO）/erythropoietin receptor （EPOR） system in neovascularization and vascular regeneration in diabetic retinopathy （DR）. Methods EPOR positive circulating progenitor cells （CPCs： CD34^＋） and endothelial progenitor cells （EPCs： CD34^＋KDR^＋） were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes （n=7）, non-prolif- erative DR （NPDR, n=7）, proliferative DR （PDR, n=8）, and PDR complicated with diabetic nephropathy （PDR-DN, n=7）. Results The numbers of EPOR^＋ CPCs and EPOR^＋ EPCs were reduced remarkably in NPDR corn pared with the control group （both P（0.01）, whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR^＋ CPCs in CPCs （NPDR vs. control, P（0.01） and that of EPOR^＋ EPCs in EPCs （NPDR vs. control, P〈0.05）. Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the impaired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR^＋ EPCs associated with ischemia.

Endocytic regulation of TGF-β signaling

Transforming growth factor-β （TGF-β） signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-β receptors are internalized into the cell, and this process plays an important regulatory role in TGF-β signaling. It is well documented that TGF-β receptors are endocytosed via clathrin-coated vesicles as TGF-β endocytosis can be blocked by potassium depletion and the GTPasedeficient dynamin K44A mutant. TGF-β receptors may also enter cells via cholesterol-rich membrane microdomain lipid rafts/caveolae and are found in caveolin-l-positive vesicles. Although receptor endocytosis is not essential for TGF-β signaling, clathrin-mediated endocytosis has been shown to promote TGF-β-induced Smad activation and transcriptional responses. Lipid rafts/caveolae are widely regarded as signaling centers for G protein-coupled recep- tors and tyrosine kinase receptors, but they are indicated to facilitate the degradation of TGF-β receptors and there- fore turnoff of TGF-β signaling. This review summarizes current understanding of TGF-β receptor endocytosis, the possible mechanisms underlying this process, and the role of endocytosis in modulation of TGF-β signaling.

Effect of JIANPI HUOXUE decoction on inflammatory cytokine secretion pathway in rat liver with lipopolysaccharide challenge

AIM： To evaluate the effect of Chinese traditional medicinal prescription, JIANPI HUOXUE decoction （JHD） on cytokine secretion pathway in rat liver induced by lipopolysaccharide （LPS）. METHODS： Twenty-four male SD rats were divided into normal group （n = 4）, model group （n = 10） and JHD group （n = 10） randomly. Rats in model group and JHD group were administrated with normal saline or JHD via gastrogavage respectively twice a day for 3 d. One hour after the last administration, rats were injected with LPS via tail vein, 50 μg/kg. Simultaneously, rats in normal group were injected with equivalent normal saline. After LPS stimulation for 1.5 h, serum and liver tissue were collected. Pathological change of liver tissues was observed through hematoxylineosin （H.E.） staining. Tumor necrosis factor alpha （TNF-α） in serum were assayed by enzyme linked immunosorbent assay （ELISA）. The protein expression of TNF-α, phosphorylated inhibit-κB （p-κB） and CD68 in liver were assayed by Western blot. The distribution of CD68 protein in liver was observed through immunohistochemical staining. The mRNA expression of TNF-α, interleukin-6 （IL-6）, CD14, toll-like receptor 2 （TLR2） and TLR4 in liver were assayed by real-time RT-PCR.RESULTS： Predominant microvesicular change, hepatocyte tumefaction and cytoplasm dilution were observed in liver tissues after LPS administration as well as obvious CD68 positive staining in hepatic sinusoidal. After LPS stimulation, serum TNF-α （31.35 ± 6.06 vs 12225.40 ± 9007.03, P 〈 0.05）, protein expression of CD68 （1.13 ± 0.49 vs 3.36 ±1.69, P 〈 0.05）, p-IκB （0.01 ±0.01 vs 2.07 ＋ 0.83, P 〈 0.01） and TNF-α （0.27 ± 0.13 vs 1.29 ± 0.37, P 〈 0.01） in liver and mRNA expression of TNF-α （1.96 ± 2.23 vs 21.45 ±6.00, P 〈 0.01）, IL-6 （4.80 ± 6.42 vs 193.50 ± 36.36, P 〈 0.01） and TLR2 （1.44 ± 0.62 vs 4.16 ± 0.08, P 〈 0.01） in liver were also increased significantly. These pathological changes were all improved in .1HD group. On the other hand, TLR4 mRNA （1.22 ± 0.30 vs 0.50 ± 0.15, P 〈 0.05） was down-regulated and CD14 mRNA increased but not significantly after LPS stimulation. CONCLUSION： JHD can inhibit cytokine secretion pathway induced by LPS in rat liver, which is probably associated with its regulation on CD68, p-IκB and endotoxin receptor TLR2.

Optimization for Production of Intracellular Polysaccharide from Cordyceps ophioglossoides L2 in Submerged Culture and Its Antioxidant Activities in vitro

Cordyceps ophioglossoides is a valuable traditional medicinal material.We have found that intracellular polysaccharide(IPS) is the major biologically active ingredient in Cordyceps ophioglossoides.This study is the first time to optimize the yield of IPS from Cordyceps ophioglossoides.The optimal medium for IPS production consists of glucose 54.50 g·L·1,yeast powder 25.50 g·L·1,NaH2PO4 0.4 g·L·1 and K2HPO4 0.4 g·L·1.The suggested culture conditions are 24 ℃,initial pH 4.5 with a rotary speed of 120 r·min·1 for 168 h.The yield of IPS is 737.93 mg·L·1,which is 50% higher than the yield under the conditions prior to optimization.The anti-oxidative activities of IPS in Cordyceps ophioglossoides L2 are also characterized using various in vitro assay.The anti-oxidative activity may explain the reason why IPS from Cordyceps ophioglossoides can be used to fight against neurodegenerative dis-eases and menopausal symptoms.

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS： Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS： Of 29 live-born recipients, 19 had the presence of human CD45^＋ cells in peripheral blood （PB） detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues （i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin） examined at the time of tissue collection, as confirmed by detecting human 132- microglobulin expression using immunohistochemistry. Tn this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CEHS-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION： Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.