Safety evaluation of tissue engineered medical devices using normal human mesenchymal stem cells

Several recent studies demonstrated the potential of bioengineering using somatic stem cells in regenerative medicine. Adult human mesenchymal stem cells (hMSCs) derived from bone marrow have the pluripotency to differentiate into cells of mesodermal origin, e.g., bone, cartilage, adipose, and muscle cells; they, therefore, have many potential clinical applications. On the other hand, stem cells possess a self-renewal capability similar to cancer cells. For safety evaluation of tissue engineered medical devices using normal hMSCs, in this study, we investigated the expression levels of several genes that affect cell proliferation in hMSCs during in vitro culture. We focused on the relationship between the hMSC proliferation and their transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and cellular senescence was observed for about 3 months. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. The mRNA expressions of Smad3 increased, but those of c-myc and nucleostemin decreased with the length hMSCs were in in vitro culture. In addition, the expression profiles of the genes which regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.     

Shh通路介导损伤后反应性星形胶质细胞获得干细胞潜能

目的 探讨Shh信号通路对大脑皮质反应性星形胶质细胞（RAS）获得神经干细胞潜能的影响。方法 体外原代培养SD大鼠大脑皮质星形胶质细胞,以肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1α和C1q三种因子诱导炎症型RAS,划痕实验制备创伤型RAS,使用免疫荧光染色法观察细胞形态及纯度。实验分为普通星形胶质细胞组（对照组）、炎症组、划痕组（创伤组）。炎症组培养至干预后 24 h,对照组和划痕组培养至第 5天进行免疫荧光染色观察巢蛋白（Nestin）阳性细胞,运用Western blot技术检测不同损伤模型的细胞中Shh蛋白的表达量。之后各组细胞培养 至第7天更换神经干细胞培养基,将前述3组各自再分为激活组（各组细胞更换神经干细胞培养基后添加Shh激活剂SAG）和抑制组（各组细胞更换神经干细胞培养基后添加Shh抑制剂环巴胺）两个亚组,培养14 d后采用qRT-PCR检测各亚组 RAS源性神经干细胞相关基因的表达量。结果 细胞损伤模型中,免疫荧光染色证明各组细胞中划痕组Nestin阳性细胞明显多于炎症组和对照组。Western blot结果显示2组损伤细胞中划痕组Shh蛋白表达量高于炎症组（P＜0.05）。更换神经干细胞培养基后,激活组中 2 组损伤细胞的干细胞相关基因 Nestin、Pax-6、Sox-2、Oct-4 和Map-2的表达水平均有所升高,其中由划痕诱导的RAS神经干细胞相关基因Nestin、Pax-6、Sox-2、Oct-4和Map-2的表达水平高于炎症诱导的 RAS神经干细胞和普通星形胶质细胞（P＜0.01）;添加 Shh抑制剂后,3组上述基因表达量皆明显降低,但划痕组的表达量仍高于其他 2组（P＜0.01）。结论 在体外,Shh信号通路可以介导损伤后大脑皮质RAS获得干细胞潜能。     

Effects of culture conditions on estrogen-mediated hepatic in vitro gene expression and correlation to in vivo responses

Refinement of in vitro systems for predictive toxicology is important in order to develop high-throughput early toxicity screening assays and to minimize animal testing studies. This study assesses the ability of mouse Hepa-1c1c7 hepatoma cell model under differing culture conditions to predict in vivo estrogen-induced hepatic gene expression changes. Custom mouse cDNA microarrays were used to compare Hepa-1c1c7 temporal gene expression profiles treated with 10 nM 17beta-estradiol (E2) in serum-free and charcoal-stripped serum supplemented media at 1, 2, 4, 8, 12, and 24 h. Stripped serum supplemented media increased the number gene expression changes and overall responsiveness likely due to the presence of serum factors supporting proliferation and mitochondrial activity. Data from both experiments were compared to a gene expression time course study examining the hepatic effects of 100 microg/kg 17alpha-ethynyl estradiol (EE) in C57BL/6 mice at 2, 4, 8, 12, 18, and 24 h. Only 18 genes overlapped between the serum-free and in vivo studies, whereas 238 genes were in common between Hepa-1c1c7 cells in stripped serum data and C57BL/6 liver samples. Stripped serum cultured cells exhibited E2-elicited gene expression changes associated with proliferation, cytoskeletal re-organization, cholesterol uptake and synthesis, increased fatty acid beta-oxidation, and oxidative stress, which correlated with in vivo hepatic responses. These results demonstrate that E2 treatment of Hepa-1c1c7 cells in serum supplemented media modulate responses in selected pathways which appropriately model estrogen-elicited in vivo hepatic responses.     

Effects of cell culture conditions on primary rat hepatocytes-cell morphology and differential gene expression

We incubated primary rat hepatocytes on collagen monolayer as well as in collagen sandwich cultures with serum-containing or serum-free medium formulations. Morphological monitoring of hepatocytes revealed that hepatocytes cultured on collagen monolayer adopted their polygonal shape and started to create aggregates earlier than sandwich-cultured cells. Bile canaliculi-like structures were observed in every cell culture system but were more prominent in serum-free cultures. Hepatocytes in collagen-sandwich configuration and serum-free medium were the most viable after 72 h of culture, still displaying polygonal shape, clear cytoplasm and stable canaliculi-like structures. Differential gene expression patterns were determined for each cell culture condition using quantitative TaqMan Low Density Arrays (LDA). Gene expression analysis revealed distinct profiles in monolayer versus sandwich cultures and in particular in serum-free versus serum-containing culture medium. The hepatocytes cultured in the collagen-sandwich with serum-free medium showed the least variation in expression values over time. Importantly, stress markers were not induced in the serum-free sandwich culture, in contrast to the monolayer and the serum-containing sandwich cultures. Additionally, expression of the investigated cytochrome P450 genes was maintained in the serum-free monolayer and the sandwich cultures. In conclusion, culturing primary rat hepatocytes in a sandwich between two layers of gelled collagen and in a serum-free medium formulation, appears to be most suitable for long-term in vitro hepatotoxicity screening.     

Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.     

Cell-based tissue engineering therapies: the influence of whole body physiology

A technology has been developed to isolate a developmentally and phenotypically homogeneous population of pluripotent human mesenchymal stem cells (hMSCs) from adult bone marrow and mitotically expand these cells in culture. These hMSCs have osteoblasts as one of their potential developmental end-stage phenotypes, and, in addition to their osteogenic potential, these hMSCs synthesize and secrete a variety of macromolecules that are known regulators of osteoclast differentiation and activity. In this review, data are presented that demonstrate the phenotypic and developmental homogeneity of the cells in hMSC cultures, as well as their ability to differentiate along multiple phenotypic pathways and serve as regulatory cells for hematopoietic and bone-resorbing cells. In addition, a logic and preliminary data are presented that support the use of hMSCs in the prevention and treatment of age-related and postmenopausal osteoporosis. Since hMSC differentiation and phenotypic expression are controlled by regulatory molecules synthesized and secreted by a variety of local and systemic mechanisms, the issue of whole organism physiology is addressed in considering tissue engineering logics.     

基因芯片技术研究Z24对人HepG2细胞基因表达的影响

目的:利用芯片技术观察Z24对人HepG2细胞基因表达的影响,以探讨其毒性作用的分子机制.方法:不同浓度处理培养的HepG2细胞,H-E染色观察细胞形态;抽提细胞RNA,利用荧光标记ddUTP逆转录制备cDNA探针,与毒理表达谱基因芯片杂交,并对Cy3,Cy5荧光信号做扫描分析.结果:Z24处理实验组细胞呈现凋亡形态;芯片扫描显示在IC20浓度情况下,Z24作用HepG2细胞有15个基因发生显著的变化,其中细胞凋亡通路有关的基因TNFRSF5发生明显上调,与肝功能有关的基因(AKR1C2和INSIG1)发生明显的下调.随Z24浓度的增加,发生改变的基因数增加到244个,其中109个基因发生明显的上调,135个基因发生明显的下调,下调的基因多与细胞增殖调控功能、氨基酸、能量和脂肪代谢有关;而细胞凋亡通路中几个重要关键蛋白(DFFB,CASP6,DR4)的基因明显上调,并且与肝脏有关的基因(INSIG1,PHKA2,HPX)也发生了明显的改变.结论:Z24可以诱导HepG2细胞凋亡,并可能是其产生毒性的重要机制之一;毒性作用的靶器官可能是肝脏.     

同型半胱氨酸对体外大鼠神经干细胞增殖及Notch信号通路相关基因表达的影响

【摘要】目的 研究同型半胱氨酸（Hcy）对体外大鼠神经干细胞(NSCs)增殖及Notch信号通路相关基因Notch1和Hes1mRNA表达的影响。方法 采用无血清培养原代培养新生大鼠NSCs,细胞分为对照组(Hcy-C)、Hcy低剂量组（Hcy-L）、Hcy中剂量组（Hcy-M）、Hcy高剂量组（Hcy-H）。采用免疫组化法检测Nestin、β-tubulin Ⅲ及GFAP的表达对NSCs进行鉴定,MTT法检测细胞增殖情况,Real-time PCR检测Notch1、Hes1基因mRNA表达。结果 无血清培养条件下,所培养细胞形成大量呈Nestin抗原阳性细胞组成的神经球;诱导分化6d后,形成β-tubulin Ⅲ表达呈阳性的神经元和GFAP表达呈阳性的星形胶质细胞;Hcy干预组细胞活力低于对照组,且有剂量依赖性(P<0.05)。Hcy干预组Hes1mRNA表达低于对照组(P<0.05)。Hcy-H组Notch1mRNA表达低于其他3组(P<0.05);Hcy-M组Notch1mRNA表达低于对照组和低剂量组(P<0.05)。结论 Hcy能抑制大鼠新生鼠NSCs的增殖,可能与下调Notch信号通路中Notch1和Hes1mRNA表达有关     

模拟微重力环境对小鼠成纤维细胞增殖和创伤修复相关蛋白基因表达的影响

目的：研究微重力细胞培养系统( RCCS)模拟微重力环境下小鼠成纤维细胞( L929细胞)增殖及创伤修复相关蛋白基因表达的变化。方法将L929细胞随机分为微重力组和正常重力对照组,分别在RCCS中培养3、5、7天,流式细胞仪检测细胞周期,实时荧光定量聚合酶链反应检测创伤修复蛋白mRNA表达水平。培养第7天取两组细胞-微载体悬液观察细胞微丝结构形态变化。结果微重力组L929细胞的G1期细胞在第3、5、7天明显少于正常重力对照组(P<0．05);与正常重力对照组相比,微重力组S期细胞在第3、5天有所增加,第7天减少,G2/M期细胞在培养第3、7天升高,培养第5天下降(P<0．05)。培养第3天微重力组Ⅰ型胶原蛋白(ColⅠ)和Ⅲ型胶原蛋白(ColⅢ)、β转化生长因子( TGF-β) mRNA表达低于正常重力对照组( P<0．05);培养第5天微重力组Caspase-3 mRNA表达低于正常重力对照组,ColⅠ、TGF-β及Bcl-2 mRNA表达均高于正常重力对照组(P<0．05);培养第7天微重力组创伤修复相关蛋白mRNA表达均高于正常重力对照组( P<0．05)。模拟微重力环境下培养7 d对L929细胞微丝结构有影响但两组间差异无明显差别。结论模拟微重力环境能改变L929细胞周期转化、增强细胞增殖能力,并影响创伤修复相关蛋白的表达。     

姜黄素对喉癌细胞增殖的作用以及对细胞Bcl-2和Bax基因表达的影响分析

目的 探讨姜黄素治疗喉癌对细胞Bcl-2和Bax基因表达的影响.方法 在姜黄素培养基中进行Hep-2细胞的培养,依据不同的培养时间,分别设为两组(24h、48h).对细胞的增殖采用MTT法进行检测,对Bcl-2、Bax基因的表达采用RT-PCR法进行检测.结果 48 h培养组与24 h培养组在不同的姜黄素浓度下相比,差异均具有显著统计学意义(P＜0.05).不同的培养时间下,三组Bcl-2以及Bax浓度具有显著差异,组间比较差异具有统计学意义(P＜0.05).结论 姜黄素对Hep-2细胞增殖有显著的时间-剂量依赖性抑制作用,其浓度越高,Bcl-2浓度越低,Bax浓度越高,可起到有效的抗癌作用.     

Biological characterization of long-term cultured human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical applications. The reason is that they may have the plasticity needed to differentiate into multiple lineages and the ability to expand ex vivo. For the therapeutic applications of hMSCs to be of practical use, it is crucial to assess the efficacy and safety of hMSCs in long-term ex vivo expansion. In this study, we cultured hMSCs by population doubling (PD) 60, and investigated their growth, osteogenic and adipogenic differential abilities, change of surface markers, telomerase activity, telomere length, and gene expression related to tumorigenesis. An in vivo tumorigenesis assay was also carried out. In long-term expanded hMSCs, the cells became aged above PD 30 and their adipogenic and osteogenic differentiation potential decreased. Telomerase activity unchanged whereas telomere length decreased and karyotypes were not changed. Gene expressions related to tumorigenesis decreased in proportion as the PD of hMSCs increased. In vivo transplantation of long-term cultured hMSCs to nude mice did not result in tumor formation. These findings suggest that diverse tests for cellular therapy should be considered during the ex vivo culture of hMSCs, particularly when a prolonged and extended propagation period is required.     

Detailed examination of cartilage formation and endochondral ossification using human mesenchymal stem cells

1. Cartilage formation is one of the most complex processes in biology. The aim of the present study was to produce a simplified in vitro system to resolve its complexities. 2. Human mesenchymal stem cells (hMSC) were maintained in alginate beads with a chondrogenesis-induction medium containing 10 ng/mL transforming growth factor (TGF)-beta3. 3. At days 0, 2, 4, 8, 12, 16 and 19 of culture, we examined the cells using a light microscope and a transmission electron microscope. We also evaluated the cells using immunocryo-ultramicrotomy. 4. The present study demonstrated that hMSC produced numerous extracellular matrices containing abnormal collagen fibres following their exposure to a chondrogenesis-induction medium in alginate beads. At this time, calcification was detected by alizarin red staining and electron-dense particles, composed of hydroxyapatite, appeared in both the cytoplasm and the extracellular spaces. 5. In addition immunocryo-ultramicrotomy revealed that collagen type II, type X and proteoglycan were prominent and that osteocalcin was detectable at day 2. During 8-16 days of culture, collagen type X maintained its strong expression and the expression of osteocalcin increased markedly. In contrast, the expression of collagen type II and proteoglycan decreased with time. 6. These findings demonstrate that hMSC rapidly differentiate into chondrocytes expressing collagen type II and proteoglycan. 7. The expression of collagen type II and proteoglycan then dropped and the activity of collagen type X was the same as before (4-8 days). As a result, the cells developed into the next cell type, so-called hypertrophic chondrocytes. Finally, both osteocalcin activity and the calcification of cell bodies and extracellular matrices became evident, indicating endochondral ossification. Thus, we conclude that hMSC rapidly differentiate into chondrocytes, followed by the development of hypertrophic chondrocytes. Endochondral ossification is the final form in this culture. 8. The findings of the present study indicate that our three-dimensional culture is a convenient in vitro model for the investigation of the regulatory mechanisms of cartilage formation and endochondral ossification.     

丹参对体外培养大鼠成纤维样滑膜细胞bcl-2和bax表达的影响

目的：探讨丹参对体外培养大鼠成纤维样滑膜细胞增殖及 bax、bcl-2表达的影响。方法取浓度为1.5＆#215;10-5个/ml的 RSC-364成纤维样滑膜细胞悬液100μl加入细胞培养板,培养24 h,加入含不同浓度丹参的培养液,采用 MTT法分析丹参对成纤维样滑膜细胞增殖抑制作用及其时效关系;免疫组化法检测 bcl-2、bax表达情况。结果丹参可明显抑制大鼠成纤维样滑膜细胞增殖,并显示出一定的量效和时效关系。丹参可显著上调大鼠成纤维样滑膜细胞 bax表达,下调 bcl-2表达,与空白对照组比较差异有统计学意义（ P 〈0.05）。结论丹参对体外培养大鼠成纤维样滑膜细胞的增殖具有抑制效应,其作用机制可能与调控 bax、bcl-2表达有关。     

Identification and characterization of unique tumoricidal genes in rat umbilical cord matrix stem cells

Rat umbilical cord matrix stem cells (UCMSC) have been shown to exhibit a remarkable ability to control rat mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in Mat B III growth attenuation, total RNA was extracted from the naive rat UCMSC alone and those cocultured with Mat B III in Transwell culture dishes. Gene expression profiles of naive rat UCMSC alone and those cocultured with Mat B III cells were investigated by microarray analysis using an Illumina RatRef-12 Expression BeadChip. The comparison of gene expression profiles between untreated and cocultured rat UCMSC identified five upregulated candidate genes (follistatin (FST), sulfatase1 (SULF-1), glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and adipocyte differentiation-related protein (ADRP)) and two downregulated candidate genes (transforming growth factor, beta-induced, 68 kDa (TGFβI) and podoplanin (PDPN)) based upon the following screening criteria: (1) expression of the candidate genes should show at least a 1.5-fold change in rat UCMSC cocultured with Mat B III cells; (2) candidate genes encode secretory proteins; and (3) they encode cell growth-related proteins. Following confirmation of gene expression by real-time PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products or addition of gene-specific siRNA's individually in cocultures of 1:20 rat UCMSC:Mat B III cells significantly increased cell proliferation, implying that these gene products are produced under the cocultured condition and functionally attenuate cell growth. Immunoprecipitation followed by Western blot analysis demonstrated that these proteins are indeed secreted into the culture medium. Individual overexpression of these three genes in rat UCMSC significantly enhanced UCMSC-dependent inhibition of cell proliferation in coculture. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes, and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumors.     

Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of alpha1- or beta-adrenoceptors, a DNA microarray was used. 2. First, the existence of alpha1- and beta-adrenoceptors in A7r5 aortic smooth muscle cells was confirmed by radioligand binding. Then, the inhibitory effects of phenylephrine (an alpha1-adrenoceptor agonist) and isoproterenol (a beta-adrenoceptor agonist) on the proliferation of A7r5 cells were determined by [3H]-thymidine incorporation. 3. The A7r5 cells were treated with 10 micromol/L phenylephrine or 1 micromol/L isoproterenol for 24 h and changes in gene expression were detected with the DNA microarray. Only 14 and 20 genes were identified after treatment of cells with phenylephrine and isoproterenol, respectively, and most genes displayed decreased expression. The changed genes could be grouped into five major functional categories: cell signalling/communication, cell structure/motility, cell/organism defence, gene/protein expression and metabolism. The gene expression profile in response to the activation of alpha1-adrenoceptors was very different from that following activation of beta-adrenoceptors. Interestingly, many phenylephrine-responsive genes were associated with metabolism, whereas many isoproterenol-responsive genes encoded cell signalling and structure proteins. This means that adrenoceptors may modulate multiple aspects of biological function in vascular smooth muscle cells. 4. Collectively, the activation of both alpha1-adrenoceptors (with phenylephrine) and beta-adrenoceptors (with isoproterenol) inhibited the proliferation of A7r5 cells, but microarray data revealed that the mechanisms may be different: the activation of alpha1-adrenoceptors could induce the expression of metabolic genes, resulting in the inhibition of proliferation, whereas activation of beta-adrenoceptors altered the expression of genes that encoded cell signalling and structure proteins to inhibit cell proliferation.     

人剪切修复基因XPD对人肝癌SMMC-7721细胞中Ets-1和Cdk6基因的调控作用

目的:观察野生型人剪切修复基因XPD转染入人肝癌细胞SMMC-7721后,细胞内Ets-1和Cdk6基因的表达变化及对SMMC-7721肝癌细胞增殖的影响。方法:将人工合成的pEGFP-N2-XPD重组质粒通过Lipofectamine 2000TM转染SMMC-7721细胞。设重组质粒转染细胞SMMC-7721-pEGFP-N2-XPD(XPD)组、空载质粒转染细胞SMMC-7721-pEGFP-N2(N2)组、脂质体组、无转染空白对照组。分别用逆转录-聚合酶链反应(RT-PCR)和蛋白印迹法(Western blot)检测细胞中XPD、Ets-1、Cdk6基因mRNA和蛋白质的表达量,并用流式细胞仪检测细胞周期变化,四甲基偶氮唑盐微量酶反应比色法(MTT)检测各组细胞的增殖活力。结果:XPD组中的XPD的mRNA和蛋白质表达较其他3组明显增高(P<0.001),而Ets-1、Cdk6 mRNA和蛋白质表达较其他3组明显减少(P<0.001)。转染pEGFP-N2-XPD重组质粒后细胞停滞在G1期,难于进入S期。转染了野生型XPD的SMMC-7721细胞增殖能力减弱。结论:XPD基因可能通过抑制Ets-1、Cdk6基因的表达影响肝癌细胞的生长。     

Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.     

PD-L1过表达在DENV-2诱导血管内皮细胞自噬和凋亡中的作用机制

目的　探讨PD-L1过表达在Ⅱ型登革病毒（DENV-2）影响血管内皮细胞自噬和凋亡中的作用及机制。方法　以血管内皮细胞EAhy926为研究对象,以含梯度DENV-2（1×108~2×109 pfu/L）的无血清培养基处理细胞24 h和48 h,同时以无血清培养基处理细胞作为对照组,无血清培养基加入空白孔作为调零孔,MTT法检测细胞增殖活力。以PD-L1过表达慢病毒液和空载慢病毒液处理细胞,分别设空载对照组和转染组。DENV-2（1.2×109 pfu/L）处理细胞12、24、48 h作为DENV-2 12 h组、DENV-2 24 h组及DENV-2 48 h组,同时设置对照组（无血清培养基处理）;另以DENV-2处理空载对照组和转染组48 h,以蛋白印迹法检测LC3B、Beclin-1及Bcl-2蛋白表达变化,流式细胞术检测各组细胞凋亡率。结果　1×108~2×109 pfu/L DENV-2可剂量依赖性抑制EAhy926细胞增殖,DENV-2 24 h和48 h的半数抑制浓度（IC50）分别是1.8×109 pfu/L和1.2×109 pfu/L。与对照组比较,DENV-2 12 h组、DENV-2 24 h组及DENV-2 48 h组LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达上调,Bcl-2蛋白表达下调,细胞总凋亡率增加（均P＜0.05）。与空载对照组比较,转染组PD-L1蛋白表达上调（P＜0.01）。DENV-2处理48 h,与空载对照组相比,转染组LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达下调,Bcl-2蛋白表达上调,细胞总凋亡率降低（均P＜0.01）。结论　DENV-2通过上调LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白表达及下调Bcl-2蛋白表达,从而诱导EAhy926细胞自噬和凋亡,而过表达PD-L1可抵抗DENV-2的诱导效应。