体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的：研究体外大鼠骨髓间充质干细胞（Bone marrow-derived mesenchymal stem cells, BMSCs）在缺血缺氧条件下发生凋亡的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞（BMSCs）在缺血缺氧条件下培养,通过Annexin V／PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组（无缺血缺氧）与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K（Phosphoinositide-3kinase）／Akt（ProteinkinaseB,PKB）信号通路被激活（P〈0．05）;同时缺血缺氧组与缺血缺氧＋PI3K／Akt抑制剂（LY294002）组比较,缺血缺氧＋PI3K／Akt抑制剂（LY294002）组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制（P〈0．05）。结论：PI3K／Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

恒磁场抑制缺血缺氧条件下大鼠骨髓间充质干细胞凋亡

目的:研究恒磁场对体外缺血缺氧培养条件下大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs) 凋亡的影响并探讨其作用机制.方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs).经形态学和流式细胞仪检测MSCs表面标志物鉴定后,将骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过TUNEL检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中特定蛋白质的变化.结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功.②缺血/缺氧组与缺血/缺氧+磁场组比较,缺血缺氧组的凋亡率显著性增加,Akt磷酸化水平显著上升(P＜0.05).提示恒磁场可以使PI3K(Phosphoinositide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活而抑制凋亡的发生.结论:恒磁场通过激活PI3K/Akt信号通路抑制体外缺血缺氧条件下培养的骨髓间充质干细胞的凋亡.     

磷脂酰肌醇3激酶抑制剂LY294002对缺血缺氧心肌细胞的作用研究

目的:明确P13K/Akt信号通路在缺血缺氧心肌细胞损害中的作用。方法:建立心肌细胞缺血缺氧模型,施加磷脂酰肌醇3激酶抑制剂LY294002干预,观察心肌细胞活力、培养液中乳酸脱氢酶(LDH)含量及碘化丙啶(PI)染色阳性细胞比例的变化。结果:模拟缺血缺氧后细胞活力下降,LDH及PI染色阳性细胞比例显著增加。LY294002干预复合缺血缺氧后,细胞活力急剧下降,LDH含量及PI染色阳性细胞比例进一步显著增加(P<0.01)。结论:应用LY294002加重了缺血缺氧对心肌细胞的损伤效应,提示PI3K/Akt通路参与了缺血缺氧心肌细胞的内源性保护反应,减轻了缺血缺氧损害。     

TGF-β1通过激活Smad信号途径抑制PI3K/AKT信号介导的BMSC成脂分化

转化生长因子β1 (TGF-β1) 是参与骨髓间充质干细胞（BMSCs）脂肪定向分化的重要调节因子,其具体的调节机制尚不清楚. 本研究证明,BMSCs在体外分化为脂肪细胞的过程中, TGF-β1的基因表达显著下调,重组TGF-β1能够抑制BMSCs体外脂肪细胞定向分化,其分化的标志蛋白C/EBPβ和αP2的表达水平显著降低. TGF-β1在激活Smad信号通路的同时,还抑制胰岛素(脂肪分化的主要诱导剂)对PI3K/Akt信号通路的激活.加入Smad特异性阻断剂后,C/EBPβ和αP2的诱导表达恢复正常,同时PI3K/Akt信号通路的活化亦得以恢复. 结果提示,TGF-β1可通过Smad信号通路干扰脂肪细胞分化的核心信号通路-PI3K/Akt的活化,从而实现对BMSCs脂肪分化的抑制.该研究结果为肥胖等导致的心血管疾病或Ⅱ型糖尿病等的临床治疗提供有价值的参考.     

胰岛素样生长因子1（IGF-I）通过PI-3K/Akt 途径对结肠癌细胞 凋亡的影响

目的:探讨胰岛素样生长因子-1(IGF-I)通过磷酯酰肌醇3-激酶/蛋白激酶B(PI-3K/Akt)信号通路对结肠癌细胞株SW480凋亡率的影响及其凋亡抑制蛋白survivin表达水平的变化。方法:培养结肠癌SW480细胞株,实验分成三组:未加IGF-I空白组、IGF-I刺激组、IGF-I+LY294002阻断组,检测阻断剂LY294002是否阻断PI-3K/Akt通路(Western Blot检测三组P-Akt表达情况);Western Blot及免疫荧光观察三组survivin蛋白表达变化;MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果:Western blot结果显示LY294002可抑制IGF-I诱导的p-Akt的表达(P<0.05);阻断IGF-I诱导的PI-3K/Akt通路后MTT显示结肠癌细胞SW480增殖抑制率升高(P<0.05),流式细胞术分析显示凋亡率明显上升(P<0.05);Western blot及免疫荧光结果显示LY294002可抑制IGF-I诱导的survivin的表达(P<0.05)。结论:IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

胰岛素生长因子-1(IGF-1)通过PI3K/Akt通路抑制骨髓间充质干细胞(BMSCs)凋亡

该研究通过构建高表达胰岛素生长因子-1(insulin-like growth factor-1,IGF-1)的异常微环境模型探讨IGF-1对骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)凋亡的影响及可能的作用机制,为BMSCs作为载体在靶向治疗肿瘤过程中的安全应用提供前期实验基础。取分离纯化的大鼠BMSCs,用流式细胞术鉴定BMSCs表面标志,将实验分为4组:BMSCs空白对照组、IGF-1刺激组、IGF-1+LY294002阻断剂组和IGF-1+MK2206阻断剂组。加药处理2周后,CCK-8法检测细胞增殖能力;Hoechst 33342染色观察细胞核形态及凋亡比例;流式细胞术检测细胞凋亡和细胞线粒体膜电位变化;RT-qPCR检测细胞Akt、Bad、Bcl-xl、c-Myc、STAT3的m RNA水平;Western blot检测细胞Akt、p-Akt、Bad、p-Bad、Bcl-xl、c-Myc、STAT3、p-STAT3的蛋白水平。结果显示:IGF-1刺激组细胞与BMSCs空白对照组比较增殖率升高,凋亡率减低;IGF-1刺激组Bad、Bcl-xl、cMyc、STAT3的mRNA的表达均显著高于BMSCs空白对照组(P0.05);IGF-1刺激组p-Akt、Bad、p-Bad、Bcl-xl、c-Myc、STAT3、p-STAT3的蛋白表达水平显著高于BMSCs空白对照组(P0.05)。而阻断剂组细胞(IGF-1+LY294002阻断剂组、IGF-1+MK2206阻断剂组)与IGF-1刺激组比较增殖率均降低,凋亡率增高,相关分子m RNA和蛋白表达水平均明显降低。以上结果表明,IGF-1能通过活化PI3K/Akt通路,激活下游增殖和凋亡相关分子,从而促进BMSCs增殖,抑制BMSCs凋亡。     

PI3K通路抑制剂LY294002对人脑胶质瘤细胞株U87细胞衰老和凋亡的影响

目的:PI3K/Akt信号通路是与胶质瘤发生发展密切相关的核心通路之一,LY294002是该通路的特异性抑制剂。本研究通过探讨PI3K通路抑制剂LY294002对U87胶质瘤细胞系细胞衰老及凋亡的影响,从而为胶质瘤患者治疗的新策略奠定理论基础。方法:将体外培养的人脑胶质瘤U87细胞株分为DMSO处理的对照组和LY294002(100μM)处理的实验组,采用β-半乳糖苷酶染色和流式细胞术的方法,分别检测并比较两组肿瘤细胞衰老和凋亡的情况。结果:LY294002处理组U87胶质瘤细胞的衰老指数(32.20±4.46%)显著高于DMSO对照组(3.40±1.61%,t=6.254,P0.001)。另外,与DMSO对照组相比,凋亡蛋白caspase-3mRNA的表达在LY294002处理组胶质瘤细胞中显著上调(t=8.923,P0.05)。LY294002处理组肿瘤细胞的凋亡指数(80.10±4.832%)明显高于DMSO对照组(4.260±1.073%,t=8.923,P0.05)。结论:LY294002既能够诱导肿瘤细胞衰老,又能够诱导肿瘤细胞凋亡,然而其诱导胶质瘤细胞凋亡的能力占据主导地位,为其发挥抗胶质瘤效应的主要途径。另外,在LY294002的持续作用下,部分衰老的肿瘤细胞或许会发生凋亡。这些结论为为临床增强胶质瘤患者的联合化疗奠定了理论基础。     

高糖和LY294002干预影响足细胞内Ⅳ型胶原表达

探讨高糖和PI3K/Akt通路对足细胞内Ⅳ型胶原(Col Ⅳ)表达的影响。体外培养小鼠足细胞,给予高糖(30mmol/L)处理后,分别于0,12,24,48h收集细胞,采用免疫细胞化学染色法和Western blot技术检测Col Ⅳ的表达;Western blot技术检测Akt的活化及LY294002对Col Ⅳ表达的抑制效应。结果表明,高糖诱导足细胞内Col Ⅳ蛋白表达增多,24h明显,各时间点与高糖刺激前相比均有统计学差异(P<0.05);高糖激活Akt蛋白磷酸化,p-Akt随刺激时间延长表达增多。PI3K/Akt通路抑制剂LY294002孵育细胞24h后,可减弱高糖诱导的足细胞内Col Ⅳ的表达(P<0.05)。因此,高糖可能通过激活PI3K/Akt通路上调足细胞内Ⅳ型胶原表达。     

IGF-1 基因转染对脂肪间充质干细胞活性的影响及机制研究

目的:探讨携带IGF-1基因慢病毒转染脂肪间充质干细胞(ADMSCs)的可行性,对其引起的细胞凋亡机制做出初步研究,并讨论其与PI3K/Akt通路的关系。方法:分离培养ADMSCs,利用脂质体将携带IGF-1基因的慢病毒载体转染入ADMSCs。实验分为Blank组,Lv-non和Lv-IGF-1三组,转染后用MTT法测定各组细胞的生长情况并绘制生长曲线,流式细胞术测定各组细胞凋亡,Western blot测定各组中IGF-1、Akt、p-Akt及凋亡相关蛋白的表达。结果:成功分离、培养了大鼠脂肪间充质干细胞;携带IGF-1慢病毒载体转染ADMSCs后,流式细胞术检测发现Lv-non和Blank组凋亡率明显高于Lv-IGF-1组(P0.05)。同时发现Lv-IGF-1组中促凋亡蛋白Caspase-3、Caspase-9蛋白和Bax的表达水平显著下调(P0.01),抗凋亡蛋白Bcl-2显著上调(P0.01)。MTT法结果显示Lv-IGF-1能促进细胞生长,在5到6天时显著高于其他两组(P0.05)。通过检测PI3K/Akt通路发现,Lv-IGF-1组PI3K和p-Akt水平显著高于Blank和Lv-non组(P0.05),通路被激活。结论:携带IGF-1基因慢病毒载体成功转染ADMSCs。在ADMSCs中过表达IGF-1蛋白可以促进细胞生长,同时抑制细胞凋亡,其机制可能与PI3K/Akt通路蛋白磷酸化相关。     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

骨髓间充质干细胞在组织工程研究中的应用新进展

骨髓间充质干细胞又称为骨髓源性间充质干细胞,是指存在于骨髓基质细胞系统中的一类干细胞,具有高度稳定的体外扩增能力和多向分化潜能等特点。骨髓间充质干细胞因其取材方便,易于分离和培养,以及在适当条件下可诱导分化为皮肤、骨骼、内脏、血液、神经等多种组织细胞的独特优势,目前被广泛应用于药物开发、免疫调节、组织修复、器官重建等多个研究领域。近年来,骨髓间充质干细胞作为种子细胞在组织工程领域有着非常诱人的潜在应用前景。本文就骨髓间充质干细胞在组织工程学研究中应用的最新进展作一综述。     

川芎嗪体外诱导小鼠骨髓间充质干细胞分化为神经元样细胞的研究

为了探讨川芎嗪体外诱导小鼠骨髓间质干细胞（BMSCs）分化为神经元样细胞的作用,以小鼠骨髓间充质干细胞为研究对象,实验分为空白对照组、β-巯基乙醇（BME）阳性对照组和川芎嗪诱导组。采用荧光免疫化学和Western blot方法,分别检测神经干细胞巢蛋白（nestin）和经元特异性烯醇化酶（NSE）的表达;RT-PCR检测诱导不同时间对神经细胞相关基因Nestin、NSE、β-微管蛋白III（β-Tubulin III）和核受体相关因子-1（Nurr1）mRNA表达的影响。结果显示川芎嗪诱导间充质干细胞24 h后,细胞形态发生显著改变,细胞突起形成且数目不等,形成神经元样细胞。细胞死亡率低于β-巯基乙醇诱导组。免疫荧光化学法和western blot结果显示：川芎嗪诱导后的细胞nes-tin和NSE蛋白表达呈阳性,且表达丰度显著高于β-巯基乙醇诱导组。川芎嗪作用不同时间的BMSCs表达神经细胞相关基因Nestin、β-Tubulin III、NSE和Nurrl。结果表明川芎嗪能定向诱导小鼠骨髓间充质干细胞分化为神经元样细胞,是较理想的诱导剂。     

兔骨髓间充质干细胞的分离、培养与鉴定

目的:寻求家兔骨髓间充质干细胞(Bone Mesenchymal Stem cells,BMSCs)培养与分离简单易行的方法,为下一步骨髓间充质干细胞在呼吸系统疾病的应用打好基础。方法:取3个月龄家兔1只,经双侧髂前上棘抽取骨髓共5.0 ml,密度梯度离心法和贴壁筛选法相结合分离、纯化获得BMSCs,倒置差显微镜观察其形态学特性,流式细胞仪鉴定CD34、CD133表达。结果:接种细胞于24小时有少量细胞贴壁,72小时多数细胞可贴壁,贴壁细胞形态多为长梭型或多边形,原代细胞呈集落生长,12-16天可达90%融合,1%胰酶消化后传代培养,培养至第三代备用。流式细胞仪鉴定90%为骨髓间充质干细胞。结论:通过密度梯度离心法与贴壁筛选法可成功分离培养出骨髓间充质干细胞,此方法简单易行,成功率比较高。     

The comparition of biological characteristics and multilineage differentiation of bone marrow and adipose derived Mesenchymal stem cells

We compared the two sources of adipose and bone marrow-derived mesenchymal stem cells (BMSCs and AMSCs ) in multiple differentiation capacity and biological characteristics to provide a theoretical basis for stem cells transplantation. We isolated bone marrow- and adipose-derived mesenchymal stem cells and compared their phenotype,cell doubling time, the secretion of factors and their ability of multi-differentiation. We also compared their differences in T lymphocyte activation, proliferation and suppression. BMSCs and AMSCs were similar in cell phenotype and the differences existed only in the expression of CD106. On the proliferation rate, AMSCs were faster than BMSCs (doubling time 28 vs. 39?h). In addition, both of these two sources of cells were able to differentiate into bone, fat and cartilage that proved their stem cells properties and the number of stem cell progenitors (CFU-F) from adipose tissue were 10 times larger than those from bone marrow. But AMSCs showed a diminished capacity for suppressing T lymphocyte proliferation and activation compared to BMSCs. Cell origin and abundance were decisive factors in stem cells applications and, in the same volume, with the same premise of AMSCs and BMSCs, adipose tissue is a more promising source of stem cells.     

Increased leptin by hypoxic-preconditioning promotes autophagy of mesenchymal stem cells and protects them from apoptosis

Autophagy is the basic catabolic progress involved in cell degradation of unnecessary or dysfunctional cellular components.It has been proven that autophagy could be utilized for cell survival under stresses.Hypoxic-preconditioning(HPC)could reduce apoptosis induced by ischemia and hypoxia/serum deprivation(H/SD)in bone marrow-derived mesenchymal stem cells(BMSCs).Previous studies have shown that both leptin signaling and autophagy activation were involved in the protection against apoptosis induced by various stress,including ischemia-reperfusion.However,it has never been fully understood how leptin was involved in the protective effects conferred by autophagy.In the present study,we demonstrated that HPC can induce autophagy in BMSCs by increased LC3-II/LC3-I ratio and autophagosome formation.Interestingly,similar effects were also observed when BMSCs were pretreated with rapamycin.The beneficial effects offered by HPC were absent when BMSCs were incubated with autophagy inhibitor,3-methyladenine(3-MA).In addition,down-regulated leptin expression by leptin-shRNA also attenuated HPC-induced autophagy in BMSCs,which in turn was associated with increased apoptosis after exposed to sustained H/SD.Furthermore,increased AMP-activated protein kinase phosphorylation and decreased mammalian target of rapamycin phosphorylation that were observed in HPC-treated BMSCs can also be attenuated by down-regulation of leptin expression.Our data suggests that leptin has impact on HPC-induced autophagy in BMSCs which confers protection against apoptosis under H/SD,possibly through modulating both AMPK and mTOR pathway.     

Proliferation and differentiation potential of human adipose-derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures

Aging has less effect on adipose-derived mesenchymal stem cells (ADSCs) than on bone marrow-derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence-associated β-galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell-based therapy, especially in elderly patients with osteoporosis.     

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.