Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Ceils and Expression of VEGF After Gene Transfection

Objectives To induce the differentiation of rabbit bone marrow mesenchymal stem cells （MSCs） to myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor （VEGF） gene in MSCs transfected by AdTrackCMV-VEGF165. Methods MSCs were isolated and purified from rabbit bone marrow by percoll （11）73 g/ml） and then cultured in low glucose DMEM with 10% FBS. AdTrackCMV-VEGF165 eukaryotic expression vector was constructed and transfected into the MSCs. After being incubated with 5-azacytidine （5- Aza）, the expression of troponin I in MSCs was assayed by immunohistochemistry and the expression of VEGF gene was identified by northern blot and western blot. The concentration of VEGF in the supernatant was measured by ELASA. Results MSCs were isolated and cultured successfully from rabbit bone marrow. The positive cTnI stain of some MSCs after the induction of 5-aza indicated that the cells were differentiated to myogenic cells. Northern blot and western blot showed that the expression of VEGF 165 mRNA was much higher in the hVEGF165 gene transfected cells than that of the control. The concentration of VEGF in the supernatant got to the peak 3-5 days after hVEGF165 gene transfection （1011- 1027 pg/ml） and decreased gradually thereafter, but still higher than that of control group or pAdTrackCMV group （349 pg/mLvs 116 pg/mL or 125pg/ml respectively, MSCs could be induced P〈 0.01）. Conclusions to differentiate to myogenic cells by 5-aza in vitro and could express VEGF by VEGF gene transfection.     

绿色荧光蛋白基因转染骨髓间质干细胞

目的 建立以增强型绿色荧光蛋白基因示踪骨髓间质干细胞的方法。方法 采用密度梯度离心法分离、培养免骨髓间质干细胞，以脂质体介导法转染增强型绿色荧光蛋白基因的表达质粒，荧光显微镜观察基因表达及转染效率。结果 绿色荧光蛋白在基因转染12h后开始表达，48～72h达高峰，并在1周内有较强的表达，以后逐渐减弱，4周左右仍有少量表达，转染效率与质粒、脂质体的浓度有关，外缘基因导入后不影响骨髓间质干细胞的生长和增殖。结论 脂质体介导的绿色荧光蛋白基因转染骨髓间质干细胞后能安全、有效地表达，转染效率为20％～30％，是一种较为理想的干细胞示踪方法。     

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.     

骨髓间充质干细胞与心肌修复

骨髓间充质干细胞 (MSC)具有多向分化潜能 ,在一定的诱导条件下可以分化为多种细胞。 5 氮胞苷可以诱导离体MSC出现心肌表型。MSC移植研究表明 ,在心肌组织微环境中 ,MSC可向心肌分化并与宿主心肌功能整合 ,从而修复损伤坏死心肌、改善心脏功能     

VEGF-expressing Bone Marrow Mesenchymal Stem Cells Transplantation Improved Heart Function of Myocardial Infarct Rabbits

Objectives To treat myocardial infarction with MSCs transplantation combined with VEGF gene therapy in rabbits and to study its mechanisms. Methods Forty-eight rabbits were randomly divided into MI group (n=12), MSCs group (n=12), VEGF group (n = 12), MSCs VEGF group (M V group, n = 12). Rabbit myocardial infarction models were founded by the ligation of left anterior descending artery. 107 MSCs were injected into the infarct-zone in four sites 2 weeks later in MSCs and M V group. phVEGF gene were injected in infarct-zone in VEGF group and MSCs transfected with phVEGF gene were injected in M V group. Heart function including LVEDP, LVSP, LVDP, -dp/dtmax dp/dtmax, were measured in vivo. The hearts were harvested at 4 weeks after transplantation and sectioned for HE stain, immunohistochemical stain of BrdU andⅧfactor antigen. Results The left ventricular hemodynamics parameters showed that heart function were improved more in M V group than MSCs group, MI group and VEGF group. The numbers of BrdU positive cells in M V group(61±8)were more than in MSCs group (44±8, P < 0.01). The numbers of vessels in infarcted zone were more in M V group (49±8) than in MSCs group (33±6, P < 0.01 ),VEGF group(30±8, P<0.01)and MI group (18±4, P<0.01). Conclusions VEGF- expressing MSCs transplantation could improve heart function after myocardial infarction, and they were more effective than sole MSCs transplantation. Keeping more MSCs survival and ameliorating the blood supply of infarct-zone might be involved in the mechanisms.     

犬骨髓间叶干细胞体外定向诱导分化心肌细胞的实验研究

目的:旨在建立骨髓间叶干细胞(MSCs)体外定向诱导分化心肌细胞的方法,为心肌疾患的移值修复治疗提供成体干细胞来源.方法:利用Percoll密度梯度法及MSCs黏附贴壁生长特性进行分离培养与扩增骨髓MSCs,并予以鉴定证实.应用5-氮胞苷对培养早期的骨髓MSCs进行定向诱导分化心肌细胞.通过细胞形态学、细胞免疫组化、透射电镜等技术观察分化细胞的肌管,心肌细胞特异性蛋白与细胞特异性超微结构以鉴定诱导分化的效果.结果:利用Percoll密度梯度法与细胞黏附贴壁生长特性,可分离培养与扩增足量骨髓MSCs.应用化学诱导剂5-氮胞苷10～20 μmol/L孵育早期培养的骨髓MSCs4～5周,可见细胞形成肌管;肌细胞特异性蛋白α-肌动蛋白,肌球蛋白以及心肌细胞特异性分子标志肌钙蛋白I免疫组化染色阳性;透射电镜可见肌丝与房性颗粒.结论:骨髓MSCs可在体外5-氮胞苷的诱导下定向转化为具有典型结构的心肌细胞.     

间充质干细胞的生物特性及其在冠心病治疗中的临床应用

间充质干细胞(m esenchym al stem cells,MSCs)是一些易于在体外扩增并且具有多种分化能力的原始细胞,它在适宜的环境中可以分化为骨、软骨、脂肪、纤维结缔组织、骨髓基质等不同细胞。已有大量试验研究MSCs的分离、纯化、培养,以及它的特性,并有临床前期的研究实验证实了MSCs对冠心病的治疗效果,现将上述国内外研究现况予以综述,并对尚未明确的相关问题进行探讨。     

自体骨髓间叶干细胞诱导分化移植重建窦房结起搏功能的实验研究

目的:探索应用骨髓干细胞治疗病态窦房结综合征的生物介入方法.方法:选取犬6只,随机分为实验组和对照组,每组3只.抽取犬自体骨髓,分离培养扩增骨髓间叶干细胞,并在体外应用5-氮胞苷进行诱导分化.应用射频技术损伤犬窦房结,建立动物病态窦房结综合征模型.将溴脱氧尿嘧啶核苷(BrdU)标记的且诱导分化的骨髓间叶干细胞自体移植到窦房结区,应用心电图、组织病理、免疫组化等技术观察干细胞移植治疗效果.结果:在动物病态窦房结综合征模型,自体移植骨髓干细胞后,心电图示窦房结功能明显改善;病理与免疫组化示移植的骨髓干细胞在窦房结区分化为拟窦房结细胞与血管内皮细胞,并与宿主细胞建立缝隙连接.结论:自体移植诱导分化的骨髓间叶干细胞可改善窦房结自律起搏功能.     

剪应力和血管内皮生长因子诱导骨髓间充质干细胞向内皮细胞分化

目的 研究血管内皮生长因子与剪应力对骨髓间充质干细胞分化的作用.方法 分离成人骨髓单个核细胞,经贴壁法获取骨髓间充质干细胞.所获细胞用血管内皮生长因子、剪应力(8和15 dyn/cm2)、血管内皮生长因子联合剪应力分别作用7天、24 h、24 h,观察细胞形态变化,并进行内皮细胞标记物假性血友病因子定性间接免疫荧光染色,进行Dil标记的乙酰化低密度脂蛋白摄取实验以鉴定内皮细胞功能.结果 在剪应力(8 dyn/cm2)、血管内皮生长因子分别作用24 h、7天后,骨髓间充质干细胞形态变得偏圆、呈多角形,血管内皮生长因子联合剪应力诱导组细胞平行于流体方向排列,假性血友病因子染色阳性,提示内皮细胞表型特征,同时Dil标记的乙酰化低密度脂蛋白摄取实验阳性,提示具有内皮细胞功能.血管内皮生长因子诱导组、15 dyn/cm2剪应力组作用24 h的骨髓间充质干细胞,假性血友病因子染色与Dil标记的乙酰化低密度脂蛋白摄取实验均阴性,提示未出现向内皮细胞分化.血管内皮生长因子和15 dyn/cm2剪应力共同作用24 h后,骨髓间充质干细胞向内皮细胞分化.结论 剪应力可诱导骨髓间充质干细胞向内皮细胞分化,且与力的大小有关.血管内皮生长因子可联合剪应力诱导骨髓间充质干细胞向内皮细胞分化.且诱导效果优于单独使用剪应力.     

转染人血管内皮生长因子165基因的骨髓间充质干细胞移植改善兔心肌梗死后心功能

目的探讨将骨髓间充质干细胞移植和血管内皮生长因子基因治疗相结合治疗兔心肌梗死的疗效及其机制。方法将48只新西兰大白兔随机分为心肌梗死组、骨髓间充质干细胞移植组、血管内皮生长因子组和骨髓间充质干细胞 血管内皮生长因子组。建立兔心肌梗死模型,在心肌梗死后2周取107个骨髓间充质干细胞移植至梗死区。移植后4周测定心功能,梗死区骨髓间充质干细胞进行鉴定及计数,进行Ⅷ因子免疫组织化学染色。结果血流动力学比较发现,骨髓间充质干细胞 血管内皮生长因子组各项参数明显优于骨髓间充质干细胞组;心肌梗死区BrdU阳性细胞数骨髓间充质干细胞 血管内皮生长因子组明显多于骨髓间充质干细胞组(61.24±8.51个/视野比44.21±7.68个/视野,P<0.01);梗死区血管的数量骨髓间充质干细胞 血管内皮生长因子组(48.75±7.96个/视野)明显多于骨髓间充质干细胞组(33.08±6.12个/视野,P<0.01)、血管内皮生长因子组(29.98±8.04个/视野,P<0.01)和心肌梗死组(18.32±3.88个/视野,P<0.01)。结论转染血管内皮生长因子基因的骨髓间充质干细胞移植可改善兔心肌梗死后的心功能,其疗效明显优于单纯骨髓间充质干细胞移植及血管内皮生长因子基因治疗,可能是通过增加骨髓间充质干细胞的存活及改善梗死区的血供而起作用的。     

高表达整合素连接激酶对大鼠骨髓间充质干细胞旁分泌功能的影响

目的 研究转染腺病毒介导的整合素连接激酶(ILK)对大鼠骨髓间充质干细胞旁分泌作用的影响.方法 构建携带人野生型整合素连接激酶质粒cDNA和人重组绿色荧光蛋白的腺病毒载体(adeno-ILK)和空载腺病毒(MSC),通过全骨髓贴壁法分离培养骨髓间充质干细胞.采用无血清培养转染adeno-ILK(MSC-ILK组)或空载腺病毒(MSC组),通过流式细胞术检测人重组绿色荧光蛋白从而测定转染效率,确定最佳的病毒感染复数.提取骨髓间充质干细胞mRNA,用Real-time PCR测定血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量.结果 MSC-ILK组旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1的基因表达量分别是MSC组的 8.2±0.4、2.6±0.2及2.4±0.2倍(P＜0.05或P＜0.01).结论 整合素连接激酶基因转染可明显促进骨髓间充质干细胞表达旁分泌因子血管内皮生长因子、成纤维细胞生长因子2和胰岛素样生长因子1.     

骨髓间充质干细胞向神经细胞分化的研究进展

骨髓间充质干细胞(BMSC)是一类具有多向分化潜能的细胞。近年来,许多学者已开始通过体内、体外实验研究BMSC是否具有定向分化为神经细胞的可能性。虽然对化学物质诱导BMSC的某些结果存在争议,但细胞移植和基因治疗的实验研究成果为未来神经系统疾病的治疗和康复展示了美好的前景。     

采用1.068 g/ml分离液对犬骨髓间充质干细胞的分离纯化与体外培养方法的探讨

目的探讨更为可靠、纯净度高的犬骨髓间充质于细胞分离、培养方法。方法将犬骨髓抽取液分别以1．068g／ml及1．073g／ml分离液进行密度梯度离心，采用贴壁培养法获得骨髓间充质干细胞（MSCs）；以LG-DMEM加15％FBS培养；应用于细胞表面标志蛋白，多向诱导分化等方法进行细胞鉴定。结果采用1．068g／ml分离液可获得纯度较高的单核细胞，接种细胞生长良好，孵育24h即可见细胞贴壁生长，平均倍增周期为1d，细胞呈纺锤状，螺旋梳状排列。连续培育8代以上，未见细胞形态、增殖特性改变。MSCs表面标志SH2阳性，CD45阴性，可定向分化为心肌细胞、成骨细胞及脂肪细胞。结论采用1．068g／m1分离液能够较好地进行犬MSCs的体外分离、培养与扩增，分离得到的细胞具备MSCs的特性。     

5-氮胞苷体外诱导成人脂肪间充质干细胞分化为心肌样细胞的初步研究

目的:探索不同浓度5-氮胞苷(5-aza)在不同诱导时间下对成人脂肪间充质干细胞(ADMSCs)分化为心肌细胞的诱导作用,确定最佳诱导条件。方法:体外分离成人脂肪组织来源的间充质干细胞并进行传代培养,流式细胞仪鉴定细胞CD44、CD34的表达,用含不同浓度5-aza(3、5、10、15、20μmol／L)培养基分别诱导12小时、24小时、48小时、72小时,倒置相差显微镜下逐日观察细胞形态变化;于诱导后14、28天时免疫细胞化学染色鉴定心肌特异性肌钙蛋白I(cTn-I)的表达,分析细胞转化率。结果:分离培养的ADMSCs表达CD44,不表达CD34;5-aza诱导后14天时免疫细胞化学未见有心肌细胞特异性cTn- I表达,诱导28天细胞免疫细胞化学显示cTn-I表达阳性,以10μmol/L 5-aza诱导24小时为最佳体外诱导条件。结论:成人脂肪组织间充质干细胞可在体外5-aza诱导下向心肌细胞分化。     

系统性红斑狼疮骨髓间质干细胞的初步研究

目的探讨系统性红斑狼疮（SLE）患者骨髓间质干细胞（MSCs）生物学功能、细胞因子的表达异常与否.方法采用密度梯度离心和贴壁筛选法对10例SLE患者和11例正常人骨髓MSCs进行分离培养,流式细胞仪鉴定MSCs表面标志物及分析MSCs细胞周期和细胞凋亡,观察MSCs形态学改变,MTT法检测MSCs的生长情况,RT-PCR检测MSCs细胞因子mRNA水平表达.结果SLE骨髓MSCs传代率（30%）低于正常（100%）（P＜0.01）,SLE患者骨髓MSCs生长较正常活跃,凋亡率（2.36%）低于正常（6.79%）,两组MSCs均表达CD29、CD44、CD105,均不表达CD14、CD34、CD45,SLE患者骨髓MSCsIL-7mRNA表达低于正常.结论SLE骨髓MSCs生物学特性及细胞因子表达存在异常.     

骨髓间充质干细胞向神经元样细胞分化中褪黑素受体和多效蛋白的表达

目的体外诱导成人骨髓间充质干细胞（MSCs）向神经元样细胞分化,并探讨分化过程中多效蛋白（PTN）和褪黑素（Mel）受体亚型MT1、MT2 mRNA的表达,以探索MSCs向神经元样细胞分化的特性和机制。方法密度梯度离心加贴壁培养法分离成人MSCs,原代和传代培养。取第6代MSCs设对照和实验组进行诱导,诱导后30min至3d,观察细胞形态并计数。免疫细胞化学法和RT—PCR法测定分化后细胞神经细胞特异性表面标志神经元特异性烯醇化酶（NSE）、微管相关蛋白-2（MAP-2）、神经胶质纤维酸性蛋白（GFAP）和诱导前、诱导后12hPTN和MT1、MT2 mRNA的表达。结果接种24h后MSCs开始贴壁,呈圆形或椭圆形。3d后可见梭状细胞呈集落状生长,10d～14d融合。第5代～第6代时呈现较均一的成纤维细胞样形态。诱导后胞体向胞核收缩;出现双极及多极细胞。12h变形细胞增多,细长突起相互连接。24h后变形细胞增多不明显。诱导后12h大部分细胞表达NSE、MAP-2,未检测到GFAP的表达。实验组诱导后12h细胞有PTN和MT1 mRNA的表达。结论PTN和Me1信号传导可能参与调控了MSCs向神经元样细胞的分化。     

血管内皮生长因子基因转染骨髓间充质干细胞移植对肺气肿大鼠的疗效

目的：观察血管内皮生长因子（VEGF）基因转染大鼠骨髓间充质干细胞（MSCs）移植对肺气肿大鼠肺泡壁细胞的修复作用。方法：pcDNA3．1-hVEGF转染雄性Lewis大鼠MSCs。雌性Lewis大鼠随机分为4组：正常对照组、肺气肿组、单纯MSCs移植组,hVEGF移植组。观察MSCs移植28d后肺组织形态学变化,支气管肺泡灌洗液细胞分类计数,ELISA法检测VEGF含量。Y染色体荧光原位杂交（Y-FISH）示踪雄性大鼠MSCs在雌性肺气肿大鼠肺组织内的植入情况;采用TUNEL法检测肺泡壁细胞凋亡,免疫组化法检测Bcl-2蛋白的表达。结果：hVEGF移植组肺气肿改变较肺气肿组、单纯MSCs移植组减轻;支气管肺泡灌洗液炎性细胞数较后2组减少;肺组织VEGF含量较后2组增多;肺泡壁细胞凋亡指数明显低于后2组;Bcl-2染色阳性细胞百分比高于后2组;Y-FISH显示hVEGF移植组、单纯MSCs移植组的雌性大鼠肺组织中有Y染色体阳性的细胞。结论：真核表达载体pcDNA3．1hVEGF可以成功地在MSCs中表达,pcDNA3．1hVEGF转染MSCs移植能够减轻大鼠肺气肿样改变,其疗效优于单纯MSCs移植治疗。

Abstract：

Objective： To evaluate the effect of mesenchymal stem cells （MSCs） transfected with human vascular endothelial growth factor （VEGF） gene for rats with pulmonary emphysema. Methods： MSCs from male lewis rats were transfected with the pcDNA3. 1-hVEGF control vector. Female Lewis rats were randomly divided into four groups： normal control group, emphysema group, emphysema ＋ MSCs transplantation group, emphysema ＋ hVEGF-transfected MSCs transplantation group. Morphologie changes of the lung tissue were observed 28 Jays after treatment. The total cell number of bronchoalveolar lavage fluid were counted, and the content of VEGF was de tected by ELISA. The engraftment of male bone marrow MSCs in female recipient lung was determined by Y chromosome fluorescent in situ hybridization （Y-FISH）. The apoptosis of the lung cells was assessed by TUNEL stai ning. The expression of Bcl-2 were determined by immunohistochemical staining. Results： Emphysematous change in the emphysema ＋ hVEGF transfeeted MSCs transplantation group was improved compared with those in emphysema group and the emphysema ＋ MSCs transplantation group. The total cell number of bronchoalveolar lavage fluid was decreased in the emphysema ＋ hVEGF transfected MSCs transplantation group compared to the emphysema group and the emphysema ＋ MSCs transplantation group. The level of VEGF was higher in emphysema ＋ hVEGF transfected MSCs transplantation group compared with those of the emphysema group and the emphysema ＋ MSCs transplantation group. The apoptotic index of the alveolar wall cells in the emphysema ＋ hVEGF transfeeted MSCs transplantation group was less than that of the emphysema group and the emphysema ＋ MSCs transplantation group. The percentage of Bcl-2 positive cells in the emphysema ＋ hVEGF transfected MSCs transplantation group was significantly higher than that of the emphysema group and the emphysema ＋ MSCs transplantation group. Y chromo some positive cells were observed in the lungs of rats from the emphysema ＋ hVEGF transfected MSCs transplantation group and the emphysema ＋ MSCs transplantation group, Conclusions： The hVEGF gene could expressed in MSCs. Trans plantation of MSCs transfected with hVEGF gene can improve emphysematous changes than single cellular therapy.     

Role of Bone Marrow Derived Mesenchymal Stem Cells and the Protective Effect of Silymarin in Cisplatin-Induced Acute Renal Failure in Rats

Background

Cisplatin is a highly effective antitumor agent whose clinical application is limited by its nephrotoxicity, which is associated with high mortality and morbidity rates. We aimed to study the protective role of silymarin and mesenchymal stem cells as a therapeutic tool of cisplatin nephrotoxicity.

骨髓间充质干细胞及其在缺血性脑血管病中的应用

骨髓间充质干细胞是骨髓中的一群非造血干细胞，具有较强的自我更新能力和多向分化潜能，在体外不同诱导条件下可分化为骨细胞、神经细胞和平滑肌细胞等多种细胞。骨髓间充质干细胞已应用于缺血性脑血管病的治疗研究。文章对骨髓间充质干细胞及其在缺血性脑血管病中的应用做了综述。