AKT1基因过表达骨髓间充质干细胞的建立

目的 使用慢病毒系统建立AKT1基因过表达的稳转骨髓间充质干细胞.方法 扩增AKT1 cDNA基因,并构建AKT1-cDNA表达载体,再结合穿梭质粒pCDH1-AKT1转染人胚肾293T细胞进行扩增后,使用慢病毒转染系统进行基因重组,并以其为载体转染猪的骨髓间充质干细胞,观察其荧光表达,采用Western blot和RT-PCR分别检测AKT1蛋白和mRNA表达水平.结果 双酶切鉴定和测序结果表明,所获取的cDNA为AKT1的蛋白质编码功能区基因.293T细胞和骨髓间充质干细胞的转染效率均达到90％以上.Western blot和RT-PCR结果表明,稳转细胞的AKT1蛋白和mRNA表达水平均明显高于原代细胞.结论 成功构建AKT1-cDNA表达载体;通过使用慢病毒系统,猪骨髓间充质干细胞能长期稳定过表达AKT1.     

慢病毒介导HO-1基因转染猪骨髓间充质干细胞的表达

目的探讨重组血红素氧合酶1(HO-1)基因体外转染猪骨髓间充质干细胞(MSCs)应用于基因治疗的可行性。方法体外分离、培养并鉴定MSCs。采用具有高效转染非分裂期细胞的慢病毒载体系统将HO-1基因导入MSCs中;采用RT-PCR和绿色荧光蛋白(GFP)荧光技术检测目的基因的表达,台盼蓝染色及MTT法检测转染后细胞的增殖能力。结果慢病毒感染MSCs的感染复数为20,最佳感染率可达80%;MSCs表面高表达CD44和CD105;GFP荧光表达自96h开始逐渐增强;转染细胞中显示目的基因mRNA的表达;转染对MSCs存活及增殖几乎无影响。结论慢病毒载体可成功转染猪MSCs,并使其HO-1表达增高,转染对MSCs存活及增殖基本无影响。     

骨髓间充质干细胞在心肌组织工程中的研究与进展

骨髓间充质干细胞因其具有高度增殖能力,多向分化潜能等优点已经引起了人们对骨髓间充质干细胞的研究热潮,尤其是做为组织工程及心血管疾病细胞移植治疗的理想种子细胞方面。常规用于房间隔或室间隔缺损修补手术的补片为涤纶补片,它具有质量轻、质地薄、生物相容性较好等优点,但近几年发现,用其修补后,如若发生术后残余漏,较易引起溶血、真菌或细菌感染。因此利用骨髓间充质干细胞的多向分化潜能在体外构建一种含有类心肌样细胞具有生物活性的补片是目前亟待解决的问题。但骨髓中的间充质干细胞含量极低,需经体外分离,纯化,扩增并吸附于一种生物相容性良好并可被机体吸收的生物材料上形成复合物,然后将其复合物植入机体组织或器官病损部位,才能达到修复缺损心肌和梗死后心肌组织的目的。     

骨髓间充质干细胞的分离、培养及生物学特性

目的 探讨大鼠骨髓间充质干细胞(BMSCs)的体外分离培养方法并鉴定其生物学特性.方法 采用全骨髓贴壁细胞培养法进行BMSCs的分离培养,倒置相差显微镜下观察细胞形态及生长特性,绘制细胞生长曲线,并检测细胞在不同诱导条件下向成脂肪细胞和成骨细胞分化的能力.结果 原代大鼠BMSCs培养24 h后镜下观察大部分细胞已经贴壁,传至第三代,细胞形态均一,呈梭形、螺旋样排列.经过约2 d的适应期,3～7 d进入对数生长期,7 d后进入平台期;成骨细胞诱导18 d,茜素红染色可见明显钙结节,成脂肪细胞诱导2周后油红O染色可见明显脂质沉淀.结论 全骨髓贴壁细胞培养法操作简单、快速,可以获取形态均一、纯度较高的BMSCs.     

miRNA-1真核表达载体的构建及转染骨髓间充质干细胞绿色荧光蛋白的表达

目的 构建大鼠miRNA-1真核表达载体,为研究miRNA-1功能提供实验基础.方法 应用技术体外合成miRNA-1前体对应的基因组片段,定向克隆到pSD11-U6/Neo/绿色荧光蛋白载体上.阳性克隆进行及DNA测序鉴定,将构建成功的重组质粒pSD11-U6/Neo/绿色荧光蛋白/miRNA-1借助脂质体转染大鼠骨髓间充质干细胞,荧光显微镜下观察绿色荧光蛋白表达.结果 PCR鉴定及DNA测序表明真核表达载体pSD11-U6/Neo/绿色荧光蛋白/miRNA-1构建成功.转染大鼠骨髓间充质干细胞后,荧光显微镜下观察到绿色荧光蛋白表达.结论 大鼠miRNA-1真核表达载体构建成功.     

骨髓间充质干细胞治疗心肌梗死的研究进展

<正>心肌梗死(myocardial infarction,MI)是指心肌的缺血性坏死,已成为当今社会严重威胁人类健康的疾病之一。尽管临床上保守的药物治疗、经皮冠状动脉介入治疗(PCI)和外科冠状动脉旁路移植手术等治疗方法的不断提高,但都无法逆转梗死期坏的心肌坏死。     

TIMP-1-shRNA基因体外转染大鼠骨髓间充质干细胞的实验研究

目的 探讨联合应用RNA干扰与干细胞移植技术在肝纤维化治疗中的应用.方法 采用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞(BMSCs),并进行流式细胞学鉴定.按使用慢病毒包装系统建立沉默基质金属蛋白酶抑制剂-1 (TIMP-1)基因不同位点分为三组:BMSCs株SH1组(LV-3-TIMP-1-rat-246),SH2组(LV-3-TIMP-1-rat-142),SH3组(LV-3-TIMP-1-rat-373).同时设立转染空载体组(LV-3-shNC,D组).观察各组细胞转染前后形态和荧光的表达;抽提蛋白,应用Western blot技术检测TIMP-1蛋白的表达.结果 原代培养大鼠BMSCs呈梭形,传代后漩涡状生长.慢病毒转染BMSCs 7 d后,各组均可检测到荧光出现,转染前后细胞形态无改变.在3个实验组中,SH3组TIMP-1蛋白表达最低(P＜0.05).结论 应用慢病毒包装系统可以建立沉默TIMP-1基因的BMSCs株,为BMSCs联合RNA干扰技术治疗肝纤维化提供了实验基础.     

超声联合一氧化氮微泡体外干预骨髓间充质干细胞安全性探讨

目的研究超声联合一氧化氮(NO)微泡体外干预大鼠骨髓间充质干细胞(BMSCs)的安全性。方法体外分离培养大鼠BMSCs,分为超声联合NO微泡组(包括1∶70,1∶50,1∶20浓度亚组)及空白对照组,体外干预后MTT法检测BMSCs增殖,PI染色流式细胞术分析细胞凋亡及细胞周期。结果与空白对照组比较,1∶20浓度组显著抑制BMSCs增殖(P<0.05);1∶50浓度组与1∶20浓度组明显诱导细胞凋亡(P<0.05)。结论超声联合NO微泡体外干预BMSCs,1∶70浓度对其是安全的。     

不同代次骨髓间充质干细胞的生物学特性

间充质干细胞来源主要有骨髓,外周血,脐带脂肪组织,外周血,胎盘羊水,其他部位如胰腺、胎肝、胎肺、骨肌腱、乳汁等[1]。其具有自我更新能力、多向和定向分化能力、免疫抑制作用和趋化性、组织修复、体内移植基因治疗等作用。干细胞研究已经取得较多成果,无论在实验室还是临床研究,在使用时,干细胞代次的选择都是首先考虑的。目前国内外研究表明各代骨髓间充质干细胞其生物学特性的异同,仍无定论[2]。故专门就实验室和动物实验的     

龟板提取物对骨髓间充质干细胞中分化抑制蛋白1表达的影响

目的观察不同剂量的龟板提取物对骨髓间充质干细胞中分化抑制蛋白1（inhibitor of differentiation 1,Id1）的作用。方法将构建成功的PGL3-Id1启动子用磷酸钙共沉淀法转染大鼠骨髓间充质干细胞,龟板提取物分别作用于转染后的骨髓间充质干细胞12、24、36 h,选取作用最为明显的36 h,每组分别加入0、1、3、30、100μg/mL龟板提取物,药物作用36 h后收集细胞分别应用萤光素酶报告基因系统、RT-PCR和Western blotting检测Id1的表达。结果早期、小剂量龟板提取物对Id1的影响不大;随着时间和剂量的增加,Id1的表达水平也逐渐升高。结论龟板提取物促进了骨髓间充质干细胞中Id1的表达,剂量越大,作用时间越长,促进作用越明显。     

Ultrasound-mediated microbubble destruction enhances the efficacy of bone marrow mesenchymal stem cell transplantation and cardiac function

1. Application of ultrasound (US) to intravascular microbubble (MB) contrast agents causes small capillary ruptures. The purpose of the present study was to examine the effects of US-mediated MB destruction on bone marrow mesenchymal stem cell (BMSC) transplantation into the infarcted myocardium and to evaluate whether this approach could improve cardiac function. 2. Ultrasound was applied to the anterior chest of rabbits after intravenous injection of MB followed by infusion of BMSC. There were four groups investigated: (i) a control group, in which neither US nor MB were used prior to infusion of BMSC; (ii) one group subjected to US alone prior to infusion of BMSC; (iii) another group injected with MB prior to infusion of BMSC; and (iv) a group in which US was applied to MB prior to the infusion of BMSC. Cardiac function was evaluated by echocardiography 24 h and 4 weeks after cell transplantation. All rabbits were killed to enable histological and immunochemical examination. 3. Echocardiography 24 h after infusion of BMSC indicated no difference in cardiac function between any of the groups, as assessed by left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimensions (LVDD), left ventricular systolic diameter (LVSD) and fractional shortening (FS%; all P > 0.05). However, 4 weeks after BMSC transplantation, there was a significant improvement in LVEF in the group subjected to US plus MB compared with the control, US alone and MB alone groups (59.5 +/- 3.5, 52.5 +/- 5.5, 52.8 +/- 5.2 and 51.1 +/- 3.5%, respectively; all P < 0.05). In addition, treatment with US plus MB significantly reduced LVDD and LVSD and increased capillary density in the infarcted area. 4. In conslusion, the results of the present study indicate that using US-mediated MB destruction prior to BMSC transplantation into the infarcted myocardium improves the effectiveness of cardiac cell therapy and cardiac function in rabbits.     

超声微泡增效骨髓干细胞移植效率的实验研究

目的在动物水平研究超声联合微泡增效骨髓干细胞(BMSCs)心肌移植的作用,为临床上提高心肌梗死后BMSCs移植效率提供一种新的思路。方法12只中国家猪分别体外分离纯化BMSCs,超顺磁性氧化铁颗粒标记;介入法建立急性心肌梗死模型,成功后14d经冠状动脉注入自体BMSCs;实验组先注入微泡后再注入BMSCs,同时行超声辐射。术后行磁共振检查;心肌做病理切光镜、透射电镜观察。结果①两组在心肌梗死区和梗死周边区磁共振上均见SPIO标记的BMSCs形成的低密度区;②实验组较对照组普鲁士蓝染色阳性细胞明显增多(P<0.01);③两组心肌超微结构无差异,但B1组见血管内皮间隙增宽。结论超声辐射微泡可增加自体BMSCs心脏的移植效率。     

成体大鼠骨髓间充质干细胞的表型研究

目的文献报道大鼠骨髓间充质干细胞（MSCs）不表达CD45,而本实验中却发发现CD45高度表达,针对这一现象做初步分析：方法成体大鼠骨髓MSCs的分离培养,对原代、第3代和第6代细胞做流式细胞仪检测MSCs的表面标记物以及染色体分析。结果体外培养的大鼠骨髓MSCs呈长梭型、有突起,排列成旋涡状。流式细胞仪检测示原代细胞：CD29（99．9％）,CD90（48．5％）,CD45（996％）,CD34（202％）;第3代细胞：CD29（100％）,CD90（97．1％）,CD45（100％）,CD34（089％）;第6代细胞：CD29（99．7％）,CD90（99．6％）,CD45（99．7％）,CD34（0．49％）。上述3代细胞的染色体分析床细胞核型为二倍体。结论体外培养的成体大鼠骨髓MSCs同时表达CD29,CD90和CD45,不表达CD34。     

PDGF-B 基因转染骨髓间充质干细胞的实验研究

目的:探讨血小板源性生长因子-B(PDGF-B)基因转染修饰大鼠骨髓间充质干细胞(bone m arrow m esenchym alstem cell,M SC)的可行性。方法:应用FAM标记重组真核表达载体系统(pcDNA3-PDGF-B),以脂质体法转染原代骨髓间充质干细胞,观察转染结果、表达情况及对靶细胞活力的影响。结果:经荧光显微镜下观察证实:成功地对骨髓间充质干细胞实现了基因转染,持续表达时间超过了8周,没有发现明显的细胞毒作用及对细胞活力的显著影响。结论:采用真核转染技术可以介导外源基因转染骨髓间充质干细胞,M SC是一种理想的基因载体细胞,可用于PDGF-B的基因治疗。     

骨髓间充质干细胞的研究进展

骨髓间充质干细胞(BMSCs)是骨髓基质中存在的非造血系的成体干细胞,具有多向分化潜能,已成为国内外基础医学和临床医学研究的热点.本文简要介绍了BMSCs的概念提出、生物学特性、培养、鉴定,并根据近年来国内外包括应用中药进行的诱导分化、组织工程、基因治疗、再生医学等相关研究及临床应用前景作一综述.     

Integral therapeutic potential of bone marrow mesenchymal stem cells

Bone marrow derived mesenchymal stem cells (MSC) are adult stem cells that reside within the bone marrow compartment. In the traditional developmental model, adult stem cells are able to differentiate only to the tissue in which they reside. Recent data have challenged the committed fate of the adult stem cells, presenting evidence for their multi-lineage differentiation potential. In addition, potential therapeutic benefits of MSC administration have been the main concern of much research, including clinical trials. These studies promote adult stem cell therapy by shedding some light on the therapeutic potential of MSC and their mechanism of action. Many doubts have found their way into MSC research. They question MSC potency and beneficial contribution. However, these obstacles should not arrest but set a challenge to MSC researchers to examine their achievements under a magnifying glass. Therapeutic benefits of MSC exogenous delivery do not run counter to its possible participation in endogenous repair. Several reports imply MSC involvement in physiological repair but no explicit data support this hypothesis. This review tries to put MSC research into perspective. Possible therapeutic applications of MSC therapy for damaged tissue replacement, tissue engineering and the underlying repair mechanisms will be discussed. In addition, reported data about MSC possible involvement in physiological multiple tissue repair, their homing to injury and site-specific differentiation will be presented.     

钙离子对骨髓间充质干细胞CXCR4表达的影响

目的 探讨钙离子对骨髓间充质干细胞(BMSCs) CXCR4表达的影响.方法 用含不同浓度钙离子的DMEM培养液体外培养大鼠BMSCs:A组为无钙离子培养基;B、C、D组分别加入1、2、4mM氯化钙;E、F和G组为C组中分别加入钙敏感受体特异性抗体、AMD3100和抗CXCR4抗体.采用RT-PCR和Western blot检测CXCR4 mRNA水平和蛋白表达,采用迁移实验测定BMSCs向基质细胞衍生因子1(SDF-1)的趋化效应.结果 CXCR4mRNA转录水平与蛋白表达水平:与A组相比,B、C、D组显著上升呈浓度依赖性(P＜0.05);与C组相比,E组显著下调(P＜0.05).与A组相比,C组向SDF-1的迁移细胞数增加;与C组相比,E、F、G组向SDF-1的迁移细胞数减少(P＜0.05).结论 钙离子可促进BMSCs的CXCR4转录和表达,并促进BMSCs向SDF-1的趋化迁移,从而可能促进BMSCs的靶向归巢.     

体外诱导大鼠骨髓间充质干细胞为血管内皮细胞的研究

目的研究骨髓间充质干细胞（mesenchymal stem cells，MSCs）定向分化为血管内皮细胞，为组织工程血管化及细胞移植修复损伤组织提供理想的细胞来源。方法贴壁法分离SD大鼠MSCs进行体外培养和连续传代，采用第四代MSCs，以血管内皮生长因子（vascular endothelial growth factor，VEGF）和碱性成纤维细胞生长因子（basic fibroblast growth facotr，bFGF）进行体外诱导，免疫荧光染色方法鉴定内皮细胞，并通过透射电镜观察内皮细胞W-P小体。结果免疫荧光染色证实实验组细胞阳性表达，透射电镜观察到内皮细胞特有的WP小体。结论MSCs可以在体外定向分化为具有血管内皮细胞特性的细胞。     

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.     

Comparison of immunomodulatory effects of placenta mesenchymal stem cells with bone marrow and adipose mesenchymal stem cells

Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerative medicine because they can be isolated from various tissues, expanded, and induced into multiple-lineages. Of note, their immunomodulatory effects maximize the therapeutic effects of stem cells engrafted on host, making them an especially attractive choice. Recently, several varieties of placenta-derived stem cells (PDSCs) including chorionic plate-derived MSCs (CP-MSCs) have been suggested as alternative sources of stem cells. However, comparative studies of immunomodulatory effects for CP-MSCs among various MSCs are largely lacking. We examined and compared immunomodulatory function of CP-MSCs with that of BM-MSCs and AD-MSCs using co-culture system with activated T-cells derived from human umbilical cord blood (UCB) exposed to anti-CD3 and anti-CD28 which are T-cell activating monoclonal antibodies. All MSCs expressed markers of stem cells and three germ layers by RT-PCR. These cells also exhibited comparable immunomodulatory effects when they were co-cultured with activated T-cells in dose-dependent manner. However, expression of HLA-ABC and HLA-G was highly positive in CP-MSCs compared to other MSCs, and higher levels of cytokines of IL-2, IL-4, IL-13, and GM-CSF were detected in dose-dependent manner in CP-MSCs. Taken together, the results of the present study suggest that while CP-MSCs, BM-MSCs, and AD-MSCs all have immunomodulatory effects, CP-MSCs may have additional advantage over the other MSCs in terms of immunomodulation. In conjunction with other previous studies, CP-MSCs are suggested to be a useful stem cell source in cell therapy.