犬骨髓间充质干细胞体外分离培养、诱导分化为心肌细胞实验研究

目的 观察犬骨髓间充质干细胞(BMSCs)分离体外培养的生长特性及诱导分化为心肌细胞.方法 利用骨髓穿刺,Percoll分离液密度梯度离心法分离穿刺骨髓细胞继而通过贴壁筛选纯化骨髓间充质干细胞进行接种培养、体外扩增经5-氮胞苷(5-azacytidine)诱导分化为心肌样细胞.倒置显微镜观察诱导前后细胞形态变化,流式细胞分析及免疫化学进行相关免疫抗原检测.结果 分离细胞流式细胞分析细胞表面分子抗原CD105(间充质干细胞抗原)阳性,CD34(造血干细胞表面抗原)阴性,CD31(内皮主细胞表面抗原)阴性.利用5-溴-2-尿嘧啶脱氧核苷(Brdu)进行细胞增殖标记,结果增殖细胞核标记率为(77.2±6.1)%.传4代后,经5-aza诱导,细胞形态发生改变,由梭形变为"心肌样",同时有自发性细胞搏动和"肌管"样结构.免疫化学检测分化细胞肌球蛋白重链(MHC)、心肌特异性肌钙蛋白I(cTnI)、连接蛋白-43(Cx-43)等心肌细胞特异蛋白表达阳性.结论 BMSCs分离纯化在体外培养条件下可以实现大量扩增,同时维持其未分化状态.与5-氮杂苷(5-azacytidine)共培养,可以诱导其分化为心肌样细胞.BMSCs是细胞移植"心肌再生"治疗缺血性心脏病的理想种子细胞来源.     

不同数量骨髓间充质干细胞静脉移植治疗老年糖尿病大鼠的疗效

目的探讨骨髓间充质干细胞(BMSCs)静脉移植治疗糖尿病的最适数量。方法体外分离、培养、扩增BMSCs,流式细胞仪鉴定其表面标志CD44和CD34。体外用bFGF、B27以及肝细胞生长因子诱导其向胰岛样细胞分化,双硫踪染色鉴定胰岛样细胞团,倒置显微镜和透射电镜观察细胞。细胞移植组经球后静脉分别注入2×104、2×105、2×106个BMSCs于注射链脲佐菌素(STZ)制备的老年糖尿病大鼠体内,移植10 d后,检测尿糖和血糖,观察疗效。结果培养、扩增的BMSCs诱导分化后,倒置显微镜和电镜下观察类似胰岛细胞,双硫踪染色鉴定为胰岛样细胞团。BMSCs细胞移植后2×106组疗效最好,血糖值由(22.35±2.13)mmol/L下降为(14.54±0.97)mmol/L,尿糖值由(21.23±1.61)g/L下降为(12.41±1.09)g/L,显著低于2×104、2×105移植组。结论一定数量的BMSCs静脉移植可治疗糖尿病。     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

干细胞及其治疗糖尿病的研究进展

糖尿病是严重威胁人类健康的代谢紊乱性疾病.干细胞具有自我更新和多向分化潜能.来源于成体干细胞和胚胎干细胞的胰岛细胞移植也许是有前途的糖尿病治疗方法.干细胞体外培养时可以自发或诱导分化为胰岛素分泌细胞,部分实验证实移植后的胰岛素分泌细胞可以纠正糖尿病小鼠的高血糖状态,这一研究为糖尿病细胞替代治疗带来希望.     

Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells

AIM： To study the capacity of bone marrow mesenchymal stem cells （BM-MSCs） trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat.
METHODS： BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. Insulin release after glucose challenge was tested with ELISA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry.
RESULTS： BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression. Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20.
CONCLUSION： Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro. Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.     

一种改良大鼠骨髓间充质干细胞培养方法

目的建立大鼠骨髓间充质干细胞(BMSCs)的分离、改良培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。方法通过改良全骨髓贴壁法对4周龄SD雄性大鼠脱颈处死,无菌条件下分离出骨髓进行原代培养、消化传代培养及纯化。对BMSCs进行形态学观察,收获第四代BMSCs进行流式细胞仪检测其细胞表面标记物CD90、CD29、CD34、CD45的表达率及向成脂方向诱导分化。结果 BMSCs的原代培养形态学观察可见骨髓细胞接种于培养皿后,细胞呈圆型,大小不一,悬浮于培养液中。24 h后部分细胞开始贴壁,呈圆形、梭形或多角形。通过换液去除未贴壁的杂质细胞,可见短梭形、星形细胞分散贴壁生长,四五天可见放射状排列的细胞集落,伸出长短不一、粗细不均的突起,梭形细胞为主,胞浆丰富,胞核大、核仁清晰。7~8 d细胞呈集落生长,融合80%~90%,呈漩涡状,同向排列,9~10 d细胞排列紧密,逐渐融合成片。传代培养可见消化传代后,传代细胞24 h完全贴壁生长。细胞形态均一,呈梭形生长,细胞生长旺盛。四至五天可传代1次。可稳定连续传代7代以上,细胞形态及生长速度未见明显变化。BMSCs表面标记物的表达通过流式细胞仪检测结果显示,培养的第4代大鼠BMSCs均一表达CD90,CD29,阳性率分别为96.9%,96.6%;而CD34,CD45,呈阴性,阳性率分别为0.395%,7.56%。BMSCs加入成脂诱导剂后18 d,诱导而成的脂肪细胞累积脂质,脂滴变大,合并呈串珠状,经油红O染色呈鲜红色。结论与传统全骨髓贴壁法相比,改良后的全骨髓贴壁法操作步骤简单,降低离心对细胞的损害,减少了污染机会,节省经费,且分离的BMSCs细胞活性高,可大量分离、纯化、扩增,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。可为组织器官缺损性疾病、恶性肿瘤等的治疗和组织工程提供充足的种子细胞来源,具有重要的现实意义。     

mHCN4基因修饰小鼠骨髓间充质干细胞重建起搏离子流通道

目的观察超极化激活的环核苷酸门控通道亚型4(mHCN4)外源基因对小鼠骨髓间充质干细胞(mMSCs)的转染及其功能表达。方法2个月大昆明小鼠拉颈处死,无菌条件下分离股、胫骨,收集骨髓细胞种植于塑料培养瓶,24h后更换培养液,保留贴壁细胞,待细胞达90%融合时胰酶消化传代培养。收集第3代贴壁细胞,用免疫磁珠法分选CD11b-细胞继续扩增培养。以pUC18、pcDNA3-mHCN4、pIRES2-EGFP、pMSCV-puro等质粒载体为架构,用不同酶类将目的片段连接构建pMSCV-mHCN4-EGFP及pMSCV-EGFP逆转录病毒载体。载体转染PT67包装细胞,收集逆转录病毒上清转染第3代扩增的mMSCs细胞,观察mMSCs的转染效果及阳性转染细胞的mHCN4通道动力学特性。结果mMSCs的转染效率约10%～20%。mHCN4基因转染组可记录到明显的时间依赖性的超极化激活内向电流(If),If的半数最大激活电压为-99.0±5.8mV,在-140mV电压时的激活时间常数为451±61ms。该电流对CsCl高度敏感;对照组细胞在超激化状态下无明显的电流出现。结论mHCN4外源基因可在mMSCs中成功表达起搏离子流通道,基因修饰后的mMSCs有可能成为一种有效的生物起搏种子细胞。     

成人骨髓源内皮祖细胞的体外分离培养及鉴定

目的探讨成人骨髓源内皮祖细胞（EPCs）体外分离培养的可行性。方法抽取成年男性骨髓,体外全骨髓培养,传代后采用免疫微珠分选方法收集CD南细胞,EGM—MV2条件培养基诱导培养EPCs,观察细胞形态、生长情况;采用流式细胞技术检测分选细胞纯度,免疫荧光法检测EPCs特殊分子标志物CD34,CD133和VE-cadherin表达,透射电子显微镜观察分选细胞超微结构,UEA-1和Dil-ac-LDL双染色法检测分选细胞的吞噬功能。结果分选后细胞培养第3天出现集落样生长,集落边缘细胞形态伸展呈梭形或多边形,传代后呈现串珠样排列;培养至5～6d,细胞连接成大片条索状结构;CD34^＋、CD133^＋细胞百分率分别为24．13％、93．29％,其表面特异性表达CD133、CD34、VE-cadherin,具有EPCs形态特征,能吞噬Dil-ac-LDL并结合UEA-1。结论成人骨髓来源的EPCs经体外培养后形态、增殖率、生存能力、表面标志表达、功能等均较为稳定,可作为心血管组织工程种子细胞或用于干细胞治疗。     

胰十二指肠同源框因子-1联合细胞因子诱导人脐带间充质干细胞分化为胰岛β样细胞的研究

目的 探讨人脐带间充质干细胞(mesenchymal stem cells,MSCs)体外诱导分化为胰岛β样细胞的方法.方法 用含胰十二指肠同源框因子-1基因(pancreatic and duodenal homeobox factor 1,PDX1)重组腺病毒(Adxsi-CMV-PDX1)感染人脐带MSCs,再联合多种细胞因子进行诱导分化.应用RT-PCR、免疫荧光染色法检测诱导后细胞的PDX1、胰岛素(insulin)、神经元素3(ngn3)、葡萄糖转运体2(glut2)、NK转录因子6.1(NKX6.1)mRNA的表达;化学发光法检测诱导后细胞培养上清中胰岛素和C肽的分泌量;流式细胞仪检测胰岛素阳性细胞率.结果 Adxsi-CMV-PDX1感染脐带MSCs并联合细胞因子诱导后,细胞从短梭形变成长梭形,并聚集形成胰岛样细胞团,经双硫腙染成亮红色.诱导17 d后的细胞表达PDX1、ngn3、NKX6.1、insulin、glut-2的mRNA;细胞胞质内有胰岛素和C肽;细胞培养上清中胰岛素和C肽含量分别为(473.1±51.5)mU/L和(1.61±0.41)ng/ml,用25 mmol/L高糖刺激1 h后,胰岛素和C肽分泌量高达(964.4±68.1)mU/L和(3.72±1.52)ng/ml;胰岛素阳性细胞率达(11.6±4.8)%.结论 PDX1转入脐带MSCs后,联合细胞因子在体外能够诱导其分化为胰岛β样细胞,胰岛素和C肽分泌水平较高,且对高糖刺激有反应,但还不是完全成熟的β细胞.     

细粒棘球蚴重组抗原B诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶的研究

目的观察细粒棘球蚴重组抗原B(rAgB)体外诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶(IDO)的情况。方法从小鼠股骨中分离出骨髓细胞,进行小鼠重组巨噬细胞集落刺激因子(rmGM-CSF)的诱导,培养并获得CD11c+树突状细胞。应用倒置显微镜和扫描电镜观察树突状细胞形态,采用流式细胞术检测其表面标志物;用混合淋巴细胞反应(MLR)观察树突状细胞对T淋巴细胞的增殖能力。培养至第6天,收集未成熟树突状细胞进行流式细胞术检测;另向部分未成熟树突状细胞中加脂多糖(LPS)刺激24 h后,收集成熟树突状细胞,进行流式细胞术检测。在获得的未成熟树突状细胞中分别加入RPMI 1640完全培养液(为阴性对照组)、重组小鼠γ干扰素(rmIFN-γ,1 000 U/ml,为IFN-γ组)和rAgB(终浓度15μg/ml,为rAgB组),培养24 h后,通过细胞免疫组织化学和蛋白质印迹(Western blot-ting)检测各组树突状细胞IDO的表达情况。结果获得纯度为80%的CD11c+树突状细胞,在倒置显微镜和扫描电镜下均观察到典型树突状细胞。经LPS刺激的成熟树突状细胞的CD40、CD80和主要组织相容性复合体(...     

Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established

The treatment of type I diabetes by islet cell transplantation, while promising, remains restricted due to the incomplete efficacy and toxicity associated with current immunosuppression, and by limited organ availability. Given reports suggesting bone marrow derived stem cell plasticity, we sought to determine whether such cells could give rise to pancreatic islet cells in vivo. In the context of autoimmune diabetes, we transplanted unfractionated bone marrow from beta-gal trangenic donor mice into NOD mice prior to, at, and two weeks beyond the onset of disease. Successful bone marrow engraftment before diabetes onset prevented disease in all mice and for 1 year after transplant. However, despite obtaining full hematopoietic engraftment in over 50 transplanted mice, only one mouse became insulin independent, and no beta-Gal positive islets were detected in any of the mice. To test whether tolerance to islets was achieved, we injected islets obtained from the same allogeneic donor strain as the hematopoietic cells into 4 transplant recipients, and 2 had a reversion of their diabetes. Thus allogeneic bone marrow transplantation prevents autoimmune diabetes and tolerizes the recipient to donor islet grants, even in diabetic animals, yet the capacity of bone marrow derived cells to differentiate into functional islet cells, at least without additional manipulation, is limited in our model.     

Efficient and controlled gene expression in mouse pancreatic islets by arterial delivery of tetracycline-inducible adenoviral vectors

Gene transfer with adenovirus vectors has been used extensively for pancreatic islet research. However, infection efficiency varies among reports. We reevaluated the infection efficiency, defined here as the percentage of islet cells expressing transgenes, in mouse islets. When the isolated islets were infected with adenoviruses, the infection efficiency was found to be 30-40% and the transduced cells were distributed in the islet periphery. Collagenase treatment of isolated islets before infection increased the infection efficiency to 70%, but with suppression of glucose-stimulated insulin secretion. To explore more efficient strategies, we employed arterial delivery of virus particles to islets in situ. Delivery of adenovirus (approximately 10(8) particles per pancreas) through the celiac and superior mesenteric arteries is highly efficient, resulting in more than 90% transduction without impairing glucose-stimulated insulin secretion. Arterial delivery of an adenovirus harboring glycerol kinase cDNA allowed us to observe glycerol-stimulated insulin secretion from mouse islets, which was not observed when we employed the conventional method. Furthermore, the arterial delivery method combined with a tetracycline-inducible adenovirus system induced efficient and controlled transgene expression. Our data provide new insights into gene transduction methods using recombinant adenoviruses in mouse islets, and are therefore anticipated to contribute to future basic and clinical islet research applications.