体外诱导人骨髓间充质干细胞分化为肝样细胞的实验研究
发布时间:2018-01-22 04:14
本文关键词: 人骨髓间充质干细胞 生物学特性 细胞培养 细胞表面标志物 骨髓间充质干细胞 肝细胞生长因子 表皮细胞生长因子 细胞分化 肝样细胞 出处:《南方医科大学》2010年硕士论文 论文类型:学位论文
【摘要】: 肝脏疾病在我国属临床常见病、多发病,严重地危害人类的健康。大量肝脏疾病患者由于缺乏理想的、有效的治疗手段而死亡。肝细胞移植是治疗终末期肝功能衰竭的有效方法之一,却面临细胞来源缺乏、体外难以大量增殖、传代后无法保持其原有特性、免疫排斥等众多因素的限制,因此寻找新的肝细胞来源尤为迫切。而人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, HMSCs)是一类具有自我增殖和分化潜能的多能干细胞,因其具有可塑性、取材方便、低免疫原性以及易于外源基因的转染和表达等优点而被广泛用于肝脏疾病的研究。近年研究表明,在适宜的微环境下,HMSCs具有向肝样细胞分化的潜能,使其在肝细胞治疗的临床应用中越来越受到人们的重视,也为临床上应用HMSCs移植治疗肝损伤和急慢性肝功能衰竭展示了一个可喜的前景。当前,HMSCs移植被认为有可能替代原位肝移植(orthotopic liver transplantation, OLT),为肝衰竭的有效治疗带来新的希望。因此,本实验主要研究HMSCs在体外定向诱导分化为肝样细胞的能力,实验内容大致分为两部分:第一部分主要通过研究HMSCs的生物学特性,建立分离培养和扩增细胞的方法,为以后的研究提供足够的细胞来源;第二部分主要研究利用肝细胞生长因子(hepatocyte growth factor, HGF)、表皮细胞生长因子(epidermal growth factor, EGF)以及HGF+EGF联合诱导HMSCs向肝细胞方向分化,并采用免疫细胞化学染色、逆转录聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)及酶联免疫吸附法(ELISA)等技术鉴定细胞分化能力,试图为自体骨髓间充质干细胞移植治疗终末期肝病的临床应用提供理论依据。 第一部分人骨髓间充质干细胞的生物学特性 目的建立HMSCs体外分离培养和扩增的方法,研究HMSCs的生物学特性,为利用HMSCs作为种子细胞治疗失代偿性肝硬化等终末期肝病提供实验基础。 方法采用密度梯度离心联合贴壁筛选法分离培养HMSCs,测定其接种贴壁率;在倒置显微镜下连续观察细胞的形态变化;并应用流式细胞仪测定细胞表面标记物鉴定所培养的细胞。 结果HMSCs体外培养生长状况良好,具有活跃的增殖能力,接种12h后细胞贴壁率达95%以上,细胞外观呈纺锤形、长梭形和多角形等,第4 d细胞数量开始增多,以克隆集落方式增殖,细胞形态呈长梭形,细胞排列成漩涡状,细胞间界限不清,10-14d铺满瓶底;培养的HMSCs阳性表达β1-整合素CD29(99.4%)和基质受体CD44(97.2%),阴性表达造血细胞标志CD34(4.7%),CD45(5.6%)。 结论HMSCs是区别于人骨髓中造血干细胞的另一类细胞,通过密度梯度离心联合贴壁筛选法可在体外培养出高纯度的HMSCs,获得的HMSCs在体外生长稳定,且增殖较快,可作为组织工程的种子细胞。 第二部分体外诱导人骨髓间充质干细胞向肝样细胞分化 目的探讨HGF和EGF体外诱导HMSCs向肝样细胞分化的可行性,为生物人工肝及肝细胞移植寻找理想的种子细胞。 方法取食管癌手术患者肋骨,采用密度梯度离心联合贴壁筛选法获取HMSCs。取第3代HMSCs,分为4组,肝细胞生长因子组(A组)加入20μg/L HGF,表皮细胞生长因子组(B组)加入20μg/L EGF,联合组(C组)同时加入20μg/LHGF和20μg/L EGF,空白对照组(D组)不加任何生长因子。倒置显微镜下观察细胞形态的变化,在诱导7、14d RT-PCR检测甲胎蛋白(alpha fetoprotein,AFP)和白蛋白(albumin, ALB) mRNA的表达,诱导7、14、21、28d免疫细胞化学染色检测AFP,细胞角蛋白18 (cytokeratin 18, CK18)及诱导7、14、21dELISA检测培养上清液中ALB水平。 结果HGF组、EGF组、联合组HMSCs诱导后向肝样细胞转化,细胞形态由长梭形变为类圆形或多角形,诱导第7,14d AFP、ALB mRNA均呈阳性表达;空白对照组未见多角形细胞,AFP、ALB mRNA均呈阴性表达;免疫细胞化学染色各诱导组均可检测出AFP和CK18表达,并以时间依赖方式产生ALB,且细胞上清液ALB的含量在A、B、C三组间的差异有显著性意义(F=9160.446,P=0.000),而空白对照组则没有检测到上述指标。 结论HGF、EGF以及二者联合均具有诱导HMSCs向肝样细胞分化的能力,以HGF+EGF诱导HMSCs分化为肝样细胞的阳性率最高,两者具有一定程度的联合作用。 通过以上实验研究,我们掌握了HMSCs的体外分离培养和扩增的方法,证明了HMSCs具有干细胞自我更新和强大复制能力的生物学特性,具备种子细胞的先决条件;而经过HGF和EGF体外诱导后的HMSCs不但在形态上转变为肝样细胞,而且还能分泌肝细胞特性蛋白,如AFP和ALB,因此,我们初步认为HMSCs具有向肝细胞分化的能力,将HMSCs用于细胞移植治疗,具有非常重要的理论研究意义和临床价值。
[Abstract]:Liver disease in China is a common clinical disease, disease, serious harm to human health. A large number of patients with liver disease due to the lack of ideal, effective treatment and death. Liver cell transplantation is one of effective methods for the treatment of end-stage liver failure, are lack of in vitro cell source, to proliferate, cannot keep its original characteristics after passage, many factors such as immune rejection, so to find a new source of liver cells is particularly urgent. But the human bone marrow mesenchymal stem cells (human bone marrow mesenchymal stem cells, HMSCs) is a kind of self proliferation and differentiation of pluripotent stem cells, because of its high plasticity material convenient, low immunogenicity and easy exogenous gene transfection and expression has been used extensively in the study of liver diseases. Recent studies show that in a suitable micro environment, HMSCs has to hepatocyte like cells Cell differentiation potential, its clinical application in liver cell therapy has been paid more attention, but also for the clinical application of HMSCs transplantation for the treatment of liver injury and chronic liver failure shows a promising prospect. At present, HMSCs transplantation is considered a possible alternative to orthotopic liver transplantation (orthotopic liver transplantation, OLT), bring new hope for the effective treatment of liver failure. Therefore, the experimental research of HMSCs in the differentiation into hepatocyte like cells, the experiment is mainly divided into two parts: the first part mainly through the study on the biological characteristics of HMSCs, a method for separation and cultivation and expansion of cells, provide enough cell sources for the future research; the second part mainly studies the use of hepatocyte growth factor (hepatocyte growth, factor, HGF), epidermal growth factor (epidermal growth, factor, and EGF) HGF+EGF induced HMSCs differentiation to hepatocytes and direction by immunocytochemical staining, reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) and enzyme-linked immunosorbent assay (ELISA) technique to identify cell differentiation ability to autologous bone marrow mesenchymal stem cell transplantation provides a theoretical basis for the clinical application of end-stage liver disease.
The biological characteristics of human bone marrow mesenchymal stem cells
Objective to establish a method for isolation, culture and amplification of HMSCs, and to study the biological characteristics of HMSCs, so as to provide experimental basis for the use of HMSCs as seed cell in the treatment of decompensated cirrhosis and other end-stage liver diseases.
Methods density gradient centrifugation combined with adherence screening method was used to isolate and culture HMSCs. The rate of inoculation adherence was detected. Morphological changes of cells were observed continuously under inverted microscope, and cell surface markers were used to identify the cultured cells.
Results HMSCs in vitro growth in good condition, with active proliferation, after inoculation with 12h cells adherent rate reached more than 95%, the appearance of cells were spindle, spindle and polygon, the number of fourth D cells began to increase, in order to clone colony proliferation, cell morphology of fusiform cells arranged in a spiral shape and between the cells is not clear, 10-14d covered the bottom of the bottle; the culture of HMSCs positive expression of 1- integrin beta CD29 (99.4%) and CD44 (97.2%), matrix receptor negative hematopoietic cell marker CD34 (4.7%), CD45 (5.6%).
Conclusion HMSCs is another kind of cell which is different from human bone marrow hematopoietic stem cells. HMSCs can be cultured in vitro by density gradient centrifugation and adhesion screening. The HMSCs obtained in vitro is stable and fast growing, and can be used as a seeding cell for tissue engineering.
Second part induced differentiation of human bone marrow mesenchymal stem cells into hepatocyte like cells in vitro
Objective to explore the feasibility of inducing HMSCs differentiation into hepatocyte like cells by HGF and EGF in vitro, and to search for ideal seed cells for the transplantation of bioartificial liver and hepatocytes.
Methods rib in patients with esophageal cancer surgery by density gradient centrifugation and adherence screening method of HMSCs. and the third generation of HMSCs, divided into 4 groups, hepatocyte growth factor group (group A) with 20 g/L HGF, epidermal growth factor group (group B) with 20 g/L EGF group (C group at the same time) with 20 g/LHGF and 20 g/L EGF, blank control group (D group) without any growth factors. Morphological changes were observed under the inverted microscope, in 7,14d induced by RT-PCR detection of alpha fetoprotein (alpha fetoprotein, AFP) and albumin (albumin, ALB) mRNA expression induced by 7,14,21,28d immunocytochemistry staining for AFP, cytokeratin 18 (cytokeratin 18, CK18) and 7,14,21dELISA induced by detection of ALB level in the culture medium.
缁撴灉HGF缁,
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