大鼠骨髓源性间充质干细胞定向诱导分化为肝细胞的实验研究

发布时间：2018-05-06 04:34

本文选题：骨髓间充质干细胞 + 肝细胞　；参考：《吉林大学》2007年硕士论文

【摘要】： 骨髓来源的间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)是骨髓内具有多种分化潜能的一类干细胞亚群,并且在一定条件下可以向多种不同组织细胞分化。本论文主要用具有脂肪细胞分化潜能的MSCs,进行向肝细胞定向诱导分化,为肝细胞移植提供可靠的种子来源。目的:分离、纯化、培养和鉴定大鼠骨髓间充质干细胞;采用损伤肝组织和血清对MSCs进行定向诱导分化,并确定MSCs体外诱导分化为肝样细胞的最佳条件。方法:无菌条件下取周龄大乳鼠四肢骨骨髓,全血培养所得的贴壁细胞用1.077g/mlFICOLL(淋巴细胞分离液)密度梯度离心去除成纤维细胞和残留血细胞,传代,通过控制消化时间得到纯度较高的骨髓MSC。取第三代细胞(P3),用成脂诱导的方法检测其分化潜能,用损伤肝组织和血清定向诱导分化MSCs,利用免疫细胞化学和RT-PCR等方法对MSCs及诱导后肝样细胞进行细胞特异性标志物的鉴定。结果:1.全血培养的骨髓原代细胞呈细沙样,换液后,贴壁细胞呈集落样生长,细胞贴壁后由于密度的不同而表现不同的形态。当密度较大时,细胞呈梭形,呈旋涡状,放射状或平行状生长。当密度较小时,细胞呈延伸态纺锤形或三角形。密度梯度离心后细胞均质性明显提高,从形态学上看具有MSC的特点。 2.传代培养的第三代(P3)MSCs经免疫细胞化学检测CD105呈阳性,提示细胞具有MSC特异性标记。 3.MSCs在20%马血清、20%马血清+氢化可的松、DX+IBMX+IS+ID,这些成脂诱导剂均能分化为脂肪细胞,阳性率达到90%以上。油红O结合苏木素染色后可见橘红色的脂肪滴和浅蓝色的细胞核。其中DX+IBMX+IS+ID诱导的脂肪细胞脂滴体积最大,诱导分化时间最短,提示虽然三组诱导环境均可检测MSCs的分化潜能,但DX+IBMX+IS+ID诱导效果最明显。 4.损伤肝组织和血清诱导MSCs后,免疫细胞化学染色ALB阳性,糖原染色阳性,RT-PCR可检测出肝特有基因AFP、ALB的表达。混合组ALB及糖原染色阳性率高于单因素组,提示混合组比单因素组能更高效的诱导MSCs向肝细胞分化。结论:1.本实验采用全血培养法和密度梯度离心法对大鼠骨髓MSCs进行分离、纯化、体外培养,同时从细胞形态和生物学特性进行观察和检测,该方法是有效、简便,可获得高纯度MSCs的可靠方法,MSCs在体外不但可进行大量培养扩增,而且其增殖和传代能力很强。 2.骨髓MSCs在成脂诱导剂诱导下,可以定向分化为脂肪细胞,阳性率均在90%以上,说明所得到的MSCs具有多向分化潜能。其中DX+IBMX+IS+ID组诱导效果最明显。 3.损伤肝组织和血清定向诱导具有脂肪细胞分化潜能MSCs(P3),分化为肝样细胞,并表达肝细胞的特异性标志物,说明损伤肝组织及血清中存在使MSCs分化为肝样细胞的相关因子或成分。结果提示混合组为MSCs向肝细胞分化提供了更适宜的诱导环境。
[Abstract]:Bone marrow derived mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) are a type of stem cell subgroup with multiple differentiation potential in bone marrow, and can differentiate into a variety of different tissue cells under certain conditions. This paper mainly uses MSCs with the potential of adipocyte differentiation to induce differentiation to liver cells and be the liver. Cell transplantation provides a reliable source of seed.
Objective: to isolate, purify, culture and identify rat bone marrow mesenchymal stem cells (MSCs), and to induce differentiation of MSCs by using damaged liver tissue and serum, and to determine the best conditions for MSCs to induce differentiation into hepatocyte like cells in vitro.
Methods: under aseptic conditions, the bone marrow of the limbs of the four limbs of the old rats was taken. The adherent cells obtained from the whole blood culture were removed by 1.077g/mlFICOLL (lymphocyte separation) density gradient centrifugation to remove the fibroblasts and residual blood cells. By controlling the digestion time, the third generation cells (P3) with high purity MSC. were obtained by controlling the digestion time, and the method of lipid induction was used. The differentiation potential was detected, the differentiated MSCs was induced by damaged liver tissue and serum, and the specific markers of MSCs and induced hepatocyst were identified by immunocytochemistry and RT-PCR.
Results: 1. the primary cells in the whole blood culture were fine sand samples. After changing the liquid, the adherent cells were colonies, and the cells displayed different forms due to the different density. When the density was large, the cells showed spindle shaped, radial or parallel growth. When the density was small, the cells showed an extended state spindle shape or triangle. After gradient centrifugation, the cell homogeneity was obviously improved, and MSC was characteristic in morphology.
2. the third generation (P3) MSCs of passage culture was positive for CD105 by immunocytochemistry, indicating that the cells had MSC specific markers.
3.MSCs in 20% horse serum, 20% horse serum + hydrocortisone, DX+IBMX+IS+ID, these lipid inducers can differentiate into adipocytes, the positive rate is more than 90%. After the oil red O combined with hematoxylin, the orange red fat drops and the light blue nuclei are visible. Among them, the fat cell lipid droplets induced by DX+IBMX+IS+ID are the largest and induce the differentiation time. The shortest suggested that although the induction potential of the three groups could detect the differentiation potential of MSCs, the induction effect of DX+IBMX+IS+ID was the most obvious.
4. after MSCs injury liver tissue and serum induced, immunocytochemical staining ALB positive, glycogen staining positive, RT-PCR can detect the expression of liver specific gene AFP, ALB expression. The positive rate of ALB and glycogen staining in the mixed group is higher than that of the single factor group, suggesting that the mixed group can induce the differentiation of MSCs to the liver cells more efficiently than the single factor group.
Conclusion: 1. the total blood culture and density gradient centrifugation were used to separate the bone marrow MSCs of rats, to be purified and cultured in vitro, and to observe and detect the morphology and biological characteristics of the cells. This method is effective and simple, and a reliable method for obtaining high purity MSCs can be obtained. MSCs can not only be expanded in vitro, but also increase in vitro. The ability to colonization and passage is very strong.
2. bone marrow MSCs can differentiate into adipocytes under the induction of lipid inducer, and the positive rate is above 90%, indicating that the obtained MSCs has multipotential differentiation potential, of which the DX+IBMX+IS+ID group has the most obvious induction effect.
3. the hepatic tissue and serum were directed to induce the differentiation potential of adipocyte MSCs (P3), differentiate into hepatocyte like cells, and express the specific markers of liver cells, indicating the existence of related factors or components that differentiate MSCs into hepatocyte like cells in the injured liver tissue and serum. The results suggest that the mixed group is more suitable for the differentiation of MSCs to liver cells. Induce the environment.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【参考文献】