骨髓间充质干细胞向肝细胞分化的诱导和肝细胞极化分子表达与调节

发布时间：2018-06-13 23:11

本文选题：骨髓间充质干细胞 + 肝细胞　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 研究背景和目的：经过20多年的发展,肝细胞移植作为肝移植的替代疗法,已经成为一种治疗急、慢性肝功能衰竭和肝脏相关遗传代谢疾病的有效方法。但是目前肝细胞移植仍面临许多问题,如肝细胞的来源短缺、移植细胞数量有限、肝细胞移植后的增殖、受损肝细胞的替代程度及免疫排斥等诸多问题。2002有人报道,BMSC可以在体外诱导成肝细胞。而BMSC以自身独特优点有望使细胞移植在肝病治疗方面取得实质性的突破,临床应用前景突出。本实验室已经掌握各种方法诱导BMSC向肝细胞分化,并取得了成功。为了临床应用方便,我们探讨了更简单、可控、成分明确的培养方法。我们用人血清白蛋白代替了FBS,用无血清的培养方法来提供肝细胞移植临床应用更好的细胞来源。肝脏生理功能的实现有赖于肝细胞极化的形成和维持。但是,目前我们对肝细胞极化的认识非常有限。肝细胞极化研究在很大程度上受限于缺乏理想的体外肝细胞极化模型。因此我们应用了骨髓间充质干细胞来源的肝细胞,对体外肝细胞的极化进行了研究。以便用骨髓间充质干细胞来源的肝细胞建立良好的体外肝细胞极化模型。熊去氧胆酸(UDCA)具有利胆、抗肝细胞凋亡和拮抗疏水性胆汁酸的细胞毒性等多种作用,在临床上用于治疗肝胆系统多种疾病。但目前对UDCA细胞内作用机制认识有限,UDCA对肝细胞极化的调节作用尚缺少研究。本研究选择了SR-B1、MRP2和NTCP三个极化分子进行检测,来研究UDCA在mRNA水平对肝细胞极化分子表达的调节作用的作用。实验方法： 1.提取成人骨髓间充质干细胞在无血清培养下向肝细胞诱导分化。通过与有血清诱导分化组和非诱导分化组比较,使用光学显微镜观察细胞分化0天、7天、14天、21天的细胞形态；使用白蛋白生化检测试剂盒检测肝细胞特异性标记物ALB的蛋白表达；使用免疫荧光染色,荧光显微镜下观察白蛋白在肝细胞内的合成、分布情况,观察无血清培养诱导的肝细胞是否具有与有血清诱导分化组细胞相似的成熟肝细胞的特征。 2.使用实时定量PCR方法来检测分化7天、14天、21天和最后7天加用50μmol/L UDCA(21+U组)诱导的肝细胞的MRP2、NTCP、SR-B1三个极化分子的表达；使用免疫荧光染色方法在共聚焦显微镜下观察MRP2、SR-B1在细胞上的定位,以此观察骨髓间充质干细胞相肝细胞诱导分化过程中MRP2、NTCP、SR-B1的mRNA表达和MRP2、SR-B1的定位情况。研究结果： 1.成人骨髓间充质干细胞在无血清培养下诱导分化21天后具有成熟肝细胞形态(无血清诱导分化组,A组),与非诱导分化组(C组)差别明显,而与有血清的诱导分化组(B组)相近。分别检测三组细胞的白蛋白表达,在分化7,14,21天的A组和B组白蛋白逐渐升高,14天后维持在较高水平,三个时间点A组和B组白蛋白表达量明显高于C组(p0.05)。A组和B组细胞在诱导分化14天后,白蛋白免疫荧光染色发现,细胞内有大量点状、颗粒状或者均匀分布的绿色荧光,较自身未加一抗的对照及C组比较明显为阳性。 2.实时定量PCR检测分化7天、14天、21天和最后7天加用50μmol/L UDCA(21+U组)诱导的肝细胞的MRP2、NTCP、SR-B1极化分子的mRNA表达发现,7天、14天、21天和21+U组四组细胞MRP2、NTCP、SR-B1的mRNA表达随时间增加而升高,14天、21天组明显高于7天组(p0.05),21+U组明显高于21天组(p0.05)。免疫荧光染色显示细胞诱导14天后MRP2、SR-B1两种极化蛋白分别定位于肝细胞膜的不同部位,呈现明显的极化特征,而对照组细胞未观察到特异性分布。研究结论： 1.无血清条件下培养骨髓间充质干细胞在特定诱导因子下可以分化为有一定功能的肝细胞,与诱导分化培养基诱导的肝细胞类似。从而为肝细胞临床移植准备了的良好的细胞来源。 2.成人骨髓间充质十细胞具有向极化肝细胞分化的潜能。骨髓问充质十细胞分化的肝细胞可以作为研究肝细胞极化形成和调节机制的良好模型。UDCA可以促进肝细胞极化分子的表达。
[Abstract]:Research background and purpose:
After more than 20 years of development, hepatocyte transplantation, as a replacement therapy for liver transplantation, has become an effective method for the treatment of acute, chronic liver failure and liver related genetic metabolic diseases. However, hepatocyte transplantation still faces many problems, such as the shortage of liver cells, the limited number of transplanted cells, and the proliferation of liver cells after transplantation. .2002 has been reported that BMSC can induce hepatocytes in vitro, and BMSC is expected to make a substantial breakthrough in the treatment of liver diseases with its own unique advantages, and the prospect of clinical application is prominent.
The laboratory has mastered various methods to induce BMSC to differentiate into hepatocytes and has achieved success. In order to be convenient for clinical application, we have explored a more simple, controllable and well-defined culture method. We use human serum albumin instead of FBS, and use serum-free culture to provide a better cell source for the clinical application of hepatocyte transplantation.
The realization of liver function depends on the formation and maintenance of hepatocyte polarization. However, our understanding of hepatocyte polarization is very limited. The study of hepatocyte polarization is largely limited to the lack of ideal model of hepatocyte polarization in vitro. Therefore, we have applied the liver cells derived from bone marrow mesenchymal stem cells to the liver in vitro. Cell polarization was studied in order to establish a good in vitro hepatocyte polarization model using bone marrow mesenchymal stem cells derived hepatocytes.
Ursodeoxycholic acid (UDCA) has many advantages, such as gallbladder, anti hepatocyte apoptosis, and antagonism to the cytotoxicity of hydrophobic bile acids. It is used in the treatment of various diseases of hepatobiliary system. However, there is limited understanding of the mechanism of intracellular action of UDCA, and the use of UDCA for the regulation of hepatocyte polarization is still lacking. This study chose SR-B1, MRP2 and NTCP. Three polarized molecules were detected to study the role of UDCA in regulating the expression of polarized molecules in hepatocytes at mRNA level.
Experimental methods:
1. the adult bone marrow mesenchymal stem cells were induced to differentiate into hepatocytes in serum-free culture. Compared with the serum induced and non induced differentiation groups, the cell morphology was observed by optical microscope for 0 days, 7 days, 14 days and 21 days by optical microscope, and the liver cell specific marker ALB was detected by the albumin biochemical test kit. The expression of protein, immunofluorescence staining, and fluorescence microscopy were used to observe the synthesis and distribution of albumin in the liver cells, and to observe whether the liver cells induced by serum-free culture have the characteristics of mature hepatocytes similar to those of the cells with serum induced differentiation.
2. using real-time quantitative PCR method to detect the expression of MRP2, NTCP, SR-B1 three molecules in the hepatocytes of 7 days, 14 days, 21 days and the last 7 days with 50 mu UDCA (21+U group), and observe the location of MRP2 and SR-B1 on the cell by using the immunofluorescent staining method under confocal microscope to observe the bone marrow mesenchymal stem cell phase. MRNA expression of MRP2, NTCP, SR-B1 and localization of MRP2 and SR-B1 during hepatocyte differentiation.
The results of the study:
1. adult bone marrow mesenchymal stem cells had mature liver cell morphology (serum free differentiation group, A group) after 21 days of serum-free induction of differentiation (group A), which was significantly different from that of non induced differentiation group (group C), and similar to that of the induced differentiation group (group B) with serum. The expression of albumin in three groups of cells was detected respectively in A and B groups of 7,14,21 days of differentiation. The protein increased gradually and maintained at a higher level in 14 days. The expression of albumin in group A and B was significantly higher than that of group C (P0.05) and group.A and B group for 14 days. Albumin immunofluorescence staining found that there were a large number of dot like, granular or uniformly distributed green fluorescence in the cells, compared with the control and C group that did not add one to one's own resistance and C group. It was obviously positive.
2. real-time quantitative PCR detection of 7 days, 14 days, 21 days and the last 7 days with 50 mol/L UDCA (21+U group) induced MRP2, NTCP, SR-B1 polarizing molecules mRNA expression, 7 days, 14 days, 21 days and 21+U groups of four cells MRP2, NTCP, SR-B1, increased with time, 14 days, 21 days group obviously higher than 7 days group obviously It was significantly higher than the 21 day group (P0.05). Immunofluorescence staining showed that the cells were induced for 14 days after MRP2, and two kinds of SR-B1 polarin were located in different parts of the liver cell membrane, showing obvious polarization characteristics, while the cells in the control group did not observe the specific distribution.
The conclusions are as follows:
1. serum free bone marrow mesenchymal stem cells can differentiate into a certain function of liver cells under specific inducible factors under the condition of serum free conditions, which are similar to those induced by differentiation medium. Thus, a good cell source for the clinical transplantation of hepatocytes is prepared.
2. adult bone marrow mesenchymal cells (ten cells) have the potential to differentiate into polarized hepatocytes. The bone marrow mesenchymal ten cell differentiated hepatocytes can be used as a good model for the study of the formation and regulation mechanism of hepatocyte polarization, which can promote the expression of polarizable molecules in liver cells.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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