骨髓基质干细胞的分离培养及定向诱导分化的实验研究
发布时间：2019-01-07 10:33
【摘要】：目的:目前肝衰竭的治疗方法主要是靠细胞替代和肝移植,但由于供肝缺乏,尚存在免疫排斥反应,因而制约了肝移植的应用。骨髓基质干细胞(BMSCs)具有容易获得,可以自我复制,高度增殖,在特定的条件下可向各胚层来源的细胞分化的特点,因而将BMSCs细胞移植应用于肝病治疗有非常重要的临床价值。本研究摸索适合BMSCs生长的培养体系,探索BMSCs诱导分化为肝细胞的适合条件和机制,为临床上通过BMSCs细胞移植治疗肝脏疾病提供理论依据。 方法:用全骨髓法从大鼠骨髓中分离出BMSCs,观察BMSCs的生长特性,MTT法比较不同的培养条件对细胞活力的影响,摸索适合BMSCs生长的培养体系;探索在体外诱导BMSCs向成骨细胞、脂肪细胞分化的条件,证实BMSCs的多向分化潜能;将BMSCs在体外传代扩增后,培养体系中加入肝细胞提取液(G)和胎肝细胞培养上清液(CM),模拟肝细胞体内生长的微环境,与加入肝细胞生长因子(HGF)、神经细胞生长因子(NGF)、维甲酸(RA)的诱导方法作比较,观察分化细胞的形态结构变化,并采用免疫细胞化学法检测肝细胞的特异性标志α_1-抗胰蛋白酶和白蛋白,ICG染色法鉴定肝细胞的生成;将BrdU标记后的BMSCs进行异体移植,免疫组织化学法检测BMSCs在肝内的迁移和分化,PAS法检测所生成肝细胞的功能。 结果:(1) 使用低糖DMEM加入20%新生牛血清比10%新生牛血清MTT细胞活性增高(P0.01),使用高糖DMEM与低糖DMEM差异无显著性(P0.05),而用RPMI-1640则MTT活性降低(P0.01)。(2) 本实验中分离的BMSCs在维生素C50μg/ml、地塞米松10~(-7)mol/L、β-甘油磷酸钠10mmol/L的诱导条件下可以向成骨细胞分化,在地塞米松10~(-8)mol/L、胰岛素10μg/ml的诱导条件下可以向脂肪细胞分化。(3) 免疫细胞化学法结果显示G、CM、NGF、HGF、RA诱导4d、14d、21d后与对照组相比α_1-抗胰蛋白酶和白蛋白表达均呈阳性(P0.01),其中G、CM、NGF组AAT、ALB表达强于HGF、RA组(P0.01)。AAT、ALB阳性的细胞形态上逐渐趋短变圆。(4) 形态上呈卵圆形的细胞PAS反应胞浆呈强阳性,有分泌储存糖原的功能。(5) 向肝细胞诱导后,卵圆形细胞能摄取ICG,呈现绿色。诱导3w后ICG阳性率比诱导2w高(P0.01)。(6) BMSCs细胞移植后1周、4周损伤组受体肝脏
[Abstract]:Objective: at present, the treatment of liver failure mainly depends on cell substitution and liver transplantation, but due to the lack of donor liver, there is still immune rejection, which restricts the application of liver transplantation. Bone marrow mesenchymal stem cells (BMSCs) have the characteristics of being readily available, self-replicating, highly proliferating, and differentiating into cells from various embryonic layers under certain conditions. Therefore, the transplantation of BMSCs cells into the treatment of liver disease has very important clinical value. This study explored the culture system suitable for the growth of BMSCs, and explored the suitable conditions and mechanism for the differentiation of BMSCs into hepatocytes, which provided a theoretical basis for the clinical treatment of liver diseases by BMSCs cell transplantation. Methods: BMSCs, was isolated from rat bone marrow by whole bone marrow method to observe the growth characteristics of BMSCs. MTT method was used to compare the effect of different culture conditions on cell viability and to explore a suitable culture system for BMSCs growth. To explore the conditions for inducing BMSCs to differentiate into osteoblasts and adipocytes in vitro and to confirm the potential of BMSCs to differentiate into osteoblasts and adipocytes. After BMSCs was subcultured and amplified in vitro, (G) and (CM), were added to the culture system to simulate the microenvironment of hepatocyte growth in vivo, and the addition of hepatocyte growth factor (HGF), to the culture system. The induction methods of (NGF), retinoic acid (RA) were compared to observe the morphological and structural changes of differentiated cells, and the specific markers 伪 _ 1-antitrypsin and albumin of hepatocytes were detected by immunocytochemistry. The formation of hepatocytes was identified by ICG staining. The BMSCs labeled with BrdU was transplanted, the migration and differentiation of BMSCs in liver were detected by immunohistochemistry, and the function of hepatocytes was detected by PAS. Results: (1) the activity of MTT cells in low glucose DMEM added 20% newborn bovine serum was higher than that in 10% newborn bovine serum (P0.01). There was no significant difference between high glucose DMEM and low glucose DMEM (P0.05). However, the BMSCs isolated by RPMI-1640 reduced MTT activity (P0.01). (2) could differentiate into osteoblasts under the condition of vitamin C 50 渭 g / ml and dexamethasone 10 ~ (-7) mol/L, 尾 -glycerophosphate 10mmol/L. When dexamethasone was induced by 10 ~ (-8) mol/L, insulin (10 渭 g/ml), adipocytes could differentiate into adipocytes. The expression of 伪 _ 1-antitrypsin and albumin were positive after 21 days compared with the control group (P0.01), and the expression of AAT,ALB was stronger in the GG CM-NGF group than in the HGF,RA group (P0.01). AAT,). (4) the PAS reaction cytoplasm of the oval cells was strongly positive, and had the function of secreting and storing glycogen. (5) after induced to hepatocytes, the oval cells could absorb ICG,. Green. The positive rate of ICG after 3 weeks of induction was higher than that of 2 weeks of induction (P0.01). (6) BMSCs cells were transplanted at 1 and 4 weeks after transplantation.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329
本文编号：2403557
[Abstract]:Objective: at present, the treatment of liver failure mainly depends on cell substitution and liver transplantation, but due to the lack of donor liver, there is still immune rejection, which restricts the application of liver transplantation. Bone marrow mesenchymal stem cells (BMSCs) have the characteristics of being readily available, self-replicating, highly proliferating, and differentiating into cells from various embryonic layers under certain conditions. Therefore, the transplantation of BMSCs cells into the treatment of liver disease has very important clinical value. This study explored the culture system suitable for the growth of BMSCs, and explored the suitable conditions and mechanism for the differentiation of BMSCs into hepatocytes, which provided a theoretical basis for the clinical treatment of liver diseases by BMSCs cell transplantation. Methods: BMSCs, was isolated from rat bone marrow by whole bone marrow method to observe the growth characteristics of BMSCs. MTT method was used to compare the effect of different culture conditions on cell viability and to explore a suitable culture system for BMSCs growth. To explore the conditions for inducing BMSCs to differentiate into osteoblasts and adipocytes in vitro and to confirm the potential of BMSCs to differentiate into osteoblasts and adipocytes. After BMSCs was subcultured and amplified in vitro, (G) and (CM), were added to the culture system to simulate the microenvironment of hepatocyte growth in vivo, and the addition of hepatocyte growth factor (HGF), to the culture system. The induction methods of (NGF), retinoic acid (RA) were compared to observe the morphological and structural changes of differentiated cells, and the specific markers 伪 _ 1-antitrypsin and albumin of hepatocytes were detected by immunocytochemistry. The formation of hepatocytes was identified by ICG staining. The BMSCs labeled with BrdU was transplanted, the migration and differentiation of BMSCs in liver were detected by immunohistochemistry, and the function of hepatocytes was detected by PAS. Results: (1) the activity of MTT cells in low glucose DMEM added 20% newborn bovine serum was higher than that in 10% newborn bovine serum (P0.01). There was no significant difference between high glucose DMEM and low glucose DMEM (P0.05). However, the BMSCs isolated by RPMI-1640 reduced MTT activity (P0.01). (2) could differentiate into osteoblasts under the condition of vitamin C 50 渭 g / ml and dexamethasone 10 ~ (-7) mol/L, 尾 -glycerophosphate 10mmol/L. When dexamethasone was induced by 10 ~ (-8) mol/L, insulin (10 渭 g/ml), adipocytes could differentiate into adipocytes. The expression of 伪 _ 1-antitrypsin and albumin were positive after 21 days compared with the control group (P0.01), and the expression of AAT,ALB was stronger in the GG CM-NGF group than in the HGF,RA group (P0.01). AAT,). (4) the PAS reaction cytoplasm of the oval cells was strongly positive, and had the function of secreting and storing glycogen. (5) after induced to hepatocytes, the oval cells could absorb ICG,. Green. The positive rate of ICG after 3 weeks of induction was higher than that of 2 weeks of induction (P0.01). (6) BMSCs cells were transplanted at 1 and 4 weeks after transplantation.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329
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