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目的:探讨大鼠骨髓间充质细胞向肝细胞样细胞的诱导分化,观察诱导分化细胞的形态和分子生物学特性,及其对急性肝损伤大鼠的治疗作用.方法:分离、培养并纯化大鼠骨髓间充质细胞,培养基中加入肝细胞生长因子(HGF)、纤维生长因子-4(FGF-4)及上皮细胞生长因子(EGF),分别于7、14、21、28d观察细胞形态变化,采用RT-PCR方法检测细胞白蛋白(ALB)、甲种胎儿球蛋白(AFP)、细胞角蛋白-18(CK-18)的mRNA表达,将诱导分化28d的细胞经DAPI染料染色后,经门静脉注入同种异体大鼠体内,经24、48、72及168h分批处死大鼠,观察大鼠肝组织内荧光细胞的分布.大鼠腹腔注射硫代乙酰胺制作急性肝衰竭模型,将1×106及5×106个诱导分化细胞经门静脉注入大鼠体内,经48、168h采血查肝功,168h处死大鼠,取肝组织病理学检查.结果:大鼠间充质细胞经上述3种细胞因子诱导后,形态和生物学行为方面都发生向肝细胞样细胞方面的转化,经门静脉注入大鼠体内后,有大量该种细胞在肝组织内分布,24h荧光细胞最多,随时间延长逐渐减少,可持续7d.骨髓间充质细胞的诱导分化细胞经门静脉注入肝损伤模型大鼠体内后,大鼠肝功能有所好转,注入1×106细胞大鼠ALT由注入前238.0±113.5U/L,48h后降至189±68.4U/L,168h后降至149.0±54.2U/L,TBIL由注入前2.9±1.6μmol/L,48h至3.0±1.4μmol/L,168h至1.3±0.3μmol/L;注入5×106个细胞者(ALT)由注入前238.0±113.5U/L,48h后降至169.7±46.0U/L,168h后降至103.7±46.0U/L,TBIL由注入前2.9±1.6μmol/L,48h至2.9±1.3μmol/L,168h至0.9±0.3μmol/L.结论:大鼠骨髓间充质细胞可在某些细胞因子的诱导作用下发生类肝细胞样转化,将转化细胞经门静脉植入同种异体大鼠体内后可在肝组织内存活并对大鼠肝损伤有一定修复作用.
OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal cells into hepatocyte-like cells and to observe the morphological and molecular biological characteristics of induced differentiated cells and their therapeutic effects on acute liver injury in rats.Methods: Isolation, culture and purification Rat bone marrow mesenchymal cells were added with hepatocyte growth factor (HGF), fibroblast growth factor (FGF-4) and epidermal growth factor (EGF). The cells were observed at 7,14,21,28d RT-PCR was used to detect the mRNA expression of albumin, ALB, AFP and CK-18, and the cells differentiated for 28 days were stained with DAPI dye , And allogeneic rats were injected through the portal vein and the rats were sacrificed at 24, 48, 72 and 168 hours in batches to observe the distribution of the fluorescent cells in the rat liver.A rat model of acute hepatic failure was induced by intraperitoneal injection of thioacetamide, 1 × 106 and 5 × 106 induced differentiated cells were injected into the rat’s body through the portal vein, and blood was collected at 48 and 168h for liver function test, and the rats were sacrificed at 168h.The pathological examination of liver tissue was performed.Results: After induction by the three cytokines, both morphological and biological behavior occurred After transformation into hepatocyte-like cells, a large number of these cells were distributed in the liver tissue through the portal vein, and the highest number of the cells were observed at 24 h, then decreased gradually with the passage of time and lasted for 7 days. The differentiation of bone marrow mesenchymal cells The rat liver function improved after the cells were injected into the liver injury model rats through portal vein. The ALT of rats injected with 1 × 106 cells decreased from 238.0 ± 113.5U / L before injection to 189 ± 68.4U / L after 48h, and 168h Then dropped to 149.0 ± 54.2U / L, TBIL was increased from 2.9 ± 1.6μmol / L, 48h to 3.0 ± 1.4μmol / L, 168h to 1.3 ± 0.3μmol / L before injection; The level of TBIL decreased from 2.9 ± 1.6μmol / L, 48h to 2.9 ± 1.3μmol / L before injection, and decreased to 169.7 ± 46.0U / L after 48h and 103.7 ± 46.0U / L after 168h, , 168h to 0.9 ± 0.3μmol / L.Conclusion: Rat bone marrow mesenchymal cells can undergo hepatocellular-like transformation under the induction of some cytokines, and the transformed cells can be implanted into allogeneic rats through the portal vein It can survive in liver tissue and has a certain repair effect on rat liver injury.