高密度培养骨髓细胞治疗大鼠肝纤维化及相关机制研究

发布时间：2018-05-11 19:28

本文选题：肝纤维化 + 细胞治疗　；参考：《上海交通大学》2014年博士论文

【摘要】：目的:建立一种稳定的wistar大鼠慢性肝纤维化模型。通过脾脏注射高密度培养骨髓细胞,观察移植细胞能否改善大鼠肝纤维化,并研究其阻止肝脏慢性纤维化进展可能的作用机制,探讨高密度培养骨髓细胞治疗慢性肝纤维化的应用价值。方法:1.通过四氯化碳(CCl4)腹腔注射,建立wistar大鼠慢性肝纤维化模型;2.将建模成功大鼠随机分成三组,高密度培养组(HD)、常规密度培养组(RD)和PBS组。经脾脏分别注射经过荧光素CM-Di L标记的2×106的骨髓HD细胞、RD细胞和200μl PBS;3.血清分析检测大鼠肝功能指标;苦味酸天狼星红(PSR)和Masson染色检测肝脏中胶原合成与分布;免疫组化检测肝脏中I型胶原、α-SMA的表达,q RT-PCR检测组织中α2-procollagen、α-SMA、fibronectin和TGF-β的基因表达;4.组织冰冻切片后行Ⅰ型胶原免疫荧光染色,荧光显微镜下观察CM-Di L阳性的移植细胞在肝脏组织中的数量及分布情况;5.肝脏组织切片行Ki-67、CD31免疫组化法染色,观察细胞增殖与血管生成;q RT-PCR检测组织中肝细胞生长因子(HGF)、血管内皮生长因子(VEGF)的基因表达情况。结果:1.重复多次腹腔注射CCl4,可以建立稳定的大鼠慢性肝纤维化模型。2.大鼠骨髓细胞经高密度培养后,细胞高表达CD31、CD133和KDR,体外成管实验显示高密度培养细胞比普通密度培养细胞具有更强的成管能力,提示高密度培养的骨髓细胞富含血管内皮前体细胞。3.造模大鼠血清谷草转氨酶和谷丙转氨酶明显增高,白蛋白降低,PBS治疗组无明显改善,细胞移植组血清谷草转氨酶和谷丙转氨酶降低,白蛋白升高,HD组优于RD组,提示高密度培养细胞的作用更好。4.造模大鼠肝脏HE染色发现肝脏重构,假小叶形成,细胞移植后假小叶减少,PSR和Masson染色发现细胞外胶原沉积减少。免疫组化分析显示细胞移植组肝脏中Ⅰ型胶原和α-SMA的表达减少,q RT-PCR检测显示细胞移植组α2-procollagen、α-SMA和fibronectin的基因表达降低,HD组优于RD组。5.肝脏组织免疫组化染色显示HD组中CD31阳性的血管多于RD组,Ki67阳性的增殖细胞数多于RD组,肝脏组织q RT-PCR检测显示促肝细胞增殖的HGF和促血管生成的VEGF基因在HD组中表达明显高于RD组。结论:骨髓高密度培养细胞对缓解大鼠肝纤维化、改善肝功能和促进肝脏再生有较好效果,这种简单、低成本的培养方式可以为骨髓细胞的未来临床应用提供一种有效的扩增方法。
[Abstract]:Objective: to establish a stable model of chronic hepatic fibrosis in wistar rats. High density bone marrow cells were injected into the spleen to observe whether the transplanted cells could improve the hepatic fibrosis in rats and to study the possible mechanism of preventing the progression of chronic hepatic fibrosis. To investigate the value of high density cultured bone marrow cells in the treatment of chronic hepatic fibrosis. Method 1: 1. The chronic hepatic fibrosis model of wistar rats was established by intraperitoneal injection of CCL 4 (CCL 4). The rats were randomly divided into three groups: high density culture group, conventional density culture group and PBS group. Splenic injection of 2 脳 10 ~ 6 bone marrow HD cells labeled with fluorescein CM-Di L and 200 渭 l / L PBS3, respectively. Serum analysis was used to detect liver function, Masson staining and picric acid Sirius) staining were used to detect collagen synthesis and distribution in the liver, and immunohistochemical staining was used to detect the expression of type I collagen, 伪 -SMA expression and 伪 2-procagenolll, 伪 -SMAfibronectin and TGF- 尾 gene expression in the liver of rats. The expression of 伪 2 procagenoll1, 伪 -SMAfifibronectin and TGF- 尾 was detected by immunohistochemistry. The number and distribution of CM-Di L positive transplanted cells in liver tissue were observed under fluorescence microscope. The expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in liver tissue were detected by Ki-67-CD31 immunohistochemical method. The expression of VEGF and VEGF was detected by cell proliferation and angiogenesis Q RT-PCR. The result is 1: 1. Repeated intraperitoneal injection of CCL 4 can establish a stable model of chronic hepatic fibrosis in rats. 2. 2. After cultured with high density, rat bone marrow cells expressed CD31, CD133 and KDR. the in vitro tube forming experiment showed that the cultured cells with high density had stronger ability of tube-forming than those cultured with normal density. These results suggest that high density cultured bone marrow cells are rich in endothelial progenitor cells. 3. Serum glutamic oxaloacetic transaminase and alanine aminotransferase were significantly increased in the model rats, but no significant improvement was found in the PBS treatment group, while the serum alanine aminotransferase and alanine aminotransferase levels were decreased in the cell transplantation group. The serum alanine aminotransferase in HD group was better than that in Rd group. The results suggest that the effect of high density cultured cells is better. 4. Liver remodeling and pseudolobule formation were found by HE staining in the liver of the model rats. PSR and Masson staining showed that extracellular collagen deposition was decreased after cell transplantation. Immunohistochemical analysis showed that the expression of type I collagen and 伪 -SMA in the liver of the transplanted group was decreased. The expression of 伪 2-procollagenase, 伪 -SMA and fibronectin in the transplanted group was lower than that in the Rd group. The expression of 伪 -SMA and 伪 -SMA gene in HD group was higher than that in Rd group. Immunohistochemical staining of liver tissue showed that the number of CD31 positive blood vessels in HD group was more than that in Rd group, and the number of proliferative cells positive in Rd group was more than that in Rd group. The expression of HGF and VEGF in HD group was significantly higher than that in Rd group. Conclusion: bone marrow high density cultured cells have better effect on relieving liver fibrosis, improving liver function and promoting liver regeneration in rats. Low-cost culture can provide an effective method for the future clinical application of bone marrow cells.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.2

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