shRNA干扰PPARγ基因表达预防兔激素性股骨头坏死的动物实验研究

发布时间：2018-05-11 09:41

本文选题：骨髓基质干细胞 + shRNA　；参考：《川北医学院》2017年硕士论文

【摘要】：目的:研究激素性股骨头坏死(steroid-induced avascular necrosis of the femoral head,SANFH)的发病机理,观察靶向PPARγ基因shRNA腺病毒载体对兔股骨头内PPARγm RNA转录及蛋白表达的影响,进一步探讨其预防兔激素性股骨头坏死(SANFH)的作用效果,为SANFH的治疗方案提供新的思路。方法:健康新西兰家兔48只,随机分为4组:单纯激素组(M组):仅肌注地塞米松;实验组(S组):在肌注地塞米松的同时,加用靶向PPARγ基因shRNA腺病毒载体感染兔活体股骨头内骨髓基质干细胞(Bone marrow stromal cells,BMSCs);无关序列组(E组):在肌注地塞米松的同时加用搭载无关序列的shRNA重组腺病毒感染兔活体股骨头内BMSCs;对照组(N组):肌注等量生理盐水。测定血清甘油三酯(TG)和胆固醇(CHO)含量。观察股骨头组织病理学改变,应用Q-PCR技术检测PPARγm RNA和Runx2m RNA表达,应用免疫组化技术检测PPARγ和Runx2蛋白表达情况。运用统计软件spss13.0对所获得的数据进行统计学分析,P0.05为差异有统计学意义。结果:1.血清学检测结果:第4、8周,M、E、S组兔血清中TG和CHO含量水平随实验进展逐渐增高,且显著高于N组正常水平,与N组相比较差异具有统计学意义(P0.05)。2.病理组织学检测结果:第8周,股骨头大体形态观察,N组和S组股骨头标本外观形态正常,关节软骨面平滑且洁净,并显示出健康的颜色;E组和M组关节软骨面光滑度较差,且股骨头右上象限表面颜色变深,软骨面可见少许剥脱,软骨下可见散在瘀斑。HE染色结果,N组和S组未见明显骨坏死,软骨下骨小梁结构完整,排列规律整齐,骨陷凹中骨细胞清晰可见;E组和M组骨坏死明显可见,骨小梁变细和稀疏,部分断裂成片状,软骨下骨板变薄,骨小梁中含大量空骨陷凹。3.Q-PCR检测结果:第4周,S组股骨头中PPARγm RNA表达量相对于M组和E组降低(P0.05),Runx2 m RNA表达量相对于M组和E组升高(P0.05),S组和N组组间比较,以及M组和E组组间比较差异不具有统计学意义(P0.05);第8周,S组PPARγm RNA表达量相对于M组和E组显著降低(P0.05),Runx2 m RNA表达量相对于M组和E组显著升高(P0.05),S组和N组组间比较,以及M组和E组组间比较差异不具有统计学意义(P0.05)。4.免疫组化结果:实验第8周,S组中PPARγ蛋白呈低表达,Runx2呈高表达,与M组和E组间差异有统计学意义(P0.05),与N组间差异无统计学意义(P0.05)。结论:1.成功构建了兔SANFH动物模型;2.长期大剂量激素摄入可致脂肪细胞增生肥大,大量空骨陷凹形成,最终诱导发生激素性股骨头坏死;3.靶向PPARγ基因shRNA腺病毒载体可有效阻断兔股骨头内PPARγm RNA表达,抑制BMSCs成脂分化;增强Runx2m RNA表达,促进其成骨分化;4.靶向PPARγ基因的shRNA腺病毒载体可预防SANFH的发生,为通过基因靶向治疗SANFH提供新思路。
[Abstract]:Aim: to study the pathogenesis of steroid-induced avascular necrosis of the femoral head SANFH (steroid-induced avascular necrosis of the femoral head) and to observe the effect of shRNA adenovirus vector targeting PPAR 纬 gene on PPAR 纬 m RNA transcription and protein expression in rabbit femoral head. To investigate the effect of SANFH on the prevention of steroid-induced femoral head necrosis in rabbits, and to provide a new idea for the treatment of SANFH. Methods: Forty-eight healthy New Zealand rabbits were randomly divided into 4 groups: group M: dexamethasone was injected intramuscularly, group S: dexamethasone was injected intramuscularly, while dexamethasone was injected intramuscularly. The shRNA adenovirus vector targeting PPAR 纬 gene was added to infect the bone marrow stromal cells of bone marrow stromal cells in the femoral head of rabbits in vivo, and group E was injected with dexamethasone simultaneously with shRNA recombinant adenovirus carrying unrelated sequences. Body femoral head BMSCs; control group N: intramuscular injection of the same amount of normal saline. Serum triglyceride (TG) and cholesterol (Cho) were determined. The histopathological changes of femoral head were observed. The expression of PPAR 纬 m RNA and Runx2m RNA was detected by Q-PCR technique, and the expression of PPAR 纬 and Runx2 protein was detected by immunohistochemical technique. The statistical software spss13.0 was used for statistical analysis of the obtained data. The result is 1: 1. The serum levels of TG and CHO increased gradually with the development of the experiment, and were significantly higher than the normal level of group N, and the difference was statistically significant compared with that of group N. Results of histopathological examination: at the 8th week, the gross morphology of femoral head was normal in group N and group S, the articular cartilage was smooth and clean, and the smooth degree of articular cartilage was poor in group E and group M. The color of the right superior quadrant of the femoral head was darkened, the cartilage surface was peeling off a little, and there was no obvious bone necrosis in group N and group S, and the trabecular structure of the subchondral bone was intact and arranged regularly, and the results showed that there was no obvious osteonecrosis in group N and group S. Osteonecrosis was clearly seen in group E and group M, bone trabeculae became thin and sparse, some of them broke into slices, and subchondral bone plates became thinner. The results of Q-PCR detection showed that the expression of PPAR 纬 m RNA in femoral head of group S was lower than that of group M and group E by Q-PCR: compared with group M and group E, the expression of PPAR 纬 m RNA in group S and group N was significantly higher than that in group M and group E. There was no significant difference between group M and group E in the expression of PPAR 纬 m RNA, and the expression of PPAR 纬 m RNA in group S was significantly lower than that in group M and group E in comparison with that in group M and group E, which was significantly higher than that in group M and group E, and that between group S and group N was significantly higher than that in group M and E. And the difference between group M and group E was not statistically significant (P 0.05. 4). The results of immunohistochemistry showed that the expression of PPAR 纬 protein in group S was lower than that in group M and group E, but there was no significant difference between group M and group E (P0.05). There was no significant difference between group S and group N in the expression of PPAR 纬 protein (P 0.05), but there was no significant difference between group E and group M (P 0.05). Conclusion 1. Rabbit SANFH animal model was successfully constructed. Long-term high dose of hormone intake can cause hypertrophy of adipocytes, formation of a large number of empty bone cavities, and eventually induce steroid-induced femoral head necrosis. ShRNA adenovirus vector targeting PPAR 纬 gene could effectively block the expression of PPAR 纬 m RNA in rabbit femoral head, inhibit the adipogenic differentiation of BMSCs, and enhance Runx2m RNA expression and promote osteogenic differentiation. The shRNA adenovirus vector targeting PPAR 纬 gene can prevent the occurrence of SANFH and provide a new idea for the gene targeting therapy of SANFH.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.8

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