本文選題：骨髓基質(zhì)干細(xì)胞 + shRNA��； 參考：《川北醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:研究激素性股骨頭壞死(steroid-induced avascular necrosis of the femoral head,SANFH)的發(fā)病機(jī)理,觀察靶向PPARγ基因shRNA腺病毒載體對兔股骨頭內(nèi)PPARγm RNA轉(zhuǎn)錄及蛋白表達(dá)的影響,進(jìn)一步探討其預(yù)防兔激素性股骨頭壞死(SANFH)的作用效果,為SANFH的治療方案提供新的思路。方法:健康新西蘭家兔48只,隨機(jī)分為4組:單純激素組(M組):僅肌注地塞米松;實(shí)驗(yàn)組(S組):在肌注地塞米松的同時(shí),加用靶向PPARγ基因shRNA腺病毒載體感染兔活體股骨頭內(nèi)骨髓基質(zhì)干細(xì)胞(Bone marrow stromal cells,BMSCs);無關(guān)序列組(E組):在肌注地塞米松的同時(shí)加用搭載無關(guān)序列的shRNA重組腺病毒感染兔活體股骨頭內(nèi)BMSCs;對照組(N組):肌注等量生理鹽水。測定血清甘油三酯(TG)和膽固醇(CHO)含量。觀察股骨頭組織病理學(xué)改變,應(yīng)用Q-PCR技術(shù)檢測PPARγm RNA和Runx2m RNA表達(dá),應(yīng)用免疫組化技術(shù)檢測PPARγ和Runx2蛋白表達(dá)情況。運(yùn)用統(tǒng)計(jì)軟件spss13.0對所獲得的數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.血清學(xué)檢測結(jié)果:第4、8周,M、E、S組兔血清中TG和CHO含量水平隨實(shí)驗(yàn)進(jìn)展逐漸增高,且顯著高于N組正常水平,與N組相比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.病理組織學(xué)檢測結(jié)果:第8周,股骨頭大體形態(tài)觀察,N組和S組股骨頭標(biāo)本外觀形態(tài)正常,關(guān)節(jié)軟骨面平滑且潔凈,并顯示出健康的顏色;E組和M組關(guān)節(jié)軟骨面光滑度較差,且股骨頭右上象限表面顏色變深,軟骨面可見少許剝脫,軟骨下可見散在瘀斑。HE染色結(jié)果,N組和S組未見明顯骨壞死,軟骨下骨小梁結(jié)構(gòu)完整,排列規(guī)律整齊,骨陷凹中骨細(xì)胞清晰可見;E組和M組骨壞死明顯可見,骨小梁變細(xì)和稀疏,部分?jǐn)嗔殉善瑺?軟骨下骨板變薄,骨小梁中含大量空骨陷凹。3.Q-PCR檢測結(jié)果:第4周,S組股骨頭中PPARγm RNA表達(dá)量相對于M組和E組降低(P0.05),Runx2 m RNA表達(dá)量相對于M組和E組升高(P0.05),S組和N組組間比較,以及M組和E組組間比較差異不具有統(tǒng)計(jì)學(xué)意義(P0.05);第8周,S組PPARγm RNA表達(dá)量相對于M組和E組顯著降低(P0.05),Runx2 m RNA表達(dá)量相對于M組和E組顯著升高(P0.05),S組和N組組間比較,以及M組和E組組間比較差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)。4.免疫組化結(jié)果:實(shí)驗(yàn)第8周,S組中PPARγ蛋白呈低表達(dá),Runx2呈高表達(dá),與M組和E組間差異有統(tǒng)計(jì)學(xué)意義(P0.05),與N組間差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.成功構(gòu)建了兔SANFH動(dòng)物模型;2.長期大劑量激素?cái)z入可致脂肪細(xì)胞增生肥大,大量空骨陷凹形成,最終誘導(dǎo)發(fā)生激素性股骨頭壞死;3.靶向PPARγ基因shRNA腺病毒載體可有效阻斷兔股骨頭內(nèi)PPARγm RNA表達(dá),抑制BMSCs成脂分化;增強(qiáng)Runx2m RNA表達(dá),促進(jìn)其成骨分化;4.靶向PPARγ基因的shRNA腺病毒載體可預(yù)防SANFH的發(fā)生,為通過基因靶向治療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 緯 gene on PPAR 緯 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 緯 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 緯 m RNA and Runx2m RNA was detected by Q-PCR technique, and the expression of PPAR 緯 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 緯 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 緯 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 緯 m RNA, and the expression of PPAR 緯 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 緯 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 緯 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 緯 gene could effectively block the expression of PPAR 緯 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 緯 gene can prevent the occurrence of SANFH and provide a new idea for the gene targeting therapy of SANFH.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R681.8

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