本文選題：ITGB5 + 慢病毒載體��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2016年博士論文

【摘要】：目的：通過(guò)干擾瘢痕相關(guān)基因整合素蛋白β5(β5-Integrin, ITGB5)的表達(dá),觀(guān)察該基因?qū)︸：鄹泶癯衫w維細(xì)胞(Keloid Fibroblast, KFb)的細(xì)胞周期、增殖、凋亡、遷移及侵襲能力的影響,闡明ITGB5基因在瘢痕疙瘩形成及發(fā)展中的相關(guān)作用,以期為瘢痕疙瘩的發(fā)病機(jī)制研究提供理論依據(jù)、為臨床探尋瘢痕疙瘩的治療靶點(diǎn)研究提供先期實(shí)驗(yàn)工作。方法：1.構(gòu)建干擾人ITGB5基因表達(dá)慢病毒載體(lentiviral vector, LV);2.將培養(yǎng)好的瘢痕疙瘩成纖維細(xì)胞隨機(jī)分為三組,分別為ITGB5干擾慢病毒載體感染瘢痕疙瘩成纖維細(xì)胞組(LV-KFb),空載病毒感染瘢痕疙瘩成纖維細(xì)胞組(LV-NC)及空白對(duì)照組(KFb),用慢病毒持續(xù)感染LV-KFb組及LV-NC組細(xì)胞,流式細(xì)胞儀篩選穩(wěn)定細(xì)胞株；3. Real-time PCR及Western Blot檢測(cè)慢病毒載體感染后各組細(xì)胞中ITGB5基因及蛋白的表達(dá)情況；4.MTT法檢測(cè)慢病毒載體感染后各組細(xì)胞的增殖情況；流式細(xì)胞儀檢測(cè)細(xì)胞周期及凋亡情況；Transwell小室檢測(cè)細(xì)胞侵襲及遷移能力變化；5.利用免疫共沉淀技術(shù)檢測(cè)轉(zhuǎn)化生長(zhǎng)因子β(Transforming Growth Factor-P, TGF-β)及ITGB5間的相互作用。結(jié)果：1.干擾人ITGB5慢病毒載體可以成功感染KFb,且在MOI=50時(shí)細(xì)胞感染率可達(dá)80%,通過(guò)FCM可獲得穩(wěn)轉(zhuǎn)細(xì)胞株；2.慢病毒感染KFb后,KFb組、LV-NC組及LV-KFb組中ITGB5 mRNA及蛋白的表達(dá)量分別為1.00±0.00、1.08±0.05及0.34+0.01和0.91±0.03、0.93+0.02及0.28±0.07,與LV-NC組及KFb組比較,LV-KFb組中ITGB5 mRNA及蛋白的表達(dá)量均明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.01)；3.慢病毒感染KFb后,各組間細(xì)胞的增殖情況在接種后24h無(wú)顯著差異,48h后,LV-KFb組的增殖較其它兩組明顯變慢(P0.01);4.慢病毒感染KFb后,KFb組、LV-NC組和LV-KFb組早期凋亡細(xì)胞所占百分比分別為5.94±0.28、6.08±0.35、13.81±0.46,晚期凋亡細(xì)胞所占百分比分別為18.75±0.93、13.36±1.35、31.30±1.67,LV-KFb組較其它兩組早期凋亡及晚期凋亡細(xì)胞數(shù)明顯增多,差異有統(tǒng)計(jì)學(xué)意義(P0.01);5.慢病毒感染KFb后,KFb組、LV-NC組和LV-KFb組S期細(xì)胞所占百分比分別為34.86±1.86、34.03±2.14、20.03±1.32,LV-KFb組較其它兩組S期細(xì)胞所占比例明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.01);6.慢病毒感染KFb后,LV-KFb組的穿膜細(xì)胞數(shù)在遷移及侵襲實(shí)驗(yàn)中均明顯少于其它兩組,差異有統(tǒng)計(jì)學(xué)意義(P0.01);7. ITGB5及TGF-β的陽(yáng)性條帶在免疫共沉淀中均能被檢測(cè)到。結(jié)論：1.成功構(gòu)建了干擾入ITGB5基因表達(dá)慢病毒載體;2.成功獲得了慢病毒感染KFb的穩(wěn)轉(zhuǎn)細(xì)胞株;3.干擾人ITGB5慢病毒能夠成功感染KFb并能有效抑制細(xì)胞中ITGB5 mRNA和蛋白的表達(dá)；4. ITGB5能夠促進(jìn)KFb的細(xì)胞分裂、增殖、遷移和侵襲能力,抑制KFb的凋亡;5. ITGB5對(duì)KFb的作用可能通過(guò)TGF-β信號(hào)通路實(shí)現(xiàn)；6. ITGB5可做為瘢痕疙瘩基因治療的重要靶點(diǎn)之一.
[Abstract]:Objective: to investigate the effects of integrin 尾 5 (ITGB5) on the cell cycle, proliferation, apoptosis, migration and invasion of keloid fibroblasts (KFb5) by interfering with the expression of integrin 尾 5 (ITGB5). To elucidate the role of ITGB5 gene in the formation and development of keloid in order to provide theoretical basis for the study of the pathogenesis of keloid and to provide preliminary experimental work for clinical research on the therapeutic targets of keloid. Method 1: 1. The expression vector of lentiviral vector interfering with human ITGB5 gene was constructed. The cultured keloid fibroblasts were randomly divided into three groups. ITGB5 interference lentivirus vector infected keloid fibroblasts group, empty virus infected keloid fibroblast group (LV-NC3) and blank control group (KFB) were used to continuously infect LV-KFb group and LV-NC group. The stable cell line was screened by flow cytometry. Real-time PCR and Western Blot were used to detect the expression of ITGB5 gene and protein after lentivirus vector infection. 4. Flow cytometry was used to detect cell cycle and apoptosis. The interaction between transforming Growth Factor-P (TGF- 尾) and ITGB5 was detected by immunoprecipitation. The result is 1: 1. Interfering human ITGB5 lentivirus vector could infect KFb successfully, and the cell infection rate could reach 80% when MOI = 50, and the stable transformed cell line could be obtained by FCM. The expression of ITGB5 mRNA and protein in LV-NC group and LV-KFb group were 1.00 鹵0.000.08 鹵0.05,0.34.01 and 0.91 鹵0.030.93 0.02 and 0.28 鹵0.07 respectively after lentivirus infection. The expression of ITGB5 mRNA and protein in LV-KFb group was significantly lower than that in LV-NC group and KFb group (P 0.01). After lentivirus infection with KFb, the proliferation of LV-KFb cells in the LV-KFb group was significantly slower than that in the other two groups at 24 h after inoculation. The percentage of early apoptotic cells and late apoptotic cells in LV-NC group and LV-KFb group were 5.94 鹵0.286.08 鹵0.35 鹵0.46 and 18.75 鹵0.93n 13.36 鹵1.35 鹵1.67 鹵1.67LV-KFb, respectively. The difference was statistically significant (P 0.01). The percentage of S phase cells in LV-NC group and LV-KFb group was 34.86 鹵1.86 鹵1.86 鹵2.14 鹵20.03 鹵1.32 渭 m LV-KFb after KFb infection, respectively, and the percentage of S phase cells in LV-KFb group was significantly lower than that in other two groups (P 0.016). The number of perforated cells in LV-KFb group after lentivirus infection was significantly lower than that in the other two groups in migration and invasion test, and the difference was statistically significant (P 0.01). ITGB5 and TGF- 尾 positive bands can be detected in immunoprecipitation. Conclusion 1. A lentiviral vector containing interfering ITGB5 gene was successfully constructed. The stable transformed cell line of lentivirus infected with KFb was successfully obtained. Interfering with human ITGB5 lentivirus can successfully infect KFb and effectively inhibit the expression of ITGB5 mRNA and protein in cells. ITGB5 can promote the cell division, proliferation, migration and invasion of KFb and inhibit the apoptosis of KFb. The effect of ITGB5 on KFb may be realized by TGF- 尾 signaling pathway. ITGB5 may be one of the important targets of keloid gene therapy.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R622

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1 顏彤彤;干擾人ITGB5基因表達(dá)對(duì)瘢痕疙瘩成纖維細(xì)胞功能的影響[D];中國(guó)人民解放軍醫(yī)學(xué)院;2016年

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本文編號(hào)：1989546