干扰人ITGB5基因表达对瘢痕疙瘩成纤维细胞功能的影响

发布时间：2018-06-07 03:28

本文选题：ITGB5 + 慢病毒载体　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：目的：通过干扰瘢痕相关基因整合素蛋白β5(β5-Integrin, ITGB5)的表达,观察该基因对瘢痕疙瘩成纤维细胞(Keloid Fibroblast, KFb)的细胞周期、增殖、凋亡、迁移及侵袭能力的影响,阐明ITGB5基因在瘢痕疙瘩形成及发展中的相关作用,以期为瘢痕疙瘩的发病机制研究提供理论依据、为临床探寻瘢痕疙瘩的治疗靶点研究提供先期实验工作。方法：1.构建干扰人ITGB5基因表达慢病毒载体(lentiviral vector, LV);2.将培养好的瘢痕疙瘩成纤维细胞随机分为三组,分别为ITGB5干扰慢病毒载体感染瘢痕疙瘩成纤维细胞组(LV-KFb),空载病毒感染瘢痕疙瘩成纤维细胞组(LV-NC)及空白对照组(KFb),用慢病毒持续感染LV-KFb组及LV-NC组细胞,流式细胞仪筛选稳定细胞株；3. Real-time PCR及Western Blot检测慢病毒载体感染后各组细胞中ITGB5基因及蛋白的表达情况；4.MTT法检测慢病毒载体感染后各组细胞的增殖情况；流式细胞仪检测细胞周期及凋亡情况；Transwell小室检测细胞侵袭及迁移能力变化；5.利用免疫共沉淀技术检测转化生长因子β(Transforming Growth Factor-P, TGF-β)及ITGB5间的相互作用。结果：1.干扰人ITGB5慢病毒载体可以成功感染KFb,且在MOI=50时细胞感染率可达80%,通过FCM可获得稳转细胞株；2.慢病毒感染KFb后,KFb组、LV-NC组及LV-KFb组中ITGB5 mRNA及蛋白的表达量分别为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,与LV-NC组及KFb组比较,LV-KFb组中ITGB5 mRNA及蛋白的表达量均明显降低,差异有统计学意义(P0.01)；3.慢病毒感染KFb后,各组间细胞的增殖情况在接种后24h无显著差异,48h后,LV-KFb组的增殖较其它两组明显变慢(P0.01);4.慢病毒感染KFb后,KFb组、LV-NC组和LV-KFb组早期凋亡细胞所占百分比分别为5.94±0.28、6.08±0.35、13.81±0.46,晚期凋亡细胞所占百分比分别为18.75±0.93、13.36±1.35、31.30±1.67,LV-KFb组较其它两组早期凋亡及晚期凋亡细胞数明显增多,差异有统计学意义(P0.01);5.慢病毒感染KFb后,KFb组、LV-NC组和LV-KFb组S期细胞所占百分比分别为34.86±1.86、34.03±2.14、20.03±1.32,LV-KFb组较其它两组S期细胞所占比例明显减少,差异有统计学意义(P0.01);6.慢病毒感染KFb后,LV-KFb组的穿膜细胞数在迁移及侵袭实验中均明显少于其它两组,差异有统计学意义(P0.01);7. ITGB5及TGF-β的阳性条带在免疫共沉淀中均能被检测到。结论：1.成功构建了干扰入ITGB5基因表达慢病毒载体;2.成功获得了慢病毒感染KFb的稳转细胞株;3.干扰人ITGB5慢病毒能够成功感染KFb并能有效抑制细胞中ITGB5 mRNA和蛋白的表达；4. ITGB5能够促进KFb的细胞分裂、增殖、迁移和侵袭能力,抑制KFb的凋亡;5. ITGB5对KFb的作用可能通过TGF-β信号通路实现；6. ITGB5可做为瘢痕疙瘩基因治疗的重要靶点之一.
[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 卤0.000.08 卤0.05,0.34.01 and 0.91 卤0.030.93 0.02 and 0.28 卤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 卤0.286.08 卤0.35 卤0.46 and 18.75 卤0.93n 13.36 卤1.35 卤1.67 卤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 卤1.86 卤1.86 卤2.14 卤20.03 卤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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R622

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