bFGF、BMP-2基因转染对羊骨髓间充质干细胞成骨功能影响的体外实验研究

发布时间：2018-10-11 12:47

【摘要】：目的：分析bFGF和BMP-2基因单独和联合转染对体外培养的羊BMSCs的增殖和骨向分化的影响,比较Collagen-I、OC、OPN等成骨相关基因于转染前后相对表达量的差别,为构建组织工程骨的种子细胞做理论依据。方法：全骨髓培养法培养羊BMSCs,构建bFGF、BMP-2慢病毒载体,分别转染羊BMSCs,流式细胞仪检测转染率,将得到bFGF组、BMP-2组、联合组,对照组细胞,提取RNA后使用实时定量PCR和Western-blot检测Collagen-I、OC、OPN等成骨相关基因的mRNA水平和蛋白水平相对表达量的变化。结果：(1)慢病毒载体转染的BMSCs的转染率达95.4%,且bFGF和BMP-2均稳定表达。(2)mRNA水平上,在Collagen-I和OC基因,bFGF主效应、BMP-2主效应、交互效应均有统计学意义(P0.05)。bFGF和BMP-2均能促进BMSCs的两基因高表达,且bFGF与BMP-2间存在协同作用。联合基因转染时Collagen-I和OC基因表达高于单一基因转染。但在OPN基因,bFGF主效应无统计学意义(P0.05)。多元析因显示,bFGF和BMP-2两因子均能增高成骨相关基因mRNA表达且存在协同的交互作用(P0.05)。(3)蛋白水平上,在Collagen-I、OC和OPN蛋白,bFGF主效应、BMP-2主效应、交互效应均有统计学意义(P0.05)。bFGF和BMP-2均能促进BMSCs的三个成骨相关基因蛋白高表达,且bFGF与BMP-2间分别存在协同作用。联合基因转染时Collagen-I和OC蛋白表达高于单一基因转染。多元析因显示,bFGF和BMP-2两因子均能增高成骨相关蛋白表达且存在协同的交互作用(P0.05)。结论：体外实验显示bFGF和BMP-2在成骨相关基因的表达上具有协同的交互作用,且联合转染组中的Collagen-I、OC、OPN等成骨相关基因mRNA水平和蛋白水平相对表达量均最高,该组细胞的成骨功能最强,适合作为组织工程骨的种子细胞。
[Abstract]:Aim: to analyze the effects of bFGF and BMP-2 gene transfection alone and co-transfection on the proliferation and bone differentiation of sheep BMSCs in vitro, and to compare the relative expression levels of Collagen-I,OC,OPN and other osteoblast related genes before and after transfection. To construct the seed cells of tissue engineering bone. Methods: bFGF,BMP-2 lentivirus vector was constructed by whole bone marrow culture of sheep BMSCs,. The transfection rate of bFGF,BMP-2 lentivirus vector was detected by flow cytometry in sheep BMSCs,. The cells of bFGF group, BMP-2 group, combination group and control group were obtained. After extracting RNA, real-time quantitative PCR and Western-blot were used to detect the relative expression of mRNA and protein in Collagen-I,OC,OPN and other osteoblast related genes. Results: (1) the transfection rate of BMSCs transfected by lentivirus vector was 95.4and the expression of bFGF and BMP-2 was stable. (2) at the mRNA level, there were significant differences in Collagen-I and OC gene, bFGF main effect, BMP-2 main effect and interaction effect (P0.05). BFGF and BMP-2 could promote the overexpression of BMSCs gene. There was a synergistic effect between bFGF and BMP-2. The expression of Collagen-I and OC genes was higher than that of single gene transfection. But in the OPN gene, the main effect of bFGF had no statistical significance (P0.05). Multiple factorial analysis showed that both bFGF and BMP-2 could increase the expression of osteoblast-associated gene mRNA and had synergistic interaction (P0.05). (3) protein level. In Collagen-I,OC and OPN protein, bFGF main effect, BMP-2 main effect. The interaction effects were statistically significant (P0.05). BFGF and BMP-2 could promote the high expression of three osteoblast-associated gene proteins in BMSCs, and there was a synergistic effect between bFGF and BMP-2. The expression of Collagen-I and OC protein in combination gene transfection was higher than that in single gene transfection. Multiple factorial analysis showed that both bFGF and BMP-2 could increase the expression of osteoblast-associated protein and had synergistic interaction (P0.05). Conclusion: in vitro experiments showed that bFGF and BMP-2 had synergistic interaction in the expression of osteoblast-related genes, and the relative expression of mRNA and protein of osteoblast-related genes such as Collagen-I,OC,OPN were the highest in co-transfection group. The cells have the strongest osteogenic function and are suitable as seed cells for tissue engineering bone.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783

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