【摘要】：目的：分析bFGF和BMP-2基因單獨(dú)和聯(lián)合轉(zhuǎn)染對(duì)體外培養(yǎng)的羊BMSCs的增殖和骨向分化的影響,比較Collagen-I、OC、OPN等成骨相關(guān)基因于轉(zhuǎn)染前后相對(duì)表達(dá)量的差別,為構(gòu)建組織工程骨的種子細(xì)胞做理論依據(jù)。方法：全骨髓培養(yǎng)法培養(yǎng)羊BMSCs,構(gòu)建bFGF、BMP-2慢病毒載體,分別轉(zhuǎn)染羊BMSCs,流式細(xì)胞儀檢測(cè)轉(zhuǎn)染率,將得到bFGF組、BMP-2組、聯(lián)合組,對(duì)照組細(xì)胞,提取RNA后使用實(shí)時(shí)定量PCR和Western-blot檢測(cè)Collagen-I、OC、OPN等成骨相關(guān)基因的mRNA水平和蛋白水平相對(duì)表達(dá)量的變化。結(jié)果：(1)慢病毒載體轉(zhuǎn)染的BMSCs的轉(zhuǎn)染率達(dá)95.4%,且bFGF和BMP-2均穩(wěn)定表達(dá)。(2)mRNA水平上,在Collagen-I和OC基因,bFGF主效應(yīng)、BMP-2主效應(yīng)、交互效應(yīng)均有統(tǒng)計(jì)學(xué)意義(P0.05)。bFGF和BMP-2均能促進(jìn)BMSCs的兩基因高表達(dá),且bFGF與BMP-2間存在協(xié)同作用。聯(lián)合基因轉(zhuǎn)染時(shí)Collagen-I和OC基因表達(dá)高于單一基因轉(zhuǎn)染。但在OPN基因,bFGF主效應(yīng)無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。多元析因顯示,bFGF和BMP-2兩因子均能增高成骨相關(guān)基因mRNA表達(dá)且存在協(xié)同的交互作用(P0.05)。(3)蛋白水平上,在Collagen-I、OC和OPN蛋白,bFGF主效應(yīng)、BMP-2主效應(yīng)、交互效應(yīng)均有統(tǒng)計(jì)學(xué)意義(P0.05)。bFGF和BMP-2均能促進(jìn)BMSCs的三個(gè)成骨相關(guān)基因蛋白高表達(dá),且bFGF與BMP-2間分別存在協(xié)同作用。聯(lián)合基因轉(zhuǎn)染時(shí)Collagen-I和OC蛋白表達(dá)高于單一基因轉(zhuǎn)染。多元析因顯示,bFGF和BMP-2兩因子均能增高成骨相關(guān)蛋白表達(dá)且存在協(xié)同的交互作用(P0.05)。結(jié)論：體外實(shí)驗(yàn)顯示bFGF和BMP-2在成骨相關(guān)基因的表達(dá)上具有協(xié)同的交互作用,且聯(lián)合轉(zhuǎn)染組中的Collagen-I、OC、OPN等成骨相關(guān)基因mRNA水平和蛋白水平相對(duì)表達(dá)量均最高,該組細(xì)胞的成骨功能最強(qiáng),適合作為組織工程骨的種子細(xì)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R783

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