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  本文選題：骨髓間充質(zhì)干細(xì)胞　切入點：人骨形成蛋白2　出處：《廣西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的 1.為大量獲得骨組織工程所需種子細(xì)胞,于體外分離、培養(yǎng)、擴增、純化兔骨髓間充質(zhì)干細(xì)胞(Bone Mesenchymal Stem Cells, BMSCs). 2.為鑒定BMSCs的干細(xì)胞多分化潛能以及成骨分化的能力,利用成骨誘導(dǎo)培養(yǎng)基對其進行培養(yǎng),誘導(dǎo)BMSCs向成骨方向分化,并鑒定其成骨分化的效果。 3.利用脂質(zhì)體介導(dǎo)攜帶有hBMP-2基因pcDNA3.1質(zhì)粒轉(zhuǎn)染BMSCs,觀察其轉(zhuǎn)染效果。 4.檢測轉(zhuǎn)染對BMSCs的增殖分化能力的影響。 方法 1.BMSCs的體外培養(yǎng)：取新生健康新西蘭大白兔,處死后于無菌條件下取出四肢長骨,完全培養(yǎng)基沖洗骨髓腔得到骨髓液,用全骨髓貼壁法進行培養(yǎng),原代培養(yǎng)6-7d后可傳代培養(yǎng),按照1：3的比例傳代,每2-3天可傳代1次。 2.BMSCs的體外成骨誘導(dǎo)分化：BMSCs傳到第3代時用p-甘油磷酸鈉,地塞米松,抗壞血酸配制而成的成骨誘導(dǎo)分化液進行成骨誘導(dǎo)14至21d,并分別用茜素紅染色,ELISA以及免疫組化的方法檢測其礦化結(jié)節(jié)的形成和ALP,BGP的表達(dá)。 3.脂質(zhì)體介導(dǎo)的hBMP-2轉(zhuǎn)染兔BMSCs:取第3代BMSCs,用脂質(zhì)體介導(dǎo)攜帶hBMP-2基因的pcDNA3.1質(zhì)粒轉(zhuǎn)染入BMSCs中,觀察GFP表達(dá)的情況,通過RT-PCR檢測瞬時轉(zhuǎn)染后hBMP-2mRNA的表達(dá)。 4.MTT法、流式細(xì)胞法檢測轉(zhuǎn)染后BMSCs的增殖能力,BMP-2免疫組化檢測細(xì)胞的分化能力。 結(jié)果 1.BMSCs體外培養(yǎng)的結(jié)果：BMSCs培養(yǎng)6-7d后即可長滿瓶底,并可傳代,傳代后細(xì)胞增殖快,生長狀態(tài)良好,可一直維持BMSCs的典型形態(tài)以及生長狀態(tài),細(xì)胞呈現(xiàn)長梭形形狀,整體呈旋渦式的排列。 2.BMSCs的體外成骨誘導(dǎo)分化的結(jié)果：BMSCs可成功向成骨方向誘導(dǎo),其中鈣結(jié)節(jié)茜素紅染色見陽性結(jié)節(jié)染色；ALP含量逐漸升高,在誘導(dǎo)2周內(nèi)增高趨勢明顯,之后趨勢減緩,各時間點與未誘導(dǎo)組有顯著差異(p0.05)；BGP免疫組化也呈陽性染色。 3.脂質(zhì)體介導(dǎo)的hBMP-2轉(zhuǎn)染兔BMSCs的結(jié)果：利用脂質(zhì)體介導(dǎo)攜帶hBMP-2的pcDNA3.1質(zhì)粒成功轉(zhuǎn)染BMSCs,轉(zhuǎn)染效率約30%；轉(zhuǎn)染24h能觀察到弱熒光表達(dá),48h后熒光強度增強；RT-PCR也檢測到了hBMP-2mRNA的表達(dá)。 4.MTT法、流式細(xì)胞周期檢測結(jié)果證實轉(zhuǎn)染對BMSCs的增殖能力無明顯影響,免疫組化結(jié)果顯示轉(zhuǎn)染hBMP-2基因后的BMSCs中BMP-2為陽性表達(dá)。 結(jié)論 1.全骨髓貼壁分離培養(yǎng)法簡便、易行,可大量擴增、純化兔骨髓間充質(zhì)干細(xì)胞。 2.本實驗所獲的細(xì)胞可于體外成功誘導(dǎo)后向成骨細(xì)胞分化。 3.通過脂質(zhì)體介導(dǎo)的方法能成功將外源hBMP-2基因?qū)隑MSCs。 4.轉(zhuǎn)染hBMP-2對BMSCs的增殖無明顯影響,但使BMSCs向成骨方向分化。
[Abstract]:Purpose. 1. In order to obtain a large number of seed cells for bone tissue engineering, bone Mesenchymal Stem cells (BMSCs) were isolated, cultured, amplified and purified from rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. 2. In order to identify the multi-differentiation potential of BMSCs stem cells and the ability of osteogenic differentiation, BMSCs was cultured in osteogenic medium to induce the differentiation of BMSCs into osteogenesis, and the effect of osteogenic differentiation was evaluated. 3. HBMP-2 gene pcDNA3.1 plasmid was transfected into BMSCs by liposome, and the transfection effect was observed. 4. The effect of transfection on the proliferation and differentiation of BMSCs was detected. Method. In vitro culture of 1.BMSCs: after being killed, the long bones of the limbs were removed under aseptic conditions. Bone marrow fluid was obtained by washing the marrow cavity in a complete culture medium, and cultured with the whole bone marrow adherent method. After 6 to 7 days of primary culture, the bone marrow fluid could be cultured by subculture. According to the 1:3 ratio of passage, every 2-3 days can be subcultured once. In vitro osteogenic differentiation of 2.BMSCs was induced by sodium pglycerophosphate and dexamethasone. The osteogenic differentiation fluid prepared by ascorbic acid was induced by osteogenesis for 14 to 21 days. The formation of mineralized nodules and the expression of ALP BGP were detected by alizarin red staining Elisa and immunohistochemistry respectively. 3. Liposome-mediated hBMP-2 transfection into rabbit BMSCs: the pcDNA3.1 plasmid carrying hBMP-2 gene was transfected into BMSCs by liposome. The expression of GFP was observed and the expression of hBMP-2mRNA after transient transfection was detected by RT-PCR. The proliferative ability of BMSCs after transfection and the differentiation ability of BMP-2 were detected by 4.MTT and flow cytometry. Results. The results of 1.BMSCs culture in vitro were as follows: after 6-7 days of culture, 1.BMSCs could grow to the bottom of the bottle and could be subcultured. After passage, the cells could proliferate quickly and grow in good condition. They could maintain the typical morphology and growth state of BMSCs, and the cells showed a long spindle shape. The whole is arranged in the form of a vortex. The results of osteogenic induction and differentiation of 2.BMSCs in vitro showed that BMSCs could be successfully induced to osteogenesis, in which calcium nodule alizarin red staining showed that the content of 2.BMSCs increased gradually, and increased significantly within 2 weeks of induction, and then slowed down. There was significant difference between the different time points and the uninduced group, and the immunohistochemical staining of BGP was also positive. 3. The results of liposome-mediated hBMP-2 transfection into rabbit BMSCs: the pcDNA3.1 plasmid carrying hBMP-2 was successfully transfected into BMSCs by liposome, the transfection efficiency was about 30%, and the expression of hBMP-2mRNA was detected by RT-PCR after 48 h of weak fluorescence expression was observed after transfection. The results of 4.MTT assay and flow cytometry showed that transfection had no significant effect on the proliferation of BMSCs. The results of immunohistochemistry showed that BMP-2 was positive in BMSCs after transfection of hBMP-2 gene. Conclusion. 1. The whole bone marrow adherent culture method is simple and easy, and can be expanded and purified from rabbit bone marrow mesenchymal stem cells. 2. The cells obtained in this experiment can differentiate into osteoblasts after successful induction in vitro. 3. Exogenous hBMP-2 gene was successfully introduced into BMSCs by liposome mediated method. 4. Transfection of hBMP-2 had no effect on the proliferation of BMSCs, but BMSCs differentiated to osteogenesis.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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