【摘要】： 目的(1)培養(yǎng)轉(zhuǎn)染所需兔骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)。(2)通過采用陽性脂質(zhì)體pcDNA3.1(-)介導(dǎo)的轉(zhuǎn)染到BMSCs方法,獲得穩(wěn)定轉(zhuǎn)染的BMSCs細(xì)胞。(3)使用免疫組化、WesternBlot法檢測pcDNA3.1-hBMP2在兔BMSCs中蛋白的表達(dá)情況。 方法(1)貼壁法培養(yǎng)擴(kuò)增所需BMSCs并從形態(tài)學(xué)、流式細(xì)胞儀測量表面標(biāo)記物。(2)將已成功構(gòu)建的pcDNA3.1-hBMP2重組質(zhì)粒進(jìn)行體外靶細(xì)胞轉(zhuǎn)染并使用G418篩選穩(wěn)定表達(dá)細(xì)胞。(3)通過免疫組化、WesternBlot法檢測其在BMSCs中的蛋白表達(dá)。 結(jié)果(1)經(jīng)培養(yǎng),擴(kuò)增兔骨髓間充質(zhì)干細(xì)胞,P2、P3可作為hBMP2轉(zhuǎn)染的靶細(xì)胞。(2)用陽離子脂質(zhì)體轉(zhuǎn)染pcDNA3.1-hBMP2的MSCs經(jīng)G418篩選后,經(jīng)免疫組化、WesternBlot法檢測,轉(zhuǎn)染pcDNA3.1-hBMP2后的兔骨髓間充質(zhì)干細(xì)胞內(nèi)有大量hBMP2 mRNA的轉(zhuǎn)錄和相關(guān)蛋白的表達(dá),而未轉(zhuǎn)染的骨髓間充質(zhì)干細(xì)胞未見陽性表達(dá)。 結(jié)論(1)兔骨髓間充質(zhì)干細(xì)胞可作為hBMP2轉(zhuǎn)染的靶細(xì)胞。(2)本實驗成功構(gòu)建hBMP2真核表達(dá)質(zhì)粒并在兔骨髓間充質(zhì)干細(xì)胞中得到相關(guān)表達(dá)。(3)本研究為進(jìn)一步研究BMP2基因轉(zhuǎn)染MSCs的體外增殖并穩(wěn)定表達(dá)和在動物試驗中來加速牽張成骨新骨形成的實驗奠定了基礎(chǔ)。
[Abstract]:Objective (1) to culture rabbit bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs). (2) for transfection to BMSCs by using positive liposome pcDNA3.1 (-)-mediated transfection. Stable transfected BMSCs cells were obtained. (3) the expression of pcDNA3.1-hBMP2 in rabbit BMSCs was detected by immunohistochemistry and Western blot. Methods (1) BMSCs was cultured and amplified by adherent method. Flow cytometry was used to measure the surface markers. (2) the successfully constructed pcDNA3.1-hBMP2 recombinant plasmid was transfected into target cells in vitro and the stable expression cells were screened by G418. (3) the protein expression in BMSCs was detected by immunohistochemistry and Western Blot. Results (1) cultured rabbit bone marrow mesenchymal stem cells (BMSCs) could be used as target cells for hBMP2 transfection. (2) MSCs transfected with pcDNA3.1-hBMP2 by cationic liposome was screened by G418 and detected by immunohistochemistry with Western blot. A large number of hBMP2 mRNA transcripts and related proteins were expressed in rabbit bone marrow mesenchymal stem cells after pcDNA3.1-hBMP2 transfection, but no positive expression was found in untransfected bone marrow mesenchymal stem cells. Conclusion (1) Rabbit bone marrow mesenchymal stem cells can be used as target cells for hBMP2 transfection. (2) the eukaryotic expression plasmid of hBMP2 was successfully constructed and expressed in rabbit bone marrow mesenchymal stem cells. (3) in order to further study the transfection of BMP2 gene in rabbit bone marrow mesenchymal stem cells. The proliferation and stable expression of MSCs in vitro and the experiment of accelerating the formation of new bone in distraction osteogenesis were established in animal experiments.
【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】：2010
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 高曉燕;金葡液在兔下頜骨牽張成骨中作用的研究[D];遼寧醫(yī)學(xué)院;2011年

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本文編號：2146702