本文選題：骨質(zhì)疏松 + 細(xì)胞分化�。� 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】：目的：構(gòu)建人真核表達(dá)的hTAZ慢病毒載體pLenti6/V5-hTAZ，轉(zhuǎn)染293FT細(xì)胞獲得重組慢病毒顆粒，研究hTAZ對前成骨細(xì)胞MC3T3-E1分化的調(diào)控作用。方法：將已經(jīng)過測序驗(yàn)證的，含有hTAZ基因慢病毒入門質(zhì)粒pENTR221-hTAZ通過LR反應(yīng)克隆到慢病毒載體pLenti6/V5-DEST中。對重組質(zhì)粒進(jìn)行酶切鑒定并在細(xì)胞中驗(yàn)證表達(dá)。將該重組載體和慢病毒包裝質(zhì)粒充分混合，用陽離子脂質(zhì)體Lipofactamine轉(zhuǎn)染293FT細(xì)胞，培養(yǎng)和待細(xì)胞完全裂解后收集富含hTAZ基因的慢病毒顆粒上清液，取適量上清液感染MC3T3 E1細(xì)胞，采用殺稻瘟菌素（Blastcidin）篩選，建立了穩(wěn)定過量表達(dá)hTAZ蛋白的MC3T3-E1/hTAZ細(xì)胞系。用條件培養(yǎng)基誘導(dǎo)MC3T3-E1和MC3T3-E1/hTAZ細(xì)胞成骨、成脂分化，von Kossa和茜素紅染色比較MC3T3-E1和MC3T3-E1/hTAZ成骨分化程度差異；油紅O染色比較其成脂分化差異，，考察hTAZ基因過表達(dá)對前成骨細(xì)胞分化的影響。結(jié)果：凝膠電泳和測序結(jié)果均證明hTAZ重組慢病毒載體pLenti6/V5-hTAZ構(gòu)建正確，并能在細(xì)胞中正確表達(dá)。與輔助質(zhì)粒共轉(zhuǎn)包裝細(xì)胞獲得慢病毒顆粒，并成功感染MC3T3-E1細(xì)胞。von Kossa染色和茜素紅染色結(jié)果表明，MC3T3-E1/hTAZ細(xì)胞成骨分化強(qiáng)于未轉(zhuǎn)染細(xì)胞，而其成脂分化受抑制。結(jié)論：本研究構(gòu)建了人真核表達(dá)的質(zhì)粒載體pLenti6/V5-hTAZ，并能在細(xì)胞中正確表達(dá)。成功包裝了hTAZ病毒并獲得過表達(dá)hTAZ的MC3T3－E1細(xì)胞。過表達(dá)hTAZ可促進(jìn)MC3T3-E1向成骨細(xì)胞分化，抑制其成脂分化。
[Abstract]:Aim: to construct hTAZ lentivirus vector pLenti6 / V5-hTAZ and transfect 293FT cells to obtain recombinant lentivirus particles, and to study the effect of hTAZ on the differentiation of preosteoblast MC3T3-E1. Methods: the primer plasmid pENTR221-hTAZ containing hTAZ gene was cloned into lentivirus vector pLenti6 / V5-DEST by LR reaction. The recombinant plasmid was digested and expressed in cells. The recombinant vector and the lentivirus packaging plasmid were fully mixed and transfected into 293FT cells by cationic liposome Lipofactamine. The supernatants of lentivirus particles rich in hTAZ gene were collected after the complete cell lysis, and the supernatants were used to infect MC3T3 and E1 cells. A MC3T3-E1 / hTAZ cell line with stable overexpression of hTAZ protein was established by Blastcidin screening. The osteogenesis of MC3T3-E1 and MC3T3-E1 / hTAZ cells was induced by conditioned medium, and the degree of osteogenic differentiation of MC3T3-E1 and MC3T3-E1 / hTAZ cells was compared with that of MC3T3-E1 and MC3T3-E1 / hTAZ cells by lipogenic von Kossa and alizarin red staining, and the effect of overexpression of hTAZ gene on the differentiation of preosteoblast was investigated. Results: the results of gel electrophoresis and sequencing proved that hTAZ recombinant lentivirus vector pLenti6 / V5-hTAZ was constructed correctly and expressed correctly in cells. The lentivirus particles were obtained by co-transfection with the co-packaged cells and infected with MC3T3-E1 cells. Von Kossa staining and alizarin red staining showed that the osteogenic differentiation of MC3T3-E1 / hTAZ cells was stronger than that of untransfected cells, but the adipogenic differentiation of MC3T3-E1 cells was inhibited. Conclusion: the plasmid vector pLenti6 / V5-hTAZ was constructed and expressed correctly in human eukaryotic cells. The hTAZ virus was successfully packaged and MC3 T 3-E1 cells expressing hTAZ were obtained. Overexpression of hTAZ could promote the differentiation of MC3T3-E1 into osteoblasts and inhibit the adipogenic differentiation of MC3T3-E1.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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