【摘要】：目的探討含有骨保護(hù)素(osteoprotegerin,OPG)的真核表達(dá)載體轉(zhuǎn)染大鼠骨髓基質(zhì)干細(xì)胞(BMSCs)表達(dá)情況并分析OPG基因轉(zhuǎn)染對BMSCs生物學(xué)行為的影響。 方法選取清潔級4周齡SD大鼠,雌雄不限,無菌條件下取脛骨和股骨,進(jìn)行骨髓基質(zhì)干細(xì)胞的分離和培養(yǎng);設(shè)立質(zhì)粒載體組、空載體組、未轉(zhuǎn)染組。質(zhì)粒載體組轉(zhuǎn)染plRES2-EGFP-OPG,空載體組轉(zhuǎn)染plRES2-EGFP,未轉(zhuǎn)染組未經(jīng)特殊處理。轉(zhuǎn)染后48小時(shí),在激光掃描共聚焦顯微鏡下檢測EGFP表達(dá);RT-PCR檢測各組骨髓基質(zhì)干細(xì)胞中OPGmRNA的表達(dá);免疫細(xì)胞化學(xué)檢測OPG蛋白的表達(dá);MTT法檢測細(xì)胞增殖能力;細(xì)胞堿性磷酸酶染色法檢測細(xì)胞向成骨細(xì)胞分化的能力。 結(jié)果1.骨髓基質(zhì)細(xì)胞分離培養(yǎng)形態(tài)觀察:初始分離的骨髓細(xì)胞呈圓形;大小不一;24 h后有少量細(xì)胞貼壁; 96 h后貼壁生長的細(xì)胞主要為梭形的成纖維樣細(xì)胞;分瓶培養(yǎng)后細(xì)胞形成克隆;傳代后細(xì)胞貼壁生長,分裂相增多,并不斷增殖分化形成均一的梭形細(xì)胞。 2.激光掃描共聚焦顯微鏡觀察結(jié)果:質(zhì)粒載體組轉(zhuǎn)染后可見綠色熒光蛋白表達(dá),未轉(zhuǎn)染組未見表達(dá)。 3. RT-PCR及免疫細(xì)胞化學(xué)檢測結(jié)果:反轉(zhuǎn)錄聚合酶鏈反應(yīng)觀察到質(zhì)粒載體組在1200bp有明顯條帶,空載體組及未轉(zhuǎn)染組未見表達(dá);免疫細(xì)胞化學(xué)檢測質(zhì)粒載體組OPG蛋白表達(dá)陽性,空載體組及未轉(zhuǎn)染組為陰性表達(dá)。 4. MTT檢測細(xì)胞增殖結(jié)果:轉(zhuǎn)染后細(xì)胞在其表面及孔內(nèi)平鋪、伸展、生長及增殖狀態(tài)良好,細(xì)胞增殖能力無明顯改變,3組比較無顯著差異(P 0.05)。 5.細(xì)胞堿性磷酸酶(ALP)染色結(jié)果:plRES2-EGFP-OPG轉(zhuǎn)染的BMSCs合成ALP的能力顯著提高,胞漿呈陽性藍(lán)染顆粒,與空載體組及未轉(zhuǎn)染組相比存在顯著差異(P 0.05)。 結(jié)論:1.成功分離培養(yǎng)大鼠BMSCs,證明了BMSCs可作為基因強(qiáng)化骨組織工程中理想的種子細(xì)胞。 2.轉(zhuǎn)染OPG基因的BMSCs后可可穩(wěn)定、高效的表達(dá)OPG,建立了OPG基因修飾的骨髓基質(zhì)干細(xì)胞瞬時(shí)基因表達(dá)體系。 3.經(jīng)plRES2-EGFP-OPG轉(zhuǎn)染后的BMSCs增殖能力未受到影響,并具有一定的生物學(xué)功能,能促進(jìn)其向成骨細(xì)胞分化。
[Abstract]:Objective to investigate the expression of (BMSCs) in rat bone marrow stromal stem cells transfected with eukaryotic expression vector containing osteoprotegerin (osteoprotegerin,OPG) and analyze the effect of OPG gene transfection on the biological behavior of BMSCs. Methods Bone marrow stromal cells (BMSCs) were isolated and cultured from the tibia and femur of 4-week-old SD rats of clean grade under sterile conditions, and the plasmid vector group, empty vector group and untransfected group were set up. Plasmid group transfected plRES2-EGFP-OPG, empty vector group transfected plRES2-EGFP, without special treatment. 48 hours after transfection, the expression of EGFP was detected by laser scanning confocal microscope, the expression of OPGmRNA in bone marrow stromal cells was detected by RT-PCR, the expression of OPG protein was detected by immunocytochemistry, the proliferative ability of cells was detected by MTT method. The ability of cells to differentiate into osteoblasts was detected by alkaline phosphatase staining. Result 1. Morphological observation of bone marrow stromal cells: the primary separated bone marrow cells were round and varied in size, a small number of cells adhered to the wall after 24 hours, and the cells that adherent to the bone marrow cells after 96 hours were mainly fusiform fibroblasts. Cell clone was formed after flask culture, cell adherent growth, mitotic phase increased after passage, and the cells proliferated and differentiated into uniform fusiform cells. 2. The results of laser scanning confocal microscopy showed that the expression of green fluorescent protein was observed in the plasmid vector group after transfection, but not in the untransfected group. Results of RT-PCR and immunocytochemistry: reverse transcriptase polymerase chain reaction showed that there were obvious bands in 1200bp in plasmid vector group, but no expression in empty vector group and untransfected group, OPG protein expression in plasmid vector group was positive by immunocytochemistry. Negative expression was found in empty vector group and untransfected group. 4. The results of MTT: after transfection, the cells were flattened, stretched, grown and proliferated well, and the ability of cell proliferation did not change obviously, but there was no significant difference among the three groups (P 0.05). The results of alkaline phosphatase (ALP) staining showed that the ability of BMSCs transfected with plRES2-EGFP-OPG to synthesize ALP was significantly increased, and the cytoplasm was blue stained, which was significantly different from that of empty vector group and untransfected group (P 0.05). Conclusion: 1. The successful isolation and culture of rat BMSCs, proved that BMSCs could be used as an ideal seed cell in gene strengthening bone tissue engineering. 2. The BMSCs transfected with OPG gene was stable and highly expressed OPG,. The transient gene expression system of bone marrow stromal cells modified with OPG gene was established. 3. 3. The proliferation of BMSCs transfected with plRES2-EGFP-OPG was not affected and had certain biological function, which could promote the differentiation of BMSCs into osteoblasts.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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