本文選題：Sox9-eGFP質(zhì)粒 + BMSCs��； 參考：《暨南大學(xué)》2015年碩士論文

【摘要】：目的:隨著人類平均壽命的延長及生活方式的變化,椎間盤退行性變導(dǎo)致的疾病無時不刻地影響著人類的健康和生活質(zhì)量。椎間盤組織之細胞表型類似軟骨細胞。本實驗研究運用的基因轉(zhuǎn)染技術(shù)是慢病毒介導(dǎo)的基因轉(zhuǎn)染,整體思路是將Sox9基因通過上述基因轉(zhuǎn)染技術(shù)導(dǎo)入到SD大鼠骨髓間充質(zhì)干細胞(Bone marrow mesenchymal stem cells,BMSCs),培養(yǎng)后檢測其基因、蛋白表達等相關(guān)指標(biāo),從而研究其成軟骨的效果,為后續(xù)人工生物椎間盤再造的研究提供種子細胞。方法:從SD大鼠骨髓腔內(nèi)提取BMSCs,隨即進行傳代培養(yǎng)。觀察骨髓間充質(zhì)干細胞形態(tài)、測定其貼壁率。用慢病毒及Sox9基因構(gòu)建Sox9-e GFP質(zhì)粒,此病毒包裝是通過轉(zhuǎn)染293T細胞來實現(xiàn)的,在此步試驗中我們會測定病毒的最佳感染復(fù)數(shù)(multiplicity of infection,MOI)值。然后將有目的基因之慢病毒轉(zhuǎn)染SD大鼠的BMSCs。用骨髓間充質(zhì)干細胞完全培養(yǎng)基常規(guī)培養(yǎng)轉(zhuǎn)染病毒的BMSCs,每天在細胞顯微鏡下密切關(guān)注細胞的生長狀態(tài)。經(jīng)過14天培養(yǎng)后,用q RT-PCR的方法檢測目的基因的相對表達量,用Westem Blot的方法測定目的基因蛋白(Sox9蛋白及Ⅱ型膠原蛋白)表達的情況。結(jié)果:從SD大鼠骨髓中提取的BMSCs貼壁狀態(tài)好,細胞形態(tài)是長條梭形、呈極性排列。細胞傳代培養(yǎng)后活力良好,細胞體積正常,細胞核清晰,細胞質(zhì)內(nèi)無大量顆粒產(chǎn)生、細胞貼壁生長、生長能力旺盛。傳至第9代時出現(xiàn)衰老,傳至12代時衰老征象十分明顯。q RT-PCR結(jié)果示:在對照組、空載組、實驗組均見表達,三組樣品Sox9 m RNA的相對表達量:1.064ug/ul,1.434ug/ul,1.456ug/ul,三組樣品間兩兩比較的結(jié)果均具統(tǒng)計學(xué)意義(P0.01)。Westem Blot結(jié)果示:通過檢測,Sox9蛋白的表達在三組細胞中都可以檢測的到,其相對表達量分別是空白組5351.69,空載組5536.347,實驗組11103.782,同樣通過統(tǒng)計學(xué)組間進行兩兩比較,結(jié)果有統(tǒng)計學(xué)意義(P0.05);II型膠原蛋白的表達在轉(zhuǎn)染Sox9組細胞中開始出現(xiàn),其余兩組此蛋白無表達。結(jié)論:綜上所述,BMSCs可以做為較為理想的種子細胞而在軟骨組織工程運用。其提取方法容易,且易于培養(yǎng)及進行體外擴增;以慢病毒做為載體轉(zhuǎn)染SD大鼠BMSCs的方法,可使目的基因得到高效而穩(wěn)定地表達,且通過實驗證明了這種方法可使BMSCs向軟骨細胞分化。轉(zhuǎn)染Sox9的骨髓間充質(zhì)干細胞可作為制備人工生物椎間盤的種子細胞。
[Abstract]:Objective: with the prolongation of life expectancy and the change of life style, the disease caused by intervertebral disc degeneration affects the health and quality of life. The cell phenotype of intervertebral disc tissue is similar to that of chondrocytes. The gene transfection technique used in this study was lentivirus-mediated gene transfection. The whole idea was to transfer Sox9 gene into SD rat bone marrow mesenchymal stem cells (BMSCs) and detect its gene after culture. Protein expression and other related indicators, so as to study the effect of cartilage formation, and provide seed cells for the subsequent study of artificial disc reconstruction. Methods: BMSCs were extracted from the medullary cavity of SD rats and then subcultured. The morphology of bone marrow mesenchymal stem cells (BMSCs) was observed and the adherence rate was measured. Sox9-e GFP plasmids were constructed with lentivirus and Sox9 gene. The virus packaging was carried out by transfection of 293T cells. In this step test, we will determine the optimal infection complex (multiplicity of infection moi of the virus. Then the lentivirus with the target gene was transfected into BMSCs of SD rats. BMSCs transfected with BMSCs were cultured on the complete culture medium of bone marrow mesenchymal stem cells (BMSCs). After 14 days of culture, the relative expression of the target gene was detected by qRT-PCR, and the expression of Sox9 protein and type 鈪，

本文編號：2105423