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(zhǎng)及生活方式的變化,椎間盤退行性變導(dǎo)致的疾病無(wú)時(shí)不刻地影響著人類的健康和生活質(zhì)量。椎間盤組織之細(xì)胞表型類似軟骨細(xì)胞。本實(shí)驗(yàn)研究運(yùn)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(guò)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(xì)胞(Bone marrow mesenchymal stem cells,BMSCs),培養(yǎng)后檢測(cè)其基因、蛋白表達(dá)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(xì)胞。方法:從SD大鼠骨髓腔內(nèi)提取BMSCs,隨即進(jìn)行傳代培養(yǎng)。觀察骨髓間充質(zhì)干細(xì)胞形態(tài)、測(cè)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(guò)轉(zhuǎn)染293T細(xì)胞來(lái)實(shí)現(xiàn)的,在此步試驗(yàn)中我們會(huì)測(cè)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(xì)胞完全培養(yǎng)基常規(guī)培養(yǎng)轉(zhuǎn)染病毒的BMSCs,每天在細(xì)胞顯微鏡下密切關(guān)注細(xì)胞的生長(zhǎng)狀態(tài)。經(jīng)過(guò)14天培養(yǎng)后,用q RT-PCR的方法檢測(cè)目的基因的相對(duì)表達(dá)量,用Westem Blot的方法測(cè)定目的基因蛋白(Sox9蛋白及Ⅱ型膠原蛋白)表達(dá)的情況。結(jié)果:從SD大鼠骨髓中提取的BMSCs貼壁狀態(tài)好,細(xì)胞形態(tài)是長(zhǎng)條梭形、呈極性排列。細(xì)胞傳代培養(yǎng)后活力良好,細(xì)胞體積正常,細(xì)胞核清晰,細(xì)胞質(zhì)內(nèi)無(wú)大量顆粒產(chǎn)生、細(xì)胞貼壁生長(zhǎng)、生長(zhǎng)能力旺盛。傳至第9代時(shí)出現(xiàn)衰老,傳至12代時(shí)衰老征象十分明顯。q RT-PCR結(jié)果示:在對(duì)照組、空載組、實(shí)驗(yàn)組均見(jiàn)表達(dá),三組樣品Sox9 m RNA的相對(duì)表達(dá)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(jì)學(xué)意義(P0.01)。Westem Blot結(jié)果示:通過(guò)檢測(cè),Sox9蛋白的表達(dá)在三組細(xì)胞中都可以檢測(cè)的到,其相對(duì)表達(dá)量分別是空白組5351.69,空載組5536.347,實(shí)驗(yàn)組11103.782,同樣通過(guò)統(tǒng)計(jì)學(xué)組間進(jìn)行兩兩比較,結(jié)果有統(tǒng)計(jì)學(xué)意義(P0.05);II型膠原蛋白的表達(dá)在轉(zhuǎn)染Sox9組細(xì)胞中開(kāi)始出現(xiàn),其余兩組此蛋白無(wú)表達(dá)。結(jié)論:綜上所述,BMSCs可以做為較為理想的種子細(xì)胞而在軟骨組織工程運(yùn)用。其提取方法容易,且易于培養(yǎng)及進(jìn)行體外擴(kuò)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(dá),且通過(guò)實(shí)驗(yàn)證明了這種方法可使BMSCs向軟骨細(xì)胞分化。轉(zhuǎn)染Sox9的骨髓間充質(zhì)干細(xì)胞可作為制備人工生物椎間盤的種子細(xì)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(hào)：2105423