【摘要】： 實(shí)驗(yàn)?zāi)康?探討大鼠脂肪干細(xì)胞(Adipose derived stem cells,ADSCs)的分離、培養(yǎng)、擴(kuò)增及表型鑒定方法。研究應(yīng)用TGF-β_1基因轉(zhuǎn)染大鼠ADSCs,誘導(dǎo)其向軟骨細(xì)胞分化,比較目的基因的分泌情況及向軟骨細(xì)胞分化效果,為軟骨組織工程學(xué)種子細(xì)胞提供新方法。 實(shí)驗(yàn)方法 先行質(zhì)粒pcDNA3.1(+)-TGF-β1擴(kuò)增、提取和酶切鑒定。取大鼠大網(wǎng)膜及腎周脂肪囊等處脂肪組織,眼科剪剪碎,加入3倍體積0.1%的Ⅰ型膠原酶,酶消化法及密度梯度離心法相結(jié)合,分離、純化SD大鼠ADSCs,倒置顯微鏡觀察培養(yǎng)各代ADSCs的形態(tài)。取第3代脂肪干細(xì)胞,免疫熒光法檢測(cè)其表面抗原CD29、CD44、CD106、CD34表達(dá)。將TGF-β1基因轉(zhuǎn)染ADSCs,按轉(zhuǎn)染情況分為4組:未轉(zhuǎn)染組(A組)、轉(zhuǎn)染空載體組(B組)、轉(zhuǎn)染TGF-β1組(C組)、轉(zhuǎn)染TGF-β1組(D組)。對(duì)轉(zhuǎn)染后細(xì)胞進(jìn)行篩選,MTT法測(cè)定篩選后細(xì)胞的增殖活性,RT-PCR檢測(cè)TGF-β1、SOX9、Aggrecan和CollagenⅡ(Col-Ⅱ)mRNA表達(dá),Western blot檢測(cè)TGF-β1、Coll-Ⅱ蛋白表達(dá)。 實(shí)驗(yàn)結(jié)果 1、TGF-β1質(zhì)粒鑒定測(cè)序結(jié)果與GenBank基因序列相符。 2、原代培養(yǎng)的脂肪干細(xì)胞1d即可在培養(yǎng)皿內(nèi)見細(xì)胞貼壁,貼壁細(xì)胞呈類圓形;2d后長梭形細(xì)胞逐漸增多;4-5d后細(xì)胞呈典型團(tuán)簇狀生長,形成集落;約7d左右細(xì)胞融合超過90%時(shí);3代后細(xì)胞形態(tài)比較均一,呈大長梭形。 3、大鼠ADSCs的鑒定免疫熒光法檢測(cè)ADSCs CD44、CD29呈陽性反應(yīng),CD106、CD34呈陰性反應(yīng)。 4、MTT法檢測(cè)轉(zhuǎn)染基因?qū)DSCs增殖活性的影響C、D組的吸光度值明顯高于A、B組,與A、B組之間的差異具有顯著性(P＜0.01),而C組和D組、A組和B組之間差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 5、RT-PCR檢測(cè)TGF-β1、SOX9、Aggrecan、Coll-ⅡmRNA表達(dá)mRNA顯示C、D組TGF-β1、SOX9、Coll-Ⅱ、和Aggrecan的mRNA表達(dá)增強(qiáng)。除TGF-β1在A、B組表達(dá)外(A、B組比較P＞0.05),余下各檢測(cè)指標(biāo)在未轉(zhuǎn)染組和轉(zhuǎn)染轉(zhuǎn)空載體組均未見表達(dá)。 6、Western blot檢測(cè)TGF-β1、Coll-Ⅱ蛋白表達(dá)C、D組的TGF-β1、Coll-Ⅱ的蛋白表達(dá)高于A、B組,差異具有顯著性(P＜0.01),A、B組間的差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 結(jié)論 大鼠ADSCs經(jīng)分離,純化,擴(kuò)增,生物學(xué)性狀穩(wěn)定。ADSCs能穩(wěn)定地表達(dá)CD29、CD44,CD106、CD34表達(dá)陰性,證明其為干細(xì)胞來源。轉(zhuǎn)染TGF-β1后ADSCs細(xì)胞的增殖能力增強(qiáng)。穩(wěn)定轉(zhuǎn)染TGF-β1后的ADSCs能夠持續(xù)表達(dá)和分泌TGF-β1基因和蛋白。轉(zhuǎn)染TGF-β1基因后的ADSCs成功表達(dá)軟骨細(xì)胞的特異性基質(zhì),具有了軟骨細(xì)胞的生化特征。采用脂質(zhì)體作為載體,安全、無毒副作用,經(jīng)基因修飾過的ADSCs攜帶了誘導(dǎo)其向軟骨細(xì)胞分化,促進(jìn)基質(zhì)分泌的細(xì)胞因子的基因,因此,經(jīng)TGF-β1基因修飾的ADSCs可以作為軟骨組織工程的種子細(xì)胞來源。
[Abstract]:Objective to investigate the isolation, culture, amplification and phenotypic identification of adipose stem cells (Adipose derived stem cells) from rats. To study the effect of TGF- 尾 1 gene transfection on ADSCsto induce ADSCsto differentiate into chondrocytes, to compare the secretion of target gene and the differentiation effect into chondrocytes, and to provide a new method for the seed cells of cartilage tissue engineering. Methods the plasmid pcDNA3.1 () -TGF- 尾 1 was amplified, extracted and identified by enzyme digestion. The adipose tissue of rat greater omentum and perirenal fat sac were cut and shredded by ophthalmology. The rats were separated by enzyme digestion and density gradient centrifugation after adding 3 times volume of type I collagenase. ADSCs were purified from SD rats. The morphology of cultured ADSCs was observed by inverted microscope. The third generation of adipose stem cells were used to detect the expression of CD29, CD44, CD106 and CD34 by immunofluorescence. TGF- 尾 1 gene was transfected into ADSCsand divided into 4 groups according to the transfection: untransfected group (A group), transfected empty vector group (B group), transfected TGF- 尾 1 group (C group), and transfected TGF- 尾 1 group (D group). The proliferative activity of the transfected cells was determined by MTT assay. The expression of TGF- 尾 1, SOX9, Aggrecan and Collagen 鈪，

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