TGF-β1转染大鼠脂肪干细胞向软骨细胞分化的实验研究

发布时间：2018-07-23 16:49

【摘要】： 实验目的 探讨大鼠脂肪干细胞(Adipose derived stem cells,ADSCs)的分离、培养、扩增及表型鉴定方法。研究应用TGF-β_1基因转染大鼠ADSCs,诱导其向软骨细胞分化,比较目的基因的分泌情况及向软骨细胞分化效果,为软骨组织工程学种子细胞提供新方法。 实验方法 先行质粒pcDNA3.1(+)-TGF-β1扩增、提取和酶切鉴定。取大鼠大网膜及肾周脂肪囊等处脂肪组织,眼科剪剪碎,加入3倍体积0.1%的Ⅰ型胶原酶,酶消化法及密度梯度离心法相结合,分离、纯化SD大鼠ADSCs,倒置显微镜观察培养各代ADSCs的形态。取第3代脂肪干细胞,免疫荧光法检测其表面抗原CD29、CD44、CD106、CD34表达。将TGF-β1基因转染ADSCs,按转染情况分为4组:未转染组(A组)、转染空载体组(B组)、转染TGF-β1组(C组)、转染TGF-β1组(D组)。对转染后细胞进行筛选,MTT法测定筛选后细胞的增殖活性,RT-PCR检测TGF-β1、SOX9、Aggrecan和CollagenⅡ(Col-Ⅱ)mRNA表达,Western blot检测TGF-β1、Coll-Ⅱ蛋白表达。 实验结果 1、TGF-β1质粒鉴定测序结果与GenBank基因序列相符。 2、原代培养的脂肪干细胞1d即可在培养皿内见细胞贴壁,贴壁细胞呈类圆形;2d后长梭形细胞逐渐增多;4-5d后细胞呈典型团簇状生长,形成集落;约7d左右细胞融合超过90%时;3代后细胞形态比较均一,呈大长梭形。 3、大鼠ADSCs的鉴定免疫荧光法检测ADSCs CD44、CD29呈阳性反应,CD106、CD34呈阴性反应。 4、MTT法检测转染基因对ADSCs增殖活性的影响C、D组的吸光度值明显高于A、B组,与A、B组之间的差异具有显著性(P＜0.01),而C组和D组、A组和B组之间差异无统计学意义(P＞0.05)。 5、RT-PCR检测TGF-β1、SOX9、Aggrecan、Coll-ⅡmRNA表达mRNA显示C、D组TGF-β1、SOX9、Coll-Ⅱ、和Aggrecan的mRNA表达增强。除TGF-β1在A、B组表达外(A、B组比较P＞0.05),余下各检测指标在未转染组和转染转空载体组均未见表达。 6、Western blot检测TGF-β1、Coll-Ⅱ蛋白表达C、D组的TGF-β1、Coll-Ⅱ的蛋白表达高于A、B组,差异具有显著性(P＜0.01),A、B组间的差异无统计学意义(P＞0.05)。 结论 大鼠ADSCs经分离,纯化,扩增,生物学性状稳定。ADSCs能稳定地表达CD29、CD44,CD106、CD34表达阴性,证明其为干细胞来源。转染TGF-β1后ADSCs细胞的增殖能力增强。稳定转染TGF-β1后的ADSCs能够持续表达和分泌TGF-β1基因和蛋白。转染TGF-β1基因后的ADSCs成功表达软骨细胞的特异性基质,具有了软骨细胞的生化特征。采用脂质体作为载体,安全、无毒副作用,经基因修饰过的ADSCs携带了诱导其向软骨细胞分化,促进基质分泌的细胞因子的基因,因此,经TGF-β1基因修饰的ADSCs可以作为软骨组织工程的种子细胞来源。
[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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