软骨源性形态发生蛋白2基因转染小鼠成肌细胞表达软骨细胞表型的实验研究

发布时间：2018-01-11 17:05

  本文关键词：软骨源性形态发生蛋白2基因转染小鼠成肌细胞表达软骨细胞表型的实验研究　出处：《辽宁医学院》2011年硕士论文　论文类型：学位论文

  更多相关文章： 成肌细胞 软骨分化 软骨源性形态发生蛋白-2

【摘要】：目的 1.研究软骨形态发生蛋白-2(cartilage-derived morphogenetic protein-2)体外对小鼠成肌细胞向软骨细胞分化的可行性。 2.构建软骨形态发生蛋白-2基因转染小鼠成肌细胞，检测转染后小鼠成肌细胞的特异性表达，探讨成肌细胞作为软骨组织工程种子细胞的可行性。 方法 对小鼠成肌细胞进行体外培养传代，取第3代细胞进行实验处理，对照组(A组)为无血清L-DMEM培养液+小鼠成肌细胞；实验组（B组）为无血清L-DMEM培养液+透明质酸钠（10g/L）+小鼠成肌细胞；实验组C组为无血清L-DMEM培养液+CDMP-2(100ng/ml)+小鼠成肌细胞；实验组D组为无血清L-DMEM培养液+CDMP-2基因转染的小鼠成肌细胞。16天后终止细胞培养，应用倒置相差显微镜观察细胞形态，进行甲苯胺蓝糖胺聚糖(GAGs)染色，行Ⅱ型胶原免疫组织化学染色、灰度值分析和Western blot检测。 结果 细胞培养16天后可见转染组和诱导组的成肌细胞形态由长梭形向软骨细胞的多角形转变。甲苯胺蓝染色显示遇甲苯胺蓝变为蓝色的糖胺聚糖(GAGs)均匀分布于细胞基质中；免疫组织化学染色显示经SP法处理后变为棕褐色的Ⅱ型胶原蛋白均匀分布于镜下，对照组与透明质酸钠组未见着色。Western blot显示转染组和诱导组Ⅱ型胶原蛋白表达阳性，且CDMP-2基因转染组细胞表达Ⅱ型胶原蛋白水平高于CDMP-2诱导组，具有统计学意义；对照组和透明质酸钠组阴性表达。 结论 CDMP-2体外能够诱导成肌细胞向软骨细胞分化，，CDMP-2基因转染的小鼠成肌细胞能够分泌软骨细胞分泌的特异性蛋白及多糖，且CDMP-2基因转染的小鼠成肌细胞能够更好的表达软骨特异性Ⅱ型胶原及糖胺多糖。
[Abstract]:Purpose 1. Study on cartilage morphogenetic protein -2cartilage-derived morphogenetic protein-2. Feasibility of differentiation of mouse myoblasts into chondrocytes in vitro. 2. Construction of chondromorphogenetic protein-2 gene transfected mouse myoblasts, detection of the specific expression of mouse myoblasts after transfection, to explore the feasibility of myoblast as seed cells for cartilage tissue engineering. Method Mouse myoblasts were cultured in vitro and treated with the third passage. The control group (group A) was serum-free L-DMEM medium. The experimental group (group B) was divided into serum-free L-DMEM medium (sodium hyaluronate 10 g / L) mouse myoblasts. Experimental group C was murine myoblast in serum-free L-DMEM medium (CDMP-2ng-1 / ml). Group D was treated with serum-free L-DMEM culture medium CDMP-2 gene transfected mouse myoblasts. After 16 days, the cultured cells were terminated, and the morphology of the cells was observed by inverted phase contrast microscope. Toluidine blue glycosaminoglycan (GAGs) staining, type 鈪

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