模拟微重力下转化生长因子β1诱导骨髓间充质干细胞向髓核样细胞分化

Differentiation of bone marrow mesenchymal stem cells（BMMSCs） to nucleus-like pulposus transfected by TGF-β1 in the mimic microgravity environment

中文关键词:  骨髓间充质干细胞  分化  髓核  TGF-β1  微重力

英文关键词:Bone marrow mesenchymal stem cells  Differentiation  Nucleus pulposus  TGF-β1  Microgravity

中文摘要:

  【摘要】 目的：观察模拟微重力条件下体外转化生长因子β1（transforming growth factor-β1，TGF-β1）诱导骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMMSCs） 向髓核样细胞表型分化的效果。方法:取成年兔骨髓间充质干细胞分离培养并增殖至第3代后建立3个培养组：微重力诱导培养组（A组），将经TGF-β1转染后的BMMSCs在藻酸钙凝胶微球及旋转式细胞培养系统内进行模拟微重力条件下动态培养；诱导培养组（B组），将经TGF-β1转染后的BMMSCs在藻酸钙凝胶微球内培养；模拟微重力自然分化组（C组），将BMMSCs在藻酸钙凝胶微球内置于旋转式细胞培养系统内培养。在培养的第3、7、14、21天分别检测以上各组BMMSCs中TGF-β1的含量变化及BMMSCs的增殖能力，应用免疫组化、甲苯胺蓝染色检测各组Ⅱ型胶原表达情况，采用RT-PCR法检测各组BMMSCs的蛋白聚糖及Ⅱ型胶原蛋白mRNA的表达。结果:培养至第14天，A、B组中已可观察到呈多角形的类髓核样细胞，C组呈不规则形。从第3天开始，上清液中TGF-β1的含量和BMMSCs中DNA的含量A组较B、C两组明显增高（P<0.05），且随时间的延长而逐渐增高。A组在免疫组化染色中可见Ⅱ型胶原蛋白染色阳性， B组呈弱阳性，C组无明显变化。RT-PCR结果显示A、B组的BMMSCs中已有蛋白聚糖和Ⅱ型胶原蛋白mRNA的表达。结论: 模拟微重力条件下TGF-β1转染的BMMSCs在一定时间内可增加合成蛋白聚糖及Ⅱ型胶原蛋白的能力。

英文摘要:

  【Abstract】 Objective：To observe the differentiation of bone marrow mesenchymal stem cells（BMMSCs） to nucleus-like pulposus phenotype transfected by TGF-β1 and cultured in the mimic microgravity in vitro. Method：Bone marrow mesenchymal stem cells（BMMSCs） obtained from adult rabbit were cultured and proliferated to the third generation，which were assigned into 3 groups as followers：micro-gravity inducing group（group A），BMMSCs transfected by TGF-β1 were cultured in calcium alginate gel beads and rotary cell culture system；inducing group（group B），BMMSCs transfected by TGF-β1 were cultured in calcium alginate gel beads；microgravity differentiation group（group C），BMMSCs were cultured in calcium alginate gel beads and rotary cell culture system.At 3，7，14，21d，the content of TGF-β1 in BMMSCs and the proliferation potency of BMMSCs were detected in all groups respectively，collagen type Ⅱ was detected by immunohistochemical and Toluidine blue staining，the expression of mRNA of proteoglycan and collagen type Ⅱ protein was detected by RT-PCR.Result：At the 14th day，the polygonal nucleus-like pulposus was observed in group A and B，and abnormal cells were observed in group C.From the 3rd day，the content of TGF-β1 in supernate and its DNA in BMMSCs in group A were higher than those in group B and C，and also increased gradually with time.Immunohistochemistry staining of collagen type Ⅱ protein in BMMSCs climbing film showed positive in group A，weak positive in group B，and negative in group C.RT-PCR results showed expression of proteoglycan and collagen type ⅡmRNA in group A and B.Conclusion：BMMSCs transfected by TGF-β1 and cultured in the mimic microgravity can increase the synthesis potential of proeoglycan and collagen type Ⅱ.