兔髓核细胞体外培养和重组人转化生长因子-β1对其代谢影响的研究

Culturing in vitro of nucleus pulposus cells and the anabolic effect induced by rhTGF-β1 in rabbit

目的:探讨体外单层和立体培养兔髓核细胞时的变化及重组人转化生长因子-β1(rhTGF-β1,10ng/ml)对其代谢的影响。方法:体外培养兔髓核细胞,分为3组。A组,单层培养组;B组,Ⅱ型胶原支架立体培养组;C组,Ⅱ型胶原支架立体培养+rhTGF-β1(10ng/ml)组。利用倒置显微镜、扫描电镜、RT-PCR、3H-proline掺入法观察髓核细胞形态学、基因表达水平和总胶原合成的变化。结果:B、C组兔髓核细胞由A组的多角形转为类圆形;与A组相比,B组Ⅱ型胶原、集聚蛋白多糖基因表达水平升高(P<0.05),总胶原合成升高(P<0.01)。与B组相比,C组Ⅱ型胶原、集聚蛋白多糖、核心蛋白多糖基因表达水平增高(P<0.01、P<0.01、P<0.05),总胶原合成升高(P<0.01)。结论:兔髓核细胞由单层培养转到Ⅱ型胶原支架上培养时其基因表达和总胶原合成增强。rhTGF-β1(10ng/ml)可增强立体培养的兔髓核细胞基因表达和总胶原合成。

Objective：To explore the changes of rabbit nucleus pulposus（NP） cells cultured in vitro and the anabolic effect introduced by rhTGF-β1 on the cells.Method：Rabbit NP cells grouped into 3 groups were cultured in vitro for one week.Group A,monolayer culture;group B,three dimensional culture in a type Ⅱ collagen scaffold;group C,three dimensional culture in the type Ⅱ collagen scaffold adding 10ng/ml rhTGF-beta1.Morphological changes were observed by inverted microscope and SEM.Gene expression levels were analyzed by half quantative RT-PCR.Total collagen synthesis was assayed by ^3H-proline incorporation.Result：Rabbit NP cells possess a round morphology when cultured in group B and group C while the cells possess a polygonal morphology when cultured in group A.Gene expression levels of type Ⅱ collagen,Aggrecan and total collagen synthesis of group B increased by 1.21,1.12,1.11 folds respectively,as compared with group A （P〈0.05,P〈0.05,P〈0.01 respectively）.Gene expression levels of type Ⅱ collagen,aggrecan,decorin and total collagen synthesis of group C increased by 1.29,1.32,1.16,1.26 folds respectively（P〈0.01,P〈0.01,P〈0.05,P〈0.01 respectively） as compared with group B.Conclusion：Gene expression levels and total collagen synthesis increase when rabbit NP cells are cultured in the type Ⅱ collagen scaffold.10ng/ml rhTGF-beta1 enhances the gene expression and total collagen synthesis of rabbit NP cells cultured in the type Ⅱ collagen scaffold.