生长激素对大鼠胫骨生长板软骨细胞胶原Ⅱ表达的影响(1)
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【摘要】 目的 探讨生长激素(growth hormone,GH)对离体培养的大鼠胫骨生长板的软骨细胞Ⅱ型胶原(collagen Ⅱ,ColⅡ)表达的影响。方法 采用两步酶消化法分离培养5~8只三周龄大鼠生长板的软骨细胞,用RTPCR和免疫组化的方法分别检测不同浓度GH对软骨细胞Ⅱ型胶原mRNA和蛋白的表达。结果 GH能促进软骨细胞ColⅡ的表达并呈浓度依赖性,GH在10~500 ng/ml之间能促进ColⅡ的表达,与对照组比较差异均有统计学意义(P<0.05),并且以100 ng/ml时表达最强,随着GH浓度的加大,其促ColⅡ表达的效应逐渐减弱,1000 ng/ml与对照组比较无差异(P>0.05)。结论 GH通过促进软骨细胞ColⅡ的表达能维持软骨细胞的表型,GH在使用过程中不会导致骨龄的增加。
【关键词】
软骨细胞;生长板;生长激素;Ⅱ型胶原
Effect of growth hormone on the expression of collagen Ⅱ of cultured growth plate chondrocytes of rats
PAN Sinian,ZHANG Chengxi.Department of Pediatrics,The Third Hospital Affiliated to Zhongshan University,Guangzhou 510630,China
【Abstract】 Objective To observe the effect of growth hormone on the expression of collagen Ⅱ of cultured growth plate chondrocytes of rats.Methods Primary chondrocytes from tibial growth plate of 5~8rats by trypsincollagenase digestion were cultured in monolayer.RTPCR and immunocytochemistry were performed to investigate the expression of collagen Ⅱ at the mRNAand protein level,respectively with different concentrations of GH.Results GHenhances the expression of collagen Ⅱ of chondrocytes in vitro in a concentrationdependent fashion.The expression of collagen Ⅱwas obviously between the GH concentration of 10 ng/ml and 500 ng/ml(P<0.05),maximally at the concentration of 100 ng/ml,and decreased again after higher concentration of GH.There was no significant difference between 1000 ng/ml group and control group(P>0.05).Conclusion GH can maintain the phenotype of chondrocytes by enhancing the expression of collagen Ⅱand it will not increase the bone age during clinical practice.
【Key words】
Chondrocyte;Epiphyseal growth plate;Growth hormone;Collagen Ⅱ
生长激素(growth hormone,GH)被广泛用于儿童生长障碍性疾病,它能改善这些患儿的成年身高。然而,GH对生长板软骨细胞的作用机制目前尚未阐明,目前国内外有关GH对离体培养软骨细胞作用的报道均较少。本文拟通过体外培养大鼠胫骨生长板的软骨细胞来探讨GH对软骨细胞胶原Ⅱ表达的影响。
1 材料与方法
1.1 实验动物 正常三周龄SpragueDawley(SD)大鼠,清洁级,雌雄不限,体质量40~50 g之间由中山大学实验动物中心提供。
1.2 主要试剂 重组人生长激素(rhGH,为CYTOLAB/PEPROTECH ASIA 产品,Israel)、DMEM/F12(1:1)培养基(Sigma)、胎牛血清(FBS,Gibco)、胰蛋白酶(1:250,AMREsCo)、Ⅱ型胶原酶、琼脂糖(Invitrogen)、SP和DAB显色试剂盒(福州迈新)、兔抗大鼠Ⅱ型胶原一抗(Neomarker)、Trizol Reagent、逆转录试剂盒和Taq酶(Toyobo)。
1.3 大鼠胫骨生长板软骨细胞的培养 将5~8只大鼠处死后在超净工作台上无菌取出胫骨近段,从骨软骨连接处分离出生长板软骨,仔细去除胫骨外的肌肉和骨膜,将软骨块剪成1 mm3大小,采用两步酶消化法(0.25%胰蛋白酶EDTA消化30 min、0 ......
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