生长激素上调性激素抑制下青春期大鼠生长板原代软骨细胞表皮生长因子的表达  

Growth Hormone Up-regulates Expression of Epidermal Growth Factor of Tibial Growth Plate Chondrocytes from Pubertal Rats Treated with GnRHa

作　　者：潘思年[1] 张成喜[2] 李燕虹[3] 马华梅[3] 朱顺叶[1] 杜敏联[3] 

机构地区：[1]中山大学附属第三医院儿科,广东广州510630 [2]中山大学附属第三医院心内科,广东广州510630 [3]中山大学附属第一医院儿科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2010年第4期496-502,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(06021298)

摘　　要：【目的】研究生长激素(GH)对性激素抑制下的离体青春期大鼠生长板软骨细胞表皮生长因子(EGF)mRNA表达及其蛋白合成、分泌的影响。【方法】3周龄雌性SD大鼠开始注射促性腺激素释放激素拟似物(GnRHa)至7周龄,随后分离培养5～8只大鼠胫骨生长板原代软骨细胞,分为时效组和量效组。采用四氮甲基唑蓝(MTT)以及免疫组化法测定增殖细胞核抗原(PCNA),观察不同剂量、不同干预时间的生长激素对性激素抑制下的离体青春期大鼠生长板软骨细胞增殖的影响。用RT-PCR法检测软骨细胞EGF mRNA的表达,酶联免疫吸附法(ELISA)测定EGF的表达。【结果】生长激素以时效和量效作用方式对雌激素受抑的离体青春期大鼠生长板软骨细胞增殖呈双相型影响。GH作用6h后软骨细胞的EGF mRNA表达显著增加(与0h比较P<0.05),且从6h到24h间存在明显的时间依赖性(组间P<0.05),在18h达到最高峰。GH作用12h后软骨细胞分泌入培养基中的的EGF表达显著增加(与0h比较P<0.05),且从12h到48h间存在明显的时间依赖性(组间P<0.05),在36h达到最高峰。量效关系显示50ng/mL浓度的GH开始能显著增加培养基中EGFmRNA和蛋白的表达,且从50ng/mL至1000ng/mL间存在明显的浓度依赖性(组间P<0.05),在100ng/mL时表达达到最高峰。【结论】生长激素能促进性激素抑制下的离体青春期大鼠生长板软骨细胞的增殖及EGF mRNA和蛋白的表达。【Objective】 To investigate whether growth hormone could up-regulate local epidermal growth factor （EGF） synthesis （EGF peptide and EGF mRNA levels） in cultured growth plate chondrocytes. 【Methods】 At 3 week of age,Sprague-Dawley rats received 2.5 mg / kg in slow-released GnRHa （triptorelin） which was repeated every 2 weeks for 2 times till 7-week old. Chondrocytes from tibial growth plate of 5 ～ 8 Sprague-Dawley rats by trypsin-collagenase digestion were cultured in monolayer. Primary chondrocytes were divided into time-effect groups and concentration-effect groups. MTT and immunohistochemical staining of proliferating cell nuclear antigen （PCNA） were conducted to observe the effect of GH on the proliferation of chondrocytes. RT-PCR and ELISA method were conducted to detect EGF expression in mRNA level and protein level respectively. 【Results】 GH enhanced the proliferation of the chondrocytes in time-course-dependent and concentration-dependent manner. Incubation with GH 100 ng / mL maximally increased expression levels of EGF mRNA （for 18 h） and EGF peptide （for 36 h） respectively,compared to the control group. The expression levels of EGF mRNA and EGF peptide by GH-stimulated synthesis was dose-dependently,maximally stimulatory concentrations of GH was 100 ng / mL. 【Conclusion】 GH enhances the proliferation of chondrocytes of pubertal female rat treated with GnRHa in vitro. GH can up-regulate local EGF synthesis （EGF mRNA and EGF peptide levels） in cultured growth plate chondrocytes.

关 键 词：软骨细胞 细胞培养 生长板 生长激素 表皮生长因子 

分 类 号：R72[医药卫生—儿科]