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重组人生长激素对体外培养的Bel-7402肝癌细胞生长激素受体的调控作用

Regulating effect of rhGH on GHR of Bel-7402 human hepatic carcinoma cell lines in vitro

摘要:

目的 了解重组人生长激素(rhGH)对体外培养的Bel-7402肝癌细胞生长激素受体(GHR)的调控作用.方法 采用放射配体法了解7402肝癌细胞生长激素受体(GHR)的表达情况,检测不同浓度(0,1,10,100,1000,10 000 ng/ml)rhGH对肝癌细胞GHR表达的影响,用辣根过氧化酶法了解7402肝癌细胞培养上清液的胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的变化,采用肿瘤细胞计数、噻唑蓝比色法了解肝癌细胞在不同浓度rhGH作用下药物敏感性,计算细胞生长率;用流式细胞计了解肝癌细胞在上述浓度rhGH作用下细胞周期各时相细胞比率、细胞增殖指数(PI)以及凋亡率变化.结果 放射配体法发现7402肝癌细胞表达GHR,在rhGH作用后24 h,7402肝癌细胞GHR的位点数量在10与100 ng/ml实验组较对照组增加(P<0.05),在10 000 ng/ml组较对照组降低(P<0.05);rhGH作用后的24 h与48 h,7402肝癌细胞上清液IGF-Ⅰ浓度在100 ng/ml组较对照组显著增高(P<0.05);与此同时,rhGH对体外培养的7402肝癌细胞增殖具有一定程度刺激作用,表现为rhGH在100 ng/ml浓度时促进肝癌细胞增殖较显著,而rhGH在其它浓度(1,10,1000,10 000 ng/ml)对肝癌细胞的增殖虽能表现出一定程度促进作用,但总体效果较rhGH在100 ng/ml浓度为弱.rhGH作用48 h流式细胞检测发现各实验组S期所占比率、PI均较对照组升高(P<0.05);G2-M细胞所占比率,10 ng/ml组与100 ng/ml组较对照组显著增高(P<0.05);各实验组凋亡率与对照组比较无显著差异.结论 rhGH可促进7402肝癌细胞DNA的合成,提高肿瘤细胞分裂增殖能力,原因可能与7402肝癌细胞表达GHR,rhGH可在一定浓度范围内上调7402肝癌细胞GHR的表达,促进肿瘤细胞合成IGF-Ⅰ有关,其确切途径仍有待进一步验证.

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abstracts:

Objective To investigate the effect of rhGH on GHR of Bel-7402 human hepatic carcinoma cell lines in vitro.Methods Radioreceptor assay was used to detect the GHR expression of the Bel-7402 human hepatic carcinoma cell lines and its relation to rhGH of different concentrations(0,1,10,100,1000,10 000 ng/ml).Horseradish peroxidase method was used to observe the changes of insulin-like growth factor Ⅰ (IGF-Ⅰ) in the supernatant of 7402 hepatic carcinoma cell line,Tumor cell count,thiazole blue chromatometry were introduced to determine the sensibility of Bel-7402 to the rhGH of different concentrations.Growth rate of tumor cells was calculated.The changes of cell proportion at each cell phase,cell proliferation index (PI) and apoptosis rate were calculated by flow cytometry.Results Radioreceptor assay confirmed that 7402 hepatic carcinoma cell line expressed GHR.Twenty-four hours after rhGH injection,GHR site quantities in the 10 ng/ml and 100 ng/ml group were significantly higher than that in the control group.Meanwhile,they were signifieantly lower in the 1000 ng/ml group than in the control group.After rhGH injection for 24 and 48 h,concentration of IGF-Ⅰ in the supernatant of 7402 hepatic carcinoma cell line was significantly higher in the 100 ng/ml group than in the control group(P<0.05).It also showed that rhGH had some effects on the growth of 7402 hepatic carcinoma cell lines.The strongest effect of enhancing proliferation of hepatic carcinoma cells was found at the concentration of 100 ng/ml rhGH (P<0.05,compared with control group) and other concentrations of it only had limited positive effects as compared with it.FCM showed that the cell ratio in S phase cell cycle and PI in study group were both significantly higher than those of the control group 48 hours after using rhGH.G2-M phase fractions in 10 ng/ml group and 100 ng/ml group were significantly higher as compared with control group.The apoptosis rate of each treatment group had no statistical differences as compared with the control group.Conclusion Definiteconcentrations of rhGH might have stimulating effects on the growth,can enhance synthesis of DNA and promote proliferation of 7402 cells line in vitro.The possible mechanism might be that rhGH can up-regulate GHR expression,which consequently enhances IGF expression and finally promotes cell line proliferation.

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作者: 刘建平 [1] 陈涛 [1] 刘波 [2] 陈小萱 [1] 区庆嘉 [1]
作者单位: 中山大学附属第二医院普外科,广州,510120 [1] 中山大学附属第三医院普外科,广州,510630 [2]
期刊: 《中华肝胆外科杂志》2009年15卷8期 603-607页 ISTICPKUCSCD
分类号: R73
栏目名称: 实验研究
DOI: 10.3760/cma.j.issn.1007-8118.2009.08.015
发布时间: 2009-10-23
基金项目:
广东省科技计划项目
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