神经生长因子对胆管癌细胞67-kDa层黏连蛋白受体表达的影响  

Effects of nerve growth factor on the expression of 67-kDa laminin receptor in human bile duct carcinoma cells

作　　者：高杨[1] 刘子沛[2] 王曙光[1] 

机构地区：[1]第三军医大学西南医院全军肝胆外科研究所、中国人民解放军西南肝胆外科医院,重庆400038 [2]解放军第三○六医院总装备部普通外科中心,北京100101

出　　处：《中华消化外科杂志》2011年第2期120-123,共4页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金（30772109）

摘　　要：目的 研究外源性神经生长因子（NGF）对QBC939细胞67-kDa层黏连蛋白受体（67LR）表达的影响,探讨胆管癌神经浸润的机制.方法 （1）细胞免疫荧光染色法检测QBC939细胞表面NGF高亲和力酪氨酸激酶受体A（TrkA）表达情况.（2）用不同浓度的重组人神经生长因子（β-NGF）对QBC939细胞进行处理,采用Real-Time PCR和Western blot检测QBC393细胞67LR mRNA和蛋白表达情况.实验分为对照组、β-NGF（1、10、100、200μg/L）组.（3）根据上述实验结果选定最佳β-NGF浓度,再加入不同浓度的TrkA阻断剂K252a,再次检测QBC939细胞67LR mRNA和蛋白表达情况.实验分为对照组、100μg/Lβ-NGF组、K252a（100、200、300 nmol/L）阻断组.数据采用单因素方差分析,两两比较采用LSD法.结果 （1）QBC939细胞膜上TrkA表达明显.（2）对照组和β-NGF（1、10、100、200 μg/L）组QBC939细胞67LRmRNA和蛋白表达水平分别为0.35±0.06、0.38±0.14、0.62±0.14、0.90±0.08、0.70±0.10和0.32±0.05、0.50±0.09、0.69±0.13、0.93±0.07、0.76±0.07,两者比较,差异有统计学意义（F=22.4,14.6,P＜0.05）,其中100ng/ml β-NGF作用最明显（t=19.0,21.0,P＜0.05）.（3）对照组、100 μg/L β-NGF组、K252a（100、200、300 nmol/L）阻断组QBC939细胞67LR mRNA和蛋白表达水平分别为0.35±0.10、0.88±0.14、0.80±0.08、0.67±0.12、0.43±0.07和0.41±0.10、0.84±0.10、0.76±0.04、0.61±0.09、0.50±0.12,两者比较,差异有统计学意义（F=14.1,8.9,P＜0.05）,其中经300 nmol/L K252a处理后,QBC939细胞67LR mRNA和蛋白表达水平与对照组比较,差异无统计学意义（t=1.02,0.85,P＞0.05）.结论 外源性NGF通过与其高亲和力受体TrkA结合,可增强QBC939细胞67LR表达,这可能是促进胆管癌细胞延外周神经纤维间隙浸润的机制之一.Objective To investigate the effects of nerve growth factor （NGF） on the expression of 67-kDa laminin receptor （67LR） in human bile duct carcinoma QBC939 cells, and study the possible mechanism of perineural invasion and metastasis of bile duct carcinoma. Methods （ 1 ） The expression of a high-affinity receptor for NGF, TrkA, was detected by immunofluorescence staining. （ 2 ） QBC393 cells were pretreated by β-NGF at different concentrations （ 1, 10, 100,200 μg/L）, and then the mRNA and protein expressions of 67LR were examined by Real-Time PCR and Western blot assay. QBC939 cells were divided into control group and β-NGF （1, 10, 100,200 μg/L） groups. （3） The ideal concentration of β-NGF was selected according to the results of previous tests, and then the mRNA and protein expressions of 67LR were re-examined by adding specific TrkA inhibitor K252a at different concentrations （ 100,200,300 nmol/L）. QBC939 cells were divided into control group, β-NGF 100 μg/L group and K252a （ 100,200,300 nmol/L） groups. All data were analyzed by one-way analysis of variance or LSD-test. Results （1） A strong expression of TrkA was detected in the membrane of QBC939 cells. （2） The mRNA and protein expressions of 67LR in QBC939 cells were 0.35 ± 0.06 and 0. 32 ± 0.05 in the control group, 0.38 ±0.14 and 0.50 ±0.09 in the β-NGF 1 μg/L group, 0.62 ±0.14 and 0. 69 ±0. 13 in β-NGF 10 μg/L group, 0.90 ± 0.08 and 0.93 ± 0.07 in the β-NGF 100 μg/L group, and 0. 70 ± 0. 10 and 0. 76 ±0.07 in the β-NGF 200 μg/L group, there were significant differences among the five groups （F = 22. 4, 14. 6,P ＜0.05）. The mRNA and protein expressions of 67LR in the β-NGF 100 μg/L group were significantly higher than those in the control group （ t = 19. 0, 21.0, P ＜ 0. 05 ）. （3） The mRNA and protein expressions of 67LR in the QBC939 cells were 0.35 ±0.10 and 0.41 ±0.10 in the control group, 0. 88 ±0. 14 and 0.84 ±0.10 in the β-NGF 100 μg/L group, 0.80±0.08 and 0.76 ±

关 键 词：胆管肿瘤 神经生长因子 层黏连蛋白受体 酪氨酸激酶受体A 神经浸润 

分 类 号：R735.8[医药卫生—肿瘤]