中华消化外科杂志
中華消化外科雜誌
중화소화외과잡지
CHINESE JOURNAL OF DIGESTIVE SURGERY
2011年
2期
120-123
,共4页
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
膽管腫瘤%神經生長因子%層黏連蛋白受體%酪氨痠激酶受體A%神經浸潤
담관종류%신경생장인자%층점련단백수체%락안산격매수체A%신경침윤
Bile duct carcinoma%Nerve growth factor%Laminin receptor%Tyrosine kinase receptor A%Perineural invasion
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.
目的 研究外源性神經生長因子(NGF)對QBC939細胞67-kDa層黏連蛋白受體(67LR)錶達的影響,探討膽管癌神經浸潤的機製.方法 (1)細胞免疫熒光染色法檢測QBC939細胞錶麵NGF高親和力酪氨痠激酶受體A(TrkA)錶達情況.(2)用不同濃度的重組人神經生長因子(β-NGF)對QBC939細胞進行處理,採用Real-Time PCR和Western blot檢測QBC393細胞67LR mRNA和蛋白錶達情況.實驗分為對照組、β-NGF(1、10、100、200μg/L)組.(3)根據上述實驗結果選定最佳β-NGF濃度,再加入不同濃度的TrkA阻斷劑K252a,再次檢測QBC939細胞67LR mRNA和蛋白錶達情況.實驗分為對照組、100μg/Lβ-NGF組、K252a(100、200、300 nmol/L)阻斷組.數據採用單因素方差分析,兩兩比較採用LSD法.結果 (1)QBC939細胞膜上TrkA錶達明顯.(2)對照組和β-NGF(1、10、100、200 μg/L)組QBC939細胞67LRmRNA和蛋白錶達水平分彆為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±0.07,兩者比較,差異有統計學意義(F=22.4,14.6,P＜0.05),其中100ng/ml β-NGF作用最明顯(t=19.0,21.0,P＜0.05).(3)對照組、100 μg/L β-NGF組、K252a(100、200、300 nmol/L)阻斷組QBC939細胞67LR mRNA和蛋白錶達水平分彆為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±0.12,兩者比較,差異有統計學意義(F=14.1,8.9,P＜0.05),其中經300 nmol/L K252a處理後,QBC939細胞67LR mRNA和蛋白錶達水平與對照組比較,差異無統計學意義(t=1.02,0.85,P＞0.05).結論 外源性NGF通過與其高親和力受體TrkA結閤,可增彊QBC939細胞67LR錶達,這可能是促進膽管癌細胞延外週神經纖維間隙浸潤的機製之一.
목적 연구외원성신경생장인자(NGF)대QBC939세포67-kDa층점련단백수체(67LR)표체적영향,탐토담관암신경침윤적궤제.방법 (1)세포면역형광염색법검측QBC939세포표면NGF고친화력락안산격매수체A(TrkA)표체정황.(2)용불동농도적중조인신경생장인자(β-NGF)대QBC939세포진행처리,채용Real-Time PCR화Western blot검측QBC393세포67LR mRNA화단백표체정황.실험분위대조조、β-NGF(1、10、100、200μg/L)조.(3)근거상술실험결과선정최가β-NGF농도,재가입불동농도적TrkA조단제K252a,재차검측QBC939세포67LR mRNA화단백표체정황.실험분위대조조、100μg/Lβ-NGF조、K252a(100、200、300 nmol/L)조단조.수거채용단인소방차분석,량량비교채용LSD법.결과 (1)QBC939세포막상TrkA표체명현.(2)대조조화β-NGF(1、10、100、200 μg/L)조QBC939세포67LRmRNA화단백표체수평분별위0.35±0.06、0.38±0.14、0.62±0.14、0.90±0.08、0.70±0.10화0.32±0.05、0.50±0.09、0.69±0.13、0.93±0.07、0.76±0.07,량자비교,차이유통계학의의(F=22.4,14.6,P＜0.05),기중100ng/ml β-NGF작용최명현(t=19.0,21.0,P＜0.05).(3)대조조、100 μg/L β-NGF조、K252a(100、200、300 nmol/L)조단조QBC939세포67LR mRNA화단백표체수평분별위0.35±0.10、0.88±0.14、0.80±0.08、0.67±0.12、0.43±0.07화0.41±0.10、0.84±0.10、0.76±0.04、0.61±0.09、0.50±0.12,량자비교,차이유통계학의의(F=14.1,8.9,P＜0.05),기중경300 nmol/L K252a처리후,QBC939세포67LR mRNA화단백표체수평여대조조비교,차이무통계학의의(t=1.02,0.85,P＞0.05).결론 외원성NGF통과여기고친화력수체TrkA결합,가증강QBC939세포67LR표체,저가능시촉진담관암세포연외주신경섬유간극침윤적궤제지일.
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 ±0.04 in the K252a 100 nmol/L group, 0.67 ±0.12 and 0.61 ± 0.09 in the K252a 200 nmol/L group, and 0. 43 ± 0.07 and 0. 50 ± 0. 12 in the K252a 300 nmol/L group, there were significant differences among the five groups ( F = 14. 1, 8. 9, P ＜ 0.05 ). There were no significant differences in the mRNA and protein expressions of 67LR between the K252a 300 nmol/L group and the control group (t =1.02, 0. 85, P＞0.05). Conclusion In bile duct carcinoma cells, NGF enhances the expression of 67LR by combining with TrkA, which might be the mechanism of NGF mediating perineural invasion of bile duct carcinoma.
