整合素α3β1对肝癌细胞与Ⅳ型胶原粘附与趋化行为的影响

目的　定量描述整合素α3β1 对肝细胞癌 (HCC)细胞与Ⅳ型胶原裱衬表面粘附力特性和HCC细胞对Ⅳ型胶原趋化行为的影响。方法　采用微吸管实验技术测定HCC细胞与Ⅳ型胶原裱衬表面的粘附力 ;进一步加入针对整合素α3β1 的单克隆抗体 (Anti α3,Anti β1 )处理HCC细胞 ,观察Anti α3、Anti β1 对细胞与Ⅳ型胶原裱衬表面的粘附力的影响。采用双微吸管实验法进行HCC细胞趋化实验 ,在微管内加入 60 0 μg/ml的Ⅳ型胶原 ,并引导微管尖端与同一细胞紧密接触 ,动态观察细胞伪足形成过程 ;在微吸管内分别加入Anti α3、Anti β1 ,观察整合素亚单位阻断对HCC细胞伪足形成的影响。利用流式细胞仪对HCC细胞表面整合素α3、β1 亚单位的表达进行分析。结果　HCC细胞与 5μg/mlⅣ型胶原裱衬表面之间的粘附力为 932± 1 34(× 1 0 - 1 0 N ,n =60 ) ,加入 5μg/mlAnti α3粘附力减小到 536± 1 2 2 (× 1 0 - 1 0 N ,n =60 ) ;加入 1 0 μg/mlAnti α3时粘附力减小到 476± 63(× 1 0 - 1 0 N ,n= 50 )。加入 5μg/mlAnti β1 粘附力减小到 449± 1 1 9(× 1 0 - 1 0 N ,n =60 ) ;加入 1 0 μg/mlAnti β1 时粘附力减小到 2 2 0± 78(× 1 0 - 1 0 N ,n =55)。双微吸管趋化实验表明 :两侧微吸管加入相同浓度Ⅳ型胶原 ,

Integrin alpha3beta1 mediates hepatocellular carcinoma cell adhesion and chemotaxis to type IV collagen

OBJECTIVE: To investigate the effects of Integrin alpha(3)beta(1) on the adhesion and chemotaxis of hepatocellular carcinoma (HCC) cells to type IV collagen (Col IV). METHODS: (1) HCC cells were culture and suspension of HCC cells was made. Anti-alpha(3) and Anti-beta(1) were added into the HCC cell suspension. Flow cytometry was used to determine the expression of integrin alpha(3)beta(1) on the surface of HCC. (2) 5 micro g/ml Col IV was used to coat a cell with the diameter of 25 mm. Digested HCC cells were added. Anti-alpha(3) and Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml respectively were added into the cell suspension. Before and after the addition of Anti-alpha(3) and Anti-beta(1), micropipette technique was used to measure the adhesion force of HCC on Col IV-coated surface, as function of the square of internal radius of micropipette and the critical negative pressure needed to detach a single HCC cell away from the substrate. (3) Col IV of the concentration of 600 micro g/ml was added into the dual micropipettes. Then the dual micropipettes were led towards the HCC cells. A HCC cell was made to seal the openings of the 2 micropipettes with different parts of the cell contacting Col IV in different micropipettes. The pseudopod protrusion was observed dynamically and recorded with tape recorder. The length of pseudopod was measured and plotted against the chemotactic time so as to obtain a pseudopod growth curve. RESULTS: (1) The expression rates of integrin subunit alpha(3) and beta(1) on the surface of HCC cells were 95.55% and 95.78% respectively. (2) The adhesion force of HCC cells to the 5 micro g/ml Col IV-coated surface was 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of the HCC cells with Anti-alpha(3) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 42% and 49%, to 536 +/- 122 (x 10(-10) N, n = 60) and 476 +/- 63 (x 10(-10) N, n = 60) respectively. Upon treatment of the HCC cells with Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 52% and 76%, to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 60) respectively. (3) The length of pseudopod increased along with the chemotactic time. The pseudopod length and growth curve were almost identical in the dual micropipettes when they were filled with Col IV. When Anti-alpha(3) or Anti-beta(1) was added into one of the dual micropipettes, the HCC cell pseudopod protrusion was almost blocked completely, while the HCC cell pseudopod in the opposite micropipette became more evident. CONCLUSION: Integrin alpha(3)beta(1) is an important constituent receptor in mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.