细胞密度对结肠癌细胞整合素αγβ6表达和基质金属蛋白酶9分泌的影响

The experimental study of cell density mediated integrin αγβ6 expression and MMP-9 secretion in colon cancer cells

目的探讨细胞密度对结肠癌细胞整合素αγβ6表达和基质金属蛋白酶9（MMP-9）分泌的影响。方法用流式细胞仪检测结肠癌细胞系WiDr和人正常角质细胞系HaCaT细胞株在高、低细胞密度培养条件下αγβ6表达的情况；用Biotrak MMP-9测定仪和明胶酶谱法分别测定不同的结肠癌细胞WiDr和SW480细胞株在高、低密度培养条件下MMP-9的活性和分泌水平。结果表达αγβ6的结肠癌WiDr细胞出现细胞密度依赖的αγβ6表达增加，而正常角质细胞系HaCaT细胞在高、低密度培养条件下αγβ6表达无改变。Biotrak MMP-9活性分析显示，每个表达αγβ6的WiDr细胞和SW480 β6细胞的MMP-9分泌量在高密度培养条件下分别是（3．3±1．2）×10^-7ng和（27．24-3．0）×10^-7ng，在低密度培养条件下分别足（1．8±0．7）×10^-7ng和（10．9±2．0）×10^-7ng，而每个缺乏αγβ6表达的SW480 wild细胞在高、低细胞密度培养条件下分别是（3．9±1．7）×10^-7ng和（3．8±0．7）×10^-7ng，MMP-9分泌水平无明显变化（t=0．47，P〉0．05）。明胶酶谱分析显示SW480 β6细胞的MMP-9活性水平在高密度培养时比低密度培养明显增加，而SWd80 wild和HaCaT细胞的MMP-9活性水平无明显改变。结论细胞高密度诱导结肠癌细胞αγβ6表达，促进MMP-9的分泌，构成了癌细胞永牛化侵袭浸润性牛长的基础。

Objective To detect the effect of cell density on both integrin αγβ6 expression and matrix metalloproteinase-9 （MMP-9） secretion in colon cancer cells. Methods Flow cytometry was applied to analyze αγβ6 expression in human WiDr colon cancer cell lines and human HaCaT keratinocyte cells, respectively, at high- and low- cell density culture. The MMP-9 activity level for various colon cancer cell lines, WiDr and SW480 cells at high- and low- cell density culture was analyzed using Biotrak MMP-9 activity assay and Gelatin Zymography assay, respectively. Results High cell density significantly enhances integrin αγβ6 expression for WiDr cells expressing αγβ6 compared with low density, but no increase was observed for human keratinocyte HaCaT cells. Biotrak MMP-9 assay indicated that the amount of MMP-9 secreted per cell for WiDr and SW480 β6 cells at high cell density culture was （3.3 ±1.2）×10^-7ng/cell and （27.2 ± 3.0）×10^-7ng/cell respectively; However, at low cell density it was （1.8 ± 0. 7）×10^-7ng/cell and （10. 9 ± 2. 0）×10^-7ng/cell,respectively. It was 2 - 3-fold higher for WiDr and SW480 β6 cells at high cell density compared with that at low cell density, but no density-dependent increase observed for SW480 wild cells lack αγβ6 expression （ t = 0. 47, P 〉 0. 05 ） , MMP-9 secretion for SW480 wild cells was （3.9 ± 1.7） ×10^-7ng/cell at high cell density and （3.8 ± 0. 7） ×10^-7ng/cell at low cell density （ P 〉 0. 05 ）, respectively. Gelatin zymography assay also indicated that the level of MMP-9 in SW480 β6 cells expressing αγβ6 was evidently higher at high density than at low density, however no density-dependent increase observed for SW480 wild cells and HaCaT cells. Conclusions High cell density induces integrin αγβ6 expression and promotes MMP-9 secretion in colon cancer ceils, which constitutes the basis for a self-perpetuating system of tumor infiltrating growth in colon cancer progression.