Cad11高和低表达肾癌细胞分选与Cad11对肾癌细胞迁移作用的影响

Sorting of Cad11+ and Cad11- cells from renal carcinoma cells and its effect on migration of renal carcinoma cells

目的分选Ⅱ型成骨细胞型钙黏附蛋白(cadherin-11,Cad11)高和低表达的肾癌细胞786-O细胞群,并证实Cad11在肾癌细胞786-O中的迁移作用。方法将质粒pBMN-Ⅰ-GFP-Cad11转染于Phoenix细胞进行包装,之后感染786-O细胞,流式细胞仪分选后扩大培养建立786-LG细胞。将786-LG细胞经Cad11抗体2C7孵育后用BSA封闭,洗涤后加入标记APC的二抗,同时以786-LG细胞(未经任何处理)和786-LG细胞(只加入标记APC的二抗处理)作为阴性对照确定分选门,绿色荧光通道和红色荧光通道进行双参数分析,流式细胞仪分选技术筛选Cad11高和低表达的786-O肾癌细胞群,蛋白质印迹法检测分选细胞Cad11的表达。786-Cad11+和786-Cad11-细胞血清饥饿24h后接种于内室(细胞数为1×104),每组设3个复孔,Transwell细胞迁移实验和细胞结晶紫染色计数穿过小室的细胞数目,以穿过膜的细胞数目评估Cad11对其肾癌细胞786-O的迁移作用。结果成功构建786-LG细胞。流式细胞仪检测结果显示,786-LG细胞中Cad11阳性细胞亚群占细胞总数的75.98%。Cad11细胞群分选后,GFP+/APC+双阳性细胞和GFP+/APC-单阳性细胞分别占细胞总数的28.0%和20.1%。蛋白质印迹法检测结果表明,Cad11相对表达量786-LG细胞为0.25±0.02,786-Cad11+细胞为0.60±0.03,786-Cad11-细胞为0.10±0.01,差异有统计学意义,F=132.32,P<0.001。与786-LG细胞相比,两个细胞亚群786-Cad11+和786-Cad11-中Cad11蛋白的表达量均有明显变化,前者蛋白表达量升高至2.41倍(P=0.007),后者蛋白表达量降低至0.41倍(P=0.004),差异均有统计学意义。Transwell迁移实验结果表明,786-LG细胞迁移数目为114.33±4.09,786-Cad11+细胞迁移数目为139.00±5.19,786-Cad11-细胞迁移数目为77.67±3.71,差异有统计学意义,F=49.65,P=0.000 2。结论成功建立了高和低表达Cad11的786-O细胞模型,并证实Cad11的表达与肾癌细胞的迁移密切相关。

OBJECTIVE To sort Cadll＋ and Cadll-- cells from renal carcinoma 786-O cell line and show the effect of Cadll on migration of renal carcinoma 786-O cell. METHODS The plasmid pBMN- I GFP-Cadll was transfect- ed into the Phoenix cells,and the virus-producing cell supernatant was harvested to infect 786-O cells. The flow cytometry was used to detect and isolate the infected 786-O cells and the 786-LG cells were established after amplification culture. The 786-LG cells were incubated with anti-Cadll antibody 2C7 and blocked with BSA. After washing several times, cells were incubated with APC conjugated secondary antibody. In order to set up sort gate, 786-LG cells （without any treat- ment） and 786-LG cells （incubated with APC conjugated secondary antibody） were used as negative controls. Based on the double parameter analysis of green and red fluorescence channels,Cadl 1 ＋ and Cadl 1- cells were sorted by the flow cy- tometry. The Cadll expression of sorted cells was detected by western blotting at protein level. 786-Cadll ＋ and 786-Cad11＋cells were serum starved for 24 h and seeded into transwell culture insert （each containing 1 × 104 cells）. There were 3 holes per group. Transwell migration was assayed and crystal violet staining was used to count cells those had passed the transwell membrane. RESULTS The 786-LG cells were established successfully. The flow cytometry showed that Cadll expression was positive in 786-LG cells （75. 98%）. After the fluorescence activating cell sorter, GFP＋/APC＋ cells（28.0%） and GFP＋/APC＋ cells （20.1%）were sorted successfully. Western blot showed that the relative expression of Cadll was obviously increased in 786-Cad11＋ cells （0.60±0.03） and decreased in 786-Cadll- cells （0.10±0.01） compared with 786-LG cells （0.25±0.02） ,F=132.32,P〈0. 001. As a result,the rates of protein ex- pression were increased to 2.41 in 786-Cadll ＋ cells （P=0. 007） and decreased to 0. 41 in 786-Cadll- cells （P= 0. 004）. Transwell migration assay showed that the number of migrated cells in 786 LG were 114.33 ± 4.09,786-Cadll ＋ cell group were 139.00± 5. 19, and 786-Cadll- cell group were 77. 67 ±3.71, differences were statistically significant （F=49.65,P=0.000 2）.CONCLUSION 786-Cad11-- and 786-Cad11＋ceils have been established and the expression of Cadll is closely related to the migration of renal carcinoma cell.