摘要
目的检测前胃泌素、胃泌素、膜联蛋白2在CD44+细胞的表达情况,研究三者对CD44+细胞生长和侵袭能力的影响。方法使用CD44免疫磁珠分选技术从胃癌BGC-823细胞中分选出CD44+细胞,流式细胞仪(FCM)和免疫荧光检测CD44+细胞中CD44的表达,以鉴定分选是否成功。FCM细胞周期分析和无血清培养,检测CD44+细胞的干细胞特性。流式细胞仪检测前胃泌素、胃泌素、膜联蛋白2在CD44+细胞、BGC-823细胞和CD44-细胞表达的差异。MTT法和Boyden小室实验研究3者对CD44+细胞生长和侵袭能力的影响。结果流式细胞仪和细胞免疫荧光结果提示:CD44+细胞中CD44阳性率为(90.20±2.24)%,显著高于BGC-823细胞[(9.57±2.35)%,P<0.01];CD44+细胞组处于G0/G1期的细胞为(71.1±5.6)%,显著高于BGC-823细胞组[(47.7±3.5)%,P<0.05];在无血清培养7 d后,CD44+细胞能够形成圆形克隆球,而CD44-细胞不能形成圆形克隆球;流式细胞仪显示前胃泌素、胃泌素、膜联蛋白2在CD44+细胞中高表达,明显高于在BGC-823细胞和CD44-细胞的表达水平(P<0.01);Pearson相关性分析显示:三者的表达率呈正相关关系(P<0.01)。高浓度的前胃泌素mAb和膜联蛋白2mAb能够抑制CD44+细胞的生长和侵袭,与对照组相比差异显著(P<0.05)。胃泌素mAb不能抑制CD44+细胞的生长和侵袭,与对照组相比差异不显著(P>0.05)。结论 CD44+细胞中高表达前胃泌素、胃泌素、膜联蛋白2,并且前胃泌素mAb和膜联蛋白2mAb能够抑制CD44+细胞的生长和侵袭能力,提示前胃泌素与膜联蛋白2能够促进CD44+细胞的生长和侵袭。
Objective To investigate the expression of progastrin, annexin 2, and gastrin in CD44 gastric cancer cells, and determine their effects on the proliferation and invasion in the CD44 ＋ cells. Methods CD44 immunomagnetic beads were used to sort CD44＋ cells from gastric cell line BGC-823, and the sorted cells were identified by flow cytometry and cell immunofluorescence for the expression of CD44. Flow cytometry- cycle analysis and serum-free culture methods were used to examine CD44 ＋ cells＇ characteristics of stem cells. The expression of progastrin, gastrin, and annexin 2 in CD44 ＋ cells, BGC-823 cells and CD44- cells were detected by flow cytometry, cell immunofluorescence and immunohistochemistry assays. The effect of them on the proliferation and invasion in the CD44 ＋ cells were detected by MTI＂ assay and Boyden chamber. Results Flow cytometry and cell immunofluorescence assay showed the expression of CD44 in CD44 ＋cells [ （90.20 ± 2. 24） % ] was markedly higher than in BGC-823 cells [ （9.57 ± 2.35） %, P 〈 0.01 ]. There were （71.1 ± 5.6） % CD44 ＋ cells arrested in Go/G1 phase, which was obviously higher than that in BGC-823 cells [ （47.7 ± 3.5 ） % , P 〈 0.05 ]. The CD44＋ cells formed circular clone balls under serum free culture for 7 d, while the CD44- cells could not. The expression of progastrin, gastrin and annexin 2 in CD44 ＋ cells was significantly higher than in BGC-823 cells and CD44 - ceils （P 〈 0.01 ）. Pearson correlation analysis showed that there was a positive correlation among their expression （ P 〈 0. 01 ）. mAb inhibited the proliferation and invasion of CD44 cells, High concentrations of progastrin mAb and annexin2 and there was statistical significance compared withcontrol group （P 〈 0. 05 ）, but gastrin mAb could not （ P 〉 0.05 ）. Conclusion CD44＋ ceils widely express progastrin, gastrin, and annexin 2. Progastrin mAb and annexin2 mAb inhibit the proliferation and invasion, which suggest that these two may promote cell proliferation and invasion.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第21期2171-2175,共5页
Journal of Third Military Medical University
