受体相互作用蛋白140对神经胶质细胞生物学行为的影响

Effects of receptor interacting protein 140 on the biological activity of giia cells

目的明确受体相互作用蛋白140（RIP140）基因过表达对小鼠小胶质瘤细胞（BV-2）增殖、凋亡、侵袭及迁移行为的影响。方法采用脂质体转染和G418筛选法构建RIP140过表达的BV-2细胞模型，用实时定量PCR及Western印迹法鉴定过表达模型构建是否成功，用四甲基偶氮唑蓝（Mrrr）法、流式细胞仪和Transwell小室检测RIPl40过表达对BV-2细胞增殖、凋亡、侵袭和迁移行为的影响。结果成功构建RIP140过表达模型BV-2—1，BV-2—1细胞RIP140mRNA与蛋白表达量均高于BV-2细胞（t=49．794，P〈0．01）。细胞增殖检测显示：BV-2细胞24、48、72h吸光度（A）值分别为1．157±0．013、1．679±0．005、2．609±0．008，BV-2—1细胞3个时间点的A值分别为0．929±0．013、1．188±0．008、1．528±0．012，BV-2，1细胞48及72h增殖率明显低于BV-2细胞（t=6．058、9．245，均P〈0．01）。凋亡检测显示：BV-2—1细胞凋亡率明显高于BV-2细胞[（5．35±0．23）％比（3．46±0．45）％，t=6．619、P=0．003]。侵袭检测显示：BV-2—1细胞侵袭平均数多于BV-2细胞[（166±43）个比（93±32）个，t=3．403，P=0．007]。迁移检测显示BV-2—1细胞迁移平均数高于BV-2细胞[（202±50）个比（101±25）个，t=4．104，P=0．002]。结论RIP140过表达抑制BV-2细胞增殖，促进其凋亡；RIP140过表达促进BV-2细胞的侵袭和迁移能力。

Objective To explore the effects of receptor interacting protein （RIP） 140 gene overexpression upon the in vitro proliferation, apoptosis, invasion and migration of microglioma cells. Methods The BV-2 RIP140 overexpression mode] （BV-2-1）was constructed by Lipofection and G418 selection, then validated by real-time PCR and Western blotting. The proliferation, apotosis, invasion and migration potencies were compared between BV-2-1 and its parents by 3- （4,5-cimethylthiazol-2-yl） -2,5- diphenyl tetrazolium （MTT） assay, flow cytometry and Transwell chamber. Results The BV-2-1 model was successfully constructed. Compared to those of the BV-2 group, the RIP140 mRNA and protein expression levels of BV-2-1 were markedly higher than those of the BV-2 group （t = 49. 794, P 〈 0. 01 ）. MTT assay showed that the absorbance values in the BV-2 group were 1. 157 ± 0. 013, 1. 679 ± 0. 005 and 2. 609 ±0. 008 at 24, 48, and 72 hours respectively. And those were 0. 929 ±0. 013, 1. 188 ±0. 008 and 1. 528 ±0. 012 in the BV-2-1 group respectively. The proliferation at the time points of 48 and 72 hours of the BV-2-1 group were significantly lower than that of the BV-2 group （t = 6. 058 and 9. 245, both P 〈 0. 01 ）. Annexin-V staining showed that there were significant differences in the apoptosis rates between the BV-2 and BV-2-1 cells [ （5.35 ± 0. 23 ） % vs （3.46 ± 0. 45 ） %, t = 6. 619, P = 0. 003 ） ]. Transwell assay showed that the invaded ceil number of the BV-2-1 group was 166 ± 43. And it was obviously higher than that of the BV-2 group （93 ± 32, t = 3.403, P = 0. 007 ）. Transwell assay also showed that the migrated cell number of BV-2 cells was 101 ± 25. And the migration potency of the BV-2-1 group （202 ± 50 ） was significantly stronger than that of the BV-2 group （t = 4. 104, P = 0. 002）. Conclusion RIP140 effectively inhibits the proliferation and facilitates the apotosis of microglioma cells. And it may effectively facilitate the in vitro invasion and migration of microglioma cells.