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中华神经创伤外科电子杂志 ›› 2017, Vol. 03 ›› Issue (03) : 159 -165. doi: 10.3877/cma.j.issn.2095-9141.2017.03.008

所属专题: 文献

基础研究

RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用
于泽奇1, 衣泰龙1, 涂悦1, 杨小飒1, 江继鹏1, 董晓煜2, 张赛1, 程世翔1,()   
  1. 1. 300162 天津,武警后勤学院附属医院脑科医院
    2. 100621 北京,北京武警总队三支队卫生队
  • 收稿日期:2017-05-05 出版日期:2017-06-15
  • 通信作者: 程世翔
  • 基金资助:
    国家自然科学基金项目(31200809); 武警部队后勤科研项目(WJHQ2012-20); 军队技术产品研究重大项目(AWS15J001); 天津市科技计划项目(15ZXLCSY00040)

Effects of necroptosis induced by RIP3 in stretch injury model of HT-22 cells

Zeqi Yu1, Tailong Yi1, Yue Tu1, Xiaosa Yang1, Jipeng Jiang1, Xiaoyu Dong2, Sai Zhang1, Shixiang Cheng1,()   

  1. 1. Neurology and Neurosurgery Hospital, Affiliated Hospital of Logistics College of Chinese People’s Armed Police Force (PAP), Tianjin 300162, China
    2. Medical Unit of Three Detachment of Beijing Armed Police Corps, Beijing 100621, China
  • Received:2017-05-05 Published:2017-06-15
  • Corresponding author: Shixiang Cheng
  • About author:
    Corresponding author: Cheng Shixiang, Email:
引用本文:

于泽奇, 衣泰龙, 涂悦, 杨小飒, 江继鹏, 董晓煜, 张赛, 程世翔. RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用[J]. 中华神经创伤外科电子杂志, 2017, 03(03): 159-165.

Zeqi Yu, Tailong Yi, Yue Tu, Xiaosa Yang, Jipeng Jiang, Xiaoyu Dong, Sai Zhang, Shixiang Cheng. Effects of necroptosis induced by RIP3 in stretch injury model of HT-22 cells[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2017, 03(03): 159-165.

目的

探讨受体相互作用蛋白3(RIP3)介导的坏死性凋亡在HT-22细胞牵张损伤模型中的作用及其机制。

方法

将HT-22细胞接种在Bioflex培养板,采用细胞损伤控制仪(CIC),设定损伤参数(阀门压力30 PSI、气体脉冲压力3.5~4.5 PSI、气体脉冲时间50 ms),建立HT-22细胞牵张损伤模型。分别采用数字全息显微镜(DHM)、乳酸脱氢酶(LDH)试剂盒、流式细胞术、western blot法检测牵张损伤后6 h Ctrl组、CIC组、GSK’872组间细胞形态差异,LDH浓度变化,细胞周期分布,RIP3/受体相互作用蛋白1(RIP1)/混合系列蛋白激酶样结构域(MLKL)、Akt/p-Akt/mTOR/p-mTOR、Caspase-8/X连锁凋亡抑制蛋白(XIAP)蛋白表达变化。

结果

与CIC组相比,应用GSK’872后细胞平均数量[(244.67±11.68)vs(190.67±15.28),t=4.865,P<0.01]、细胞平均面积[(260.14±16.81)μm2 vs(175.91±15.00)μm2t=6.476,P<0.01]有所增加,细胞平均厚度有所减小[(6.12±0.47)μm vs(8.04±0.48)μm,t=4.942,P<0.01];LDH浓度有所下降[(222.74±11.06)ng/l vs(275.93±12.26)ng/l,t=5.581,P<0.01];细胞周期有所恢复[Sub-G1:(0.33±0.15)% vs(6.51±0.63)%,t=16.530,P<0.01;G0/G1:(46.67±2.96)% vs(33.04±7.07)%,t=3.085,P<0.05];能够降低RIP3[(0.73±0.04)vs(1.09±0.09),t=6.239,P<0.01]、RIP1[(0.75±0.05)vs(0.91±0.05),t=4.211,P<0.05]、MLKL[(0.56±0.03)vs(0.70±0.04),t=4.785,P<0.01]、Akt[(0.49±0.05)vs(0.77±0.05),t=6.763,P<0.01]、p-Akt[(0.88±0.05)vs(1.06±0.05),t=4.509,P<0.05]、mTOR[(0.81±0.02)vs(0.90±0.05),t=2.813,P<0.05]、p-mTOR[(0.65±0.05)vs(1.00±0.05),t=8.413,P<0.01]、XIAP[(0.50±0.05)vs(0.73±0.05),t=5.814,P<0.01]蛋白表达,并可促进Caspase-8蛋白表达持续升高[(0.96±0.05)vs(0.75±0.05),t=5.351,P<0.01],差异具有统计学意义。

结论

RIP3介导的坏死性凋亡在HT-22细胞牵张损伤模型中起到重要作用,应用GSK’872可减轻HT-22细胞牵张损伤的程度,提示RIP3有可能成为将来临床上治疗颅脑创伤新的靶点。

Objective

To investigate the effects of necroptosis induced by receptor-interacting protein 3 (RIP3) on stretch injury model of HT-22 cells and its mechanisms.

Methods

HT-22 cell lines were seeded with Bioflex cell plate, and stretch injuries with 30 psi for the regulator with a 50 msec pulse resulting in 3.5~4.5 peak injury pressure by cell injury controller Ⅱ (CIC) instrument. The morphological changes of HT-22 cells were assessed by digital holographic microscopy (DHM), the degree of injury was detected by lactate dehydrogenase (LDH) assay, the cell cycle was detected with Flow cytometry and the expression of RIP3/RIP1/mixed lineage kinase domain like (MLKL), Akt/p-Akt/mTOR/p-mTOR and Caspase-8/X-linked inhibitor of apoptosis protein (XIAP) were detected by Western Blot assay at 6 h post-CCI among Ctrl, CIC and GSK’872 groups.

Results

Compared with the CIC group, cells treated with GSK’872 exhibited an increase in number [(244.67±11.68) vs (190.67±15.28), t=4.865, P<0.01] and area [(260.14±16.81) μm2 vs (175.91±15.00) μm2, t=6.476, P<0.01], however, a reduction in thickness [(6.12±0.47) μm vs (8.04±0.48) μm, t=4.942, P<0.01] with DHM. In the presence of GSK’872, the leakage of LDH was sharply decreased compared with the CIC group [(222.74±11.06) ng/l vs (275.93±12.26) ng/l, t=5.581, P<0.01]. What’s more, GSK’872 could convert the alteration of cell cycle and cell death [Sub-G1: (0.33±0.15)% vs (6.51±0.63)%, t=16.530, P<0.01; G0/G1 (46.67±2.96)% vs (33.04±7.07)%, t=3.085, P<0.05] compared to the CIC group. Furthermore, GSK’872 treatment could significantly decrease the levels of RIP3 [(0.73±0.04) vs (1.09±0.09), t=6.239, P<0.01], RIP1 [(0.75±0.05) vs (0.91±0.05), t=4.211, P<0.05], MLKL [(0.56±0.03) vs (0.70±0.04), t=4.785, P<0.01], Akt [(0.49±0.05) vs (0.77±0.05), t=6.763, P<0.01], p-Akt [(0.88±0.05) vs (1.06±0.05), t=4.509, P<0.05], mTOR [(0.81±0.02) vs (0.90±0.05), t=2.813, P<0.05], p-mTOR [(0.65±0.05) vs (1.00±0.05), t=8.413, P<0.01], XIAP [(0.50±0.05) vs (0.73±0.05), t=5.814, P<0.01], but increase the level of Caspase-8 [(0.96±0.05) vs (0.75±0.05), t=5.351, P<0.01] compared with CIC group.

Conclusion

Necroptosis induced by RIP3 may play an important role in stretch injury model of HT-22 cells, what’s more, GSK’872 could reduce the degree of injury after CIC, which reveals that RIP3 may be a new target for clinical treatment of TBI in the future.

图1 光镜观察细胞牵张损伤后6 h Ctrl组、CIC组、GSK’872组细胞形态学变化(×100)
图2 数字全息显微镜观察细胞牵张损伤后6 h Ctrl组、CIC组、GSK’872组细胞形态学变化
图3 细胞牵张损伤后6 h Ctrl组、CIC组、GSK’872组乳酸脱氢酶浓度变化
表1 细胞牵张损伤对HT-22细胞周期的影响(±s,%)
图4 流式细胞术检测细胞周期
图5 RIP3、RIP1、MLKL蛋白表达变化
图6 Akt、p-Akt、mTOR、p-mTOR蛋白表达变化
图7 Caspase-8、XIAP蛋白表达变化
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