大江 发表于 2014-10-1 14:50:13

枪弹压力波损伤内皮细胞的膜信号机制

         
       赖西南 邵银初

  【摘要】 目的 探讨在投射物侵彻撞击时产生的压力波作用下,血管内皮细胞肌醇磷脂信号通路的变化及其在继发损伤中的作用。 方法 以军用7.62 mm枪弹侵彻介质水所产生的强压力波作用于培养的血管内皮细胞,测定压力波作用后细胞内三磷酸肌醇(IP3)、游离钙含量以及蛋白激酶C(PKC)、培养液中乳酸脱氢酶(LDH)活性,并以特异的肌醇磷脂代谢阻断剂新霉素预作用于培养内皮细胞后,观察压力波作用后上述参数变化。 结果 压力波作用后2 h内IP3明显升高,游离钙水平和PKC 活性在4 h内显著增加,培养液内升高的LDH活性与上述磷酸肌醇代谢激活一致。压力波作用前采用新霉素预作用培养内皮细胞可明显抑制肌醇磷脂代谢激活,细胞浆和LDH泄漏。 结论 血管内皮细胞肌醇磷脂信号通路可能是传导投射物侵彻撞击时压力波效应,启动继发损伤的重要化学信号通路。
  【关键词】 创伤和损伤,枪击; 磷脂酰肌醇类; 内皮,血管; 信号传递

Signal transduction of pressure wave injured endothelial cells after bullet impact

LAI Xinan, SHAO Yinchu.
Research Institute of Surgery/Daping Hospital, The Third Military Medical University, Chongqing 400042, China

  【Abstract】 Objective To study the change of phosphoinositide signaling pathway in the vascular endothelial cells exposed to the pressure wave created by bullet impact and its role in the delayed injury of those cells.  Methods Temporal pressure wave created by military bullets(7.62 mm in calibre) penetrating water affected the cultured vascular endothelial cells, which was taken as an experimental model in vitro in the present study .The intracellular inositol-1,4,5,-triophosphate(IP3) , free calcium dynamics,protein kinase C(PKC)activity,which are the paraments of activating phosphoinositide metabolism,were measured after the cultured cells were exposed to the pressure wave. The lactate dehydrogerase(LDH) activity in the cultured mediage were assayed at the same time.To further elucidate the biochemical signaling pathway,cultured cells were pretreated with neomycin,a special blocker for phosphoinositide metabolism,before exposure and the above paraments were assayed after exposure.  Results Exposure of the cells to the pressure wave resulted in marked increase of IP3 within 2h and free calcium and PKC activity within 4h. The increase of LDH leakage from the cells of exposure were parallel with the change of phosphoinositide metabolism.As the cells were pretreated by neomycin before exposure, the activation of phosphoinositide metabolism was suppressed and the LDH leakage weas eliminated in some degrees after exposure.  Conclusions The phosphoinositide signaling pathway may be an important component in transduction of pressure wave stimutation into delayed injury to the endothelial cells after projectile penetrating impact.
  【Key words】 Wounds and injuries, firearms;  Phosphatidylinositols;   Endothelium,vascular;  Signal transduction

  枪弹伤发生瞬时空腔和冲击波效应产生的强压力波,可损伤远离伤道的血管内皮细胞。这种损伤不仅表现在压力波作用即刻因细胞应变超过损伤阈值而发生的细胞破裂、撕脱等,而且在压力波作用停止后数小时,内皮细胞仍出现渐进性的结构和功能改变,即延迟效应。以往研究注意到,这种延迟损伤可能与激活细胞磷酸肌醇代谢有关,但尚缺乏直接的实验证据[1,2]。
  笔者采用枪弹侵彻水所产生的强压力波致伤的培养血管内皮细胞为实验模型,测定压力波作用后细胞内三磷酸肌醇(IP3)、游离钙[Ca2+]i以及蛋白激酶C(PKC)活性的变化,探讨磷酸肌醇信号通路在介导压力波损伤内皮细胞的延迟效应中的作用。

材料与方法

  1. 实验模型:将培养的传代人脐静脉内皮细胞(细胞株 ECV 304),细胞数约105/cm2,活细胞数>90%,连同培养皿装入薄乳胶囊中,将囊置于水温37℃ 1 m×1 m×1 m的水箱内,培养皿面朝弹道方向,并在同样位置布放YH-1A压阻式压力传感器。以56式7.62 mm 制式枪弹射击水箱,在密度和声阻抗与人体软组织相似的水中产生峰值为(160±32) kPa,持续时间为1.0~2.0 ms,振荡频率为5.6 kHz的压力波,以此压力波致伤培养的内皮细胞,然后将培养皿取出,放入CO2培养箱中继续培养。
  2. 实验分组:压力波致伤组(PG),阻断磷酸肌醇组(NPG),假致伤组(CG)。NPG为致伤前30 min在培养液加入磷脂酶C(PLC)抑制剂新霉素5 mmol/L,预孵育后致伤,CG除了不以压力波致伤外,余处理和观察指标同PG。
  3. 检测指标:压力波作用后10 min和2,4,6 h分别测定内皮细胞内IP3(阳离子交换层析分离法),[Ca2+]i(Fura-2/AM荧光探针法)和胞膜PKC活性(同位素酶解测定法),以比色法测定培养液内乳酸脱氢酶(LDH)的活性。
  4. 统计学处理:所有数据均用±s表示,并用t检验进行统计学分析。

结  果

  压力波作用后10 min~2 h。PG培养内皮细胞内IP3较CG明显升高(P<0.01),伤后4~6 h下降接近CG水平(表1)。压力波作用后细胞内游离钙变化与IP3相似(表2),二者呈明显正相关(r=0.98)。PKC活性变化表现为压力波作用后10 min升高,2 h达峰值,4 h仍高于CG(表3)。预采用新霉素可明显抑制压力波作用后内皮细胞内IP3、游离钙浓度和PKC活性升高。
  PG细胞培养液中LDH活性在压力波作用后2 h持续升高,新霉素能明显减轻胞浆LDH泄漏(表4)。

表1 培养脐静脉内皮细胞内IP3含量变化(min-1/106 cells,±s)


组别 标本数 伤后时间(h)
0.5 2 4 6
CG 6
87.08±5.32
88.10±8.17
85.50±9.39
89.50±10.43

PG 12 170.84±10.90**△△ 118.33±11.45*△ 85.90±5.28 83.80±7.05

    更多相关文档请点击>>细胞(6192)培养(952)损伤(1815)压力(168)信号(278)活性(686)作用(2694)内皮(279)机制(351)                   
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