【作者】 贺教江；

【导师】 刘良明；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 创伤性失血性休克、感染\脓毒性休克等各型休克的后期均存在血管低反应性,即血管对舒、缩血管药物的反应性显著下降甚至不反应,这种血管低反应性严重影响着休克的发生、发展和治疗,是导致休克后期血压难以回升,最后导致多脏器功能不全综合征的重要原因之一。关于休克血管低反应性的发生机制,研究表明它可能与血管平滑肌细胞膜受体失敏、细胞膜超极化等因素有关。针对这些机制采用相应的预防措施可部分改善休克后的血管低反应性。我们在研究失血性休克血管低反应性的发生机制时发现失血性休克后血管平滑肌细胞存在钙失敏(calcium desensitization),钙失敏参与了失血性休克血管低反应性的形成。内毒素休克是否也存在钙失敏,钙失敏是否也参与了内毒素休克后血管低反应性的发生?内毒素休克以全身炎症反应综合征为主要病生变化特征,大量炎性因子的失控性表达参与了内毒素休克的重要病理生理过程,研究表明利用TNF-α可以诱导出脓毒样休克模型。细胞因子TNF-α是否参与了内毒素休克血管平滑肌细胞钙敏感性的调节,参与了内毒素休克血管低反应性的形成?这方面目前国内外尚缺乏研究。据此,本研究利用家兔内毒素休克模型,以肠系膜上动脉(SMA)为研究对象,研究了内毒素休克后血管平滑肌是否存在钙失敏,钙失敏是否在内毒素休克血管低反应性的发生中起重要作用,内毒素休克血管平滑肌细胞(VSMC)钙敏感性是否受TNF-α的调节及其调节的可能机制。主要实验方法:取正常和内毒素休克家兔肠系膜上动脉(SMA),利用离体血管环张力测定技术,用血管环对梯度浓度去甲肾上腺素(NE)的收缩力反映血管反应性,用去极化状态下(120 mmol/L K+)血管环对梯度浓度Ca2+的收缩力反映血管的钙敏感性,用western blot测定MLC20磷酸化水平的变化,用ELISA法测定血浆炎性因子TNF-α的水平和Pho-激酶的活性。实验分三部分:第一部分实验观察内毒素休克低反应性血管是否存在钙敏感性的降低,以及通过观察钙敏感性调节剂精氨酸血管加压素(arginine vasopression,AVP)和胰岛素(insulin)是否可以通过调节钙敏感性来调节血管反应性,以证实钙失敏在内毒素休克血管低反应形成中的作用。第二部分观察家兔静注LPS后各时间点血浆中TNF-α浓度的变化及不同浓度的TNF-α对肠系膜上动脉钙敏感性的影响,以证实TNF-α对内毒素休克血管钙敏感性的调节作用。第三部分观察Rho激酶特异性抑制剂Y-27632、PKC的抑制剂Staurosporine对低浓度的TNF-α对肠系膜上动脉钙敏感性及Rho激酶活性的影响,以初步探讨TNF-α对内毒素休克血管钙敏感性调节的可能机制。主要研究结果:1.内毒素休克后肠系膜上动脉血管(SMA)存在钙失敏,表现为家兔LPS(1mg/kg)静注后,早期(即LPS后30min和1h)SMA血管环对NE和钙的反应性升高,量-效曲线左移,最大收缩力(Emax)升高(LPS1h后p<0.05或p<0.01);随着时间的延长,SMA血管环对NE和钙的反应性逐渐下降,到LPS后6h明显降低,其量-效曲线明显右移,最大收缩力(Emax)明显降低(p<0.01)。休克早期(LPS后0.5,1h)肠系膜上动脉MLC20磷酸化水平增加(P>0.05),LPS后2h MLC20磷酸化水平大幅度下降(p<0.01),而休克晚期(LPS后4,6h)又有小幅回升(p<0.05)。说明内毒素休克血管存在钙敏感性的降低。2.具有钙敏感性增强作用的AVP(1×10-9mmol/L)可使LPS后6hSMA血管环对NE和钙的量效曲线左移,使NE和Ca2+的Emax增高(p<0.05或p<0.01);而具有钙敏感性抑制作用的胰岛素则可使LPS后1h血管环对NE和钙的量效曲线右移,使NE和Ca2+的Emax降低(p<0.05或p<0.01),提示钙失敏在内毒素休克血管低反应性的发生中起重要作用。3.家兔LPS(1mg/kg)静脉注射后2h,血清中TNF-α的水平逐渐升高,4h达到高峰,6h有所降低但仍高于正常组(P <0.05,P <0.01)。其变化趋势与血管钙敏感性变化呈一定负相关关系。低浓度的TNF-α(20ng/ml)可明显升高SMA对钙的反应性,高浓度的TNF-α(200ng/ml)可明显降低SMA对钙的反应性(P <0.05,P <0.01)。提示TNF-α对内毒素休克血管的钙敏感性有重要的调节作用。4.Rho激酶特异性抑制剂Y-27632可明显降低低浓度TNF-α升高SMA对钙的反应性(P <0.05,P <0.01);而PKC的抑制剂Staurosporine无拮抗作用(P>0.05)。内毒素休克早期(即LPS后30min和1h)Rho激酶的活性升高(P <0.05);晚期(即LPS后6h)明显降低(P <0.01)。同时低浓度的TNF-α(20ng/ml)可明显升高肠系膜上动脉Rho激酶的活性(P <0.01)和MLC20磷酸化水平(P <0.05),高浓度的TNF-α(200ng/ml)可明显降低肠系膜上动脉Rho激酶的活性(P <0.05)和MLC20磷酸化水平(P <0.01),Rho激酶特异性抑制剂Y-27632可明显降低低浓度的TNF-α升高肠系膜上动脉Rho激酶的活性(P <0.01)MLC20磷酸化水平(P <0.01)。提示TNF-α对内毒素休克血管的钙敏感性的调节作用主要与Rho激酶有关,与PKC可能关系不大。结论:1.内毒素休克血管平滑肌细胞存在钙敏感性降低(钙失敏);血管平滑肌细胞的钙敏感性降低在内毒素休克血管低反应性的发生中起重要作用。2. TNF-α对内毒素休克血管平滑肌钙敏感性有重要的调节作用,低浓度TNF-α可明显升高SMA对钙的反应性,高浓度TNF-α可明显降低SMA对钙的反应性。3. TNF-α对内毒素休克血管平滑肌细胞的钙敏感性的调节与Rho-激酶有关。更多还原

【Abstract】 There is vascular hyporeactivity in the prolonged period of various shock, including hemorrhagic shock and septic shock. Vascular hyporeactivity is that blood vessels are refractory to vasoconstrictors or vasodilatators, which plays an important role in the incidence,development and therapy of shock.Meanwhile, it is associated with the failure of boosting pressure leading to multiple organ dysfunction syndrome. The occurrence of vascular hyporeactivity after shock may be related to receptor desensitization, or the membrane hyperpolarization of vascular smooth muscle cell(VSMC). However, recovering the receptor sensitivity and improving the polarization state of the cell membrane only partially ameliorate the vascular reactivity. Our previous study showed that calcium desensitization existed in VSMC following hemorrhagic shock, and played important role in vascular hyporeactivity. Whether there is calcium desensitization and it also plays an important role in vascular hyporeactivity in endotoxic shock is not clear. The characteristic of the pathophysiology in endotoxic shock is systemic inflammatory response syndrome(SIRS). The continuing express of a great number of cytokines took part in the Pathophysiology of endotoxic shock.Previous study have demonstrated that sepsis-like shock can be induced by TNF-α.There is no report whether TNF-αtakes part in the regulation of calcium desensitization and the development of vascular hyporeactivity following endotoxic shock is not determined. To elucidate above issues we used endotoxic shock model of rabbits to investigate the role of calcium desensitization of vascular smooth muscle in vascular hyporeactivity, the regulatory effects and the possible mechanisms of TNF-αon calcium sensitivity following endotoxic shock.Methods:The superior mesenteric artery(SMA) from normal and LPS induced endotoxic shock rabbits was adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine(NE) and Ca2+ under depolarizing conditions(120mmol/LK+)with isolated organ perfusion system.Meanwhile,the phosphorylation of MLC20 was measured by western blot. Rho kinase activity and plasma TNF-αlevels were measured by ELISA. The experiments were conducted in three parts. In the first part, we observed whether calcium desensitization was existed in the hyporesponsive blood vessels following endotoxic shock or not, and if calcium desensitization of blood vessel played an important role in vascular hyporeactivity by observing AVP and insulin, the calcium sensitivity regulating agents could regulate the vascular reactivity through regulating the calcium sensitivity. In the second part,in order to confirm the regulatory effect of TNF-αon the calcium sensitivity of blood vessels following endotoxic shock,we observed serum TNF-αlevels from rabbits at different time after 1mg/kg LPS intravenous injection and the regulatory effect of different concentration of TNF-αon calcium sensitivity of SMA. In the third part, in order to study the possible mechanisms of TNF-αon calcium sensitivity of blood vessels following endotoxic shock, we observed the role of Y-27632,a specific inhibitor of Rho-kinase, and PKC inhibitor Staurosporine on calcium sensitivity of SMA incubating with low concentration of TNF-αand effects of TNF-αon the activity of Rho kinase .Results:1. As compared with the normal control group , the vascular reactivity of SMA to NE and Ca2+ was increased in the early period at 30 min and 1 hour following 1mg/kg LPS intravenous injection, the cumulative dose-response curves of SMA to NE and Ca2+ were shifted to the left, the maximal contraction (Emax) of NE and Ca2+ was significantly increased (P<0.05 or p<0.01).But the vascular reactivity of SMA to NE and Ca2+ was decreased in the later period, the cumulative dose-response curves of SMA to NE and Ca2+ were shifted to the right, the maximal contraction (Emax) of NE and Ca2+ were significantly decreased (p<0.01).Meanwhile, the phosphorylation of MLC20 to SMA was increased in the early period at 30 min and 1 hour following 1mg/kg LPS intravenous injection, the phosphorylation of MLC20 to SMA was greatly decreased at 2 hour after 1mg/kg LPS intravenous injection(P<0.01),but was slightly increased in the later period at 4 hour and 6 hour following 1mg/kg LPS intravenous injection(P<0.05).All these results suggested that the calcium sensitivity was decreased following endotoxic shock.2. Calcium sensitivity regulating agent AVP(1×10-9mol/L) made the cumulative dose-response curve of SMA to NE and Ca2+ shift to the left, and increased the maximal contraction (Emax) of SMA to NE and Ca2+(p<0.05 or p<0.01);however, insulin(1×10-7mol/L) made the cumulative dose-response curve of SMA to NE and Ca2+ shift to the right and decreased the contractile response of NE and Ca2+(p<0.05 or p<0.01). These results suggested that calcium desensitization played an important role in the development of vascular hyporeactivity.3. As compared with the normal control group,serum TNF-αbegan to increase at 2 hours and reached the peak at 4 hour after LPS administration and almost decreased to the normal control level at 6 hour following LPS administration (P<0.05,P<0.01). Lower concentration of TNF-α(20ng/ml) incubated with SMA significantly increased the vascular reactivity of SMA to Ca2+, while higher concentration of TNF-α(200ng/ml) significantly decreased the vascular reactivity of SMA to Ca2+ (P<0.05,P<0.01).These results showed that TNF-αcan regulate the calcium sensitivity of vascular smooth muscle in endotoxic shock rabbits.4. Rho-kinase inhibitor Y-27632 could abolish TNF-α(20ng/ml) induced increase of vascular reactivity of SMA to Ca2+. PKC inhibitor Staurosporine had no effect on this effect of TNF-α(20ng/ml) (P>0.05). As compared with the normal control group, the Rho kinase activity of SMA was increased in the early period (at 30 min and 1 hour) following 1mg/kg LPS intravenous injection(P<0.05), but the Rho kinase activity of SMA was significantly decreased in the later period (at 6 hour) following 1mg/kg LPS intravenous injection(P<0.01). Meanwhile,lower concentration of TNF-α(20ng/ml) significantly increased the Rho kinase activity and phosphorylation of MLC20 of SMA(P<0.05,P<0.01),but higher concentration of TNF-α(200ng/ml) significantly decreased the Rho kinase activity and phosphorylation of MLC20(P<0.05,P<0.01), meanwhile Y-27632(10-5mol/L) abolished the TNF-α(20ng/ml) induced increase of Rho kinase activity and MLC20 phosphorylation(P<0.01).These results suggested the regulatory effect of TNF-αon the calcium sensitivity of SMA following endotoxic shock rabbits were closely related to Rho-kinase but not to PKC.Conclusion:1. Calcium desensitization existed in the vascular smooth muscle following endotoxic shock,which played an important role in vascular hyporeactivity endotoxic shock.2. TNF-αplayed an important role in the regulation of calcium sensitivity of vascular smooth muscle following endotoxic shock. Lower concentration of TNF-αsignificantly increased the vascular reactivity of SMA to Ca2+, while higher concentration of TNF-αsignificantly decreased the vascular reactivity of SMA to Ca2+.3. The regulatory effect of TNF-αon the calcium sensitivity of vascular smooth muscle following endotoxic shock rabbits was closely related to Rho-kinase.更多还原