硕士学位论文钙敏感性受体在创伤失血性休克大鼠血管反应性调节中的作用及机制彭小勇指导教师 刘良明 教授 导师组成员 李涛 教授、杨光明 副教授培养单位 陆军特色医学中心研究部申请学位类别 硕士学术学位 专业名称 外科学（野战外）答辩委员会主席 付小兵 院士答辩委员 李涛教授 王翔教授 尹一兵教授 周元国教授论文答辩时间 2019年11月论文提交时间 2019年12月二〇一九年十一月分类号 R826 密级 公开 学号 2002015643 学校代码91012目录缩略语表 (1)英文摘要 (3)中文摘要 (7)论文正文钙敏感性受体在创伤失血性休克大鼠血管反应性调节中的作用及机制 (10)第一章前言 (10)第二章钙敏感性受体在创伤失血性休克大鼠血管反应性调节中的作用及机制 (12)2.1 材料与方法 (12)2.2 实验结果 (30)2.3 讨论 (39)2.4 小结 (42)全文结论 (43)参考文献 (44)文献综述钙敏感性受体及其配体在心血管系统的研究进展 (49)参考文献 (55)攻读学位期间的研究成果 (59)致谢 (60)缩略语表英文简称英文全称中文AP Ammonium Persulfate过硫酸铵BSA Albumin from Bovine Serum牛血清白蛋白Cal Calhex2314-氯苯甲酰基1,2-二氨基环己烷CaSR Calcium Sensing Receptor钙敏感性受体DMSO Dimethyl Sulfoxide二甲基亚砜DTT Dithiothreitol二硫苏糖醇EDTA Ethylenediaminetetraacetic Acid乙二胺四乙酸EGTA Ethylenebis(oxyethylenenitrilo)乙二醇双(2-氨基乙基醚)四乙酸Tetraacetic Acidh hour小时ICU Intensive Care Unit重症监护室ISO Isoprenaline Hydrochloride异丙肾上腺素L Littre升LR Lactated Ringer’s Solution乳酸林格氏液LVSP Left Intraventricular Systolic Pressure左室收缩末压MAP Mean Arterial Blood Pressure平均动脉压mg Milligram毫克miR MicroRNA微小核糖核酸ml Milliliter毫升mm Millimeter毫米MLC Myosin Light Chain肌球蛋白轻链MA Mesenteric Arteriole肠系膜微动脉mRNA Messenger RNA信使RNANC Negative Control阴性对照NE Norepinephrine去甲肾上腺素NO Nitric Oxide 一氧化氮PBS Phosphate Buffer solution磷酸盐缓冲液p-MLC Phospho-Myosin Light Chain磷酸化肌球蛋白轻链PSS Physiological Salt Solution生理盐溶液PTH Parathyroid Hormone甲状旁腺素qPCR quantitative Polymerase Chain Reaction荧光定量聚合酶链式反应RIPA Radio Immune-Precipitation Assay lysis蛋白裂解缓冲液bufferRPM Revolutions Per Minute转每分SB Sample Loading Buffer上样缓冲液SDS Sodium Dodecyl Sulphate十二烷基磺酸钠siRNA Small interfering RNA小分子干扰RNASMA Superior Mesenteric Artery肠系膜上动脉TBST Tris Buffered Saline Tween-20三羟甲基氨基甲烷缓冲盐吐温溶液TEMED Tetramethylethylenediamine四甲基乙二胺VEC Vascular Endothelium Cell血管内皮细胞VSMC Vascular Smooth Muscle Cell血管平滑肌细胞WB Western Blot蛋白免疫印迹±Dp /Dt max maximal rising and declining rate of left左室压力最大上升/下降速率intraventricularpressureEffects of calcium sensing receptor on vascular reactivity and its mechanisms after traumatichemorrhagic shock in ratsAbstractTraumatic hemorrhagic shock is a series of clinical syndromes with bleeding, edema and exudation, caused by severe trauma such as vascular injury, rupture of solid viscera, and fracture. Vascular hyporeactivity is often seen after traumatic hemorrhagic shock, which is characterized by a reduced response of blood vessels to vasoactive drugs. Vascular hyporeactivity plays important role in the therapy and outcome of shock, which is the important factor that influences the rising of blood pressure after resuscitation and the successful treatment of shock. About 30%-40% of ICU patient death is related to vascular hyporeactivity. Hence, it is important to clarify the mechanism of vascular hyporeactivity after traumatic hemorrhagic shock.Calcium sensing receptor (CaSR), a member of G-protein-coupled receptor family, was first found in the bovine parathyroid gland. The primary role of CaSR is maintaining systemic Ca2+ homeostasis. Recent studies showed that CaSR also plays important role in cardiovascular system, such as vascular calcification and apoptosis of cardiomyocytes. It has been reported that CaSR can regulate blood pressure via vascular endothelium cells. However, whether CaSR takes part in the regulation of vascular reactivity after traumatic hemorrhagic shock, and the possible mechanism remains unclear.Thus, the present study is to investigate the role of CaSR in vascular hyporeactivity and the possible mechanism after traumatic hemorrhagic shock, using traumatic hemorrhagic shock rats and primary cultured vascular smooth muscle cell (VSMC). The study included two parts: ○1whether or not CaSR inhibitor Calhex231 (Cal) exerts protective effect on traumatic hemorrhagic shock by improving vascular reactivity; ○2the mechanism of Cal in the regulation of vascular reactivity. This study aims to further explore the mechanisms of vascular hyporeactivity, and provide experimental basis for new treatment of traumatic hemorrhagic shock.Methods:The traumatic hemorrhagic shock model of rats was adopted. Superior mesenteric artery (SMA) rings and the isolated cardiac papillary muscle from rats were used to observe the contractile response induced by vasoactive drug with isolated organ perfusion system. The contractile response of mesenteric arteriole (MA) was observed by a video camera and the contractile response of single VSMC was observed by the laser scanning confocal microscope. The hemodynamics were determined with a polygraph physiologic recorder. The blood flow of liver and kidney was measured by Laser-speckle contrast analysis. The cardiac output was measured by cardiac output meter and the contractile function of single cardiomyocyte was measured by cell contractility recording system. The level of parathyroid hormone (PTH) in plasma was measured by ELISA kits. The expression of all proteins was measured by WB. The relative expression microRNA(miR) was quantified with qPCR.Experimental Protocols:Part I. The protective effect of Cal on traumatic hemorrhagic shock rats and its relationship to regulation of vascular reactivity1. The experiment was divided into 6 groups: normal group, shock group, lactated Ringer’s solution (LR) group, LR+Cal 0.1mg/kg group, LR+Cal 1mg/kg group and LR+Cal 5mg/kg group, the effect of different dose of Cal on animal survival and hemodynamics was observed.2. The appropriate dose of Cal was chosen according to the results above. The experiment was divided into 4 groups: normal group, shock group, LR group and LR+Cal 1 mg/kg group. The effect of Cal on blood flow of liver and kidney was observed. The cardiac output, contractile response of cardiac papillary muscle and single cardiomyocyte were measured. The contractile response of SMA, MA and single VSMC were also measured respectively.Part II. The relationship between Cal regulating vascular reactivity of traumatic hemorrhagic shock rats and the phosphorylation of MLC, adrenergic receptor (AR) and miR1. The experiment was divided into 4 groups as described above, the blood and SMA tissues were collected. The content of PTH in plasma was measured, and the expression of CaSR, MLC, p-MLC, α1-AR and β1-AR in SMA was measured and the relative expression of miRs (miR-200b, miR-200c, miR-429, miR-208a and miR-1) was also quantified.2. The effects of Cal and CaSR-siRNA on the expression of CaSR and the relative expression of miRs in rat VSMC were observed.Results:Part I. The protective effect of Cal on vascular reactivity and traumatic hemorrhagic shock1. All rats subjected to hemorrhagic shock died within hours，the administration of LR slightly prolonged the survival time of shock rats. Cal significantly improved the survival rate and survival time of shock rats. Hemodynamics including MAP, LVSP and ±Dp/Dtmax were significantly decreased after shock, LR slightly improved hemodynamics, Cal treatment significantly improved hemodynamics, and 1mg/kg of Cal had the best effect.2. The blood flow of liver and kidney was significantly decreased in traumatic hemorrhagic shock rats. LR slightly increased blood flow. Cal significantly increased the blood flow of liver and kidney. The cardiac output, contractile response of cardiac papillary muscle and single cardiomyocyte all decreased in traumatic hemorrhagic shock rats. LR significantly improved these indicators, however, there was no significant difference between the Cal group and the LR group. Traumatic hemorrhagic shock caused severe vascular hyporeactivity. The contractile response of SMA, MA, and acutely isolated VSMC all reduced. LR slightly increased the contractile response. Cal significantly improved the vascular reactivity and the contractile response of VSMCs.Part II. The mechanisms of Cal regulating vascular reactivity after traumatic hemorrhagic shock1. The level of PTH in plasma significantly increased after traumatic hemorrhagic shock, and it continued to increase after LR treatment. Cal significantly decreased the level of PTH. The expression of p-MLC and α1-AR in SMA significantly decreased after shock. Cal increased the expression of p-MLC and α1-AR. But there were no significant changes in the expression of MLC and β1-AR.2. The expression of CaSR in SMA increased after traumatic hemorrhagic shock, and the relative expression of miR-429, miR-208a and miR-1 decreased. Cal significantly reduced the expression of CaSR and increased the relative expression of miR-429 and miR-208a. While the relative expression of miR-200b and miR-200c had no significant changes during the process. Similar changing trend was observed in primary cultured VSMC experiments. Caland CaSR-siRNA reduced the expression of CaSR and increased the relative expression of miR-429 and miR-208a.Conclusion:1. The cardiac function and vascular reactivity significantly decreased after traumatic hemorrhagic shock. LR infusion significantly improved the cardiac function, but had no significantly improvement on vascular hyporeactivity. Cal exerted a protective effect on traumatic hemorrhagic shock via improving the vascular hyporeactivity.2. Cal reduced the level of PTH in blood, and increased the expression of α1-AR, thereby increased the phosphorylation level of MLC, and improved the vascular reactivity after traumatic hemorrhagic shock. In addition, Cal decreased the expression of CaSR and increased the levels of miR-429 and miR-208a in vascular tissues and VSMCs, which may be involved in the effects of Cal.Key words：calcium sensing receptor; traumatic hemorrhagic shock; vascular hyporeactivity; Calhex231；α1adrenergic receptor; myosin light chain phosphorylation; miR-429; miR-208a钙敏感性受体在创伤失血性休克大鼠血管反应性调节中的作用及机制摘要创伤失血性休克是指由于各种创伤所致血管损伤、实质脏器破裂、骨折等引起机体出血、水肿以及渗出等一系列临床综合征。
