脓毒症大鼠创面愈合过程中MCP-1和MIP-1α变化的研究

发布时间：2018-01-07 08:28

本文关键词：脓毒症大鼠创面愈合过程中MCP-1和MIP-1α变化的研究　出处：《暨南大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分脓毒症动物模型的建立目的探索建立脓毒症大鼠模型的可行方法。方法体重200-250g雄性SD大鼠40只，随机分成两组，分别为脓毒症组（CLP组）和假手术组（Sham组），每组20只。CLP组大鼠盲肠远端1/3行结扎手术，并用12号穿刺针在结扎段的中点垂直肠系膜方向对穿一次，挤出少量粪便入腹腔。术后按5ml/100g立即给予皮下注入生理盐水进行液体复苏。Sham组除不行盲肠结扎并穿刺以外，其余手术步骤均一致。术后6h、12h、24h及48h各组分别随机取5只大鼠观察其一般情况、死亡率、内脏病理学变化、内毒素水平；术后12h行血培养检查。实验结果用Levene test行方差齐性检验，用两个独立样本比较的T检验进行分析。结果CLP组大鼠术后12h均出现了烦躁、寒战竖毛、进食饮水减少等症状。而Sham组无异常变化。CLP组大鼠术后12h-24h间死亡4只，24h后死亡1只，共死亡5只，死亡率为25%。而Sham组大鼠未出现死亡。CLP组大鼠各脏器淤血明显，部分脏器与腹壁粘连，腹腔有脓性渗液，伴恶臭味。胃与小肠积气扩张。盲肠末端肿胀明显，呈紫黑色样缺血性坏死。而Sham组未见明显的异常变化。CLP组大鼠肺基本纹理结构可见，但是肺泡壁明显增厚，部分肺泡塌陷，肺间质中见大量炎性细胞浸润；肝细胞肿胀、坏死明显，肝细胞索解离，肝窦内广泛炎症细胞浸润，均以术后12h-24h炎性改变最为明显。CLP组大鼠血浆内毒素水平在各时间点均高于假手术组(p0.01)，在12h达到峰值（4.839±0.050Eu/ml）之后逐渐下降。CLP组术后12h血培养检测到有粪肠球菌、大肠埃希菌及其他肠杆菌属，而Sham组均未发现异常病原菌。结论采用盲肠远端1/3结扎并用12号穿刺针在结扎段的中点对穿一次的方法,可以在动物中观察到符合临床脓毒症患者的病理生理变化，因此是建立脓毒症动物模型的可行方法。第二部分脓毒症对大鼠创面愈合过程的影响目的观察脓毒症模型大鼠的创面愈合过程，研究脓毒症对创面愈合的影响。方法大鼠背部制作2个1.5cm×1.5cm大小的全层皮肤缺损创面，2天以后（相当于烧伤休克期），分成三组，分别为空白对照组（N组）、假手术组（Sham组）以及盲肠结扎穿刺的脓毒症组（CLP组），每组25只。于制创后3d、5d、7d、10d及14d各组随机取大鼠5只，观察创面愈合时间、记录创面面积的大小，用创面评分标准对创面愈合情况进行评分，检测创面组织学形态、胶原形成及血管生成情况。采用SNK test行方差齐性检验，若方差齐，行完全随机设计资料的方差分析；若方差不齐则采用多个独立样本比较的Kruskal-Wallis H检验。结果CLP组创面愈合时间（13±1.87d）较Sham组及N组明显延长（P0.05）。在制创后3d和5d，CLP组创面愈合率明显低于N组（P0.05），但与Sham组比较无差异；但在制创后7d和10d，CLP组创面愈合率（52±8%，76±10%）明显低于Sham组（71±10%，94±2%）（P0.05），14d时三组比较无差异。在制创后3d，CLP组创面评分与Sham组和N组比较无差异（P0.05），而其余时间点均低于两组对照组。除制创后3d外，其余各时间点CLP组创面胶原形成面积和新生血管生成较Sham组及N组明显减少（P0.05）。结论脓毒症使其模型大鼠的创面愈合明显延迟。第三部分脓毒症大鼠创面愈合过程中MCP-1和MIP-1α的变化目的通过检测脓毒症大鼠创面组织中趋化因子MCP-1和MIP-1α蛋白及其mRNA的表达，分析其与脓毒症创面愈合是否存在相关性。方法大鼠创面及脓毒症的制作方法如第二部分。于各时间点取材，用Elisa试剂盒检测创面组织MCP-1和MIP-1α蛋白的表达，，并用RT-PCR检测其mRNA的表达。采用SNK test行方差齐性检验，若方差齐，行完全随机设计资料的方差分析；若方差不齐则采用多个独立样本比较的Kruskal-Wallis H检验。结果各组MCP-1表达的水平均在制创后3d达到最大值，之后呈逐渐下降的趋势，10d以后处于相对恒定状态,制创后3d和5d，CLP组MCP-1水平与Sham组比较无差异，而明显高于N组（P0.05），其余时间点均高于Sham组和N组。各组MIP-1α表达水平也在制创后3d达到最高水平，且CLP组明显高于N组，但与Sham组比较无差别（P0.05），制创后5d时，CLP组MIP-1α水平低于Sham组和N组（P0.05），而7d时三组之间进行比较，差异无统计学意义（P0.05）。但制创后10d，CLP组出现反跳性升高（208.94±65.33pg/ml），且明显高于Sham组及N组（P0.05），而之后急剧下降，至14d时明显低于Sham组和N组。MCP-1mRNA与MIP-1α mRNA表达水平与其相应的蛋白表达一致。结论脓毒症大鼠创面愈合过程中MCP-1呈早期高表达和后期表达下降的表达紊乱是影响脓毒症创面愈合的原因；而MIP-1α可能不是影响脓毒症大鼠创面愈合的关键因素。
[Abstract]:Establishment of animal model of first partial sepsis Objective To explore a feasible method to establish a rat model of sepsis . Methods Forty - four male SD rats weighing 200 - 250 g were randomly divided into two groups : sepsis group ( CLP group ) and sham operation group ( Sham group ) . Results There were no abnormal changes in the lungs of CLP group . There were no obvious abnormal changes in the lungs of CLP group . Conclusion It is feasible to establish an animal model of sepsis by using 1 / 3 ligation of the distal end of the cecum and the puncture needle in the middle point of the ligation section . Effect of second partial sepsis on wound healing process in rats Objective To observe the wound healing process of sepsis model rats and study the effect of sepsis on wound healing . Methods The rats were divided into three groups : blank control group ( N group ) , sham operation group ( Sham group ) and cecal ligation puncture group ( CLP group ) after 2 days ( equivalent to burn shock stage ) . Results Compared with Sham group ( P 0.05 ) , the wound healing rate of CLP group was significantly lower than that in Sham group ( P 0.05 ) , but the wound healing rate of CLP group ( 52 卤 8 % , 76 卤 10 % ) was significantly lower than that in Sham group ( 71 卤 10 % , 94 卤 2 % ) ( P0.05 ) . Conclusion The sepsis leads to a significant delayed wound healing in the model rats . Changes of MCP - 1 and MIP - 1伪 during wound healing in third partial sepsis rats Objective To investigate the expression of MCP - 1 and MIP - 1伪 protein in wound tissue of septic rats and their mRNA expression . Methods The expression of MCP - 1 and MIP - 1伪 in wound tissue was determined by ELISA kit . The expression of MCP - 1 and MIP - 1伪 protein was detected by ELISA kit . Results The levels of MCP - 1 in each group reached the maximum at 3 d and then decreased gradually . After 10d , the level of MCP - 1 in CLP group was higher than Sham group and N group ( P0.05 ) . However , the level of MIP - 1伪 in CLP group was significantly higher than Sham group and N group ( P0.05 ) . Conclusion The early and late expression of MCP - 1 during wound healing of sepsis is the cause of wound healing in sepsis . MIP - 1伪 may not be a key factor affecting wound healing in sepsis rats .

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R459.7

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