HGF基因对高糖环境下大鼠抗氧化应激和促血管新生的实验研究

发布时间：2018-05-24 12:12

本文选题：pUDKH + 肝细胞生长因子　；参考：《兰州大学》2012年硕士论文

【摘要】：目的 1.探究HGF基因对高糖(High Glucose, HG)环境下大鼠肢体缺血模型的抗氧化应激作用。 2.探讨HGF基因对高糖环境下大鼠肢体缺血后促进血管生成情况。方法 1.建立后肢动脉缺血糖尿病(Diabetes mellitus, DM)大鼠动物模型。 2.将DM大鼠模型随机组合后分为三组：高浓度治疗组、低浓度治疗组和安慰剂组(即对照组)。建模后24h内高浓度组注射pUDKH200μg/只,低浓度组注射pUDKH100μg/只,对照组注射等体积医用注射用水。15d后处死所有动物,取标本待测。 3.检测方法：大体观察；HE染色法观察DM大鼠后肢缺血后组织的病理改变；Elisa法检测缺血局部组织中总ROS(reactive oxygen species, ROS)以及HGF、β-Gal表达；利用流式细胞术(Flow Cytometry, FCM)检测缺血区组织中EPCs数目生成情况。 4.所有数据统计学分析采用均数±标准差(x±s)表示,使用SPSS17.0统计软件对数据进行处理,采用t检验和单因素方差分析进行相应的统计学分析。P0.05表示差异性显著。结果 1.一般情况比较：对比纳入实验组各组糖尿病模型血糖浓度,经统计学分析,P0.05,表明组间血糖无差异,可以进行后续试验。大体观察所见,在缺血造模24h内,所有大鼠均出现跛行。48h内,安慰剂组1只大鼠发生死亡,各组大鼠活动如常。72h内,治疗组大鼠伤口无渗出,呈干燥状；一周后,治疗组伤口结痂,有缝合线头脱落；说明HGF治疗组术后恢复优于安慰剂组。 2.组织学观察：HE病理切片所见,高浓度和低浓度治疗组肌纤维及肌筋膜结缔组织间均有丰富的小血管网形成；高浓度治疗组血管腔形成完整、分布密集,低浓度治疗组仅有散在的管腔形成：高浓度治疗组肌纤维横纹较低浓度治疗组保持完整,高浓度肌间隙接近正常对照侧；对照组病理显示无管腔形成,肌纤维断裂,横纹消失,肌浆凝固,肌间隙变宽,有灶性炎性细胞浸润。 3. Elisa检测结果表明,局部缺血组织匀浆液中,高浓度治疗组的ROS生成量少于安慰剂组,P0.05；HGF在缺血局部组织中的表达量高于安慰剂组,P0.05；FCM检测EPCs细胞数生成情况显示,CD34和CD133抗体双标阳性区的数目在高浓度治疗组的要高于其它两组。结论 1.携带HGF基因重组质粒pUDKH对糖尿病大鼠具有刺激血管生成与抗炎作用。 2.HGF基因能抑制高糖诱导的大鼠氧化应激；HGF能恢复高糖作用下大鼠体内ROS的生成/清除平衡,具有保护作用。 3.HGF基因有利于动员EPCs向糖尿病肢体局部缺血组织募集,并刺激新生血管生成。
[Abstract]:Purpose 1. Objective: to investigate the antioxidant stress effect of HGF gene on limb ischemia model of rats under high glucose (HG) environment. 2. To investigate the effect of HGF gene on angiogenesis after limb ischemia in rats with high glucose. Method 1. An animal model of diabetic rats with hindlimb artery ischemia diabetes mellitus (DMMS) was established. 2. The diabetic rats were randomly divided into three groups: high concentration group, low concentration group and placebo group. Within 24 hours after modeling, pUDKH200 渭 g / mouse was injected into high concentration group, pUDKH100 渭 g / mouse was injected into low concentration group, and all animals were killed in the control group after injection of equal volume of medical injection water for 15 days. 3. Methods: the histopathological changes of hind limbs of diabetic rats after ischemia were observed by HE staining. The expression of total ROS(reactive oxygen species, ROS) and HGF, 尾 -Gal in ischemic local tissues were detected by Elisa. Flow Cytometry (FCM) was used to detect the number of EPCs in ischemic tissue. 4. All the data were analyzed by mean 卤standard deviation (x 卤s), SPSS17.0 software was used to process the data, and t test and single factor ANOVA were used to analyze the statistical data. Result 1. General comparison: the blood glucose concentration of diabetic model was compared and analyzed statistically (P0.05), which showed that there was no difference in blood glucose between the groups, so the follow-up test could be carried out. Gross observation showed that within 24 hours after ischemia, all rats had claudication. Within 48 hours, one rat in the placebo group died, and the rats in each group moved as usual within 72 hours. The wound of the treatment group had no exudation and was dry, and one week later, the rats in the treatment group had no exudation. In the treatment group, the wound was scabbed and the suture was off, which indicated that the recovery of HGF group was better than that of the placebo group. 2. Histopathological observation showed that there were abundant small vascular networks between muscle fiber and myofascial connective tissue in high concentration and low concentration treatment groups, while in high concentration treatment group, the vascular lumen formed intact and distributed intensively, while in the high concentration treatment group, there was abundant small vascular network between muscle fiber and myofascial connective tissue. In the low concentration treatment group, the muscle fiber transverse stria remained intact, the high concentration muscle gap was close to the normal control side, the control group showed no lumen formation, the muscle fiber was broken, and the transverse striae disappeared, while in the low concentration treatment group, the muscle fiber transverse striae remained intact, and the high concentration muscle gap was close to the normal control side. The myoplasm coagulates, the muscle space becomes wider, has the focal inflammatory cell infiltration. 3. The results of Elisa showed that, in the homogenate of ischemic tissue, The amount of ROS in the high concentration treatment group was lower than that in the placebo group. The expression of P0. 05 ROS in the ischemic tissue was higher than that in the placebo group. The number of EPCs cells in the control group was higher than that in the placebo group. The number of double labeled positive areas of CD34 and CD133 antibody in the high concentration treatment group was higher than that in the placebo group. Higher than the other two groups. Conclusion 1. The recombinant plasmid pUDKH carrying HGF gene can stimulate angiogenesis and prevent inflammation in diabetic rats. 2.HGF gene can inhibit the oxidative stress induced by high glucose and restore the balance of ROS production / clearance in rats with high glucose. 3.HGF gene is helpful to mobilize EPCs from diabetic limb ischemic tissue and stimulate neovascularization.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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