糖尿病大鼠主动脉中HMGA1表达变化的研究

发布时间：2018-07-17 00:28

【摘要】：目的应用高糖高脂饲料喂养联合小剂量链脲佐菌素(Streptozotocin,STZ)腹腔注射构建大鼠2型糖尿病模型,观察糖尿病状态下大鼠主动脉血管组织中高迁移率族蛋白A1(high mobility group A1,HMGA1)m RNA和蛋白的表达水平,并探讨可能影响其变化的病理因素,试图为糖尿病血管并发症的发病机制及防治研究提供新的方向。方法4周龄雄性SD大鼠按随机分组方法分为:正常对照组(normol contol group,NC)、高糖高脂喂养组(high-sucrose-high-fat diets group,HSFDs)、糖尿病造模组(diabetes mellitus group,DM)。NC组懫用普通标准大鼠饲料喂养,HSFDs组及DM组均懫用高糖高脂饮食喂养,起始及喂养4周后分别测量体重(body weight,BW)并检测空腹血糖(fasting blood glucose,FBG)、非空腹血糖(nonfasting blood glucose,NFBG)、血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白(low density lipoprotein,LDL)、高密度脂蛋白(high density lipoprotein,HDL)及空腹胰岛素(fasting insulin,FINS)等相关生化指标的水平。4周末,DM组大鼠腹腔注射STZ(30mg/Kg),3天后开始每周检测模型组大鼠NFBG,观察血糖升高水平。第8周末处死所有大鼠,采集血液标本检测FPG、NFBG、TC、TG、LDL、HDL、FINS及糖化血红蛋白(Glycosylated hemoglobin A1c,HBA1c)等生化指标;留取腹主动脉血管组织,HE染色法观察血管组织形态学改变,q RT-PCR、Western Blot及免疫组化法分别检测血管中HMGA1 m RNA和蛋白表达水平。利用Spearman法分别对FPG、NFBG、TC、TG、LDL、HDL和FINS与HMGA1 m RNA及蛋白表达水平进行相关性分析。结果(1)起始时及喂养4周后,各组大鼠BW、FBG、NFBG、FINS及胰岛素敏感指数(insulin sensitivity index,ISI)等指标均无明显差异(P0.05),起始时各组大鼠TG、TC、LDL及HDL水平亦无显著差异(P0.05)。注射STZ后第3、10及21天测DM组大鼠NFBG均明显升高(11.1mmol/l)。第8周末,DM组FBG、NFBG、Hb A1c、TG、TC、LDL及HDL水平较NC及HSFD组均明显增加,而BW、FINS及ISI则明显下降;HSFDs组的BW、FINS、TC及LDL水平较NC组明显增加,ISI则明显下降,以上差异均有统计学意义(P0.05)。(2)荧光定量PCR结果显示,DM组大鼠腹主动脉HMGA1的m RNA表达水平明显高于NC及HSFDs组(P0.05);HSFDs组HMGA1的m RNA表达水平稍高于NC组,但差异无统计学意义(P0.05)。(3)Western-blot及免疫组化结果均显示,DM组大鼠腹主动脉HMGA1蛋白表达水平明显高于NC及HSFDs组(P0.05);HSFDs组HMGA1蛋白表达水平稍高于NC组,但差异无统计学意义(P0.05);此外,免疫组化结果还显示,HMGA1蛋白主要定位于血管平滑肌细胞(vascular smooth muscle cells,VSMCs)核中。(4)HE染色结果显示,NC组血管内膜平整、光滑,血管平滑肌细胞排列整齐、紧密;DM组血管内膜欠光滑,血管中膜增厚,平滑肌细胞质基质疏松;HSFDs组血管形态介于前二者之间。对各组血管内-中膜厚度(intimia-media thickness,IMT)进行测量,结果显示,HSFDs组血管内-中膜厚度较NC组增加,DM组较NC及HSFDs组增加,差异均有统计学意义(P0.05)。(5)对FPG、NFBG、TC、TG、LDL、HDL及FINS与HMGA1 m RNA及蛋白表达水平进行Spearman秩相关分析,结果显示FBG、NFBG、TC及LDL水平均与大鼠主动脉中HMGA1的表达水平呈正相关性(FBG:HMGA1m RNA:r=0.424,P=0.039,HMGA1 protein(IOD):r=0.549,P=0.005;NFBG:HMGA1 m RNA:r=0.584,P=0.003,HMGA1 protein(IOD):r=0.646,P=0.001;TC:HMGA1 m RNA:r=0.544,P=0.006,HMGA1protein(IOD):r=0.726,P=0.000;LDL:HMGA1 m RNA:r=0.636,P=0.001,HMGA1 protein(IOD):r=0.756,P=0.000)。结论(1)高糖高脂饲料喂养联合小剂量链脲佐菌素腹腔注射可成功构建大鼠2型糖尿病模型。(2)糖尿病大鼠主动脉可发生中膜增厚,平滑肌细胞质基质疏松等形态改变;主动脉组织中HMGA1m RNA及蛋白表达水平均增加。(3)FBG、NFBG、TC及LDL均与HMGA1表达水平呈正相关性,可能是影响动脉血管组织中HMGA1表达变化的病理因素。
[Abstract]:Objective to construct rat model 2 diabetes by intraperitoneal injection of high glucose and high fat diet combined with Streptozotocin (STZ), and to observe the expression level of high mobility group protein A1 (high mobility group A1, HMGA1) m RNA and protein in the aortic vascular tissue of rats with diabetes, and to explore the possible influence on the change of the expression level of the protein. The pathological factors, try to provide new direction for the pathogenesis and prevention and control of diabetic vascular complications. Methods 4 weeks male SD rats were divided into normal control group (normol contol group, NC), high glucose and high fat feeding group (high-sucrose-high-fat diets group, HSFDs), diabetic model group (diabetes mellitus group,) DM) group.NC was fed with normal standard rat feed, group HSFDs and group DM were fed with high sugar and high fat diet. After 4 weeks of starting and feeding, the body weight (body weight, BW) was measured and fasting blood glucose (fasting blood glucose, FBG), non fasting blood glucose (nonfasting), glycerin, glycerin, and glycerol were three. Triglyceride, TG, low density lipoprotein (low density lipoprotein, LDL), high density lipoprotein (high density lipoprotein, HDL), and fasting insulin (fasting insulin) were injected into the abdominal cavity for the weekend. 3 days later, the rat model group was detected to observe the blood glucose increase. All rats were killed at the end of the eighth week, and blood samples were collected to detect the biochemical indexes such as FPG, NFBG, TC, TG, LDL, HDL, FINS and glycosylated hemoglobin (Glycosylated hemoglobin A1c, HBA1c), and to observe the vascular tissue of the abdominal aorta and observe the vascular histomorphology by HE staining. RNA and protein expression level. FPG, NFBG, TC, TG, LDL, HDL and FINS and HMGA1 m RNA and protein expression levels were analyzed by Spearman. Results (1) starting and feeding for 4 weeks, there were no significant differences between each group. There was no significant difference in TG, TC, LDL and HDL levels in each group (P0.05). The NFBG in DM group was significantly increased in 3,10 and 21 days after STZ injection (11.1mmol/l). At the end of the eighth week, DM group FBG. In addition, ISI decreased significantly, and the above differences were statistically significant (P0.05). (2) fluorescence quantitative PCR showed that the expression level of M RNA in abdominal aorta of DM rats was significantly higher than that in NC and HSFDs group (P0.05), but the level of HMGA1 in HSFDs group was slightly higher than that in the group, but there was no statistical significance (3) and immunohistochemical results. The expression of HMGA1 protein in abdominal aorta in DM rats was significantly higher than that in group NC and HSFDs (P0.05), and the expression level of HMGA1 protein in group HSFDs was slightly higher than that in NC group, but the difference was not statistically significant (P0.05). Moreover, the immunohistochemical results also showed that HMGA1 protein was mainly located in the nucleus of smooth muscle cells of the blood tube (vascular smooth). (4) The E staining showed that the vascular intima of the NC group was smooth and smooth and the vascular smooth muscle cells were arranged neatly and closely. The vascular intima in the DM group was not smooth, the membrane was thickened and the smooth muscle cytoplasm matrix was loose; the vascular morphology in the group HSFDs was between the first two. The blood vessel thickness (intimia-media thickness, IMT) was measured in each group, and the results showed HS The thickness of intravascular and middle membrane in group FDs was higher than that in group NC, and in group DM was higher than that in group NC and HSFDs, the difference was statistically significant (P0.05). (5) FPG, NFBG, TC, TG, LDL, HDL and expressions were correlated with the level of protein expression. FBG:HMGA1m RNA:r=0.424, P=0.039, HMGA1 protein (IOD): r=0.549, P=0.005, NFBG:HMGA1 m RNA:r=0.584. Intraperitoneal injection of small dose streptozotocin could successfully construct model 2 diabetic rats. (2) the aorta of the aorta could be thickened and the smooth muscle cytoplasm matrix was loose, and the expression level of HMGA1m RNA and protein in the aorta increased. (3) FBG, NFBG, TC and LDL were all positively correlated with the expression of HMGA1. Sex may be a pathological factor affecting the expression of HMGA1 in arterial tissue.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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