本文選題：pUDKH + 肝細胞生長因子�。� 參考：《蘭州大學》2012年碩士論文

【摘要】：目的 1.探究HGF基因對高糖(High Glucose, HG)環(huán)境下大鼠肢體缺血模型的抗氧化應激作用。 2.探討HGF基因對高糖環(huán)境下大鼠肢體缺血后促進血管生成情況。 方法 1.建立后肢動脈缺血糖尿病(Diabetes mellitus, DM)大鼠動物模型。 2.將DM大鼠模型隨機組合后分為三組：高濃度治療組、低濃度治療組和安慰劑組(即對照組)。建模后24h內高濃度組注射pUDKH200μg/只,低濃度組注射pUDKH100μg/只,對照組注射等體積醫(yī)用注射用水。15d后處死所有動物,取標本待測。 3.檢測方法：大體觀察；HE染色法觀察DM大鼠后肢缺血后組織的病理改變；Elisa法檢測缺血局部組織中總ROS(reactive oxygen species, ROS)以及HGF、β-Gal表達；利用流式細胞術(Flow Cytometry, FCM)檢測缺血區(qū)組織中EPCs數(shù)目生成情況。 4.所有數(shù)據(jù)統(tǒng)計學分析采用均數(shù)±標準差(x±s)表示,使用SPSS17.0統(tǒng)計軟件對數(shù)據(jù)進行處理,采用t檢驗和單因素方差分析進行相應的統(tǒng)計學分析。P0.05表示差異性顯著。 結果 1.一般情況比較：對比納入實驗組各組糖尿病模型血糖濃度,經(jīng)統(tǒng)計學分析,P0.05,表明組間血糖無差異,可以進行后續(xù)試驗。大體觀察所見,在缺血造模24h內,所有大鼠均出現(xiàn)跛行。48h內,安慰劑組1只大鼠發(fā)生死亡,各組大鼠活動如常。72h內,治療組大鼠傷口無滲出,呈干燥狀；一周后,治療組傷口結痂,有縫合線頭脫落；說明HGF治療組術后恢復優(yōu)于安慰劑組。 2.組織學觀察：HE病理切片所見,高濃度和低濃度治療組肌纖維及肌筋膜結締組織間均有豐富的小血管網(wǎng)形成；高濃度治療組血管腔形成完整、分布密集,低濃度治療組僅有散在的管腔形成：高濃度治療組肌纖維橫紋較低濃度治療組保持完整,高濃度肌間隙接近正常對照側；對照組病理顯示無管腔形成,肌纖維斷裂,橫紋消失,肌漿凝固,肌間隙變寬,有灶性炎性細胞浸潤。 3. Elisa檢測結果表明,局部缺血組織勻漿液中,高濃度治療組的ROS生成量少于安慰劑組,P0.05；HGF在缺血局部組織中的表達量高于安慰劑組,P0.05；FCM檢測EPCs細胞數(shù)生成情況顯示,CD34和CD133抗體雙標陽性區(qū)的數(shù)目在高濃度治療組的要高于其它兩組。 結論 1.攜帶HGF基因重組質粒pUDKH對糖尿病大鼠具有刺激血管生成與抗炎作用。 2.HGF基因能抑制高糖誘導的大鼠氧化應激；HGF能恢復高糖作用下大鼠體內ROS的生成/清除平衡,具有保護作用。 3.HGF基因有利于動員EPCs向糖尿病肢體局部缺血組織募集,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 鹵standard deviation (x 鹵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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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