生肌玉红胶原促进大鼠后肢缺血组织血管新生的实验研究

目的探讨生肌玉红胶原在缺血组织中促进血管新生的作用效果及其作用机理。方法 Wistar大鼠48只按随机配对方法随机均分为空白组、对照组（胶原）及实验组（生肌玉红胶原）3组;在大鼠后肢缺血模型基础上于大鼠右后肢缺血组织中植入不同的胶原制剂,分别于模型制作术后3,7、14及28 d取埋植的胶原制剂标本及标本周围约0.5 cm范围的缺血肌肉组织。通过激光散斑成像系统观察大鼠右后肢缺血模型制作术后各时点的血流灌注情况,采用光度计法测定胶原制剂（生肌玉红胶原）内的血红蛋白含量,采用免疫组化染色方法检测标本周围肉芽组织中的微血管计数,采用实时荧光定量RT-PCR方法检测缺血肌肉组织中各时点低氧诱导因子（HIF）-1αmRNA以及血管内皮生长因子（VEGF）mRNA的表达。结果大鼠右后肢缺血模型制作成功即刻大鼠患肢呈现明显的缺血状态,其血流灌注值明显低于术前（P〈0.01）;在术后3 d却呈明显的充血状态,其血流灌注值明显升高,且略高于术前水平,但差异无统计学意义（P〉0.05）;在术后7～14 d大鼠患肢的血流灌注值呈逐渐下降趋势,至术后28 d又开始回升,该3个时点实验组大鼠患肢的血流灌注值均高于空白组（P〈0.05）,对照组与空白组比较其差异均无统计学意义（P〉0.05）;术后7及14 d实验组大鼠肢体的血流灌注值高于对照组（P〈0.05）。各时点实验组胶原标本内血红蛋白含量均高于对照组（P〈0.01）。实验组微血管计数于术后7及14 d高于对照组（P〈0.05）;实验组术后各时点HIF-1αmRNA及VEGF mRNA的表达水平均高于对照组和空白组（P〈0.05）,而对照组与空白组间比较差异无统计学意义（P〉0.05）。结论生肌玉红胶原通过诱导HIF-1αmRNA及VEGF mRNA血管新生相关基因的表达而改善缺血组织的血流灌注,促进微血管新生,从而达到促进组织修复的功效。

Experimental Research on Promoting Angiogenesis on Rat Hind Limb Ischemia Tissues with Local Implantation of Shengji Yuhong Collagen

Objective To evaluate the effects of Shengji Yuhong collagen on promoting angiogenesis of the ischemia tissues and probe the possible mechanisms. Methods Forty-eight Wistar rats were divided by random method of paired into blank group, control group （collagen）, and experimental group （Shengjiyuhong collagen）. After made the rats hind limb ischemia model, collagens with or without the extracts of Shengji Yuhong Gao were randomly paired implanted locally in hind limb ischemia tissues of rats in experimental group or control group. The samples of collagens and tissues about 0. 5 cm large surrounding the collagen were explanted respectively on day 3,7, 14, and 28 for detected the hemog-lobin contents in colagen, microvascular counting by using CD34 markers, and the expressions of HIF- 1 ct mRNA and VEGF rnRNA by using real-time fluorescent quantitative RT-PCR. The blood perfusion of the ischemic tissues at each time were determined by using laser speckle imaging system of Moor-FLPI. Results The results of Moor- FLPI showed that the obvious ischemia condition after model made, the blood perfusion was significantly lower than that before operation （P〈0. 01）. On day 3 after operation it showed obvious congestion in the isehemic tissues, and from day 7 to day 14, it showed the isehemia state locally till day 28 after operation which showed improved situation of ischemie. Except for the day 3, the blood perfusion of experimental group were higher than those of blank group （P〈0. 05）. There was no statistical significance between the blank group and control group （P〉0. 05）. The blood perfusion on day 7 and day 14 after operation of experimental group were higher than those of control group （P〈0. 05）. The hemoglobin contents of each time point in the experimental group were higher than those in the control group （P 〈 0. 01）. The mierovascular counting on day 7 and day 14 in experimental group were higher than those of control group （P 〈0. 05）. The expressions ofHIF-lct mRNA and VEGF mRNA at each time point of experimental group were higher than those of control group and blank group （P〈0. 05）, and there was no significant differences between the control group and blank group （P〉0. 05）. Conelusl0n The effects on promoting angiogenesis of rat hind limb isehemia tissues with Shengii Yuhong collagen may though inducing the expressions ofHIF-1α mRNA and VEGF rnRNA locally.