内皮抑素抑制兔耳瘢痕增生的实验研究

发布时间：2018-02-24 20:29

  本文关键词： 增生性瘢痕 内皮抑素 兔耳瘢痕模型 靶向治疗　出处：《浙江大学》2013年博士论文　论文类型：学位论文

【摘要】：研究背景： 增生性瘢痕的发生发展与血管形成密切相关,抑制伤口愈合后的过度新生血管有助于减少增生性瘢痕的形成。因此,寻找一种能够靶向作用于瘢痕组织抑制瘢痕形成的药物有一定的意义。文献报道内皮抑素局部注射能抑制瘢痕增生,但是全身用药能否抑制瘢痕增生尚未见文献报道；内皮抑素抑制瘢痕增生的作用机制目前仍不清楚。 目的： 研究内皮抑素全身用药对兔耳瘢痕的作用并初步探讨其可能的作用机制。 研究方法： 选取体重在2.2-2.5kg的新西兰大白兔共8只,随机分为实验组和对照组,每组4只,分笼饲养两周。静脉麻醉后用角膜钻在8只新西兰白兔的耳朵腹侧建立直径7mm的创面(每耳6个,共96个)。棉球压迫止血,待伤口自行愈合,一周后麻醉下用镊子剥去痂皮,造成新的伤口,自行愈合。在兔耳钻孔术后两周开始,实验组每天腹腔注射内皮抑素,对照组每天腹腔注射等量的生理盐水,连续用药20天。在药物注射当天和注射后第6,13,20天(即伤后15,21,28,35天)观察兔耳瘢痕颜色、质地变化,并用游标卡尺测量瘢痕厚度；在以上各个时点分别用激光多普勒血流仪对实验组与对照组瘢痕组织微循环血流灌注进行检测,研究分析内皮抑素对兔耳瘢痕微循环血流灌注的影响。在给药后20天(即伤后第35天),将实验动物麻醉,分别切取实验组与对照组瘢痕组织。从瘢痕组织顶部正中将瘢痕用利刀切为两半,一半放入10%甲醛溶液中保存留作HE染色及]masson染色,另一半放入液氮瓶中并保存在-80℃的深低温冰箱内留作分子生物学检测Ⅰ型胶原和凋亡抑制基因bcl-2蛋白。 结果： 1大体检测 1.1瘢痕颜色 对照组的瘢痕充血明显,呈紫色或暗红色,而实验组瘢痕充血不明显,呈浅红色或偏苍白。 1.2瘢痕厚度 伤后15天两组瘢痕厚度达到高峰,两组厚度之间在统计学上没有显著性差异。之后,随着时间的推移,两组瘢痕厚度均逐渐下降。但是,实验组厚度下降更快,在伤后第21天及35天两组的瘢痕厚度在统计学上具有显著差异(P0.05)。 1.3激光多普勒测定瘢痕血流灌注 对照组兔耳瘢痕内的血流灌注指标PU值呈先上升后逐渐下降,实验组的PU值则呈逐渐下降走势,在伤后21天两者的PU值相比在统计学上有显著性差异(P0.05)。 2组织学检测 2.1瘢痕增生指数(SEI) 实验组的瘢痕增生指数(1.094±0.19)低于对照组(1.364±0.28),两组SEI相比在统计学上具有显著性差异(P0.01) 2.2瘢痕微血管密度(MVD) 通过HE染色下微血管计数,连续计数5个400倍视野中的微血管数,取平均值,计算微血管密度。实验组的微血管密度为1.734±0.94个/高倍视野,对照组为5.634±1.78个/高倍视野,实验组MVD低于对照组,在统计学上具有显著性差异(P0.01) 2.3瘢痕Masson染色 实验组瘢痕组织内胶原较细、排列整齐,对照组胶原粗大且排列紊乱。 3分子生物学检测 3.1Ⅰ型胶原检测 实验组瘢痕组织中Ⅰ型胶原的表达低于对照组。 3.2凋亡抑制基因bcl-2蛋白检测 实验组的bcl-2表达低于对照组。 结论： 内皮抑素全身用药可以抑制兔耳增生性瘢痕的形成,表现为同一时点较对照组瘢痕厚度减小、瘢痕充血减轻、微血管密度减少、胶原排列较规则,其机制可能是内皮抑素通过下调bcl-2促进瘢痕组织内皮细胞凋亡进而抑制瘢痕新生血管形成,进一步使内皮细胞释放的促进纤维化的细胞因子减少从而间接抑制瘢痕增生；然而,内皮抑素对成纤维细胞作用的具体作用机制尚待进一步实验研究。
[Abstract]:Research background:
Closely related with the occurrence and development of vascular hypertrophic scar formation, inhibit excessive neovascularization after wound healing can help reduce scar formation. Therefore, to find a way to have a certain significance of targeting drugs to inhibit scar formation of scar tissue. Reported local injection of endostatin can inhibit scar hyperplasia, but systemic medication can inhibit scar hyperplasia has not been reported; mechanism of endostatin inhibit scar hypertrophy remains unclear.
Objective:
The effect of endostatin on the rabbit ear scar was studied and its possible mechanism was preliminarily discussed.
Research methods:
Select the weight in 2.2-2.5kg New Zealand white rabbits were 8, were randomly divided into experimental group and control group, 4 rats in each group, divided into cages for two weeks. After intravenous anesthesia with corneal wound to establish drill diameter 7mm in 8 New Zealand white rabbits ear ventral (6 per ear, a total of 96). SpongeBob oppression check the blood, until the wound healed after a week under anesthesia with tweezers stripped crust, resulting in a new wound, healing. At the beginning of the two week after the rabbit ear drilling operation, the experimental group received intraperitoneal injection of endostatin, the control group normal saline was injected every day, 20 days of continuous use. In the day of drug injection and the first 6,13,20 days after injection (15,21,28,35 days after injury) observation of rabbit ear scar color, texture changes, and use vernier caliper to measure the thickness of the scar; above each time point respectively by laser Doppler flowmetry in experimental group and control group. The scar tissue perfusion Measurement, analysis of effects of endostatin on rabbit ear scar microcirculation perfusion. After administration for 20 days (i.e., thirty-fifth days after injury), experimental animal anesthesia, were harvested for the experimental group and control group. The scar tissue from the center of the top of the scar scar tissue with a sharp knife cut in half, half in the left HE staining and]masson staining to save 10% Formaldehyde Solution, the other half bottle and saved in liquid nitrogen for type I collagen and apoptosis detection of molecular biology of suppressor gene Bcl-2 protein in deep low temperature refrigerator -80 degrees.
Result锛，

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