【作者】 武元元；

【导师】 张选奋；

【作者基本信息】 兰州大学 ， 外科学， 2013， 硕士

【摘要】 背景与目的内皮抑素(ES)作为最重要的血管生成抑制因子,已被广泛应用于抗肿瘤血管生成治疗。既往研究表明ES可抑制瘢痕增生,但目前尚未见局部外用ES对创面愈合影响及蛋白激酶C(PKC)-a是否介导其作用的研究报道。本实验探讨PKC-a在ES调控创面愈合及血管增生中的信号作用。材料与方法选用健康成年新西兰大耳白兔36只,雌雄不限,体重2.0-2.5kg。所有动物适应性饲养1周,3%戊巴比妥钠(1.0ml/kg)耳缘静脉麻醉后,分别在每只兔耳腹侧做4个直径为10.0mm,间距15.0mm的全层皮肤缺损创面。将36只大耳白兔随机分为6组：Rh-ES组、PMA组、Go6983组、Rh-ES+PMA组、Rh-ES+Go6983组和PBS对照组,每组48个创面,分别湿敷Rh-ES (10μg/ml)、 PMA (10nM)、Go6983(1nM)、Rh-ES+PMA (10μg/ml+10nM)、Rh-ES+Go6983(10μg/ml+1nM)和等量的PBS液。于伤后3d、7d、11d和上皮化(12d-16d)时观察创面愈合情况,记录创面愈合时间和创面愈合率,并切取创面标本行免疫组化染色检测CD34、PKC-a表达并计算微血管密度(MVD)。所有数据用均数±标准差(x±s)表示。采用SPSS17.0软件包进行统计处理：方差齐性时多组间均数比较采用方差分析,组间两两比较采用LSD法；方差不齐时采用Kruskal-Wallis检验,P<0.05为差异有统计学意义。结果1.创面愈合时间：各组间创面愈合时间存在显著差异(F=9.241,P=0.000)。Rh-ES组、Go6983组、Rh-ES+G66983组愈合时间分别为(14.33±1.15)d、(14.58±1.24)d、(15.08±1.08)d,较对照组(13.36±1.21)d延长(P<0.05),而PMA组愈合时间(12.36±0.67)d缩短(P＜0.05), Rh-ES+PMA组(13.45±1.13)d与对照组相似(P＞0.05)。Rh-ES+Go6983组愈合时间较Rh-ES组延长(P<0.05),而Rh-ES+PMA组缩短(P<0.05)。2.创面愈合率：伤后第3d, Rh-ES+Go6983组创面愈合率低于PMA组和对照组(P<0.05),其余各组间创面愈合率无差异(P>0.05)。伤后第7d和第11d,Rh-ES组、Go6983组、Rh-ES+Go6983组创面愈合率低于对照组(P<0.05),但两两之间无差异(P>0.05)；PMA组创面愈合率较其余各组增加(P<0.05)。Rh-ES+PMA组各时间点创面愈合率与对照组相似(P>0.05)。3.MVD：伤后第3d, Rh-ES+Go6983组MVD低于对照组(P<0.05),其余各组与对照组相似(P>0.05)。自伤后第7d起Rh-ES组和Go6983组MVD低于对照组(P<0.05)而PMA组高于对照组(P<0.05),Rh-ES+PMA组MVD高于Rh-ES组、Go6983组和Rh-ES+Go6983组(P<0.05),但与对照组无差异(P>0.05),Rh-ES+Go6983组MVD低于对照组(P<0.05)。4. PKC-α表达：Rh-ES组各时间点PKC-a表达与对照组无差异(P>0.05)。自伤后第7d起PMA组和Rh-ES+PMA组PKC-a表达均高于其余各组(P<0.05),而Go6983组和Rh-ES+Go6983组PKC-a表达均低于其余各组(P<0.05),但PMA组和Rh-ES+PMA组、Go6983组和Rh-ES+Go6983组组间无差异(P>0.05)。结论1.内皮抑素可显著降低肉芽组织微血管密度,并延迟创面愈合。2.PKC-a可能不介导内皮抑素处理创面抑制血管增生的作用。3.PKC活化剂(PMA)促进创面愈合,PKC抑制剂(G66983)延迟创面愈合。更多还原

【Abstract】 Background and objective Endostatin(ES),as the most important angiogenesis inhibitor,has been widely used in anti-tumor angiogenesis therapy. Previous study showed that ES can inhibit scar hyperplasia,but it lack of the experimental report about the effect of local application of ES on wound healing and whether protein kinase C(PKC)-alpha involved in this process at present. This study is to investigate the roles of PKC-alpha in the process of angiogenesis of ES on wound healing.Materials and methods Thirty-six adult, healthy New Zealand white rabbits had been chosed weighing from2.0to2.5kg,without thinking of sex. Each rabbit was housed in a separate cage and was feeded for one week. After anesthetization with sodium pentobarbital through the auricular vein (1.Oml/kg body weight),four standardized full-thickness skin wounds of10.0mm in diameter extending to the cartilage were made on the ventral side of each rabbit ear. All the rabbits were randomly divided in to the Rh-ES treated group(10μg/ml), the PMA treated-group(10nM),the Go6983treated-group(1nM), the Rh-ES+PMA treated-group(10μg/ml+10nM),the Rh-ES+Go6983treated-group(10μg/ml+1nM) and the control group (PBS treatment). On the3rd,7th,11th post-wound day (PWDs) and epithelization(12th-16th PWDs),the wound healing rate and wound healing time were recorded and the wounds tissues were collected.The microvessel density(MVD) of wound tissues was counted according to the result of CD34immunohistochemistry and the expressions of PKC-alpha were determined.The data,expressed with(x±s), were assessed by the analysis of variance when homogeneity of variance among groups was satisfied, otherwise by the Kruskal-Wallis Test,and their further multiple comparison by LSD(least significant different) method. Data were analyzed using SPSS17.0for Windows, p<0.05was considered statistically significant.Results1. The wound healing time:There was significant differences in wound healing time among6groups (F=9.241, P=0.000).The wound healing time of Rh-ES-treated group(14.33±1.15)d, Go6983treated-group (14.58±1.24)d, Rh-ES+Go6983treated-group(15.08±1.08)d were longer than control group[(13.36±1.21)d, P＜0.05], while PMA treated-group(12.36±0.67)d was shorter(P＜0.05). The wound healing time of Rh-ES+PMA treated-group(13.45±1.13)d had no difference compared with control group(P>0.05).The wound healing time of Rh-ES+Go6983treated-group were longerer than Rh-ES treated-group(P<0.05),while Rh-ES+PMA treated-group was shorter (P＜0.05).2. The wound healing rate:The wound healing rate of Rh-ES+Go6983treated-group was lower than PMA treated-group and control group(P＜0.05), and other groups’ wound healing rates had no different on3rd PSDs(P>0.05).Compared with control group, the wound healing rate of Rh-ES treated group,Go6983treated-group, Rh-ES+Go6983treated-group were decreased signifieantly on the7th and11th PWDs (P＜0.05),but there was no difference between the any of two out of three groups(P＞0.05);While the wound healing rate of PMA treated-group was inereased significantly compared with other groups(P＜0.05). And the wound healing rate had no difference between Rh-ES+PMA treated-group and control group in any PWDs(P＞0.05).3. Microvessel density (MVD):The MVD of Rh-ES+Go6983treated-group was lower than control group(P＜0.05), and other groups had no difference compared with control group on3rd PSDs(P＞0.05). Since the7th PWDs, the MVD of Rh-ES treated group and Go6983treated-group were lower than control group (P＜0.05),while PMA treated-group were higher than control group(P＜0.05). The MVD of Rh-ES+PMA treated-group was higher than Rh-ES treated group,Go6983treated-group and Rh-ES+Go6983treated-group,but had no difference compared with control group at the same time (P>0.05). The MVD of Rh-ES+Go6983treated-group was lower than control group (P＜0.05).4. Expression of PKC-alpha:There was no differences to the expression of PKC-alpha in Rh-ES treated-group compared with control group in any PWDs(P＞0.05). Since the7th PSDs,the expression of PKC-alpha increased significantly in all groups.The expression levels of PKC-alpha was higher in PMA treated-group or Rh-ES+PMA treated-group than in other groups(P＜0.05),while lower in Go6983treated-group or Rh-ES+G66983treated-group than in other groups (P＜0.05). There was no significant difference between PMA treated-group and Rh-ES+PMA treated-group,as well as Go6983treated-group and Rh-ES+Go6983treated-group(P>0.05).Conclusions1. Endostatin might reduce the microvessel density of the granulation tissue and delay wound healing.2. PKC-alpha might not mediate the anti-angiogenesis effect of endostatin on wound healing.3.PKC activator (PMA) might promote wound healing, and PKC inhibitor (Go6983) was just the opposite.更多还原