【作者】 潘丽；

【导师】 张选奋；

【作者基本信息】 兰州大学 ， 外科学（专业学位）， 2013， 硕士

【摘要】 目的研究PKC-a在重组血管抑素(Recombinant Human Angiostatin, rh-AS)(K1-3)影响兔耳皮肤创面愈合和血管增生中的作用。方法构建兔耳腹侧皮肤创面愈合模型。创面分别给予PBS液(对照组)、rh-AS(K1-3)(rh-AS组)、PMA (PMA组)、Go6983(Go6983组)、rh-AS联合PMA (rh-AS+PMA组)和rh-AS联合Go6983(rh-AS+Go6983组)湿敷,每日1次,连续13-16天(上皮化)。分别于伤后3天、7天、11天,采集创面图像,用Image-Pro Plus6.0图像处理软件计算创面愈合率,并记录各组创面愈合时间。于伤后第3天、7天、11天、上皮化时切取创面标本,采用CD34免疫组化染色检测微血管密度变化,用免疫组化方法半定量检测各时间点创面组织中PKC-a表达情况。创面愈合时间、创面愈合率、微血管密度及PKC-a阳性细胞比例均采用单因素方差分析,组间两两比较采用LSD法检验,若方差不齐则采用Kruskal-Wallis检验；P<0.05表示差异有统计学意义。结果1rh-AS组和G66983组创面愈合时间长于对照组而PMA组创面愈合时间短于对照组(P<0.05)；rh-AS组创面愈合时间长于rh-AS+PMA组,但较rh-AS+Go6983组缩短(P<0.05)；PMA组创面愈合时间短于rh-AS+PMA组(P<0.05)；G66983组创面愈合时间较rh-AS+Go6983组缩短(P<0.05)。2.伤后第3天各组间创面愈合率无差异(P>0.05)。伤后第7天：rh-AS组、G66983组创面愈合率小于对照组,而PMA组则高于对照组(P＜0.05); rh-AS组创面愈合率低于rh-AS+PMA组,而高于rh-AS+Go6983组(P＜0.05); PMA组创面愈合率高于rh-AS+PMA组(P<0.05)；G66983组创面愈合率高于rh-AS+Go6983组(P<0.05)。伤后第11天：rh-AS组、G66983组创面愈合率低于对照组(P<0.05),PMA组高于对照组(P<0.05)；rh-AS组创面愈合率高于rh-AS+Go6983组(P<0.05)；PMA组创面愈合率高于rh-AS+PMA组(P<0.05)；Go6983组创面愈合率高于rh-AS+Go6983组(P<0.05)。3.微血管密度测定：伤后3天、7天、11天各组创面比较,rh-AS组、G66983组、rh-AS+PMA组、rh-AS+Go6983组微血管密度小于对照组(P<0.05),PMA组血管增生显著高于对照组及其余各组(P<0.05)。各组创面上皮化时,新生血管也随之退化：各组微血管密度下降,但PMA组微血管密度仍高于各组(P<0.05)。4.PKC-α广泛表达于创面组织细胞,随创面愈合过程进展,各组PKC-α表达增强,阳性细胞(成纤维细胞、内皮细胞、表皮细胞)比例增加。同一时间点组间比较,rh-AS组PKC-α表达水平与对照组无差异(P>0.05),PMA组、rh-AS+PMA组各时间点PKC-α阳性细胞比例均高于对照组和rh-AS组,Go6983组、rh-AS+Go6983组低于对照组和rh-AS组(P<0.05)。结论1.血管抑素抑制创面愈合过程中血管增生,延迟创面愈合。2. PKC-α不介导血管抑素对创面愈合及血管增生的抑制作用。3.蛋白激酶C活化剂PMA促进创面愈合,而抑制剂Go6983延迟创面愈合。更多还原

【Abstract】 Objective To research the roles of PKC-a in recombinant human angiostatin(K1-3) inhibition on wound healing and angiogenesis in the ventral skin of rabbit ears.Methods The wound healing model was built on the ventral skin of rabbit ears.The wounds were respectively treaed with PBS, recombinant human angiostatin, PMA, G56983, recombinant human angiostatin combined with PMA or Go6983once a day until epithelization(13-16days).Wound images were collected to calculate wound healing rates with Image-Pro Plus6.0software on3d,7d and11d.And wound healing time was recorded.Microvessel density(MVD) and the expression of PKC-α in wound healing process was detected by immunohistochemical staining with vascular endothelial cell-specific marker CD34and PKC-α polyclonal antibody.The wound healing time, the wound healing rates, the MVD and the expression of PKC-α were expressed with Mean±tandard difference (x±s),which were assessed by one-way ANOVA when the homogeneity of variance between groups and their further multiple comparison by LSD(least significant different) method, using the Kruskal-Wallis Test when heterogeneity of variance.Results1.The wound healing time in rh-AS group and Go6983group was longer than control group but PMA group was shortened compared with control group(P＜0.05).The wound healing time in rh-AS group was longer than rh-AS+PMA group,but shorter compared with rh-AS+Go6983group(P＜0.05).The wound healing time in PMA group was shorter in rh-AS+PMA group(P＜0.05).And the wound healing time in Go6983group was shorter compared with rh-AS+Go6983group(P＜0.05).2.There were differences on wound healing rates in every group at different time:Wound healing time on the3rd day after injury in every group was no significant difference(P＞0.05).On the7th and11th day after injury,the wound healing rates in rh-AS group and Go6983group were lower than control group,but PMA group was higher than control group(P＜0.05).Rh-AS group had lower wound healing rates than rh-AS+PMA group,and higher compared with rh-AS+Go6983group(P＜0.05).The wound healing rates in PMA group and Go6983group was higher than in rh-AS+PMA group and rh-AS+Go6983group,respectively(P＜0.05).3.Microvessel density:The microvessel density of rh-AS group, Go6983group and rh-AS+Go6983group was lower than control group on the any day after injury (P<0.05) whereas that of PMA group was higher than the other groups(P＜0.05). The microvessel density in every group declined with the wound epithelialization, but that in PMA group was higher than other groups(P＜0.05).4.The expression levels of PKC-α:PKC-α was widely expressed in all kinds of cells the wound such as fibroblasts,endothelial cells,epidermal cells. The expression of PKC-a and the proportion of positive cells(fibroblasts,endothelial cells,epidermal cells) increased in every group with the wound healing progress.On the same time, the expression levels of PKC-a in rh-AS group was no significant difference with control group(P＞0.05).The proportion of positive cells in PMA group or rh-AS+PMA group was higher than control group and rh-AS group.The expression levels of PKC-a in Go6983group and rh-AS+Go6983group was lower compared with control group or rh-AS group(P＜0.05).Conclusions1. The angiostatin might inhibit the angiogenesis in the wound healing and delays wound healing.2. PKC-a might not mediate the inhibition effects of angiostatin on the wound healing and angiogenesis.3. PMA might be conducive to the wound healing whereas Go6983might delay wound healing.更多还原