钙敏感受体在大鼠轴型皮瓣缺血再灌注损伤模型中的表达和意义

Expression of calcium-sensing receptor in ischemia/reperfusion injury model of rat axial skin flaps transplantation and its significance

目的:研究钙敏感受体(calcium-sensing receptor,Ca SR)在轴型皮瓣血管内皮细胞的表达,并探讨Ca SR在皮瓣缺血再灌注损伤过程中的重要作用。方法:构建以腹壁下浅动、静脉为蒂的大鼠轴型皮瓣,使用微动脉夹夹闭血管3 h建立轴型皮瓣缺血再灌注(ischemia/reperfusion,I/R)损伤模型。随后大鼠被随机分为3组(每组10只),分别通过股静脉给予Ca Cl2、NPS2143+Ca Cl2和生理盐水(normal saline,NS)治疗。再灌注2 h和7 d后,采用计算机辅助面积测量法评估皮瓣坏死;HE染色观察皮瓣血管形态学变化;TUNEL染色检测细胞凋亡;RT-PCR、免疫组化法分别检测血管内皮细胞中Ca SR、Cleaved Casepase-3、Bax和Bcl-2的表达情况。结果:与NS对照组和NPS2143+Ca Cl2组相比,Ca Cl2组皮瓣坏死率增多的同时[(14.87±1.02)%,P=0.000],TUNEL染色阳性细胞增多(2 h:46.960±1.965,7 d:48.855±3.550,P=0.000),Ca SR(2 h:0.157±0.011,7 d:0.129±0.015,PIHC=0.000;PPR-PCR=0.000)、Cleaved Casepase-3(2 h:0.101±0.020,7 d:0.098±0.011,PNS=0.046,PNPS2143+Ca Cl2=0.000)表达升高,并且Bax存在明显棕黄色染色,而Bcl-2几乎无表达。但是NPS2143+Ca Cl2组与NS对照比相比,结果刚好相反。结论:Ca SR在大鼠轴型皮瓣的血管内皮细胞中具有功能性表达,其表达水平与轴型皮瓣I/R损伤程度密切相关,并且抑制Ca SR表达能减轻轴型皮瓣I/R损伤。

Objective：To investigate the expression of calcium sensing receptor（CaSR） in vascular endothelial cells and the role of CaSR expression in ischemia-reperfusion （I/R） injury model of rat axial flaps transplantation. Methods：I/R injury model was estab- lished on SD rats axial skin flaps based on superficial inferior epigastric vessels. After 3 h ischemia, all flaps were randomly divided into 3 groups（n=10 per group） ：CaC12,NPS2143＋CaCl2 and normal saline（NS）. The necrosis ratios of skin flaps were evaluated;mor- phological changes were observed by liE staining; cell apoptosis was. assessed by TUNEL staining. The expressions of CaSR, Cleaved Casepase-3, Bax and Bcl-2 were detected by RT-PCR and immunohistochemistry, respectively, at 2 h and on 7 d after reperfusion.Results：Compared with＂ the NS control group and NPS2143 ＋ CaC12 group,CaCl2 group acquired significantly more flap necrosis[（ 14.87 ± 1.02）% ,P=0.000],TUNEL staining-positive cells（2 h：46.960 ± 1.965,7 d：48.855 ± 3.550,P=0.000）, consistent with higher protein expression of CaSR（2 h：0.157± 0.011,7 d：0.129 ±0.015 ,PIHC=0.000;PPR-PCR=0.000） ,Cleaved Casepase-3（2 h：0.101 ± 0.020,7 d：0.098 ＋ 0.011 ,PNS=0.046, PNHS2143＋CaCl2=0.000 ） and obvious brown staining of Bax, while almost no expression of Bcl-2. However compared with the NS control, NPS2143＋CaC12 group manifested an anti-injury effect. Conclusion ： The activation of CaSR in the vascular endothelial cells may aggravate the I/R injury of of axial skin flaps while inhibi- tion of CaSR could deprive skin flaps of I/R injury.