复方虫草制剂延长猪同种异体移植皮存活期的实验研究

复方虫草制剂延长猪同种异体移植皮存活期的实验研究 复方虫草制剂延长猪同种异体移植皮存活期的实验研究 作者:程代薇，邹勇，钱宁，王朝亮，田应彪，王达利，赵桂香，高 振宇【关键词】 冬虫夏草 ,摘要, 目的:在猪同种异体皮肤移植 中，采用由冬虫夏草和昆明山海棠制成的复方虫草制剂(compound of Cordyceps sinensis and Tripterygium hypoglaucum, CSTHC)进行 移植皮片的孵化和局部外涂，观察CSTHC对移植皮片存活时间的影 响，并探讨其作用机制。方法:采用CSTHC水煎剂浸泡孵化供体猪皮 片，然后进行皮片移植，局部外涂CSTHC软膏。以环孢素A (cyclosporine A, CsA)和生理盐水作为对照，观察移植皮片的存 活情况。移植术前及术后检测外周血白细胞计数、肝肾功能;观察移 植皮片及睾丸的组织学表现;采用免疫组织化学法检测各组移植皮片 CD4+、CD8+ T细胞的表达。结果:CSTHC组移植皮片的存活时间为 (28.50?3.26)d，较模型组(10.60?1.52)d明显延长，差异有 统计学意义;与CsA组(28.33?3.50)d比较，则无统计学差异。 移植术后第7、14天，CSTHC组移植皮片局部CD4+、CD8+ T细胞浸 润表达低于模型组，差异有统计学意义;与CsA组比较则无统计学差 异。移植术后第7天，模型组外周血白细胞计数明显高于CSTHC组及 CsA组，差异有统计学意义。结论:局部应用CSTHC可以明显延长移 植皮片的存活时间，且无明显的毒副作用。其抑制移植皮片免疫排斥 反应的机制可能与减少移植皮片局部炎性细胞尤其是CD4+、CD8+ T 细胞的浸润有关。 ,关键词, 冬虫夏草; 同种异体移植; 皮肤 移植; 组织存活; 免疫排斥 Effects of compound preparation of Cordyceps sinensis and Tripterygium hypoglaucum on survival time of pigskin after allogeneic transplantation ABSTRACT Objective: To investigate the effects of compound preparation of Cordyceps sinensis and Tripterygium hypoglaucum (CSTHC) on survival time of grafted pigskin after allogeneic transplantation and its mechanism. Methods: The pigskin was treated with CSTHC solution before allogeneic transplantation, and CSTHC ointment was applied for external use on the grafted pigskin after skin transplantation. Cyclosporine A (CsA) and normal saline were served as control. The survival time, the appearance and the histomorphological changes of the grafted pigskin were observed. The histomorphological changes of testicles in pigs were also examined. The CD4 and CD8 expressions in the grafted pigskins were measured by immunohistochemical method. The white blood cell count in peripheral blood and the liver and renal functions were also examined. Results: The survival time of the grafted pigskin in the CSTHCtreated group was (28.50?3.26) d, which was much longer as compared with (10.60?1.52) d in the untreated group (P,0.01). The survival time of the grafted pigskin in the CsAtreated group was (28.33?3.50) d, and there was no remarkable difference in the survival time of the grafted pigskin between the CsAtreated group and the CSTHCtreated group. The expressions of CD4 and CD8 were lower in the CSTHCtreated group than those in the untreated group on the 7th and 14th day after skin graft (P,0.05), while there was no significant difference in the indices between the CSTHCtreated group and the CsAtreated group. The WBC count was higher in the untreated group than that in the CSTHCtreated group or CsAtreated group on the 7th day after skin graft (P,0.05). Conclusion: CSTHC can prolong the survival time of allogeneic grafted pigskin. Its mechanism of inhibiting the immunological rejection may relate to decreasing the expressions of CD4+ and CD8+ in the grafted pigskin and reducing the local inflammatory reaction. KEY WORDS Cordyceps sinensis; allograft; skin transplantation; tissue survival; immunological rejection 异体皮肤移植是烧 伤早期进行创面覆盖的重要方法，如何减轻免疫排斥反应、延长移植 物的存活时间一直是研究的热点。本研究采用冬虫夏草(Cordyceps sinensis, CS)和昆明山海棠(Tripterygium hypoglaucum, TH)制 成虫草复方制剂(compound preparation of Cordyceps sinensis and Tripterygium hypoglaucum, CSTHC)，在猪同种异体皮肤移植过程中用于孵化皮片和局部外涂，通过观察CSTHC对移植物存活时间的影响及检测移植皮肤局部CD4+及CD8+ T细胞的浸润表达情况，探讨CSTHC延长猪同种异体移植皮片存活期的的作用机制。 1 与方法1 实验动物及分组 供体猪:健康家猪1只，雄性，体质量78 kg。受体猪:贵州小型白香猪17只，3月龄，体质量(15?2.5)kg。17只白香猪随机分为3组:CSTHC组(6只)、环孢素A(cyclosporine A, CsA)组(6只)和模型组(5只)。2 药剂制备 选用青海产野生型CS 30 g及贵州独山产TH 60 g，制成水煎剂600 ml，分别含CS 5%及TH 10%。取300 ml加入氮酮(azone)3 ml，用NaCl调节渗透压后制成孵化液。另300 ml加入氮酮及丙二醇各10 g，再加入软膏基质混合制成300 g软膏。另按上法配制0.1 % CsA孵化液及CsA软膏。采用生理盐水配制模型组孵化液及软膏。3 皮肤移植 取供体猪中厚皮片3份，在各组预热的孵化液中孵化1 h后，移植于各组受体猪的一侧胸腹部，每猪移植9片，每片4.0 cm×4.0 cm。移植术后第8天起，各组受体猪采用相应软膏外涂植皮区，1次/d。4 移植皮片存活情况观察 移植皮片红润有光泽，视为皮片成活;若失去光泽呈苍白或暗褐色，且干枯面积,2/3，视为皮片坏死。5 标本采集及指标检测 移植术前及术后第7、14、28天分别抽取受体猪外周血，检测外周血白细胞(white blood cell, WBC)计数、丙氨酸氨基转移酶及肌酐的水平。移植术后第14天取各猪移植皮片组织制成石蜡块，于,80 ?保存，每个石蜡块制成5张切片，其中1张采用苏木素伊红(hematoxylin and eosin, HE)染色进行组织学观察;另4张用丙酮(4 ?)固定后，采用CD4、CD8单抗分型试剂盒(购自武汉博士德生物工程公司)进行检测，镜下阳性表达表现为细胞膜、细胞质内呈现棕黄色颗粒，显微镜下(×400)每张切片选取4个视野，进行阳性细胞计数，取其平均数作为阳性表达值。移植术后第28天取雄性受体猪睾丸组织，制成石蜡切片，采用HE染色进行组织学观察。6 统计学方法 采用SPSS 10.0软件进行统计学分析，计量资料均数用x?s表示，同组内比较行自身对照t检验，两组之间采用组间比 较t检验。 2 结果 2.1 移植皮片存活情况及组织学观察 移植术后第9天起，模型组皮片逐渐坏死，至第13天全部坏死;此时，CSTHC组及CsA组移植皮片存活情况良好。移植术后第3,4周起，CSTHC组及CsA组移植皮片相继出现免疫排斥反应，分别于术后第34天及第35天全部坏死。CSTHC组移植皮片平均存活时间为(28.50?3.26)d，较模型组(10.60?1.52)d明显延长，差异有统计学意义;CSTHC组移植皮片平均存活时间与CsA组(28.33?3.50)d比较，则无统计学差异。移植术后第14天，模型组移植皮片有大量炎性细胞浸润，组织结构不清;CSTHC组及CsA组移植皮片炎性细胞浸润少，皮肤结构清晰