大鼠施万细胞与成纤维细胞联合移植对大鼠失神经支配穿支皮瓣神经再生的影响及其机制  

Effects of combined transplantation of rat Schwann cells and fibroblasts on nerve regeneration of denervated perforator flaps in rats and the mechanism

作　　者：陈伟[1] 魏在荣[1] 吴必华[1] 杨成兰[1] 金文虎[1] 龚飞宇[1] 孙广峰[1] 聂开瑜[1] 王达利[1] Chen Wei;Wei Zairong;Wu Bihua;Yang Chenglan;Jin Wenhu;Gong Feiyu;Sun Guangfeng;Nie Kaiyu;Wang Dali(Department of Burns and Plastic Surgery,Affiliated Hospital of Zunyi Medical College,Zunyi 563003,China)

机构地区：[1]遵义医学院附属医院烧伤整形外科,563003

出　　处：《中华烧伤杂志》2019年第2期134-142,共9页Chinese Journal of Burns

基　　金：国家自然科学基金(81360295、81560315、81760347).

摘　　要：目的 探讨大鼠施万细胞与成纤维细胞(Fb)联合移植对大鼠失神经支配穿支皮瓣神经再生的影响及其机制. 方法 (1)从2只孕14 ~16 d SD大鼠胚胎躯干分离培养Fb并观察细胞形态,取第3代细胞用于后续实验.免疫组织化学法观察细胞纤维粘连蛋白和Ephrin-B2蛋白的表达,实时荧光定量反转录PCR法检测细胞Ephrin-B2 mRNA的表达(样本数为3).(2)从45只出生1~3dSD大鼠双侧坐骨神经和臂丛神经分离培养施万细胞并观察细胞形态,取第3代细胞用于后续实验.免疫荧光法及流式细胞仪检测S100阳性细胞率,样本数分别为9、3.(3)于DMEM高糖培养基中,取1×105个/mL Fb、施万细胞各1 mL共培养,设为施万细胞+Fb共培养组;另取1×105个/mL施万细胞2 mL单独培养,设为施万细胞单独培养组.每组5孔细胞.培养6、24 h于倒置相差显微镜下观察2组施万细胞的成簇群情况并计数,另用免疫荧光法观察培养24 h时施万细胞+Fb共培养组施万细胞的成簇群情况,蛋白质印迹法检测培养24 h时2组施万细胞EphB2、Sox2和N-钙黏蛋白的蛋白表达(样本数为20).(4)取100只8周龄雄性SD大鼠,每只大鼠腹壁制成1个原位回植失神经支配穿支皮瓣,按随机数字表法分为单纯皮瓣组、Fb单独移植组、施万细胞单独移植组、施万细胞+Fb共移植组,每组25只.Fb单独移植组、施万细胞单独移植组大鼠皮瓣分别注射0.4 mL Fb、施万细胞(均为2×106个),施万细胞+ Fb共移植组大鼠皮瓣注射0.4 mL Fb与施万细胞混合细胞(共2×106个,细胞数量比为1∶1),单纯皮瓣组大鼠皮瓣注射等体积的DMEM高糖培养基.注射后2、5、7、9、14 d,按随机数字表法每组分别取5只大鼠,取皮瓣组织,免疫荧光法观察再生神经数量、直径及排列.对数据行完全随机设计t检验、重复测量方差分析、t检验及Bonferroni校正. 结果 (1)从大鼠胚胎躯干分离培养的第3代细胞大小、形态较均一,呈长梭形,细胞核�Objective To explore the effects of combined transplantation of the rat Schwann cells and fibroblasts (Fbs) on the nerve regeneration of denervated perforator flaps in rats and the mechanism.Methods (1) Fbs were isolated from the trunk of 2 Sprague-Dawley (SD) rats embryos of 14-16 days' pregnancy and cultured,and the morphology of the cells was observed.The third passage of cells were used for subsequent experiments.The protein expressions of fibronectin and Ephrin-B2 were observed by immunohistochemical method.The mRNA expression of Ephrin-B2 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (n =3).(2) Schwann cells were isolated from the bilateral sciatic nerves and brachial plexus nerves of 45 SD rats born for 1-3 days and cultured,and the morphology of the cells was observed.The third passage of cells were used for subsequent experiments.The rate of S100 positive cells was detected by immunofluorescence method and flow cytometer,with sample numbers of 9 and 3 respectively.(3) In Dulbecco's modified Eagle medium (DMEM) high glucose medium,1 mL Fbs and 1 mL Schwann cells both in the concentration of 1 × 105 cells/mL were co-cultured as Schwann cells + Fbs co-culture group,and 2 mL Schwann cells in the concentration of 1 × 105 cells/mL were cultured alone as Schwann cells alone culture group,with 5 wells in each group.The clusters of Schwann cells in the two groups were observed and counted under inverted phase contrast microscope at post culture hour (PCH) 6 and 24 respectively.The clusters of Schwann cells in Schwann cells + Fbs co-culture group were observed by immunofluorescence method at PCH 24 too.The protein expressions of EphB2,Sox2,and N-cadherin in Schwann cells of two groups at PCH 24 were detected by Western blotting (n =20).(4) Totally 100 8-week-old male SD rats were selected,and an in situ replanted peritoneal denervated perforator flap was made in each rat.According to the random number table,the rats were divided into simple flap group,Fbs alon

关 键 词：外科皮瓣 成纤维细胞 神经再生 施万细胞 共培养 

分 类 号：R62[医药卫生—整形外科]