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

Chen Wei; Wei Zairong; Wu Bihua; Yang Chenglan; Jin Wenhu; Gong Feiyu; Sun Guangfeng; Nie Kaiyu; Wang Dali

摘要

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 alone transplantation group,Schwann cells alone transplantation group,Schwann cells + Fbs co-transplantation group,with 25 rats in each group.Flaps of rats in Fbs alone transplantation group and Schwann cells alone transplantation group were injected with 0.4 mL Fb and 0.4 mL Schwann cells respectively (2 × 106 cells each).Flaps of rats in Schwann cells + Fbs co-transplantation group were injected with 0.4 mL Fbs and Schwann cells mixed cells (totally 2 × 106 cells,cell number ratio:1 ∶ 1),and flaps of rats of simple flap group were injected with the same volume of DMEM high glucose medium.On post injection day (PID) 2,5,7,9,and 14,5 rats in each group were selected respectively according to the random number table.The flap tissue was collected,and the number,diameter,and arrangement of regenerated nerves were observed by immunofluorescence method.Data were processed with completely random designed t test,analysis of variance for repeated measurement,t test,and Bonferroni correction.Results (1) The third passage of cells isolated and cultured from the rat embryo trunks were uniform in size and shape,long spindle-shaped,with a large proportion of nuclei.Strong positive expressions of fibronectin and Ephrin-B2 protein in cells were observed,and the mRNA expression of Ephrin-B2 was 0.004 1 ± 0.000 8.The cells were identified as Fbs.(2) After 5 days of culture,the primary cells isolated from the sciatic nerves and brachial plexus nerves of neonatal rats were elongated in cell bodies and grew in nest,fence,or vortex-like shape.The third passage of cells were detected by immunofluorescence method and flow cytometer,and the corresponding S100 positive cell rates were (95.9 ± 1.0) ％ and (95.8 ± 1.1) ％ respectively.The cells were identified as Schwann cells.(3) At PCH 6 and 24,the cluster numbers of Schwann cells in Schwann cells + Fbs co-culture group were significantly higher than those of Schwann cells alone culture group (t =6.500,10.614,P ＜ 0.01).At PCH 24,the Schwann cells in Schwann cells + Fbs co-culture group aggregated into clusters,Fbs dispersed around the Schwann cell clusters,and the protein expressions of EphB2,N-cadherin,and Sox2 in Schwann cells were significantly higher than those in Schwann cells alone culture group (t =2.975,19.717,11.159,P ＜0.05 or P ＜0.01).(4) On PID 2,a small number of scattered,disordered,short,and thin nerve fibers were observed in the flap tissue of rats in the four groups.From PID 5 to 14,the number of nerve fibers in the flap tissue of rats of Schwann cells + Fbs co-transplantation group increased gradually,and the nerve fibers were with long diameter and arranged orderly.The number of nerve fibers in the flap tissue of rats of Schwann cells alone transplantation group increased,but the nerve fibers were with short diameter and arranged disorderly,and the number was smaller than that of Schwann cells + Fbs co-transplantation group.In simple flap group and Fbs alone transplantation group,the nerve fibers in the flap tissue of rats gradually degenerated with gradually decreased number or even disappeared.Conclusions The combined transplantation of Fbs and Schwann cells in rats can regulate Schwann cells migration and clustering by activating Ephrin/Eph-Sox2-N-cadherin signaling pathway,thus promoting the orderly nerve regeneration of denervated perforator flaps in rats.%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,细胞核所占比例较大;细胞内纤维粘连蛋白及Ephrin-B2蛋白呈强阳性表达,Ephrin-B2 mRNA的表达量为0.004 1 ±0.0008.细胞鉴定为Fb.(2)从新生大鼠坐骨神经和臂丛神经分离的原代细胞培养5d后可见胞体拉长,呈巢状、栅栏状或旋涡状生长;第3代细胞经免疫荧光法及流式细胞仪检测显示,S100阳性细胞率分别为(95.9±1.0)％和(95.8±1.1)％.细胞鉴定为施万细胞.(3)培养6、24 h,施万细胞+Fb共培养组施万细胞成簇群数均明显高于施万细胞单独培养组(t=6.500、10.614,P＜0.01).培养24 h,施万细胞+Fb共培养组施万细胞聚集成簇群,Fb分散围绕在施万细胞簇群周围,施万细胞EphB2、N-钙黏蛋白和Sox2蛋白表达均明显高于施万细胞单独培养组(t=2.975、19.717、11.159,P＜0.05或P＜0.01).(4)注射后2d,4组大鼠皮瓣组织内均见少量散在、排列较杂乱、较短而细的神经纤维.注射后5 ～14 d,施万细胞+Fb共移植组大鼠皮瓣组织中神经纤维数量逐渐增多,且直径较粗大、排列有序;施万细胞单独移植组大鼠皮瓣组织内神经纤维数量有所增加,但直径细小、排列杂乱,数量较施万细胞+Fb共移植组少;单纯皮瓣组和Fb单独移植组大鼠皮瓣组织内神经纤维逐渐发生溃变,数量逐渐减少甚至消失. 结论大鼠Fb与施万细胞联合移植可通过激活Ephrin/Eph-Sox2-N-钙黏蛋白信号通路调控施万细胞迁移使其聚集成簇群,从而促进大鼠失神经支配穿支皮瓣神经有序再生.

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

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