替米沙坦/胶原蛋白/聚己内酯神经导管的构建及其修复大鼠坐骨神经缺损效果的实验研究  

Experimental study on the construction of telmisartan/collagen/polycaprolactone nerve conduit and its repair effect on rat sciatic nerve defect

作　　者：武小煜 李海波 尹健健 刘纯 吴思宇 刘均 马佳义 戴婷 赵红斌 WU Xiaoyu;LI Haibo;YIN Jianjian;LIU Chun;WU Siyu;LIU Jun;MA Jiayi;DAI Ting;ZHAO Hongbin(Medical Research Centre,the Affiliated Changzhou No.2 People’s Hospital,Nanjing Medical University,Changzhou Jiangsu,213164,P.R.China)

机构地区：[1]南京医科大学附属常州第二人民医院中心实验室,江苏常州213164

出　　处：《中国修复重建外科杂志》2022年第3期352-361,共10页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：常州市科技支撑社会发展项目(CE20185047)。

摘　　要：目的构建替米沙坦/胶原蛋白/聚己内酯(polycaprolactone,PCL)神经导管,观察其修复大鼠坐骨神经缺损的效果。方法制备质量浓度为60%的胶原蛋白/六氟异丙醇混合液、40%PCL/六氟异丙醇溶液并混匀后,分别将0、5、10、20 mg替米沙坦溶于10 mL混合液。利用高压静电纺丝技术构建替米沙坦/胶原蛋白/PCL神经导管;扫描电镜观察京尼平交联前后神经导管结构;体外缓释方法检测药物释放率。取RAW264.7细胞与脂多糖培养使其致炎后,与负载不同浓度替米沙坦的神经导管共培养24 h,实时荧光定量PCR检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和精氨酸酶1(Arginase 1,Arg-1)mRNA的表达。将40只成年Wistar大鼠随机分成A、B、C、D组(n=10),制备坐骨神经15 mm长缺损后,分别以交联后负载0、5、10、20 mg替米沙坦的神经导管桥接修复缺损。术后观察大鼠一般情况,检测坐骨神经运动功能指数(sciatic functional index,SFI),大体观察神经导管与坐骨神经桥接情况以及导管完整性,HE染色观察神经导管内组织生长和材料降解情况,免疫组织化学染色观察新生组织中M1型巨噬细胞标志分子CD86和M2型巨噬细胞标志分子CD206、髓磷脂碱基蛋白(myelin basic protein,MBP)和髓磷脂蛋白0(myelin protein 0,P0)的表达,免疫荧光染色观察新生组织中神经丝蛋白200(neurofilament 200,NF200)和S-100β表达。结果大体观察示制备的神经导管内径为1.8 mm、外径2.0 mm,呈白色;交联后纳米纤维变粗,结构更致密。药物缓释检测示神经导管负载的替米沙坦能实现缓释效应。实时荧光定量PCR检测示,随着替米沙坦浓度增加,iNOS mRNA相对表达量下调,Arg-1mRNA相对表达量上调,其中20 mg组与其他各组比较差异均有统计学意义(P<0.05)。动物体内实验观测示,术后各组大鼠均存活至实验完成。术后各时间点,C、D组SFI均高于A、B组(P<0.05),且6个月时D组高于C组(P<0.0Objective To construction the telmisartan/collagen/polycaprolactone(PCL)nerve conduit and assess its effect on repairing sciatic nerve defect in rats.Methods The 60%collagen/hexafluoroisopropanol(HFIP)solution and 40%PCL/HFIP solution were prepared and mixed(collagen/PCL solution).Then the 0,5,10,and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution,respectively.Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology.The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope(SEM).The drug release efficiency was detected by in vitro sustained release method.RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation,and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours.The mRNA expressions of inducible nitric oxide synthase(iNOS)and Arginase 1(Arg-1)were detected by using real-time fluorescence quantitative PCR.Forty adult Wistar rats were randomly divided into 4 groups(n=10).After preparing 15-mm-long sciatic nerve defect,the defect was repaired by cross-linked nerve conduits loaded with 0,5,10,and 20 mg telmisartan in groups A,B,C,and D,respectively.After operation,the general condition of rats was observed after operation;the sciatic function index(SFI)was tested;the bridging between the nerve conduit and sciatic nerve,and the integrity of nerve conduit were observed;the tissue growth in nerve conduit and material degradation were observed by HE staining;the expressions of CD86(M1 macrophage marker),CD206(M2 macrophage marker),myelin basic protein(MBP),and myelin protein 0(P0)in new tissues were also observed by immunohistochemical staining;the expressions of neurofilament 200(NF-200)and S-100βin new tissues were assessed by immunofluorescence staining.Results The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm.After cross-linking by genipin,the nanofiber became

关 键 词：替米沙坦 胶原蛋白 聚己内酯 巨噬细胞极化 神经再生 大鼠 

分 类 号：R965[医药卫生—药理学]