舌下神经植入组织工程骨骼肌的体内实验研究  

IN VIVO STUDY ON TISSUE ENGINEERED SKELETAL MUSCLE WITH HYPOGLOSSAL NERVE IMPLANTATION

作　　者：唐休发[1] 张富贵[2] 冯扬[2] 周伟[2] 刘济远[2] 华成舸[1] 

机构地区：[1]四川大学华西口腔医院头颈肿瘤外科 [2]四川大学华西口腔医院口腔疾病研究国家重点实验室

出　　处：《中国修复重建外科杂志》2012年第3期359-364,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30872890)~~

摘　　要：目的构建高表达胶质源性神经营养因子(glial cell derived neurotrophic factor,GDNF)的成肌细胞(myoblast,Mb)并作为种子细胞,以去细胞胶原海绵为支架,体内构建组织工程骨骼肌,观察构建组织能否与舌下神经断端发生连接。方法取7只2日龄雄性Lewis大鼠四肢肌肉体外分离培养Mb,采用携带GDNF基因的重组腺病毒Ad-GDNF转染第3代Mb(MbGDNF)。将Mb及MbGDNF与去细胞胶原海绵支架体外复合培养构建细胞支架复合物,24 h后扫描电镜观察细胞黏附情况。取8周龄雌性Lewis大鼠54只,解剖分离舌下神经,取1.0～1.5 cm舌下神经离断其远心端,用Mb支架复合物(Mb组,n=27)和MbGDNF支架复合物(MbGDNF组,n=27)包裹并固定其近心端,术后1、6及12周分别行HE染色,myogenin、slow skeletal myosin及乙酰胆碱受体α1(actylcholine receptorα1,AchRα1)免疫组织化学染色检测植入物生长情况,并行霍乱毒素B标记的过氧化物酶逆行示踪染色检测损伤后舌下神经核运动神经元存活情况。结果构建的MbGDNF能高表达转染基因。Mb及MbGDNF在支架上黏附和生长状态良好。HE染色:术后12周,两组植入物与周围组织紧密连接,有新生肌纤维从周围正常肌组织长入,与正常组织分界渐不明显。免疫组织化学染色:术后1、6及12周均可检测到细胞质呈myogenin及slow skeletal myosin阳性的肌源性细胞,以及AchRα1呈弥散性阳性细胞。Y染色体染色阳性细胞随时间延长而减少。术后1、6及12周,MbGDNF组阳性标记的神经元数量分别为261.0±6.6、227.3±8.5及173.3±9.1;Mb组分别为234.7±5.5、196.0±13.5及166.7±11.7;术后1、6周MbGDNF组神经元数量多于Mb组(P<0.05),术后12周两组差异无统计学意义(P>0.05)。结论构建的组织工程骨骼肌与舌下神经断端发生了直接物质联系,MbGDNF产生的重组GDNF能通过逆行运输方式保护损伤后舌下神经核团内运动神经元的存活。Objective To construct tissue engineered skeletal muscle in vivo using glial cell derived neurotrophic factor（GDNF） genetically modified myoblast（Mb） on acellular collagen sponge with hypoglossal nerve implantation,and to observe whether structural or functional connection could be established between engineered tissue and motor nerve or not.Methods Mbs were isolated from 7 male Lewis rats at age of 2 days,cultured and genetically modified by recombinant adenovirus carrying GDNF cDNA（MbGDNF）.Calf skin-derived acellular collagen sponge was used as scaffold;cell adhesion was detected by scanning electron microscope after 24 hours.Hypoglossal nerve was implanted into Mb-scaffold complex（Mb group,n=27） or MbGDNF-scaffold complex（MbGDNF group,n=27） in 54 female Lewis rats at age of 8 weeks.HE staining was performed at 1,6,and 12 weeks postoperatively,and immunohistochemistry staining and fluorescence in situ hybridization were used.Results MbGDNF could highly expressed GDNF gene.Mb and MbGDNF could adhere to the scaffold and grew well.HE staining showed tight junctions between implant and peripheral tissue with new muscle fiber and no distinguished line at 12 weeks in 2 groups.Immunohistochemistry staining showed that positive cells of myogenin and slow skeletal myosin were detected,as well as positive cells of actylcholine receptor α1 at 1,6,and 12 weeks.The positive cells of Y chromosome decreased with time.At 1,6,and 12 weeks,the positive neurons were 261.0 ± 6.6,227.3 ± 8.5,and 173.3 ± 9.1,respectively in MbGDNF group,and were 234.7 ± 5.5,196.0 ± 13.5,and 166.7 ± 11.7,respectively in Mb group;significant differences were found between 2 groups at 1 and 6 weeks（P 0.05）,no significant difference at 12 weeks（P 0.05）.Conclusion Connection can be established between engineered tissue and implanted hypoglossal nerve.Recombinant GDNF produced by MbGDNF might play a critical role in protecting central motor neurons from apoptosis by means of retrograde transportation.

关 键 词：组织工程骨骼肌 胶质源性神经营养因子 去细胞胶原海绵 舌下神经 成肌细胞 大鼠 

分 类 号：R782[医药卫生—口腔医学]