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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 myoblast (Mb) with high expression of glial cell derived neurotrophic factor (GDNF) as seed cells and to construct tissue-engineered skeletal muscle in vivo by using acellular collagen sponge as a scaffold. Whether the tissue can connect with the hypoglossal nerve. Methods Mb were isolated and cultured from the muscle of four limbs of 7 male Lewis rats at 2 days of age. The third generation Mb (MbGDNF) was transfected with recombinant adenovirus Ad-GDNF carrying GDNF gene. Mb and MbGDNF and acellular sponge scaffold were cultured in vitro to construct the scaffold complex, and the cell adhesion was observed by scanning electron microscopy after 24 h. Fifty-four female Lewis rats aged 8 weeks were sacrificed and the hypoglossal nerve was dissected. The hypoglossal nerve was dissected from 1.0 to 1.5 cm. The distal end of the hypoglossal nerve was dissociated. Using Mb scaffold complex (Mb group, n = 27) and MbGDNF scaffold complex (MbGDNF group, n = 27) .After 1, 6 and 12 weeks after operation, HE staining, myogenin, slow skeletal myosin and actylcholine receptorα1 (AchRα1) immunohistochemical staining Implant growth was observed with cholera toxin B labeled peroxidase retrograde tracing staining to detect the survival of hypoglossal motor neurons. As a result, MbGDNF was highly expressed in transfection. Mb and MbGDNF adhere well to the scaffold and grow well. HE staining: Twelve weeks after operation, the implants of both groups were closely connected with the surrounding tissues. The new-born myofibers grew in surrounding normal muscular tissue and gradually disappeared from the normal tissues. Immunohistochemical staining: myogenin and slow skeletal myosin-positive myogenic cells were detected at 1, 6 and 12 weeks after surgery, and AchRα1 was diffuse positive cells. Y chromosome staining positive cells decreased with time. The numbers of neurons positive in MbGDNF group were 261.0 ± 6.6, 227.3 ± 8.5 and 173.3 ± 9.1 respectively at 1, 6 and 12 weeks after operation; 234.7 ± 5.5, 196.0 ± 13.5 and 166.7 ± 11.7 respectively in Mb group; The number of neurons in MbGDNF group was more than that in Mb group at 6 weeks (P <0.05). There was no significant difference between the two groups after 12 weeks (P> 0.05). Conclusion The constructed tissue engineering skeletal muscle has a direct physical connection with the hypoglossal nerve segment. The recombinant GDNF produced by MbGDNF can protect the motoneurons in the hypoglossal nucleus after trauma by retrograde transport.