骨髓间充质干细胞移植治疗巨结肠的实验研究  

A preliminary study on the treatment of experimental aganglionosis by mesenchymal stem cells transplantation into colon in vivo

作　　者：金先庆[1] 徐纪荣[2] 陈小章[3] 

机构地区：[1]重庆医科大学附属儿童医院外科,400014 [2]山西省儿童医院外科 [3]香港中文大学上皮细胞生物学研究中心

出　　处：《中华小儿外科杂志》2007年第9期481-485,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(编号:30100198);香港创新科技基金(2001~2004)

摘　　要：目的在体外诱导骨髓间充质干细胞(MSCs)的神经细胞分化基础上,探讨移植治疗实验性巨结肠的可能性。方法8～9周龄SD大鼠用1%苯扎氯铵(BAC)制作巨结肠模型。贴壁筛选法进行MSCs原代培养、扩增后用碱性全反式视黄酸(ATRA)和碱性成纤维生长因子(FGF2)诱导法收获Nestin、NF表达阳性的MSCs细胞,显微注射法将来源于雄性大鼠,Nestin、NF阳性表达的MSCs植入雌性实验性巨结肠模型鼠病变肠段。实验分对照组(PBS组)及实验组(MSCs组),分别于术后1、2、4周进行大体观察,钡灌肠X线检查,病理学检查、平滑肌收缩功能以及采用短路电流技术检测上皮离子转运的变化。结果BAC处理后1周,实验组大鼠出现腹胀,BAC处理段出现狭窄。MSCs被纯化并被诱导为神经细胞。MSCs组免疫组化结果显示移植后1、2、4周Nestin及NF呈阳性表达,但在PBS组未观察到阳性表达。MSCs组腹胀较轻,梗阻近端少量大便,MSCs移植组大鼠结肠上皮第一周跨膜电压升高,但第四周其基础电流下降,钠离子吸收减少;MSCs移植后第一、四周Forskolin所引起的氯离子分泌电流均较PBS组高。结论MSCs可在实验性巨结肠模型鼠结肠壁内分化为神经细胞,部分恢复结肠神经调节作用。Objective To explore the feasibility of the treating of experimental aganglionosis by mesenchymal stern cells （MSCs） transplantation into the colon. Methods Sprague Dawley rats,8-9 weeks old,were applied 0. 1 % benzalkonium chloride （BAC） to establish rat models of experimental aganglionosis. MSCs were cultured and proliferated by adhesive-screening method. The MSCs with expressions of Nestin and NF were obtained after induced by ATRA and FGF2, and then were transplanted into female experimental aganglionosis rats colon by microinjection. Microscopic observation, contrast roentgenography of colon,detections of Nestin and NF by immunochemistry, pharmacologic examinations for contraction of colonic smooth muscle, and detection of ion transport of colonic epithelium by the short-circuit current were performed at an interval of 1w,2w and 4w after MSCs transplantation. Results One week after BAC treatment, the rats had abdominal distention. Roentagenographic examination and autopsy revealed a narrowed segment. MSCs with positive expression of Nestin, purified by an adhesive-screening method,differentiated into neuronal cells in normal rats colon. Immunochemistry showed sprinkled positive cells with expressions of Nestin and NF at lw, 2w and 4w after MSCs transplantation. Nevertheless, no positive cells were observed after PBS transplantation. Less abdominal distention and less feces filled in distended proximal colon was observed in MSCs transplantation group compared with PBS transplantation group. The short-circuit current produced by colonic epithelium increased at lw after MSCs transplantation. However, basal short-circuit current and aminoride-sensitive short-circuit current decreased at 4w. The forskolinqnduced short-circuit current increased significantly compared with PBS transplantation group. Conclusions MSCs can differentiate into neuron cells in colon of rats with experimental aganglionosis and partially repair neuromuscular modulation of colon.

关 键 词：间充质干细胞移植 巨结肠 先天性 大鼠 

分 类 号：R726.5[医药卫生—儿科]