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目的建立一种适合骨髓间充质干细胞(mesenchymal stem cells,MSCs)移植的实验性无神经节细胞症大鼠模型,并探讨模型大鼠结肠上皮离子转运的变化。方法8~9周龄SD大鼠80只,随机分为二组。氯胺酮麻醉下开腹,实验组用0.1%苯扎氯铵(benzalkonium chloride,BAC)处理大鼠降结肠浆膜40min,温盐水冲洗后关腹,对照组用生理盐水代替苯扎氯铵。分别于术后1、2、3、4、8周进行大体观察、钡灌肠X线检查,结肠测压、取处理段结肠行组织学检查,利用短路电流技术检测结肠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,处理段结肠狭窄,反射性收缩消失,近端结肠扩张,且随着时间延长症状加重。组织学检查发现BAC处理后1周结肠神经节细胞明显减少、空泡变,3周后完全消失。短路电流检测提示,与对照组比较,实验组大鼠结肠上皮各时间点跨膜电压、基础电流明显降低,而钠离子吸收电流占基础电流的百分比升高。另外,由Fors-kolin所引起的氯离子分泌电流也明显高于对照组,但二苯胺-2,2’-二羧酸阻断Forskolin所引起的Isc的百分比下降且小于1,而结肠跨膜电阻二组之间无差异。结论①采用0.1%BAC处理降结肠浆膜成功建立了实验性无神经节细胞症大鼠模型,为进一步结肠内MSCs移植分化为神经细胞的研究奠定了基础;②实验性无神经节细胞症大鼠模型结肠黏膜屏障功能未受损伤,结肠上皮离子转运功能紊乱,提示巨结肠大便排空困难除与失神经之后的平滑肌和括约肌功能失调有关外,可能还与结肠离子转运的变化有关。
OBJECTIVE: To establish a rat model of gangliocytic ganglion for transplantation of mesenchymal stem cells (MSCs) and to investigate the changes of ion transport in colonic epithelium of model rats. Methods 80 SD rats aged 8-9 weeks were randomly divided into two groups. The rats in the experimental group were treated with benzalkonium chloride (BAC) 0.1% benzalkonium chloride for 40 minutes, rinsed with warm saline solution and then abdomen. The rats in the control group were treated with saline instead of benzalkonium chloride. General observations were made at 1, 2, 3, 4, and 8 weeks after operation. Barium enema X-ray and colonic manometry were performed. Colonic histological examination was performed. The changes of ion transport in colon epithelium were detected by short-circuit current technique. Results One week after BAC treatment, rats in the experimental group developed abdominal distension, the colon of the treatment segment was stenosed, the reflex contraction disappeared, the proximal colon was dilated, and the symptom was aggravated with time. Histological examination found that after 1 week of BAC treatment, the number of colonic ganglion cells was significantly reduced, vacuolar change, completely disappeared after 3 weeks. The short-circuit current detection suggested that compared with the control group, the transmembrane voltage and the basal current of the colonic epithelium of the experimental group were significantly decreased at each time point, while the percentage of the sodium ion absorption current in the basal current was increased. In addition, the chloride current secreted by Fors-kolin was also significantly higher than that of the control group, but the percentage of Isc blocked by diphenylamine-2,2’-dicarboxylic acid blocking Forskolin decreased by less than 1, No difference between the two resistance groups. Conclusions ① The model of rat gangliocytic degeneration was successfully established by treating the descending colon serosa with 0.1% BAC, which lays the foundation for the further study on the differentiation of colon MSCs into neural cells. ② The experimental ganglion cell-free Colon mucosal barrier function was not impaired in mouse model, and the ion transport function of colonic epithelium was disordered, suggesting that the difficulty of emptying the colonic bowel movement was related to the dysfunction of smooth muscle and sphincter muscle after denervation, and possibly the changes of colonic ion transport.