T型钙离子通道α1H亚型在先天性巨结肠大鼠模型病变肠段的分布及功能变化  

Expression and function of T-type α1H Ca^2＋ channels in the rat model of Hirschsprung disease

作　　者：张妮妮[1] 田姣[1] 林燕[1] 张薇[1] 虎崇康[1] 曾令超[1] 杨洁[1] 王宝西[1] 舒震[2] 江逊[1] 

机构地区：[1]第四军医大学唐都医院儿科,西安710038 [2]第四军医大学药学生物技术中心

出　　处：《中华儿科杂志》2016年第5期354-359,共6页Chinese Journal of Pediatrics

基　　金：国家自然科学基金（81170331、81370490）

摘　　要：目的研究T型钙离子通道d1H亚型（Cav3．2）在先天性巨结肠大鼠模型发生发展中的作用。方法将80只6～8dSD乳鼠通过随机数字表法分为两组，每组40只。模型组经肛门灌注0．2％苯扎氯铵建立先天性巨结肠模型，对照组用生理盐水作为替代。分别在4、6、8周每组随机抽取10只乳鼠处死，通过大体形态学观察、组织病理学观察、免疫荧光的方法来鉴定先天性巨结肠模型是否建立成功。同时通过免疫组织化学、蛋白免疫印迹和免疫荧光双标技术检测模型大鼠病变肠段中Cav3．2的分布变化及其与Cajal间质细胞特异性标志物c—kit共变情况；离体肌条组织电生理检测模型大鼠病变肠段及干预Cav3．2后的变化。组间数据资料采用t检验。结果处理后8周通过大体形态学观察，组织病理学观察，免疫荧光方法鉴定先天性巨结肠模型建立成功，即处理肠段肌问神经节细胞的减少甚至缺如。免疫组织化学检测Cav3．2在模型大鼠病变肠段的分布，对照组结肠组织Car3．2主要分布于环行肌与纵行肌之间，多呈连续分布；模型组随着处理时间的延长，狭窄段组织Cav3．2的分布逐渐减少，连续性遭到破坏。蛋白免疫印迹检测处理后大鼠Cav3．2蛋白表达的变化，结果与免疫组化检测一致，随着处理时间的延长，Cav3．2的表达逐渐减少，8周时减少最明显。处理后4、6、8周模型组Car3．2的相对表达量分别为0．63±0．06、0．38±0．06、0．26±0．07；对照组分别为0．63±0．06、0．62±0．09、0．63±0．06，两组在处理后6、8周相比，差异均有统计学意义（t值分别为5．27和8．63，P值均〈0．05）；Cav3．2和c—kit共变情况分析显示，与对照组相比，狭窄段组织c—kit和Cav3．2的共同阳性区域明显减少甚至消失。离体肌条组织电生理结果发现：与对照组相比，模型组狭窄段组织的自发性节律性�[ Abstract] Objective To investigate the role pathogenesis of Hirschsprung disease （ HD ）. Method of T-type alH Ca2± channels（ Cav 3.2） in the Eighty neonatal SD rats 6 to 8 days of age were randomly divided into 2 groups, 40 in each. Microinjector catheters were carefully placed into the bowl of one group, and 0. 2% benzalkonium chloride （ BAC ） solution was injected to establish HD rat model. Control group was treated with saline instead of benzalkonium chloride. At postoperative 4, 6 and 8 weeks, ten rats were sacrificed randomly and examined through general observation, histopathological observation and immunofluorescent staining of PGP 9. 5 to identify the animal models. Meanwhile, the distribution of Car 3.2 in abnormal colon of HD rat model was studied by immunohistochemical staining, Western blot, and the co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Besides, function of Cav 3.2 was surveyed with the model rats tensile force and frequency of colonic muscle in vitro. Result After 8 weeks of BAC treatment, the rat model was successfully established according to the results of histopathological and immunofluorescent staining, which showed decrease or lack of ganglion cells within the area of BAC treatment. The distribution of Cav 3.2 was detected by immunohistochemical staining. In the normal colon, Cav 3.2 were mainly distributed between circular muscle and longitudinal muscle, and showed continuous distribution. However, in the narrow segment of HD rat model, the distribution of Car 3.2 was decreased significantly, and its continuity was destroyed . The results of Western blot were quite consistent with immunohistochemistry staining, in the narrow segment of HD rat model,the expression of Cav 3.2 was decreased gradually after the BAC treatment, especially at postoperative 8 weeks. The relative expression of Cav 3.2 of the control group was 0. 63 ± 0. 06, 0. 62 ± 0. 09, 0. 63 ± 0.06 at postoperative 4, 6 and 8 weeks respectively. While that of HD grou

关 键 词：HIRSCHSPRUNG病 大鼠 钙通道 

分 类 号：R725.7[医药卫生—儿科] R-332[医药卫生—临床医学]