先天性巨结肠结肠组织钾离子通道的表达变化

目的 通过离子通道芯片及Western blot实验证实先天性巨结肠(Hirschsprungdisease,HD)病变肠段的钾离子通道表达与正常的结直肠存在差异.方法 2012年8月到2013年12月在哈尔滨医科大学附属第一医院手术治疗先天性巨结肠患儿30例,通过病史、直肠测压、钡剂灌肠造影、术中冰冻及术后病理等确诊为HD,术中结肠切除完成后,分别取正常及病变狭窄段肠组织,大小为1.0 cm×1.0 cm;标本置入液氮中冻存以待进一步实验.取其中6组标本,应用人类神经离子通道PCR芯片(neuronal ion channels PCR array)进行筛选表达具有差异性的钾离子通道;全部30例标本应用Western blot检测所筛选出钾离子通道的表达情况.结果采用Image-Pro Plus魔棒选取蛋白条带,利用IOD算法计算条带灰度.结果 我们将表达变化在2倍以上的基因认定为是差异表达基因,从而通过芯片筛选出表达具有差异性的钾离子通道1 5个,其中无神经节肠段表达增强1个,表达降低14个,共包括电压门控性的钾离子通道9个、大电导Ca2+激活的K+通道2个、小电导Ca2+激活的K+通道2个、内向整流性钾离子通道2个.利用Westernblot验证大电导Ca2+激活的K+通道的Maxi K通道及电压门控性钾离子通道的Kv11.1通道a亚基蛋白的表达.并经统计学分析,认定差异具有显著差异性.结论 先天性巨结肠结肠组织中确实存在表达下调的钾离子通道.

Differential expression of potassium ion channel in Hirschsprung's disease

Objective To confirm the differential expression of potassium ion channel between Hirschsprung's disease (HD) and non-HD colorectal tissues by neuronal ion chips polymerase chain reaction (PCR) array and Western blot.Methods A total of 30 patients were definitely diagnosed as HD by case history,anorectal menometry,barium enema radiography and intraoperative biopsy at our hospital during August 2012 and December 2013.Normal and stenotic colonic tissues (1.0 cm× 1.0 cm apiece) were harvested after partial colonic removal.PCR Array chips were used for screening the expression differences of K+ channel for 6 random specimens and Western blot for verification.The protein bands were selected by Image-Pro Plus and gray levels calculated.Results The changes of gene expression were significant when the ratio was more than 2 folds.Fifteen channels were screened for differential expression,including 9 voltage-gated potassium channel,2 large-conductance calciumactivated potassium channels,2 small-conductance calcium-activated potassium channels and 2 inward rectifier potassium channels.And Western blot was used for verifying the expression difference in protein level of Maxi K channel together with Kv11.1.Through statistical analysis,the expressions of Maxi K channel and Kv11.1 decreased in HD colorectal tissues.Conclusions The expression of potassium ion channel is down-regulated in HD.