大鼠胰岛beta细胞截短型P糖蛋白表达的初步鉴定  

Preliminary identification of mini-P-glycoprotein in rat pancreatic beta cells

作　　者：申翔 杨蔚[2,3] 李代清[2] SHEN Xiang;YANG Wei;LI Daiqing(Intensive Care Unit,Tianjin Union Medical Center,Tianjin 300000;Dicbetes Laboratory,Zhu Xianyi Memorial Hospital,Tianjin Medical University;Department of Endocrinology,First People’s Hospital of Changde City)

机构地区：[1]天津市人民医院重症医学科,天津300000 [2]天津医科大学朱宪彝纪念医院糖尿病研究室 [3]常德市第一人民医院内分泌科

出　　处：《山西医科大学学报》2020年第12期1355-1359,共5页Journal of Shanxi Medical University

基　　金：国家自然科学基金面上项目(81270891)。

摘　　要：目的初步探究大鼠胰岛beta细胞截短型P糖蛋白(mini-P糖蛋白,65 kD)表达的存在。方法采用实时荧光定量PCR(RT-qPCR)检测雄性Wistar大鼠胰岛、肝脏和INS-1细胞中P糖蛋白主要调控基因abcb1b N端、C端及abcb1a的表达情况。提取INS-1细胞的总RNA,设计abcb1b基因3′末端和5′末端引物,逆转录合成对应的cDNA,PCR产物经过地高辛-dUTP标记,获得Northern blot检测探针。提取大鼠胰岛总RNA,通过甲醛变性电泳分离RNA并转至尼龙膜,将探针与尼龙膜上RNA进行杂交,检测abcb1b基因的选择性剪切。提取INS-1细胞蛋白,分为蛋白酶抑制剂组(加入蛋白酶抑制剂)和无蛋白酶抑制剂组,利用Western blot技术检测截短型P糖蛋白的表达差异。结果实时荧光定量PCR结果显示大鼠胰岛、INS-1细胞及肝的abcb1b表达量远大于abcb1a,差异具有统计学意义(P<0.05)。大鼠胰岛、INS-1细胞的abcb1b C端拷贝数大于N端(约1.7倍),差异具有统计学意义(P<0.05)。Northern blot杂交结果为单条带,分子量与全长P糖蛋白符合,未检测到abcb1b基因选择性剪切条带。Western blot结果显示无蛋白酶抑制剂组仅有65 kD截短型P糖蛋白的表达,而蛋白酶抑制剂组主要表达分子量为170 kD的全长P糖蛋白,截短型P糖蛋白表达显著减低,与无蛋白酶抑制剂组相比,截短型P糖蛋白的表达差异具有统计学意义(P<0.05)。结论初步认为在大鼠胰岛beta细胞探测到的截短型P糖蛋白可能为蛋白降解产物,并非abcb1b基因选择性剪切的结果。Objective To explore the existence of mini-P-glycoprotein(65 kD)in rat pancreatic beta cells.Methods Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of abcb1a and the N-terminus and C-terminus of abcb1b in male Wistar rat islets,liver and insulinoma cells(INS-1),respectively.The total RNA of INS-1 cells were extracted and cDNA was synthesized using the primer of abcb1b.The PCR products were labeled by digoxin dUTP as the probe for Northern blot.The total RNA of isolated rat islets was extracted and separated by formaldehyde modified electrophoresis.Hybridization of the probe was performed when the RNAs were transferred to nylon membrane.INS-1 cell protein was extracted and divided into protease inhibitor group(treated with protease inhibitor)and non-protease inhibitor group.The expression of mini-P-glycoprotein in two groups was detected by Western blot.Results RT-qPCR results showed that the expression of abcb1b was significantly higher than that of abcb1a in islets,INS-1 cells and liver(P<0.05).The C-terminal copy number of abcb1b in rat islets and INS-1 cells was more than that in N-terminal(about 1.7 times),and the difference was statistically significant(P<0.05).Northern blot showed a single band with molecular weight consistent with the full-length P-glycoprotein.The hybridization of Northern blot was failed to detect any alterntive splicing band of abcb1b gene.Western blot results showed that 65 kD mini-P-glycoprotein was only detected in non-protease inhibitor group,while the mainly expressed protein was full-length P-glycoprotein(170 kD)in the protease inhibitor group,and the expression of 65 kD mini-P-glycoprotein was significantly decreased.There was statistical difference between the two groups regarding mini-P-glycoprotein expression(P<0.05).Conclusion It could be concluded that the 65 kD mini-P-glycoprotein detected in rat islet beta cells might be protein degradation,but not the products alternative spliced by abcb1b gene.

关 键 词：P糖蛋白 截短型P糖蛋白 abcb1b基因 

分 类 号：R587[医药卫生—内分泌]