人工诱导突变的尿素转运蛋白B在5/6肾切除大鼠肠上皮细胞表达及功能研究

Urea transporter(UT), expressed on gastrointestinal epithelial cells and functioning as the mediator of urea transmembrane transportation, plays an important role in urine concentration and regulation of nitrogen balance. The impairment of renal function will reduce the urine excretion of urea and then the concentration of urea will elevate in the blood. The increase of intestinal excretion of urea through UT can help relieve the overload of urea in the body. Accordingly, we aim to firstly construct the recombinant adenovirus vector expressing artificial induced mutational UT-B(UT-Bm) with the ability of increasing urea transportation, then transfect the human intestinal epithelial cell line Caco-2. The mRNA and protein expression of UT-Bm in Caco-2 will be determined by RT-PCR and Western blot, and the urea transportation ability of Caco-2 after transfection will be tested through isotope-labeling method. In the in vivo study, through the injection of recombinant adenovirus vector with UT-Bm into intestine of 5/6 nephrectomized rat, alterations of mRNA and protein expression of UT-Bm in intestine epithelial cell, the intestinal urea excretion level, serum creatinine and BUN will be tested to determine whether intestine epithelial cell with UT-Bm can increase urea excretion. This innovative study aims to find a new treatment for patients with chronic kidney disease.

尿素转运蛋白(UT)介导尿素跨膜转运，在尿浓缩和调节氮平衡中起重要作用。胃肠道粘膜上皮细胞有UT表达，当肾脏功能受损时，尿液中的尿素排泄减少，血液中尿素浓度增加，尿素向肠腔内分泌增加，有助于减轻体内氮负荷。本课题通过人工诱导UT-B基因突变，筛选可提高尿素转运效率的UT-B突变基因(UT-Bm)，并构建带有突变UT-Bm的重组腺病毒载体,在体外转染结肠上皮细胞株Caco-2,采用RT-PCR、Western blot检测UT-Bm mRNA和蛋白的表达，同位素标记法检测Caco-2细胞尿素转运功能的改变；将带有UT-Bm基因的重组腺病毒注入5/6肾切除的大鼠结肠，检测UT-Bm mRNA和蛋白在肠组织的表达及肠道尿素排泄量、大鼠血肌酐、尿素氮浓度改变，明确带有UT-Bm的肠上皮细胞是否能更有效地清除尿毒症毒素，为慢性肾衰竭患者寻找新的治疗途。

尿素转运蛋白(UT)介导尿素跨膜转运，在尿浓缩和调节氮平衡中起重要作用。哺乳动物的UT起源于两种基因：UT-A和UT-B。UT-A家族有多种cDNA，而UT-B仅有一种cDNA，且UT-B的尿素转运效率远高于UT-A。UT-B在体内分布广泛，胃肠道粘膜上皮细胞也有 UT-B 表达。慢性肾脏病的发病率不断上升，对慢性肾功能不全患者，在进入终末期肾衰竭需肾脏替代治疗前多给予口服胃肠透析治疗，通过肠道排泄毒素、改善代谢紊乱，但效果有限。肠粘膜血管壁为半透膜，胃肠粘膜血流丰富，肠粘膜上皮细胞面积广，如能通过基因转染方法将UT-B大量转入肠上皮细胞，则可极大提高肠上皮细胞的尿素转运功能，提高口服透析效能。为实现这一目标，我们首先克隆了SD大鼠UT-B的CDS全长基因并构建了含大鼠UT-B基因的真核表达载体和重组腺病毒载体，以真核表达载体体外转染结肠上皮细胞株 Caco-2，分别采用RT-PCR、Western blot 检测UT-B mRNA 和蛋白的表达，并以14C同位素标记法检测 Caco-2 细胞尿素转运功能。结果发现，重组腺病毒载体转染Caco-2细胞后，Caco-2细胞中UT-B mRNA和蛋白的表达明显增高，同时尿素转运效能明显增强。为观察UT-B基因转染对肾功能的影响，我们采用5/6肾切除大鼠肾功能不全动物模型，经肠道给予重组腺病毒载体，以不含UT-B的腺病毒空载体为对照，检测大鼠肠组织UT-B mRNA和蛋白的表达及血中BUN和Cr水平。结果发现，大鼠5/6肾切除8周后血中BUN和Cr明显增高，经肠道给予重组腺病毒载体后，大鼠肠组织UT-B mRNA和蛋白的表达明显增强，而血中BUN明显降低，Cr也有一定程度下降。本研究经体外和体内实验证实，UT-B基因转染可明显提高肠粘膜上皮细胞UT-B mRNA和蛋白的表达并能提高尿素转运效能，降低血中BUN和Cr，改善肾功能，为肾功能不全的基因治疗提供了理论依据。