中文摘要：

目的探讨高渗刺激下渗透压反应增强子结合蛋白（OREBP）对热休克蛋白（HSP）70的转录调控及对黏液分泌的影响。方法采用体外培养的人气道上皮细胞HBE16为实验对象，分时程施加高渗条件，Western印迹检测OREBP和HSP70—2蛋白水平，ELISA检测培养上清黏蛋白（MUC）5AC分泌；进一步构建系列截短和定点突变的HSP70-2启动子荧光素酶报告基因质粒，将其与OREBP的小片段干扰RNA（siRNA）共转染HBE16细胞，求证渗透压反应增强子（ORE）在HSP70-2基因启动子上游的结合位点及对MUC5AC分泌的影响。结果600mOsm／L高渗盐水培养HBE16细胞3h后，OREBP、HSP70-2和培养上清MUC5AC蛋白相对含量分别为（0．54±0．07，0．20±0．08，0．17±0．04），与对照组（0．21±0．05，0．15±0．06，0．13±0．04）相比显著增加到2．57、1．33和1．31倍（均P〈0．05），且随高渗培养时间延长而增加，6h时分别增加到2．81、3．07、3．77倍（均P〈0．01），9h时分别增加到3．57、5．13、4．00倍（均P〈0．01），12h时分别增加到4．24、5．33、4．54倍（均P〈0．01）；siRNA抑制OREBP表达48h后，高渗刺激下的OREBP、HSP704和上清MUC5AC蛋白相对含量为（0．36±0．08，0．33±0．08，0．24±0．05），和对照组（0．95±0．27，0．75±0．22，0．58±0．22）相比显著减少（蛋白抑制率分别为62％、56％和59％）（P〈0．05，P〈0．01，P〈0．05）；荧光素酶测定表明HSP70—2启动子上游-353至-66区域存在一个关键的ORE位点；定点突变失活-93区域的ORE位点可引起高渗诱导条件下HSP70-2启动子的活性显著降低（P〈0．01）。结论高渗盐水诱导OREBP通过与HSP704启动子上-93位ORE结合而激活HSP70—2转录并促进HBE16细胞MUC5AC高分泌。

英文摘要：

Objective To investigate the transcriptioncal regulation of osmotic response element binding protein （OREBP） to heat shock proteins （HSP）70 and its effect on mucus secretion under hypertonic conditions. Methods Human bronchial epithelial HBE16 cells were cultured in vitro in hypertonic medium for different times. Western blot was used to analyze the levels of OREBP and HSP70-2 protein. The mucin （MUC）SAC protein content in supernatant were detected by enzyme linked immunosorbent assay （ELISA）. Different deletions and mutants of HSP70-2 promoter in upstream region were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with OREBP siRNA and the promoter activities detected with dual luciferase assay to study the ORE site in the HSP70-2 promoter and its impact on MUC5AC. Results Compared with control group （0. 21 ± 0. 05, 0. 15 ±0. 06, 0. 13 ± 0.04）, the levels of OREBP, HSP70-2 and MUC5AC in supernatant significantly increased （0. 54 ± 0. 07, 0. 20 ± 0. 08, 0. 17 ± 0. 04） after HBE16 cells were exposed to 600 mOsm/L hypertonic mediums for 3 h （2. 57, 1.33 and 1.31 fold in protein respectively） （all P 〈 0. 05）, and their expression contents increased in＇a time-dependent manner, for 6 h （2. 81, 3.07 and 3.77 fold） （ all P 〈 0. 01 ）, for 9 h （3.57, 5.13 and 4. 00 fold） （ all P 〈 0. 01 ）, for 12 h （4. 24, 5.33 and 4. 54 fold） （ all P 〈 0. 01 ）. After a knock-down of OREBP by RNAi for 48 h, the levels of OREBP, HSPT0-2 and MUCSAC significantly decreased （0. 36 ± 0.08 ;0. 33 ±0. 08 ; 0. 24 ± 0. 05 ） versus the control group （0. 95 ±0. 27, 0. 75 ± 0. 22, 0. 58 ±0.22） （protein inhibition ratio at 62%, 56% and 59% respectively） （P 〈 0. 05, P 〈 0. 01, P 〈 0. 05 ）. Luciferase assay indicated that an important ORE site in HSPT0-2 promoter was in the region from -353 to -66. The inactivation of ORE site at -93 by site-directed mutagenesis led to a complete loss of HSPT0-2 promoter activity （