中文摘要：

目的：探讨低氧能否激活成纤维细胞转化为肌成纤维细胞，以及低氧环境对肾皮质肌成纤维细胞表达Ⅰ型胶原（Col—Ⅰ）的影响及其可能的信号通路。方法：（1）采用正常大鼠肾成纤维细胞系（NRK-49F），应用Western blotting方法检测低氧诱导因子-1α（HIF-1α）的表达；比较低氧和正常氧条件下α-平滑肌肌动蛋白（α—SMA）的蛋白水平。（2）原代培养正常大鼠肾皮质肌成纤维细胞，Western blotting方法检测低氧和正常氧条件下，HIF—1α和Ⅰ型胶原蛋白水平以及ERKI／2的活化及其特异阻断剂PD98059的阻断效应；RT—PCR方法检测Ⅰ型胶原mRNA表达水平；细胞免疫化学法检测HIF-1α胞内表达部位的变化；明胶酶谱法检测细胞上清中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的活性。结果：（1）低氧刺激6h，NRK-49F细胞和肌成纤维细胞胞内和核内均有HIF-1α蛋白表达，核内更明显。（2）低氧培养12h，NRK-49F细胞表达α-SMA蛋白明显增高，是正常氧组的187％±32％（P〈0．05），验证了低氧可引起成纤维细胞表型转化。（3）低氧刺激原代培养的正常大鼠。肾皮质肌成纤维细胞6h、12h上清中Ⅰ型胶原蛋白水平增加，分别为正常氧组的171％±27％（P〈0．05）和256％±61％（P〈0．05）；低氧刺激4h、6h，Ⅰ型胶原mRNA表达增加，为正常氧组的189％±28％（P〈0．05）和221％±44％（P〈0．05）。（4）低氧刺激肌成纤维细胞6h、12h、24h，培养上清液中MMP-9、MMP-2活性无明显变化。（5）低氧刺激肌成纤维细胞15min即可使ERK1／2活化，阻断实验显示，PD98059可以使低氧12h引起Ⅰ型胶原增加（低氧刺激组为正常氧对照组的273％±51％，P〈0．05）显著减少（PD98059＋低氧组为正常氧组的108％±19％，P〉0．05）。结论：低氧促肾纤维化可能与其诱导肾成纤维细胞转化为肌成纤维细胞并?

英文摘要：

AIM： To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts, and associated signaling of hypoxia on the production of collagen Ⅰ in cultured rat renal cortical myofibroblasts. METHODS： The study is composed of two relevant parts. In the first part, a normal rat renal interstitial fibroblast cell line NRK -49F was treated with hypoxia （1% O2 ） or normoxia （21% O2 ） for 6 h, 12 h and 24 h. The expression of hypoxia inducible factor -1α （HIF - 1α） was examined by Western blotting in order to make sure the hypoxic condition is reliable. The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin （α-SMA）. In the second part, the object was done on the primary cultured rat renal cortical myofibroblasts. Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times. The levels of HIF - 1α in cell lysates and collagen I protein in supematant culture medium and the activation of extracellular signal - regulated ki- nase （ERK） 1/2 MAPK pathway were analyzed by Western blotting. RT - PCR was carried out to measure the levels of collagen Ⅰ mRNA at different time points （2 h, 4 h and 6 h）. The distribution of HIF - 1α in myofibroblasts was demonstrated by immunocytochemistry. The changes of collagen I production were detected after PD98059, a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation. The activity of gelatinase matrix metalloproteinase -2 （MMP-2） and MMP- 9 in the supernatant medium from the cultured cells were assayed by gelatin zymography. RE- SULTS ： Significant increased levels of HIF - 1 α protein appeared in cell lysates under hypoxia for 6 h. Furthermore, HIF - 1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia. The levels of α - SMA protein increased in NRK -49F under hypoxia for 12 h （ 187% ±32%, P 〈0. 05）. The level of collagen I pr