Guo Li-ping, Huang Hai-chang, Li Jing-zi
Department of Nephrology, Peking University First Hospital, Institute of Nephrology, Peking University, Beijing 100034, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2007 Feb 18;39(1):67-71.
To investigate whether hypoxia can affect the expression and secretion of connective tissue growth factor(CTGF) and fibronectin(FN) in primary cultured rat renal cortical myofibroblasts .
The primary cultured rat renal cortical myofibroblasts were subjected to hypoxic (1%O(2)) or normoxic (21% O(2)) conditions for a variety of times. The protein levels of HIF-1alpha, CTGF and FN protein were analyzed by Western blotting in both the whole cell lysates and supernatant culture medium 6 h, 12 h and 24 h after incubation, respectively. RT-PCR was carried out to measure the levels of FN mRNA at different time points (2 h,3 h,6 h and 12 h). The activity of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell medium was assayed by gelatin zymography.
The expression of HIF-1alpha was induced at h6 in cells under hypoxia incubation. The levels of cellular CTGF protein were increased in hypoxia treated myofibroblasts at h6 (175%+/-52%),significantly elevated at h12 (347%+/-67%, P<0.05 ) , and sustained the high levels by 24 h (143%+/-27%). The protein level of CTGF in supernatant culture medium reached 3.48 times higher than that in normoxic group of cells at h24 (348%+/-99% , P<0.05 ). The levels of secreted FN by myofibroblasts were elevated under hypoxia at h6 (187%+/-42%), h12 (199%+/-51%) and reached the peak level at h24 (210%+/-29%, P<0.05), whereas the levels of cellular FN was declined at the same time points. Furthermore, we found the expression of FN mRNA was increased in cells under hypoxia condition at h3, reached the peak level at h6(135%+/-13%, P<0.05), and then decreased to the comparable level of cells in normoxic group at h12. The activities of MMP-2 and MMP-9 in the supernatant cultured medium were not significantly changed along with the experimental time points.
Hypoxia could potentiate renal interstitial fibrosis through stimulating the expression and secretion of CTGF and FN in cultured cortical myofibroblasts.
研究缺氧是否能影响原代培养的大鼠肾皮质肌成纤维细胞中结缔组织生长因子（CTGF）和纤连蛋白（FN）的表达及分泌。
将原代培养的大鼠肾皮质肌成纤维细胞置于缺氧（1%O₂）或常氧（21%O₂）条件下不同时间。分别在孵育6小时、12小时和24小时后，通过蛋白质印迹法分析全细胞裂解物和上清培养基中HIF-1α、CTGF和FN蛋白的水平。进行RT-PCR以测量不同时间点（2小时、3小时、6小时和12小时）的FN mRNA水平。通过明胶酶谱法检测培养细胞培养基上清中明胶酶MMP-2和MMP-9的活性。
缺氧孵育6小时时，细胞中HIF-1α的表达被诱导。缺氧处理的肌成纤维细胞中细胞CTGF蛋白水平在6小时时升高（175%±52%），12小时时显著升高（347%±67%，P<0.05），并在24小时时维持在高水平（143%±27%）。在24小时时，上清培养基中CTGF的蛋白水平比常氧组细胞高3.48倍（348%±99%，P<0.05）。肌成纤维细胞分泌的FN水平在缺氧6小时（187%±42%）、12小时（199%±51%）时升高，并在24小时达到峰值（210%±29%，P<0.05），而细胞FN水平在相同时间点下降。此外，我们发现缺氧条件下细胞中FN mRNA的表达在3小时时增加，在6小时达到峰值（135%±13%，P<0.05），然后在12小时降至与常氧组细胞相当的水平。上清培养基中MMP-2和MMP-9的活性随实验时间点无明显变化。
缺氧可通过刺激培养的皮质肌成纤维细胞中CTGF和FN的表达及分泌来增强肾间质纤维化。
