低氧促进肌成纤维细胞表达和分泌结缔组织生长因子及纤维连接蛋白

Hypoxia induces the expression and secretion of connective tissue growth factor and fibronectin by cultured renal cortical myofibroblasts

目的:观察低氧对肾肌成纤维细胞合成和分泌结缔组织生长因子(connectivetissuegrowthfactor,CTGF)和细胞外基质纤维连接蛋白(fibronectin,FN)的影响,以了解肌成纤维细胞在肾间质纤维化过程中的作用。方法:(1)采用原代培养正常大鼠肾皮质肌成纤维细胞,以密闭的细胞培养罐造成低氧环境(1%O2,体积分数),应用Westernblot方法检测低氧和正常氧(21%O2,体积分数)条件下低氧诱导因子-1α(hypoxiainducedfactor-1α,HIF-1α)蛋白的表达,验证所营造的低氧条件是可靠的。(2)用Westernblot方法检测在低氧和正常氧条件下,各时间点上细胞及上清液中CTGF和FN蛋白水平。(3)用RT-PCR方法检测低氧和正常氧条件下,各时间点FNmRNA表达的变化。(4)用明胶酶谱法检测细胞上清液中基质金属蛋白酶-2(matrixmetalloprotei-nase-2,MMP-2)和基质金属蛋白酶-9(matrixmetalloproteinase-9,MMP-9)的活性。结果:正常氧组HIF-1α蛋白表达微弱,低氧组有较高水平HIF-1α蛋白表达。低氧作用于原代培养的肌成纤维细胞6h,12h和24h后细胞内的CTGF蛋白均比正常氧组增高,分别为175%±52%,347%±67%和143%±27%,以12h最明显(P<0.05);低氧刺激24h时上清液中CTGF蛋白显著增加,是正常氧组的348%±99%(P<0.05);低氧刺激肌成纤维细胞6h,12h和24h后上清液中FN蛋白水平增加,分别是正常氧组的187%±42%,199%±51%和210%±29%,以24h最明显(P<0.05),而对应时间点上清液中MMP-9和MMP-2活性无明显变化,但细胞内FN蛋白减少,低氧剌激6h为正常氧组的64%±13%。低氧刺激肌成纤维细胞3h,FN的mRNA水平开始上升,6h达到正常氧组的135%±13%(P<0.05),12h下降接近正常氧水平。结论:低氧可通过促进肌成纤维细胞表达CTGF和FN参与肾间质纤维化的进展。

Objective： 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 . Methods： The primary cultured rat renal cortical myofibroblasts were subjected to hypoxic （1% O2 ） or normoxic （21% 02 ） conditions for a variety of times. The protein levels of HIF - 1 α, 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. Results： The expression of HIF - 1 α was induced at h6 in ceils under hypoxia incubation. The levels of cellular CTGF protein were increased in hypoxia treated myofibroblasts at h6 （175% ± 52% ）, significandy 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 ceils 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 1124 （210% ± 2 9 % , P 〈 0.0 5 ） , 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 inthe supematant cultured medium were not significantly interstitial changed along with the experimental time points. Conclusion： Hypoxia could potentiate renal fibrosis through stimulating the expression and secretion of CTGF and FN in cultured cortical myofibroblasts.