【摘要】：目的:觀察體外高糖環(huán)境下大鼠近端腎小管上皮細(xì)胞脂聯(lián)素受體基因及其蛋白表達(dá)變化。方法:正常大鼠近端腎小管上皮細(xì)胞細(xì)胞株(NRK-52E)常規(guī)培養(yǎng)于含10%滅活新生胎牛血清的低糖DMEM培養(yǎng)液傳代培養(yǎng),待細(xì)胞生長(zhǎng)至亞融合狀態(tài)時(shí),更換為無胎牛血清低糖DMEM培養(yǎng)基培養(yǎng)24h使其同步化,將NRK-52E細(xì)胞分為5組:A組為正常對(duì)照組,低糖DMEM完全培養(yǎng)液(含D-葡萄糖5.5mmol/L);B組為高糖刺激12h組:高糖DMEM完全培養(yǎng)液(含D-葡萄糖25mmol/L)刺激12h;C組為高糖刺激24h組:高糖DMEM完全培養(yǎng)液刺激24h;D組為高糖刺激48h組:高糖DMEM完全培養(yǎng)液刺激48h;E組為高糖刺激72h組:高糖DMEM完全培養(yǎng)液刺激72h。然后分別提取各組總RNA和總蛋白,分別用RT-PCR檢測(cè)各組目的基因AdipoR 1/2mRNA的表達(dá),Western印跡法檢測(cè)各組目的蛋白AdipoR 1/2蛋白的表達(dá)。結(jié)果:脂聯(lián)素受體1(AdipoR1)和脂聯(lián)素受體2(AdipoR2)在NRK-52E細(xì)胞中均有表達(dá);在高糖刺激情況下,隨著刺激時(shí)間的延長(zhǎng),目的基因AdipoR 1/2mRNA的表達(dá)均有先增高后降低的趨勢(shì),在刺激24h時(shí)升高到最高點(diǎn),和對(duì)照組相比有統(tǒng)計(jì)學(xué)意義(P0.05);與正常對(duì)照組相比,高糖刺激后各時(shí)間點(diǎn)AdipoR 1/2蛋白的表達(dá)有上升的趨勢(shì),但差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:NRK-52E細(xì)胞AdipoR1/2蛋白的表達(dá)不受高糖刺激的影響;高糖影響NRK-52E細(xì)胞AdipoR1/2mRNA的表達(dá),并隨著刺激時(shí)間的延長(zhǎng)表現(xiàn)出先增高后降低。高糖通過影響脂聯(lián)素受體基因的表達(dá)從而影響脂聯(lián)素發(fā)揮其生物學(xué)作用,以致影響大鼠近端腎小管上皮細(xì)胞的功能。
[Abstract]:Aim: to observe the changes of adiponectin receptor gene and its protein expression in proximal tubular epithelial cells of rats under high glucose environment in vitro. Methods: normal proximal tubular epithelial cell line (NRK-52E) was cultured in low glucose DMEM medium containing 10% inactivated fetal bovine serum. The NRK-52E cells were divided into 5 groups (n = 5) as normal control group (n = 5), and cultured in the medium containing fetal bovine serum low glucose DMEM for 24 h, and NRK-52E cells were divided into 5 groups as normal control group. Low glucose DMEM complete culture medium (containing DMEM 5.5 mmol / L) group B was treated with high glucose stimulation for 12 h, high glucose DMEM complete culture medium (including D- glucose 25 mmol / L) for 12 h and high glucose stimulation group for 24 h; high glucose DMEM complete culture medium for 24 h; high glucose group for 48 h; Group C for 12 h; Group C for 24 h; Group D for 48 h; Group D for 24 h; High glucose DMEM complete culture medium was stimulated with high glucose for 72 h and high glucose DMEM for 72 h. The expression of AdipoR 1 / 2 mRNA was detected by RT-PCR and the expression of AdipoR 1 / 2 protein was detected by Western blot. Results: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) were expressed in NRK-52E cells. Compared with the control group, the expression of AdipoR 1 / 2 protein increased at 24 hours after high glucose stimulation (P0.05), but the difference was not statistically significant (P0.05). Conclusion the expression of AdipoR1 / 2 protein in NRK-52E cells was not affected by high glucose stimulation, but the expression of AdipoR1 / 2 mRNA in NRK-52E cells was increased first and then decreased with the prolongation of stimulation time. High glucose affects the biological function of adiponectin by affecting the expression of adiponectin receptor gene, thus affecting the function of proximal tubular epithelial cells in rats.
【作者單位】： 武漢大學(xué)中南醫(yī)院腎內(nèi)科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(編號(hào):163411)
【分類號(hào)】：R363

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