【摘要】：目的探討miR-302a對(duì)葉酸缺乏小鼠胚胎干細(xì)胞(mESC)增殖和凋亡的影響。方法 mESC分為完全培養(yǎng)基組(對(duì)照組),無葉酸培養(yǎng)基組(無葉酸組),無葉酸培養(yǎng)基+miR-302amimic組(miR-302a組),通過RT-PCR進(jìn)行檢測miR-302a在完全培養(yǎng)基及無葉酸培養(yǎng)基中的表達(dá)。構(gòu)建miR-302amimic,后轉(zhuǎn)染到無葉酸培養(yǎng)基mESC中,采用MTT法檢測miR-302a mimic對(duì)mESC活力的影響,Annexin V-FITC/PI流式細(xì)胞術(shù)檢測miR-302amimic對(duì)mESC細(xì)胞凋亡的影響;流式細(xì)胞術(shù)檢測miR-302amimic對(duì)mESC細(xì)胞周期的影響;Western blot檢測及磷脂酰肌醇3羥激酶(PI3K)/蛋白激酶B(Akt)/哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)信號(hào)通路激活情況,以及下游分子細(xì)胞周期蛋白D1(CyclinD1)、p21、p27表達(dá)的影響。結(jié)果 RT-PCR證實(shí)無葉酸組中miR-302a表達(dá)量下降(P0.01)。與完全培養(yǎng)基比較,無葉酸培養(yǎng)基中,mESC細(xì)胞活力下降,細(xì)胞凋亡增加,細(xì)胞周期阻滯在G1期,Akt及mTOR磷酸化水平下降,CyclinD1表達(dá)下調(diào),p21及p27表達(dá)上調(diào),差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。與無葉酸組比較,miR-302a組中,mESC細(xì)胞活力上升,凋亡下降,G1期縮短,AKT及mTOR磷酸化水平提高,CyclinD1表達(dá)上調(diào),p21及p27表達(dá)下調(diào),差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論 miR-302a類似物能顯著抑制缺乏葉酸mESC凋亡,能促進(jìn)其增值,與PI3K/AKT/mTOR信號(hào)通路有關(guān)。
[Abstract]:Objective to investigate the effect of miR-302a on proliferation and apoptosis of embryonic stem cells (mESC) in folic acid deficient mice. Methods mESC was divided into complete medium group (control group), folic acid free medium group (folic acid free group) and folic acid free medium miR-302amimic group (miR-302a group). The expression of miR-302a in complete medium and folic acid free medium was detected by RT-PCR. MiR-302amimic, was constructed and transformed into folic acid free medium mESC. The effect of miR-302amimic on mESC activity was detected by MTT assay, the effect of miR-302amimic on apoptosis of mESC cells was detected by Annexin V-FITC/PI flow cytometry, and the effect of miR-302amimic on mESC cell cycle was detected by flow cytometry. Western blot was used to detect the activation of phosphatidylinositol 3-hydroxykinase (PI3K) / protein kinase B (Akt) / mammal rapamicin target protein (mTOR) signaling pathway, and the expression of downstream molecular cell cycle proteins D1 (CyclinD1), p21 and p27. Results RT-PCR confirmed that the expression of miR-302a in folic acid group decreased (P 0.01). Compared with the complete culture medium, the activity of mESC cells decreased, the apoptosis increased, the cell cycle arrest was in G 1 phase, the phosphorylation level of Akt and mTOR decreased, the expression of CyclinD1 was down-regulated, and the expression of p21 and p27 was up-regulated in folic acid-free medium, the difference was statistically significant (P 0.01). Compared with folic acid group, the activity of mESC cells was increased, apoptosis decreased, G 1 phase was shortened, AKT and mTOR phosphorylation level was increased, CyclinD1 expression was up-regulated, p21 and p27 expression was down-regulated in miR-302a group, the difference was statistically significant (P 0.01). Conclusion miR-302a analogues can significantly inhibit apoptosis of folic acid deficient mESC and promote its increment, which is related to PI3K/AKT/mTOR signaling pathway.
【作者單位】： 四川省醫(yī)學(xué)科學(xué)院/四川省人民醫(yī)院兒科;
【分類號(hào)】：R321

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