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作 者:史春雪[1,2] 常爽[1] 双婷[1] 接智慧[1] 吴建磊[1] 王敏[1]
机构地区:[1]中国医科大学附属盛京医院妇产科,辽宁沈阳110004 [2]沈阳市妇婴医院,辽宁沈阳110014
出 处:《中国实用妇科与产科杂志》2013年第2期142-145,共4页Chinese Journal of Practical Gynecology and Obstetrics
基 金:国家自然科学基金(编号30973189)
摘 要:目的探讨miR-17~92抑制质粒转染对紫衫醇耐药卵巢癌细胞的影响和作用机制,为卵巢癌耐药的研究提供理论依据。方法2011年中国医科大学附属盛京医院,病毒介导法转染miR-17~92抑制质粒,Real—timePCR及Western—Blot法检测转染前后miR-17—92及其靶基因表达,荧光素酶报告基因检测系统证明miR-17—92与靶基因3’uTR的直接结合作用。M33'比色法及流式细胞技术检测转染后耐药卵巢癌细胞增殖及细胞周期变化。结果耐药卵巢癌细胞SKOV3-TR30中miR-17~92高表达。转染miR-17—92-PTIP—Spongeall抑制质粒于耐药卵巢癌细胞抑制miR-17—92表达使细胞增殖受到抑制,紫杉醇浓度为100nM抑制作用最明显,抑制率为40.4%,P〈0.05。不同浓度紫杉醇作用于转染的耐药细胞,使细胞阻滞在G2/M期,当其作用浓度为100nM时阻滞作用更明显,P〈0.05。抑制miR-17~92表达且随着紫衫醇浓度的增加均可使BIM蛋白表达升高,P均〈0.05,PTEN蛋白的表达虽均呈上升趋势,但均无统计学意义。结论miR-17—92是与卵巢癌耐药最相关的miRNAs之-,抑制miR-17~92基因簇表达可以抑制耐药卵巢癌细胞的增殖,其影响卵巢癌耐药的机制是通过对BIM转录后水平的调节实现的。Objective Transduct miR-17 - 92 inhibitory plasmid to ovarian cancer drug resistance cells and investigate the relative mechanism to provide a theoretical basis on the formation of ovarian cancer drug resistance. Methods Shengjing Hospital Affiliated to China Medical University in 2011. With retroviral infection, paclitaxel resistant SKOV3- TR30 was transducted with the miR-17 - 92 inhibitory plasmid and miR-17 - 92 empty plasmid. Before and after the transduetion, the expression of miR-17-92 was validated by real-time PCR, PTEN and BIM were tested by Western-Blot. Luciferase reporter assays was applied to clarify the directly binding site of miR-17 - 92 to the 3 "UTR of target gene. MTI" assay and Flow Cytometry were used to detect drug-resistant ovarian cancer cell proliferation and cell cycle changes after transduction. Results MiR-17 - 92 is obvious overexpressed in SKOV3-TR30. Expression of miR-17 - 92 within SKOV3-TR30 was inhibited by the transduction of miR-17 - 92 inhibitory plasmid and cell proliferation was inhibi- ted, and the inhibitory effect of the paclitaxel with concentration of 100nM was the most obvious with 40.4% inhibition rate (P 〈 0.05 ). Paclitaxel with different concentration can cause cell arrest in the G2/M phase to SKOV3-TR30 which is transdueted with miR-17 - 92 inhibitory plasmid and this effect is most obvious when the concentration is 100nM( P 〈 0.05 ). Inhibiting the expression of miR-17 - 92 can cause overexpression of BIM, while the expression of PTEN protein is not affected. Conclusion Through overexpression of BIM instead of PTEN, inhibition of miR-17 - 92 expression in-hibit the proliferation of drug-resistance ovarian cancer cells and can cause cell arrest at the G2 / M phase.
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