【摘要】：目的構(gòu)建穩(wěn)定過(guò)表達(dá)人表皮生長(zhǎng)因子受體2(HER2/ErbB2)基因的黑素瘤細(xì)胞株,研究其對(duì)黑素瘤細(xì)胞的增殖、侵襲和遷移的影響。方法將pCMV3-ErbB2(人源)重組質(zhì)粒通過(guò)脂質(zhì)體法轉(zhuǎn)染至黑素瘤B16細(xì)胞株,用潮霉素B篩選出耐藥陽(yáng)性克隆。實(shí)時(shí)定量PCR(qRT-PCR)檢測(cè)各單克隆ErbB2 mRNA相對(duì)表達(dá)量,免疫熒光技術(shù)檢測(cè)ErbB2蛋白的表達(dá)。MTT法檢測(cè)轉(zhuǎn)染后細(xì)胞增殖能力,TranswellTM實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力。結(jié)果實(shí)時(shí)定量PCR檢測(cè)結(jié)果表明,通過(guò)脂質(zhì)體法成功構(gòu)建過(guò)表達(dá)HER2的B16細(xì)胞株,其HER2的表達(dá)顯著高于空載體對(duì)照細(xì)胞。TranswellTM等實(shí)驗(yàn)顯示HER2過(guò)表達(dá)增強(qiáng)黑素瘤細(xì)胞增殖、侵襲和遷移的能力。結(jié)論過(guò)表達(dá)HER2促進(jìn)黑素瘤細(xì)胞的增殖、侵襲和遷移。
[Abstract]:Objective to construct a melanoma cell line stably expressing human epidermal growth factor receptor 2 (HER2/ErbB2) gene and study its effect on the proliferation, invasion and migration of melanoma cells. Methods pCMV3-ErbB2 recombinant plasmid was transfected into melanoma B16 cell line by liposome method, and hygromycin B was used to screen the drug resistant clones. The relative expression of each monoclonal ErbB2 mRNA was detected by real-time quantitative PCR (qRT-PCR), the expression of ErbB2 protein was detected by immunofluorescence, the proliferative ability of transfected cells was detected by MTT assay, the ability of cell invasion was detected by TranswellTM assay, and the ability of cell migration was detected by scratch assay. Results the results of real-time quantitative PCR analysis showed that the expression of HER2 in B16 cell line which overexpressed HER2 was significantly higher than that in blank vector control cells by liposome method. TranswellTM and other experiments showed that HER2 overexpression enhanced the proliferation of melanoma cells. The ability to invade and migrate. Conclusion overexpression of HER2 promotes the proliferation, invasion and migration of melanoma cells.
【作者單位】： 新鄉(xiāng)醫(yī)學(xué)院生命科學(xué)技術(shù)學(xué)院河南省高校合成生物改造工程與應(yīng)用重點(diǎn)實(shí)驗(yàn)室;
【基金】：河南省重大科技專(zhuān)項(xiàng)研究課題(161100310900) 新鄉(xiāng)醫(yī)學(xué)院研究生科研創(chuàng)新支持計(jì)劃(YJSCX201634Y)
【分類(lèi)號(hào)】：R739.5

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