腫瘤特異性Surivivin啟動(dòng)子調(diào)控的FAKshRNA靶向治療肝癌的實(shí)驗(yàn)研究

鐘寶元+劉清泉+鄧龍穎

【摘要】 目的：探討腫瘤特異性Surivivin啟動(dòng)子調(diào)控的FAK shRNA靶向治療肝癌的療效。方法：根據(jù)GenBank中Surivivin基因序列設(shè)計(jì)引物，在上下游引物5端引入相應(yīng)酶切位點(diǎn)，以HepG2基因組DNA為模板擴(kuò)增Surivivin啟動(dòng)子基因片段。體外合成最小polyA轉(zhuǎn)錄中止信號(hào)，以pSUPER.retro.puro載體為媒介構(gòu)建新的干涉載體pS-surP-shRNA-mpA（pS/surP），完成干涉靶點(diǎn)的選取并完成干涉載體的構(gòu)建和鑒定；建立細(xì)胞培養(yǎng)和轉(zhuǎn)染及穩(wěn)定轉(zhuǎn)染細(xì)胞系，完成FAK RNAi的干擾效應(yīng)性檢測(cè)，進(jìn)行體外細(xì)胞增殖及凋亡實(shí)驗(yàn)。結(jié)果：重組質(zhì)粒完成DNA提取后，PCR擴(kuò)增，經(jīng)瓊脂糖凝膠電泳后獲得1條預(yù)期大小的特異性皮帶，質(zhì)粒Surivivin啟動(dòng)子調(diào)控的FAK shRNA涉及符合要求。將目的片段與腫瘤特異性Surivivin啟動(dòng)子調(diào)控的FAK shRNA慢性載體表達(dá)載體連接的陽性克隆序列，獲得DNA序列證實(shí)含有目的序列片段，提示質(zhì)粒構(gòu)建成功；PCR儀結(jié)果顯示：Surivivin啟動(dòng)子調(diào)控的FAK shRNA在肝癌細(xì)胞中表達(dá)水平較低，抑制率能達(dá)到73.33%；Western blotting法結(jié)果顯示：轉(zhuǎn)染后，與未轉(zhuǎn)染的HepG2細(xì)胞相比，轉(zhuǎn)染后肝癌細(xì)胞Surivivin含量明顯下降；流式細(xì)胞儀結(jié)果表明：細(xì)胞轉(zhuǎn)染后，腫瘤特異性Surivivin啟動(dòng)子調(diào)控的FAK shRNA細(xì)胞凋亡率為24.39%，與未轉(zhuǎn)染細(xì)胞的4.12%比較，差異有統(tǒng)計(jì)學(xué)意義（P<0.05）。結(jié)論：腫瘤特異性Surivivin啟動(dòng)子調(diào)控的FAK shRNA靶向治療肝癌可明顯抑制腫瘤細(xì)胞的增殖，控制腫瘤的侵襲與轉(zhuǎn)移。

【關(guān)鍵詞】 腫瘤特異性； Surivivin啟動(dòng)； FAK shRNA； 靶向治療

【Abstract】 Objective：To investigate the therapeutic effects of tumor specific Surivivin promoter targeting FAK and shRNA on hepatocellular carcinoma.Method：Primers were designed according to the Surivivin gene sequence in GenBank，and the corresponding restriction sites were introduced at the 5th and the end of primer DNA.The promoter fragment of Surivivin was amplified by HepG2 genomic DNA template.In vitro synthesis of polyA transcription termination signal to the minimum，pSUPER.retro.puro vector mediated interference vector pS-surP-shRNA-mpA （pS/surP），a new interference target selection and complete the construction and identification of interference vector.Establish the cell culture and transfection and stable transfection cell lines，to complete the interference effect of detecting FAK RNAi，cell proliferation and apoptosis experiments in vitro.Result：After DNA extraction，the recombinant plasmid was amplified by PCR，and 1 expected size specific bands were obtained by agarose gel electrophoresis.FAK shRNA regulated by plasmid Surivivin promoter met the requirement.FAK shRNA chronic carrier of the fragment and tumor specific Surivivin promoter and expression of positive clone sequence vector，DNA sequence was confirmed with the objective fragment，suggesting that plasmid was successfully constructed.PCR instrument showed that Surivivin promoter FAK shRNA in HCC cells expression level is low，the inhibition rate can reach 73.33%.Western blotting method showed：after transfection，compared with untransfected HepG2 cells，transfected Surivivin cells was significantly decreased.Flow cytometry results showed that FAK cells after transfection，shRNA cell apoptosis of tumor specific Surivivin promoter was 24.39%，and there was significant difference compared with 4.12% of non transfected cells（P<0.05）.Conclusion：Tumor specific Surivivin promoter targeting FAK shRNA targeting HCC can significantly inhibit the proliferation of tumor cells and control tumor invasion and metastasis.endprint

【Key words】 Tumor specificity； Surivivin promoter； FAK shRNA； Targeted therapy

First-authors address：The First Affiliated Hospital of Gannan Medical College，Ganzhou 341000，China

doi：10.3969/j.issn.1674-4985.2017.29.007

原發(fā)性肝癌是臨床上常見的惡性腫瘤，而我國(guó)是原發(fā)性肝癌發(fā)病率較高的國(guó)家[1]。有報(bào)道顯示，我國(guó)每年惡性腫瘤發(fā)病率為35萬，每年死亡人數(shù)約32萬，嚴(yán)重影響我國(guó)居民健康與生活[2]。目前，臨床上對(duì)于原發(fā)性肝癌以手術(shù)、放化療及介入等治療為主，這些方法雖然能改善患者癥狀，但是長(zhǎng)期療效欠佳，不能從根本上改善患者的預(yù)后[3-4]。……