【摘要】：研究目的：腫瘤的發(fā)生伴隨著癌基因的激活或者抑癌基因的失活，研究這些基因的功能，有助于腫瘤的靶向治療。ARHI是母源性腫瘤抑制印跡基因，在卵巢癌中失表達(dá)。本課題研究卵巢癌細(xì)胞過表達(dá)ARHI對(duì)細(xì)胞增殖的抑制作用及機(jī)制。 方法：本研究以質(zhì)粒pcDNA3.0-ARHI為模板PCR擴(kuò)增ARHI基因編碼序列，獲得重組質(zhì)粒所需目的基因，選用pIRES2-EGFP質(zhì)粒為載體，通過EcoRI和BamHI雙酶切及T4連接酶連接方法把擴(kuò)增的目的基因插入到真核生物載體pIRES2-EGFP的MCS區(qū)，兩者的粘性末端連接，并后續(xù)完成構(gòu)建后酶切、電泳及測(cè)序鑒定。QPCR法檢測(cè)不同卵巢癌細(xì)胞株中ARHI基因的表達(dá)情況，將pIRES2-EGFP-ARHI重組質(zhì)粒轉(zhuǎn)染至ARHI基因低表達(dá)的人卵巢癌細(xì)胞內(nèi)實(shí)現(xiàn)ARHI基因表達(dá)，并設(shè)立pIRES2-EGFP空載體轉(zhuǎn)染組和未轉(zhuǎn)染組細(xì)胞作為對(duì)照， CCK-8法檢測(cè)pIRES2-EGFP-ARHI重組質(zhì)粒表達(dá)對(duì)轉(zhuǎn)染細(xì)胞的生長(zhǎng)抑制率，流式細(xì)胞儀分析細(xì)胞周期分布并檢測(cè)細(xì)胞凋亡率，并用Western blotting檢測(cè)微管相關(guān)蛋白輕鏈Ⅱ（LC3-Ⅱ）、細(xì)胞內(nèi)MAPK/ERK1/2及JAK-STAT3信號(hào)轉(zhuǎn)導(dǎo)通路功能蛋白P-ERK和P-STAT3的表達(dá)水平變化。 結(jié)果：QPCR結(jié)果顯示所檢測(cè)卵巢癌細(xì)胞均出現(xiàn)不同程度的ARHI失表達(dá)，SKOV3細(xì)胞在所檢測(cè)卵巢癌細(xì)胞株中ARHI表達(dá)最低。SKOV3細(xì)胞經(jīng)不同處理后，，實(shí)驗(yàn)組細(xì)胞培養(yǎng)24h、48h、72h、96h及120h的生長(zhǎng)抑制率分別為64.69%、70.17%、67.01%、66.87%、67.70%，均明顯高于質(zhì)粒對(duì)照組（P㩳0.01）。培養(yǎng)48h時(shí)實(shí)驗(yàn)組、質(zhì)粒對(duì)照組、空白對(duì)照組S期細(xì)胞的比例分別為64.18%、38.43%及15.15%；凋亡率分別為47.97%、26.53%及9.33%；培養(yǎng)72h時(shí)實(shí)驗(yàn)組、質(zhì)粒對(duì)照組、空白對(duì)照組S期細(xì)胞的比例分別為43.95%、12.37%及10.89%；凋亡率分別為51.34%、20.55%及4.39%；實(shí)驗(yàn)組細(xì)胞培養(yǎng)48h時(shí)LC3-Ⅱ表達(dá)水平增加，而P-ERK和P-STAT3的表達(dá)水平下降，實(shí)驗(yàn)組與對(duì)照組間有明顯差異。 結(jié)論：ARHI基因在卵巢癌中失表達(dá)，過表達(dá)ARHI基因可以抑制SKOV3細(xì)胞生長(zhǎng)，使SKOV3細(xì)胞阻滯于S期，誘導(dǎo)細(xì)胞凋亡及自體吞噬。可能機(jī)制是ARHI抑制了ERK1/2和STAT3的磷酸化激活，阻斷了細(xì)胞增殖信號(hào)通路，從而誘導(dǎo)的SKOV3細(xì)胞凋亡。
[Abstract]:Objective: tumorigenesis is accompanied by activation of oncogenes or inactivation of tumor suppressor genes. Studying the function of these genes may be helpful to target therapy of tumor .ARHI is a maternal tumor inhibitory imprinting gene, which is inexpressed in ovarian cancer. The aim of this study was to investigate the inhibitory effect and mechanism of overexpression of ARHI on ovarian cancer cells. Methods: in this study, the coding sequence of ARHI gene was amplified by PCR using plasmid pcDNA3.0-ARHI as template, and the target gene of recombinant plasmid was obtained. PIRES2-EGFP plasmid was selected as vector. The amplified target gene was inserted into the MCS region of eukaryotic vector pIRES2-EGFP by EcoRI and BamHI double digestion and T4 ligase ligation. Electrophoretic and sequencing methods were used to detect the expression of ARHI gene in different ovarian cancer cell lines. PIRES2-EGFP-ARHI recombinant plasmid was transfected into human ovarian cancer cells with low ARHI gene expression to achieve ARHI gene expression. PIRES2-EGFP empty vector transfected cells and untransfected cells were used as control. CCK-8 assay was used to detect the growth inhibition rate of transfected cells by pIRES2-EGFP-ARHI recombinant plasmid expression. Flow cytometry was used to analyze cell cycle distribution and apoptosis rate. The expression levels of microtubule-associated protein light chain 鈪

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