本文選題：卵巢癌 + 羅勒多糖　； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：背景與目的卵巢癌是女性生殖系統(tǒng)常見惡性腫瘤之一,其死亡率居于婦科惡性腫瘤的首位；腫瘤侵襲轉(zhuǎn)移是致死的主要原因。目前手術(shù)、放療及化療仍是卵巢癌的主要治療手段,但其療效仍相對有限。部分傳統(tǒng)中醫(yī)藥在惡性腫瘤治療中展現(xiàn)出較好的療效和獨(dú)特的優(yōu)勢；本論文主要研究傳統(tǒng)活血化瘀方成分之一的提取物羅勒多糖對卵巢癌SKOV3細(xì)胞侵襲能力的影響,并從羅勒多糖對血管內(nèi)皮生長因子受體表達(dá)的調(diào)控作用這一角度探討其可能的參與機(jī)制。 方法Transwell侵襲試驗(yàn)研究、檢測不同濃度羅勒多糖對SKOV3細(xì)胞侵襲能力的影響；酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測羅勒多糖對SKOV3細(xì)胞分泌血管內(nèi)皮生長因子(VEGF)的影響,實(shí)時定量PCR (qRT-PCR)、流式細(xì)胞術(shù)(FCM)檢測羅勒多糖對VEGF受體(VEGFRs)在SKOV3細(xì)胞表達(dá)的調(diào)控作用；設(shè)計針對VEGFR2/KDR的shRNA及陰性對照shRNA,并構(gòu)建攜帶不同shRNA的pLL3.7慢病毒表達(dá)載體,采用293T細(xì)胞包裝攜帶shRNA的慢病毒；慢病毒感染SKOV3細(xì)胞后,流式細(xì)胞術(shù)分選其中的GFP陽性細(xì)胞,qRT-PCR、流式細(xì)胞術(shù)驗(yàn)證shRNA對VEGFR2表達(dá)的敲減作用；Transwell試驗(yàn)檢測穩(wěn)定敲減VEGFR2在SKOV3細(xì)胞表達(dá)對羅勒多糖侵襲抑制能力的影響。結(jié)果Transwell侵襲試驗(yàn)表明羅勒多糖可顯著抑制SKOV3細(xì)胞的體外侵襲能力,并具有一定的濃度依賴性；羅勒多糖并未影響SKOV3細(xì)胞VEGF分泌水平的表達(dá),對VEGFR1、VEGFR3在SKOV3的表達(dá)也無顯著影響,但可明顯上調(diào)VEGFR2在SKOV3細(xì)胞mRNA水平和膜表面蛋白水平的表達(dá)；成功感染穩(wěn)定表達(dá)針對VEGFR2/KDR shRNA的慢病毒后,qRT-PCR及流式細(xì)胞術(shù)檢測結(jié)果表明VEGFR2在SKOV3細(xì)胞的表達(dá)顯著下調(diào)；進(jìn)一步研究發(fā)現(xiàn)羅勒多糖對敲減VEGFR2表達(dá)的SKOV3細(xì)胞侵襲抑制能力顯著降低。 結(jié)論羅勒多糖這一傳統(tǒng)中藥提取物可顯著抑制卵巢癌細(xì)胞SKOV3細(xì)胞的侵襲能力,其抑制作用部分是通過上調(diào)VEGFR2在SKOV3細(xì)胞的表達(dá)介導(dǎo)的。
[Abstract]:Background & objective Ovarian cancer is one of the most common malignant tumors in female reproductive system. Surgery, radiotherapy and chemotherapy are still the main treatment for ovarian cancer, but the efficacy is still relatively limited. Part of traditional Chinese medicine has shown good curative effect and unique advantage in the treatment of malignant tumor. This paper mainly studied the effect of basil polysaccharide, one of the components of traditional Huoxue Huayu recipe, on the invasion ability of ovarian cancer SKOV3 cells. From the perspective of the regulation of basil polysaccharide on the expression of vascular endothelial growth factor receptor, the possible mechanism of its involvement was discussed. Methods Transwell invasion assay was used to detect the effect of basil polysaccharides at different concentrations on the invasiveness of SKOV3 cells, and the effects of basil polysaccharides on the secretion of vascular endothelial growth factor (VEGF) in SKOV3 cells were detected by Elisa assay. The regulation of basil polysaccharides on the expression of VEGF receptor VEGFRsin SKOV3 cells was detected by real-time quantitative PCR qRT-PCRG and flow cytometry, shRNA targeting VEGFR2/KDR and negative control shRNAs were designed, and pLL3.7 lentivirus expression vectors carrying different shRNA were constructed. The lentivirus carrying shRNA was packaged with 293T cells. After lentivirus was infected with SKOV3 cells, Flow cytometry (FCM) was used to detect the inhibitory effect of shRNA on VEGFR2 expression in SKOV3 cells. Flow cytometry was used to detect the effect of stable knockout VEGFR2 expression on the invasion inhibition of basil polysaccharides in SKOV3 cells. Results Transwell invasion test showed that basil polysaccharide could significantly inhibit the invasiveness of SKOV3 cells in vitro in a dose-dependent manner, and that basil polysaccharides did not affect the expression of VEGF secretion in SKOV3 cells, nor did it affect the expression of VEGFR1 and VEGFR3 in SKOV3. However, the expression of mRNA and membrane surface protein in SKOV3 cells could be upregulated significantly by VEGFR2, and the expression of VEGFR2 in SKOV3 cells was significantly down-regulated by QRT-PCR and flow cytometry after successful infection with stable expression of lentivirus against VEGFR2/KDR shRNA. Further studies showed that basil polysaccharides significantly inhibited the invasion of SKOV3 cells expressing knockout VEGFR2. Conclusion basil polysaccharide, a traditional Chinese medicine extract, can significantly inhibit the invasion of ovarian cancer cell line SKOV3, which is mediated in part by up-regulating the expression of VEGFR2 in SKOV3 cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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