罗勒多糖对卵巢癌SKOV3细胞血管内皮生长因子受体表达的调控作用及对其侵袭能力的影响

发布时间：2018-05-09 16:50

本文选题：卵巢癌 + 罗勒多糖　；参考：《山东大学》2014年硕士论文

【摘要】：背景与目的卵巢癌是女性生殖系统常见恶性肿瘤之一,其死亡率居于妇科恶性肿瘤的首位；肿瘤侵袭转移是致死的主要原因。目前手术、放疗及化疗仍是卵巢癌的主要治疗手段,但其疗效仍相对有限。部分传统中医药在恶性肿瘤治疗中展现出较好的疗效和独特的优势；本论文主要研究传统活血化瘀方成分之一的提取物罗勒多糖对卵巢癌SKOV3细胞侵袭能力的影响,并从罗勒多糖对血管内皮生长因子受体表达的调控作用这一角度探讨其可能的参与机制。方法Transwell侵袭试验研究、检测不同浓度罗勒多糖对SKOV3细胞侵袭能力的影响；酶联免疫吸附试验(ELISA)检测罗勒多糖对SKOV3细胞分泌血管内皮生长因子(VEGF)的影响,实时定量PCR (qRT-PCR)、流式细胞术(FCM)检测罗勒多糖对VEGF受体(VEGFRs)在SKOV3细胞表达的调控作用；设计针对VEGFR2/KDR的shRNA及阴性对照shRNA,并构建携带不同shRNA的pLL3.7慢病毒表达载体,采用293T细胞包装携带shRNA的慢病毒；慢病毒感染SKOV3细胞后,流式细胞术分选其中的GFP阳性细胞,qRT-PCR、流式细胞术验证shRNA对VEGFR2表达的敲减作用；Transwell试验检测稳定敲减VEGFR2在SKOV3细胞表达对罗勒多糖侵袭抑制能力的影响。结果Transwell侵袭试验表明罗勒多糖可显著抑制SKOV3细胞的体外侵袭能力,并具有一定的浓度依赖性；罗勒多糖并未影响SKOV3细胞VEGF分泌水平的表达,对VEGFR1、VEGFR3在SKOV3的表达也无显著影响,但可明显上调VEGFR2在SKOV3细胞mRNA水平和膜表面蛋白水平的表达；成功感染稳定表达针对VEGFR2/KDR shRNA的慢病毒后,qRT-PCR及流式细胞术检测结果表明VEGFR2在SKOV3细胞的表达显著下调；进一步研究发现罗勒多糖对敲减VEGFR2表达的SKOV3细胞侵袭抑制能力显著降低。结论罗勒多糖这一传统中药提取物可显著抑制卵巢癌细胞SKOV3细胞的侵袭能力,其抑制作用部分是通过上调VEGFR2在SKOV3细胞的表达介导的。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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