二氢丹参酮Ⅰ抑制卵巢癌增殖与转移作用的机制研究

发布时间：2018-04-21 00:26

本文选题：二氢丹参酮I + 卵巢癌　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景:卵巢癌是卵巢肿瘤的一种恶性肿瘤,其早期诊断比较困难,晚期不易治疗,死亡率极高。目前,治疗卵巢癌的有效手段是放疗、化疗和手术切除等。而化疗是目前治疗卵巢癌最广泛,最主要的治疗手段。临床常用的化疗药物为顺铂,顺铂通过血液循环到达全身的组织和器官,从而造成DNA损伤杀死癌细胞。但顺铂对人体正常细胞也会产生一系列的细胞毒性,破坏人体的免疫系统,此外,癌细胞会对顺铂产生耐药性,因此,寻找一种高效且对正常细胞没有毒性的抗卵巢癌药物对卵巢癌的治疗具有重要意义。二氢丹参酮I是丹参根茎的提取物,丹参是临床中最常用的活血化瘀的中药,相关文献报道丹参具有增加冠脉血流量、扩张冠状动脉、防止心肌缺血等心血管作用,以及用于治疗胃癌癌、肝癌、宫颈癌等多种疾病。二氢丹参酮I是丹参中最主要的抗肿瘤成分,并且二氢丹参酮I对人体正常细胞毒性较小,但关于二氢丹参酮I抗肿瘤的作用机制尚不明确。目的:本研究主要是检测二氢丹参酮I对卵巢癌的抑制作用机制,证明二氢丹参酮I抑制卵巢癌的增殖及转移,为临床治疗卵巢癌提供了新的策略。方法:1.采用MTT法检测体外培养的A2780和OV2008细胞的生长与增殖情况。2.采用划痕和侵袭实验检测二氢丹参酮I对A2780和OV2008细胞转移能力的影响。3.采用基因芯片技术检测A2780细胞经过二氢丹参酮I处理后的基因变化情况。4.根据目的基因PIK3CA的碱基序列,设计特异的过表达质粒及无关(NC)序列,通过lipofectamin2000脂质体法瞬时转染A2780细胞,获得PIK3CA表达上调的细胞模型,Western blot方法检蛋白表达情况。5.q PCR和Western blot方法检测细胞中目的基因的m RNA及蛋白质水平的表达情况。6.采用MTT法和克隆形成实验比较二氢丹参酮I对A2780细胞与上调PIK3CA的A2780细胞生长和增殖得变化情况。7.采用划痕和侵袭实验比较二氢丹参酮I对A2780细胞与上调PIK3CA的A2780细胞的转移能力。结果:1.MTT验证了二氢丹参酮I对A2780和OV2008细胞的增殖抑制作用,其抑制作用具有时间和剂量依赖关系。2.划痕与侵袭实验证明二氢丹参酮I对A2780与OV2008的转移具有抑制作用。3.基因芯片结果显示经二氢丹参酮I处理后A2780细胞中PIK3CA、DDB2、HGF等增殖与转移相关基因下调。4.利用脂质体瞬时转染技术,成功获得PIK3CA表达上调的细胞模型,PIK3CA表达上调组细胞受二氢丹参酮I后的生长和增殖抑制作用明显低于对照组细胞。5.PIK3CA表达上调组细胞受二氢丹参酮I后的转移抑制作用明显低于对照组细胞。结论:研究结果表明二氢丹参酮I通过下调PIK3CA的表达影响PI3K/AKT信号通路抑制卵巢癌细胞的增殖与转移。二氢丹参酮I治疗癌症具有潜在的临床意义。
[Abstract]:Background: ovarian cancer is a malignant tumor of ovarian tumor, its early diagnosis is difficult, the late stage is difficult to treat, the mortality rate is extremely high. At present, the effective treatment of ovarian cancer is radiotherapy, chemotherapy and surgical resection. Chemotherapy is the most extensive and main treatment for ovarian cancer. The commonly used chemotherapeutic drug is cisplatin, which passes through the blood circulation to the tissues and organs of the body, causing DNA damage to kill cancer cells. But cisplatin also produces a series of cytotoxicity to human normal cells, destroying the body's immune system. In addition, cancer cells develop resistance to cisplatin, so, It is important to find an effective anti-ovarian cancer drug with no toxicity to normal cells for the treatment of ovarian cancer. Dihydrotanshinone I is an extract from the rhizome of Salvia miltiorrhiza. Salvia miltiorrhiza is the most commonly used Chinese medicine for promoting blood circulation and removing blood stasis. It is reported that Salvia miltiorrhiza has cardiovascular effects such as increasing coronary blood flow, expanding coronary artery, preventing myocardial ischemia, etc. And for the treatment of gastric cancer, liver cancer, cervical cancer and other diseases. Dihydrotanshinone I is the most important antitumor component in Danshen, and the toxicity of dihydrotanshinone I to human normal cells is relatively small, but the mechanism of dihydrotanshinone I anti-tumor is unclear. Objective: to investigate the mechanism of dihydrotanshinone I inhibiting ovarian cancer, and to prove that dihydrotanshinone I inhibits the proliferation and metastasis of ovarian cancer and provides a new strategy for clinical treatment of ovarian cancer. Method 1: 1. The growth and proliferation of cultured A2780 and OV2008 cells were detected by MTT assay. The effect of dihydrotanshinone I on metastasis ability of A2780 and OV2008 cells was detected by scratch and invasion assay. The gene changes of A2780 cells treated with dihydrotanshinone I were detected by gene chip technique. According to the base sequence of the target gene PIK3CA, a specific overexpression plasmid and an unrelated sequence were designed. A2780 cells were transiently transfected with lipofectamin2000 liposome. A cell model with up-regulated expression of PIK3CA was obtained. The expression of protein was detected by Western blot. 5.q PCR and Western blot were used to detect the expression of m RNA and protein level of the target gene. 6. The effects of dihydrotanshinone I on the growth and proliferation of A2780 cells and PIK3CA up-regulated A2780 cells were compared by MTT assay and clone formation assay. The metastatic ability of dihydrotanshinone I on A2780 cells and PIK3CA up-regulated A2780 cells was compared by scratch and invasion assay. Results 1. The inhibitory effects of dihydrotanshinone I on the proliferation of A2780 and OV2008 cells were confirmed by MTT. The inhibitory effects were in a time-and dose-dependent manner. Scratch and invasion tests showed that dihydrotanshinone I inhibited the metastasis of A2780 and OV2008. The microarray results showed that the proliferation and metastasis related genes such as PIK3CAG DDB2HGF were down-regulated in A2780 cells treated with dihydrotanshinone I. Using liposome transient transfection technology, The growth and proliferation inhibitory effect of PIK3CA up-regulated cells after dihydrotanshinone I was significantly lower than that of control cells. 5. PIK3CA expression upregulation group was inhibited by dihydrotanshinone I metastasis. The effect was significantly lower than that in the control group. Conclusion: dihydrotanshinone I inhibits the proliferation and metastasis of ovarian cancer cells by down-regulating the expression of PIK3CA. Dihydrotanshinone I has potential clinical significance in the treatment of cancer.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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