疏肝益肾方对TAM耐药乳腺癌HER2-MAPK-ERα通路的作用

发布时间：2018-05-18 03:14

本文选题：MCF-7细胞 + 耐药　；参考：《北京中医药大学》2014年硕士论文

【摘要】：乳腺癌是女性最常见的恶性肿瘤,近年来,中国女性乳腺癌发病率有明显的上升,且呈现年轻化趋势。随着现代手术、化疗、放疗、内分泌治疗、靶向治疗及生物治疗技术的进步,乳腺癌的死亡率呈下降趋势。每年,在中国约有50%,国外约有70%的乳腺癌患者,激素受体表达呈阳性。目前,拮抗雌激素受体(三苯氧胺)的内分泌治疗依然是雌激素受体阳性(ER+)乳腺癌的重要治疗策略。然而,经过最初有效的治疗后,最后仍约有1/4患者会出现不可避免的耐药而导致治疗无效,出现复发转移甚至死亡。耐药严重影响了乳腺癌的远期疗效。因此探讨乳腺癌内分泌治疗耐药的相关机制及延迟／逆转乳腺癌内分泌治疗耐药的有效方法,已引起越来越多学者的关注,成为当今肿瘤研究的热点之一。目前的资料强烈支持HER2/ERα/EGFR/SRC的交互作用及其下游基因MAPK的通路的活化是耐药产生的重要机制,ER α在耐药链中起着核心作用,蛋白激酶及其磷酸化在耐药过程中也发挥着重要作用。课题组前期实验结果显示,疏肝益肾方联合内分泌治疗可以提高内分泌治疗的疗效,推迟进行化疗的时间,减轻潮热、骨痛等副作用,延长无进展生存期,与内分泌耐药最相关的LuminalB患者的受益提示该药可能会对HER2有作用。体外研究结果显示：疏肝益肾方对MCF-7TAM耐药细胞的增殖、迁移力有抑制作用,与耐药相关的基因芯片分析现示：疏肝益肾方逆转MCF-7TAM耐药细胞耐药的机制可能与抑制MAPK通路有关,但与MAPK通路的哪条通路关系最为密切,又与HER2、ERα有着怎么样的关系,需要进一步的研究。目的：研究疏肝益肾方对三苯氧胺耐药的乳腺癌细胞MCF-7HER2-MAPK-ERα通路的作用,并明确其发挥作用的机制。方法： 1.雌性大鼠卵巢去势模型的制备：雌性大鼠麻醉后,常规剃毛、消毒,于腰部脊柱两侧约卵巢位置,做2个纵行切口,分离肌肉、脂肪、大肠等组织,结扎并剪下被白色脂肪团包裹的鲜红色颗粒状卵巢,并缝合术区。 2.制备含药血清：将雌性大鼠去势后,分别用疏肝益肾方高剂量水煎剂、TAM(三苯氧胺)溶液、疏肝益肾高剂量水煎剂+三苯氧胺(等效剂量)溶液、生理盐水(空白)灌胃,制备实验用大鼠含药血清及空白血清。 3.MTT法观察疏肝益肾方组、TAM组、联合组的含药血清及空白组大鼠血清对野生型MCF-7WT细胞和耐药型乳腺癌MCF-7LCC9细胞体外增殖的抑制作用。 4Wound healing法测定疏肝益肾方组、TAM组、联合组的含药血清及空白组大鼠血清对野生型MCF-7WT细胞和耐药型乳腺癌MCF-7LCC9细胞的横向迁移能力的影响。 5.通过RT-PCR法观察疏肝益肾方组、TAM组、联合组的含药血清及空白组大鼠血清对耐药型乳腺癌MCF-7LCC9细胞HER2-MAPK-ERα通路相关基因mRNA表达量的影响。 6.通过Western blot法观疏肝益肾方组、TAM组、联合组的含药血清及空白组大鼠血清对耐药型乳腺癌MCF-7LCC9细胞HER2-MAPK-ER a通路相关蛋白表达量的影响。结果： 1.与空白组相比,疏肝益肾方、TAM、疏肝益肾方+TAM联合组均能抑制MCF-7WT细胞的增殖(p0.05),且疏肝益肾方+TAM联合组在48h时抑制作用最明显(p0.01)。与空白组相比,疏肝益肾方、疏肝益肾方+TAM联合组对MCF-7LCC9细胞增殖的抑制作用也是在48h时最显著(p0.05),以疏肝益肾方+TAM联合组抑制作用为优,但与疏肝益肾方组相比,抑制作用不显著(p0.05),而TAM组对MCF-7LCC9细胞的抑制作用不明显(p0.05)。 2.除TAM组对MCF-7LCC9细胞的抑制作用不甚明显外(p0.05),疏肝益肾方、疏肝益肾方+TAM联合组对MCF-7LCC9细胞迁移力有不同程度的抑制作用,且作用48h时,抑制作用最显著(p0.05)。疏肝益肾方、TAM、疏肝益肾方+TAM联合组,对MCF-7WT细胞迁移力均有一定的抑制作用(p0.05)。 3RT-PCR结果显示：与MCF-7WT细胞相比,MCF-7LCC9耐药细胞的ESR1(ERα) mRNA表达量降低(p0.05),ERBB2(HER2) mRNA表达量轻微上升(p0.05),MAPK3(ERK1/2)、MAPK14(P38mapk) mRNA表达量上升(p0.05)；疏肝益肾方、疏肝益肾方+TAM联合组作用后,ESR1mRNA表达量显著上升(p0.05),疏肝益肾方+TAM联合组较疏肝益肾方上升多,但无显著差异(p0.05)；ERBB2、MAPK3、MAPK14mRNA表达量显著下降(p0.05),疏肝益肾方+TAM联合组较疏肝益肾方下降多,但无显著差异(p0.05)。而MAPK9(JNK/SAPK) mRNA表达量各组之间变化不明显,无显著差异(p0.05)。 4western blot结果显示：与MCF-7WT细胞相比,MCF-7LCC9耐药细胞的ERα的蛋白含量降低(p0.05),HER-2的蛋白表达轻微上升(P0.05),p-p44/42mapk(磷酸化ERK1/2)、p-p38MAPK(磷酸化p38MAPK)的蛋白含量升高显著(p0.05),p44/42mapk (ERK1/2)、p38MAPK (p38MAPK)的蛋白含量升高不显著(P0.05)；在疏肝益肾方、疏肝益肾方+TAM联合组作用后ER α的蛋白含量显著上升(p0.05),HER-2、p-p44/42mapk、p-p38MAPK的蛋白含量显著降低(p0.05),p44/42mapk、 p38mapk的蛋白含量降低不显著(P0.05)。疏肝益肾方+TAM联合组作用较疏肝益肾方作用显著,但二者相比,P0.05,无统计学意义。JNK变化不显著(P0.05)。结论： 1.疏肝益肾方具有抑制MCF-7WT、MCF-7LCC9的增殖能力、迁移能力的作用,且疏肝益肾方+TAM联合组抑制作用最强,表明疏肝益肾方可以增强TAM的细胞毒作用,且对TAM耐药性产生延迟／逆转作用。 2.疏肝益肾方可以提高三苯氧胺耐药乳腺癌细胞MCF-7LCC9的ER α的表达,降低HER-2、ERK1/2MAPK、P38MAPK在细胞中的表达,而对JNK/SAPK的作用不是很显著。实验结果表明：疏肝益肾方通过下调HER-2基因的表达,抑制MAPK通路中ERK1/2MAPK、P38MAPK两条通路,来提高细胞对乳腺癌MCF-7LCC9细胞ERα的表达,起到逆转耐药的作用。
[Abstract]:Breast cancer is the most common malignant tumor in women . In recent years , the incidence of breast cancer in Chinese women has increased significantly , and there is a tendency to decrease the mortality of breast cancer .

Objective : To study the role of Shugan Yiren Recipe on MCF - 7HER2 - MAPK - ER伪 pathway in breast cancer cell line with tamoxifen resistance and to clarify its mechanism .

Method :

1 . Preparation of ovarian castration model in female rats : After the female rats were anesthetized , regular shaving and disinfection were performed on both sides of the lumbar spine . Two longitudinal incisions were made to separate tissue such as muscle , fat , large intestine and the like , ligated and cut off the fresh red granular ovary surrounded by white fat mass , and the operation area was sutured .

2 . Preparation of drug - containing serum : After the female rats were removed , the serum and blank serum of the drug - containing serum and blank serum of the experimental rats were prepared by dispersing stagnated liver and kidney - side high - dose water decoction , TAM ( tamoxifen ) solution , dispersing stagnated liver - benefiting kidney high - dose water decoction + tamoxifen ( equivalent dose ) solution and physiological saline ( blank ) .

3 . MTT assay was used to observe the inhibition of the proliferation of wild type MCF - 7WT cells and drug - resistant breast cancer MCF - 7LCC9 cells by MTT assay .

4Wound healing method was used to determine the effect of serum on cross - migration of wild - type MCF - 7WT cells and MCF - 7LCC9 cells .

5 . The expression of HER2 - MAPK - ER伪 in MCF - 7LCC9 cells of breast cancer MCF - 7LCC9 cells was investigated by RT - PCR .

6 . The effects of Western blot on the expression of HER2 - MAPK - ER a pathway in MCF - 7LCC9 cells of breast cancer MCF - 7LCC9 cells were investigated by Western blot .

Results :

1 . Compared with the blank group , the inhibition of the proliferation of MCF - 7WT cells was inhibited by the combined group of Shugan YiShen Fang , TAM , Shugan and Shenfang + TAM ( P < 0.01 ) .

2 . In addition to TAM group , the inhibition of MCF - 7 LCC9 cells was not significantly inhibited ( p < 0.05 ) .

Compared with MCF - 7WT cells , the expression of ESR1 ( ER伪 ) mRNA in MCF - 7LCC9 - resistant cells decreased ( P < 0.05 ) , and the expression of ERBB2 ( HER2 ) mRNA increased slightly ( P < 0.05 ) , and the mRNA expression level of MAPK3 ( 1 / 2 ) and MAPK14 ( P38mapk ) increased ( p < 0.05 ) .
The expression of ESR1mRNA was significantly increased ( p < 0.05 ) after the combined action of Shugan YiShen Fang and Shugan Yi Shen Fang + TAM combined group , but there was no significant difference ( p < 0.05 ) .
The expressions of ERBB2 , MAPK3 and MAPK14 mRNA were significantly decreased ( p < 0.05 ) , but there was no significant difference between the two groups ( p < 0.05 ) .

Compared with MCF - 7WT cells , the protein content of MCF - 7LCC9 - resistant cells decreased ( P0.05 ) , the protein expression of HER - 2 increased slightly ( P0.05 ) , the protein content of p - p38MAPK ( phosphorylated p38MAPK ) increased significantly ( p0.05 ) , p44 / 42mapk ( 1 / 2 ) and p38MAPK ( p38MAPK ) increased significantly ( P0.05 ) ;
The protein content of ER 伪 increased significantly ( P < 0.05 ) , HER - 2 , p - p44 / 42mapk , p - p38MAPK decreased significantly ( P < 0.05 ) , p44 / 42mapk and p38mapk decreased significantly ( P0.05 ) .

Conclusion :

1 . The effect of dispersing stagnated liver and kidney - tonifying recipe on proliferation and migration of MCF - 7WT and MCF - 7LCC9 is the strongest , which indicates that Shugan - benefiting kidney can enhance the cytotoxicity of TAM and delay / reverse TAM drug resistance .

2 . The expression of ER伪 in breast cancer cell line MCF - 7LCC9 can be improved by soothing the liver and tonifying the kidney , and the expression of HER - 2 , 1 / 2 MAPK and P38MAPK in the cells was reduced . The results showed that the expression of the HER - 2 gene was downregulated by the Shugan YiShen prescription .
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R273

【参考文献】