本文選題：MCF-7細胞 + 耐藥��； 參考：《北京中醫(yī)藥大學》2014年碩士論文

【摘要】：乳腺癌是女性最常見的惡性腫瘤,近年來,中國女性乳腺癌發(fā)病率有明顯的上升,且呈現(xiàn)年輕化趨勢。隨著現(xiàn)代手術、化療、放療、內(nèi)分泌治療、靶向治療及生物治療技術的進步,乳腺癌的死亡率呈下降趨勢。每年,在中國約有50%,國外約有70%的乳腺癌患者,激素受體表達呈陽性。目前,拮抗雌激素受體(三苯氧胺)的內(nèi)分泌治療依然是雌激素受體陽性(ER+)乳腺癌的重要治療策略。然而,經(jīng)過最初有效的治療后,最后仍約有1/4患者會出現(xiàn)不可避免的耐藥而導致治療無效,出現(xiàn)復發(fā)轉移甚至死亡。耐藥嚴重影響了乳腺癌的遠期療效。因此探討乳腺癌內(nèi)分泌治療耐藥的相關機制及延遲／逆轉乳腺癌內(nèi)分泌治療耐藥的有效方法,已引起越來越多學者的關注,成為當今腫瘤研究的熱點之一。目前的資料強烈支持HER2/ERα/EGFR/SRC的交互作用及其下游基因MAPK的通路的活化是耐藥產(chǎn)生的重要機制,ER α在耐藥鏈中起著核心作用,蛋白激酶及其磷酸化在耐藥過程中也發(fā)揮著重要作用。課題組前期實驗結果顯示,疏肝益腎方聯(lián)合內(nèi)分泌治療可以提高內(nèi)分泌治療的療效,推遲進行化療的時間,減輕潮熱、骨痛等副作用,延長無進展生存期,與內(nèi)分泌耐藥最相關的LuminalB患者的受益提示該藥可能會對HER2有作用。體外研究結果顯示：疏肝益腎方對MCF-7TAM耐藥細胞的增殖、遷移力有抑制作用,與耐藥相關的基因芯片分析現(xiàn)示：疏肝益腎方逆轉MCF-7TAM耐藥細胞耐藥的機制可能與抑制MAPK通路有關,但與MAPK通路的哪條通路關系最為密切,又與HER2、ERα有著怎么樣的關系,需要進一步的研究。 目的：研究疏肝益腎方對三苯氧胺耐藥的乳腺癌細胞MCF-7HER2-MAPK-ERα通路的作用,并明確其發(fā)揮作用的機制。 方法： 1.雌性大鼠卵巢去勢模型的制備：雌性大鼠麻醉后,常規(guī)剃毛、消毒,于腰部脊柱兩側約卵巢位置,做2個縱行切口,分離肌肉、脂肪、大腸等組織,結扎并剪下被白色脂肪團包裹的鮮紅色顆粒狀卵巢,并縫合術區(qū)。 2.制備含藥血清：將雌性大鼠去勢后,分別用疏肝益腎方高劑量水煎劑、TAM(三苯氧胺)溶液、疏肝益腎高劑量水煎劑+三苯氧胺(等效劑量)溶液、生理鹽水(空白)灌胃,制備實驗用大鼠含藥血清及空白血清。 3.MTT法觀察疏肝益腎方組、TAM組、聯(lián)合組的含藥血清及空白組大鼠血清對野生型MCF-7WT細胞和耐藥型乳腺癌MCF-7LCC9細胞體外增殖的抑制作用。 4Wound healing法測定疏肝益腎方組、TAM組、聯(lián)合組的含藥血清及空白組大鼠血清對野生型MCF-7WT細胞和耐藥型乳腺癌MCF-7LCC9細胞的橫向遷移能力的影響。 5.通過RT-PCR法觀察疏肝益腎方組、TAM組、聯(lián)合組的含藥血清及空白組大鼠血清對耐藥型乳腺癌MCF-7LCC9細胞HER2-MAPK-ERα通路相關基因mRNA表達量的影響。 6.通過Western blot法觀疏肝益腎方組、TAM組、聯(lián)合組的含藥血清及空白組大鼠血清對耐藥型乳腺癌MCF-7LCC9細胞HER2-MAPK-ER a通路相關蛋白表達量的影響。 結果： 1.與空白組相比,疏肝益腎方、TAM、疏肝益腎方+TAM聯(lián)合組均能抑制MCF-7WT細胞的增殖(p0.05),且疏肝益腎方+TAM聯(lián)合組在48h時抑制作用最明顯(p0.01)。與空白組相比,疏肝益腎方、疏肝益腎方+TAM聯(lián)合組對MCF-7LCC9細胞增殖的抑制作用也是在48h時最顯著(p0.05),以疏肝益腎方+TAM聯(lián)合組抑制作用為優(yōu),但與疏肝益腎方組相比,抑制作用不顯著(p0.05),而TAM組對MCF-7LCC9細胞的抑制作用不明顯(p0.05)。 2.除TAM組對MCF-7LCC9細胞的抑制作用不甚明顯外(p0.05),疏肝益腎方、疏肝益腎方+TAM聯(lián)合組對MCF-7LCC9細胞遷移力有不同程度的抑制作用,且作用48h時,抑制作用最顯著(p0.05)。疏肝益腎方、TAM、疏肝益腎方+TAM聯(lián)合組,對MCF-7WT細胞遷移力均有一定的抑制作用(p0.05)。 3RT-PCR結果顯示：與MCF-7WT細胞相比,MCF-7LCC9耐藥細胞的ESR1(ERα) mRNA表達量降低(p0.05),ERBB2(HER2) mRNA表達量輕微上升(p0.05),MAPK3(ERK1/2)、MAPK14(P38mapk) mRNA表達量上升(p0.05)；疏肝益腎方、疏肝益腎方+TAM聯(lián)合組作用后,ESR1mRNA表達量顯著上升(p0.05),疏肝益腎方+TAM聯(lián)合組較疏肝益腎方上升多,但無顯著差異(p0.05)；ERBB2、MAPK3、MAPK14mRNA表達量顯著下降(p0.05),疏肝益腎方+TAM聯(lián)合組較疏肝益腎方下降多,但無顯著差異(p0.05)。而MAPK9(JNK/SAPK) mRNA表達量各組之間變化不明顯,無顯著差異(p0.05)。 4western blot結果顯示：與MCF-7WT細胞相比,MCF-7LCC9耐藥細胞的ERα的蛋白含量降低(p0.05),HER-2的蛋白表達輕微上升(P0.05),p-p44/42mapk(磷酸化ERK1/2)、p-p38MAPK(磷酸化p38MAPK)的蛋白含量升高顯著(p0.05),p44/42mapk (ERK1/2)、p38MAPK (p38MAPK)的蛋白含量升高不顯著(P0.05)；在疏肝益腎方、疏肝益腎方+TAM聯(lián)合組作用后ER α的蛋白含量顯著上升(p0.05),HER-2、p-p44/42mapk、p-p38MAPK的蛋白含量顯著降低(p0.05),p44/42mapk、 p38mapk的蛋白含量降低不顯著(P0.05)。疏肝益腎方+TAM聯(lián)合組作用較疏肝益腎方作用顯著,但二者相比,P0.05,無統(tǒng)計學意義。JNK變化不顯著(P0.05)。 結論： 1.疏肝益腎方具有抑制MCF-7WT、MCF-7LCC9的增殖能力、遷移能力的作用,且疏肝益腎方+TAM聯(lián)合組抑制作用最強,表明疏肝益腎方可以增強TAM的細胞毒作用,且對TAM耐藥性產(chǎn)生延遲／逆轉作用。 2.疏肝益腎方可以提高三苯氧胺耐藥乳腺癌細胞MCF-7LCC9的ER α的表達,降低HER-2、ERK1/2MAPK、P38MAPK在細胞中的表達,而對JNK/SAPK的作用不是很顯著。實驗結果表明：疏肝益腎方通過下調(diào)HER-2基因的表達,抑制MAPK通路中ERK1/2MAPK、P38MAPK兩條通路,來提高細胞對乳腺癌MCF-7LCC9細胞ERα的表達,起到逆轉耐藥的作用。
[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偽 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偽 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偽 ) 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 偽 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偽 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 .
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R273

【參考文獻】

相關期刊論文 前10條

1 甄林林;朱旬;鄭偉;王萱儀;武正炎;;表皮生長因子受體信號通路在MCF-7細胞對三苯氧胺產(chǎn)生耐藥中的作用[J];癌癥;2006年07期

2 孫莉娜;孟靜巖;孫曉霞;;中藥血清藥理學方法相關問題的思考[J];西部中醫(yī)藥;2012年11期

3 韓艷春;劉魯英;張騫;曹璋;;乳腺癌組織中VEGF-C和p38MAPK的表達及與伴淋巴結轉移的關系[J];中國組織化學與細胞化學雜志;2013年01期

4 萬芳,鐘剛,何福仙,陳敏,關艷梅;ERK/MAPK信號通路激活與乳腺癌細胞浸潤性生長的關系[J];醫(yī)學研究生學報;2004年06期

5 王玉華,趙謹瑤,葛常輝,楊佩滿;雄黃對乳腺癌MCF-7/ADM細胞多藥耐藥的逆轉及機制研究[J];解剖科學進展;2003年02期

6 隆霜;沈宜;謝瑩珊;范維珂;姜蓉;陳黎;;乳腺癌MDA-MB-231細胞源Exosomes對內(nèi)皮細胞增殖的影響及MAPK/ERK和PI3K/Akt信號通路的作用[J];解放軍醫(yī)學雜志;2012年11期

7 秦英剛;;花寶金教授以調(diào)肝健脾法治療乳腺癌經(jīng)驗[J];中醫(yī)學報;2013年01期

8 吳健宇,穆靜,李儀奎;血清藥理學方法體外實驗系統(tǒng)的血清添加量問題[J];上海中醫(yī)藥雜志;2001年09期

9 劉麗麗;劉艷娥;房國濤;;三七總皂苷逆轉乳腺癌細胞MCF-7/ADM多藥耐藥的實驗研究[J];時珍國醫(yī)國藥;2008年04期

10 王威;何永志;史紅;劉友剛;王超;鄭紡;楊琳;顧志敏;鮑麗穎;胡利民;;不同性別大鼠中藥含藥血清對成骨細胞增殖的影響[J];天津中醫(yī)藥;2009年01期

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