【摘要】：研究背景： 卵巢癌是女性生殖系統(tǒng)常見的三大惡性腫瘤之一，其高死亡率位居?jì)D科惡性腫瘤的首位。在引起女性死亡最主要10類惡性腫瘤中，卵巢癌排在第5位，年死亡率大約在9.5/10萬[1]。嚴(yán)重威脅著女性的生命與健康。目前卵巢癌的治療方案是以首選手術(shù)治療，，術(shù)后再輔以相應(yīng)的化學(xué)藥物治療或放射治療為常規(guī)治療方案。但由于化療藥物本身或其溶媒顯著的毒副作用、化療價(jià)格昂貴以及原發(fā)性與獲得性耐藥等原因，化學(xué)治療往往很難達(dá)到理想的效果。腫瘤細(xì)胞對(duì)化療藥物的耐藥性嚴(yán)重影響了卵巢癌患者的化療效果及生存質(zhì)量，使得患者的5年生存率大大下降，是造成治療失敗的主要原因之一[2]。因此，挖掘新的、低毒、高效的抗癌藥物或化療增敏藥物是改善卵巢癌預(yù)后的關(guān)鍵。 白藜蘆醇（Resveratrol,Res）是一種含有芪類結(jié)構(gòu)的非黃酮類多酚化合物，廣泛存在于葡萄科、百合科、豆科、蓼科等70多種植物中，具有抗炎、抗菌、抗氧化、抗病毒、抗動(dòng)脈粥樣硬化、免疫調(diào)節(jié)及神經(jīng)保護(hù)等多種生物學(xué)功能，而其中最重要的藥理作用則是白藜蘆醇可以通過不同途徑對(duì)腫瘤細(xì)胞的生長(zhǎng)起到促進(jìn)凋亡和抑制增殖的作用。據(jù)報(bào)道白藜蘆醇對(duì)肝癌、卵巢癌、白血病、喉鱗癌、乳腺癌、前列腺癌、甲狀腺癌和皮膚鱗癌等都有治療作用[3]。白藜蘆醇被認(rèn)為是一種很有潛力的腫瘤抑制劑，國內(nèi)外在白血病、乳腺癌、肝癌、肺癌、大腸癌、宮頸癌等腫瘤中研究較多，但對(duì)卵巢癌的相關(guān)研究報(bào)道甚少。因此，研究白藜蘆醇對(duì)卵巢癌細(xì)胞增殖及凋亡的影響，探討該凋亡的發(fā)生機(jī)制，有重要的臨床價(jià)值，以期在卵巢癌的預(yù)防與臨床治療方面提供新的突破點(diǎn)。 目的： 研究白藜蘆醇對(duì)人卵巢癌細(xì)胞SKOV-3的誘導(dǎo)凋亡作用，探討該凋亡的發(fā)生機(jī)制。以期為白藜蘆醇有望成為卵巢癌新的治療藥物提供靶點(diǎn)和理論依據(jù)。 方法： 1、細(xì)胞培養(yǎng)：以常規(guī)方法復(fù)蘇人卵巢癌SKOV-3細(xì)胞，將其接種于含10%小牛血清的RPMI-1640培養(yǎng)基中，置于37℃含5%CO2的細(xì)胞培養(yǎng)箱中培養(yǎng)。2.5g/L胰蛋白酶和0.2g/L EDTA消化、傳代； 2、細(xì)胞增殖抑制實(shí)驗(yàn)（MTT比色法）：用不同濃度的白藜蘆醇復(fù)合培養(yǎng)液（400μmol/L、200μmol/L、100μmol/L、50μmol/L、25μmol/L），作用于人卵巢癌SKOV3細(xì)胞，通過MTT法檢測(cè)白藜蘆醇對(duì)人卵巢癌SKOV-3細(xì)胞增殖的影響； 3、透射電子顯微鏡：通過該儀器觀察對(duì)照組、不同濃度的白藜蘆醇組作用于卵巢癌SKOV-3細(xì)胞后，細(xì)胞超微結(jié)構(gòu)的改變； 4、流式細(xì)胞儀分析：以不同濃度的白藜蘆醇復(fù)合培養(yǎng)液作用于人卵巢癌SKOV-3細(xì)胞48小時(shí)，用流式細(xì)胞儀定量分析各組細(xì)胞周期和細(xì)胞凋亡率的變化情況； 5、RT-PCR法：以不同濃度的白藜蘆醇復(fù)合培養(yǎng)液培養(yǎng)人卵巢癌SKOV-3細(xì)胞48小時(shí)，用RT-PCR法檢測(cè)SKOV-3細(xì)胞Bcl-2和Bax的mRNA表達(dá)。 6、Westernblot法：以不同濃度的白藜蘆醇復(fù)合培養(yǎng)液培養(yǎng)人卵巢癌SKOV-3細(xì)胞48小時(shí)，用Westernblot法檢測(cè)SKOV-3細(xì)胞Bcl-2和Bax的蛋白表達(dá)。 結(jié)果： 1、不同濃度白藜蘆醇對(duì)卵巢癌SKOV-3細(xì)胞均有抑制作用，并呈劑量時(shí)間依賴關(guān)系。 2、透射電鏡下不同濃度的白藜蘆醇作用的各組卵巢癌SKOV-3細(xì)胞均可見典型的凋亡細(xì)胞形態(tài)改變。 3、流式細(xì)胞儀檢測(cè)結(jié)果表明：經(jīng)不同濃度白藜蘆醇作用48小時(shí)后，白藜蘆醇誘導(dǎo)SKOV-3細(xì)胞凋亡的作用隨著白藜蘆醇濃度的增加，細(xì)胞凋亡率增高，且細(xì)胞周期發(fā)生明顯變化：G0/G1期細(xì)胞百分比明顯降低，S期細(xì)胞百分比上升，提示白藜蘆醇通過抑制細(xì)胞DNA合成而誘導(dǎo)細(xì)胞發(fā)生凋亡。 4、RT-PCR檢測(cè)Bcl-2和Bax的mRNA表達(dá)：不同濃度白藜蘆醇（25、50、100、200、400μmol/L）作用48h后，RT-PCR檢測(cè)發(fā)現(xiàn)SKOV-3細(xì)胞Bcl-2和Bax的mRNA表達(dá)呈陽性，并且Bcl-2mRNA條帶密度隨濃度的增加而遞減，Bax mRNA條帶密度隨濃度的增加而增強(qiáng)。這一結(jié)果表明白藜蘆醇可能通過下調(diào)Bcl-2的表達(dá)和上調(diào)Bax的表達(dá)而誘導(dǎo)細(xì)胞發(fā)生凋亡。 5、Westernblot檢測(cè)Bcl-2和Bax的蛋白表達(dá)：分別用25、50、100、200、400μmol/L白藜蘆醇作用SKOV-3細(xì)胞48h，Westernblot檢測(cè)發(fā)現(xiàn)隨白藜蘆醇濃度的增加，Bcl-2蛋白帶逐漸變細(xì)，Bax蛋白帶逐漸變粗。結(jié)果表明，白藜蘆醇可能通過下調(diào)Bcl-2的表達(dá)和上調(diào)Bax的表達(dá)而誘導(dǎo)細(xì)胞發(fā)生凋亡。 結(jié)論： 1、白藜蘆醇能夠抑制卵巢癌細(xì)胞SKOV-3的增殖并誘導(dǎo)其凋亡； 2、白藜蘆醇可在S期阻斷卵巢癌SKOV-3細(xì)胞的增殖； 3、白藜蘆醇誘導(dǎo)人卵巢癌SKOV-3細(xì)胞凋亡的機(jī)制可能與抑制抗凋亡因子Bcl-2的表達(dá)，增強(qiáng)凋亡因子Bax的表達(dá)有關(guān)。
[Abstract]:Research background:
Ovarian cancer is one of the three most common malignant tumors in the female reproductive system, and its high mortality rate ranks first in gynecological malignancies. Ovarian cancer ranks fifth in the 10 most common types of malignant tumors causing female deaths, with an annual mortality rate of about 95/100,000 [1]. It seriously threatens the lives and health of women. Surgical treatment is preferred, followed by chemotherapy or radiotherapy. However, due to the significant toxic and side effects of chemotherapy drugs or their mediators, the high cost of chemotherapy, and the primary and acquired drug resistance, chemotherapy is often difficult to achieve the desired results. Tumor cells are resistant to chemotherapy drugs. Drug-resistance seriously affects the chemotherapy effect and quality of life of ovarian cancer patients, and greatly reduces the 5-year survival rate of patients, which is one of the main causes of treatment failure.
Resveratrol (Res) is a non-flavonoid polyphenolic compound with stilbene structure. It is widely distributed in more than 70 species of plants such as Vitis, Liliaceae, Leguminosae and Polygonaceae. Resveratrol has many biological functions, such as anti-inflammatory, antibacterial, antioxidant, antiviral, anti-atherosclerosis, immune regulation and neuroprotection, among which the most important pharmacological actions are as pharmacology. Resveratrol has been reported to have therapeutic effects on liver cancer, ovarian cancer, leukemia, laryngeal squamous cell carcinoma, breast cancer, prostate cancer, thyroid cancer and skin squamous cell carcinoma [3]. Tumor inhibitors have been extensively studied in leukemia, breast cancer, liver cancer, lung cancer, colorectal cancer, cervical cancer at home and abroad, but few studies have been reported on ovarian cancer. Therefore, it is of great clinical value to study the effects of resveratrol on proliferation and apoptosis of ovarian cancer cells and to explore the mechanism of apoptosis in order to prevent ovarian cancer. It will provide new breakthroughs in clinical treatment.
Objective:
Objective To study the apoptosis-inducing effect of resveratrol on human ovarian cancer cell line SKOV-3 and to explore the mechanism of apoptosis.
Method:
1. Cell culture: Human ovarian cancer SKOV-3 cells were resuscitated by routine methods, inoculated into RPMI-1640 medium containing 10% calf serum, and cultured in a cell culture box containing 5% CO 2 at 37 C. 2.5g/L trypsin and 0.2g/L EDTA were digested and passaged.
2. Cell proliferation inhibition assay (MTT colorimetric method): Different concentrations of resveratrol (400 micromol/L, 200 micromol/L, 100 micromol/L, 50 micromol/L, 25 micromol/L) were used to treat human ovarian cancer SKOV3 cells, and the effects of resveratrol on the proliferation of human ovarian cancer SKOV-3 cells were detected by MTT method.
3. Transmission electron microscopy: The ultrastructural changes of ovarian cancer SKOV-3 cells treated with resveratrol at different concentrations were observed by this instrument.
4. Flow cytometry analysis: Human ovarian cancer SKOV-3 cells were treated with resveratrol at different concentrations for 48 hours, and the changes of cell cycle and apoptosis rate were quantitatively analyzed by flow cytometry.
5. RT-PCR: Human ovarian cancer SKOV-3 cells were cultured in different concentrations of resveratrol for 48 hours. The mRNA expressions of Bcl-2 and Bax in SKOV-3 cells were detected by RT-PCR.
6. Western blot: Human ovarian cancer SKOV-3 cells were cultured in different concentrations of resveratrol for 48 hours. The expression of Bcl-2 and Bax proteins in SKOV-3 cells was detected by Western blot.
Result:
1, different concentrations of resveratrol inhibited ovarian cancer SKOV-3 cells in a dosed time-dependent manner.
2. Typical morphological changes of apoptotic cells were observed in ovarian cancer SKOV-3 cells treated with different concentrations of resveratrol under transmission electron microscopy.
3. The results of flow cytometry showed that the apoptosis rate of SKOV-3 cells induced by resveratrol increased with the increase of resveratrol concentration 48 hours after resveratrol treatment, and the cell cycle changed significantly: the percentage of cells in G0/G1 phase decreased significantly, the percentage of cells in S phase increased, suggesting that resveratrol could induce apoptosis of SKOV-3 cells. Alcohol induces apoptosis by inhibiting cell DNA synthesis.
4. RT-PCR detection of Bcl-2 and Bax mRNA expression: 48 hours after resveratrol (25,50,100,200,400 micromol/L) treatment, RT-PCR detection found that SKOV-3 cells Bcl-2 and Bax mRNA expression was positive, and Bcl-2 mRNA band density decreased with the increase of concentration, Bax mRNA band density increased with the increase of concentration. Alcohol may induce apoptosis by downregulating the expression of Bcl-2 and upregulated Bax expression.
5. Western blot analysis of Bcl-2 and Bax protein expression: Resveratrol 25,50,100,200,400 micromol/L was used to treat SKOV-3 cells for 48 hours. Western blot analysis showed that with the increase of resveratrol concentration, the Bcl-2 protein band gradually thinned and the Bax protein band gradually thickened. The results showed that resveratrol may down-regulate the expression of Bcl-2 and up-regulate the expression of Bax. Induction of apoptosis.
Conclusion:
1, resveratrol can inhibit the proliferation and induce apoptosis of ovarian cancer cell SKOV-3.
2, resveratrol can inhibit the proliferation of ovarian cancer SKOV-3 cells in S phase.
3. The mechanism of resveratrol-induced apoptosis in human ovarian cancer SKOV-3 cells may be related to inhibiting the expression of anti-apoptotic factor Bcl-2 and enhancing the expression of apoptotic factor Bax.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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