白藜芦醇诱导人卵巢癌SKOV-3细胞凋亡机制的研究
发布时间：2018-09-10 06:13
【摘要】：研究背景： 卵巢癌是女性生殖系统常见的三大恶性肿瘤之一，其高死亡率位居妇科恶性肿瘤的首位。在引起女性死亡最主要10类恶性肿瘤中，卵巢癌排在第5位，年死亡率大约在9.5/10万[1]。严重威胁着女性的生命与健康。目前卵巢癌的治疗方案是以首选手术治疗，，术后再辅以相应的化学药物治疗或放射治疗为常规治疗方案。但由于化疗药物本身或其溶媒显著的毒副作用、化疗价格昂贵以及原发性与获得性耐药等原因，化学治疗往往很难达到理想的效果。肿瘤细胞对化疗药物的耐药性严重影响了卵巢癌患者的化疗效果及生存质量，使得患者的5年生存率大大下降，是造成治疗失败的主要原因之一[2]。因此，挖掘新的、低毒、高效的抗癌药物或化疗增敏药物是改善卵巢癌预后的关键。 白藜芦醇（Resveratrol,Res）是一种含有芪类结构的非黄酮类多酚化合物，广泛存在于葡萄科、百合科、豆科、蓼科等70多种植物中，具有抗炎、抗菌、抗氧化、抗病毒、抗动脉粥样硬化、免疫调节及神经保护等多种生物学功能，而其中最重要的药理作用则是白藜芦醇可以通过不同途径对肿瘤细胞的生长起到促进凋亡和抑制增殖的作用。据报道白藜芦醇对肝癌、卵巢癌、白血病、喉鳞癌、乳腺癌、前列腺癌、甲状腺癌和皮肤鳞癌等都有治疗作用[3]。白藜芦醇被认为是一种很有潜力的肿瘤抑制剂，国内外在白血病、乳腺癌、肝癌、肺癌、大肠癌、宫颈癌等肿瘤中研究较多，但对卵巢癌的相关研究报道甚少。因此，研究白藜芦醇对卵巢癌细胞增殖及凋亡的影响，探讨该凋亡的发生机制，有重要的临床价值，以期在卵巢癌的预防与临床治疗方面提供新的突破点。 目的： 研究白藜芦醇对人卵巢癌细胞SKOV-3的诱导凋亡作用，探讨该凋亡的发生机制。以期为白藜芦醇有望成为卵巢癌新的治疗药物提供靶点和理论依据。 方法： 1、细胞培养：以常规方法复苏人卵巢癌SKOV-3细胞，将其接种于含10%小牛血清的RPMI-1640培养基中，置于37℃含5%CO2的细胞培养箱中培养。2.5g/L胰蛋白酶和0.2g/L EDTA消化、传代； 2、细胞增殖抑制实验（MTT比色法）：用不同浓度的白藜芦醇复合培养液（400μmol/L、200μmol/L、100μmol/L、50μmol/L、25μmol/L），作用于人卵巢癌SKOV3细胞，通过MTT法检测白藜芦醇对人卵巢癌SKOV-3细胞增殖的影响； 3、透射电子显微镜：通过该仪器观察对照组、不同浓度的白藜芦醇组作用于卵巢癌SKOV-3细胞后，细胞超微结构的改变； 4、流式细胞仪分析：以不同浓度的白藜芦醇复合培养液作用于人卵巢癌SKOV-3细胞48小时，用流式细胞仪定量分析各组细胞周期和细胞凋亡率的变化情况； 5、RT-PCR法：以不同浓度的白藜芦醇复合培养液培养人卵巢癌SKOV-3细胞48小时，用RT-PCR法检测SKOV-3细胞Bcl-2和Bax的mRNA表达。 6、Westernblot法：以不同浓度的白藜芦醇复合培养液培养人卵巢癌SKOV-3细胞48小时，用Westernblot法检测SKOV-3细胞Bcl-2和Bax的蛋白表达。 结果： 1、不同浓度白藜芦醇对卵巢癌SKOV-3细胞均有抑制作用，并呈剂量时间依赖关系。 2、透射电镜下不同浓度的白藜芦醇作用的各组卵巢癌SKOV-3细胞均可见典型的凋亡细胞形态改变。 3、流式细胞仪检测结果表明：经不同浓度白藜芦醇作用48小时后，白藜芦醇诱导SKOV-3细胞凋亡的作用随着白藜芦醇浓度的增加，细胞凋亡率增高，且细胞周期发生明显变化：G0/G1期细胞百分比明显降低，S期细胞百分比上升，提示白藜芦醇通过抑制细胞DNA合成而诱导细胞发生凋亡。 4、RT-PCR检测Bcl-2和Bax的mRNA表达：不同浓度白藜芦醇（25、50、100、200、400μmol/L）作用48h后，RT-PCR检测发现SKOV-3细胞Bcl-2和Bax的mRNA表达呈阳性，并且Bcl-2mRNA条带密度随浓度的增加而递减，Bax mRNA条带密度随浓度的增加而增强。这一结果表明白藜芦醇可能通过下调Bcl-2的表达和上调Bax的表达而诱导细胞发生凋亡。 5、Westernblot检测Bcl-2和Bax的蛋白表达：分别用25、50、100、200、400μmol/L白藜芦醇作用SKOV-3细胞48h，Westernblot检测发现随白藜芦醇浓度的增加，Bcl-2蛋白带逐渐变细，Bax蛋白带逐渐变粗。结果表明，白藜芦醇可能通过下调Bcl-2的表达和上调Bax的表达而诱导细胞发生凋亡。 结论： 1、白藜芦醇能够抑制卵巢癌细胞SKOV-3的增殖并诱导其凋亡； 2、白藜芦醇可在S期阻断卵巢癌SKOV-3细胞的增殖； 3、白藜芦醇诱导人卵巢癌SKOV-3细胞凋亡的机制可能与抑制抗凋亡因子Bcl-2的表达，增强凋亡因子Bax的表达有关。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
本文编号：2233614
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
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