刚地弓形虫培养上清对卵巢癌细胞SKOV-3的影响
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摘要
研究背景及目的:卵巢癌是死亡率最高的妇科恶性肿瘤,及早发现可以有效提高病人生存率,但由于发病隐匿,无明显症状,往往病情会被忽视,因此,卵巢癌又称为“沉默杀手”。据统计,在100 000个妇女中,就有3.8位死于此病。虽然近20年来在卵巢癌治疗方面取得了长足进展,但均未能显著改善患者的长期生存率。因此,针对能有效抑制肿瘤细胞生长的新抗癌药的不断开发应用,是十分迫切的任务。目前抑制肿瘤细胞的生长主要通过两个方面:一方面可以通过改变细胞的周期,使其不能进行正常的有丝分裂,另外一方面可以通过促进细胞凋亡,通过改变凋亡相关蛋白表达水平来增加了肿瘤细胞凋亡率,抑制肿瘤细胞的增殖和集落形成能力,提高肿瘤细胞化疗药物和放疗的敏感性。
弓形虫是一种专性细胞内寄生的原虫,其寄生于宿主细胞过程中,虫体与细胞的生物学特性均会发生改变:研究二者间的联系,一直是国内外关注的焦点。业已证实的是弓形虫在寄生过程中可以抑制宿主细胞增值,引起细胞周期改变,并且诱导宿主细胞凋亡。本文利用液氮保种小鼠传代及CF-11纤维素纯化的方法提取制作弓形虫培养上清液,将其视为一种生物制剂在体外条件下人卵巢癌细胞SKOV-S细胞共培养,来观察弓形虫培养上清液对SKOV-S细胞生物学行为影响。
方法:
1.采用四甲基氮噻唑蓝(MTT)法检测空白对照组与刚地弓形虫培养上清处理组的吸光度(A490值)并按公式计算卵巢癌细胞SKOV-3细胞增殖抑制率。
2. PI染色后,上流式细胞仪技术检测空白对照组与刚地弓形虫培养上清处理组的细胞周期。
3. Annexin-v-FITC/PI染色后,上流式细胞仪技术检测空白对照组与刚地弓形虫培养上清处理组的细胞凋亡率。
4.荧光显微镜观察空白对照组与刚地弓形虫培养上清处理组的细胞在不同时项形态改变情况。
5.采用琼脂糖凝胶电泳观察培养上清处理组的凋亡DNA条带。
6.采用western blot,对培养上清处理前后,卵巢癌细胞SKOV-3细胞周期蛋白cyclinB1、cdc2及凋亡蛋白bcl-2及bax表达量进行检测。
结果:
1. MTT法检测结果显示卵巢癌细胞SKOV-3细胞增殖抑制率随着刚地弓形虫速殖子培养上清浓度和作用时间增加均明显增大,弓形虫速殖子培养试验组抑制率与对照组之间比较差异均有统计学意义(p﹤0.05)。
2.培养上清作用于卵巢癌细胞SKOV-3细胞48h后,对照组与3×107、6×107、12×107 / mL数量的弓形虫速殖子培养上清组的卵巢癌细胞SKOV-3细胞G2/M期细胞比例分别为(22.43±3.78)%、(35.56±5.22)%、(38.76±3.19)%、(44.95±4.74)%,以12×107 / mL数量组增加最为明显,差异具有显著性意义(n=5, p<0.05),提示培养上清具有诱导卵巢癌细胞SKOV-3细胞G2/M期停滞的作用,且有一定剂量依赖性。
3.以浓度为12×107 / mL弓形虫速殖子培养上清作用后24、36、48、72h后的卵巢癌细胞SKOV-3细胞用Annexin-v-FITC与PI双染后,FCM分析细胞凋亡,结果表明其凋亡率分别为15.23℅、16.12℅、18.23℅、17.64℅,与空白对照组0.64℅、0.83℅、1.56℅、2.67℅比较有显著差异(p﹤0.05)。
4.选择与数量为12×107 / ml弓形虫速殖子培养上清作用不同时间段的细胞置荧光显微镜下观察,可见2种细胞形态:早期凋亡细胞,细胞膜着绿色,核拒染;凋亡中晚期细胞与坏死细胞,核着红色,细胞膜着绿色,空白对照组的正常细胞不着色。
5.以浓度为12×107/ml弓形虫速殖子培养上清作用细胞不同时间后的琼脂糖凝胶电泳均出现了相差约180-200bp的典型的细胞凋亡的DNA梯形条带。
6.弓形虫速殖子培养上清作用前卵巢癌细胞SKOV-3细胞可见周期蛋白cyclinB1、cdc2与凋亡蛋白Bcl-2、Bax的表达的表达。上清作用48h后,与对照组相比,不同处理组的cyclinB1表达量(表达积分光密度值,下同)为0.86+0.04、0.73+0.06、0.60+0.05 ;cdc2蛋白表达量为0.98+0.02, 0.84+0.05、0.65+0.06;cyclinB1蛋白表达量为0.97+0.05, 0.81+0.02和0.78+0.05;cdc2蛋白表达量为1.23+0.03, 1.54+0.04、1.75+0.02,与对照组相比均有显著性差异(p<0.05),以12×107 / mL数量的弓形虫速殖子组最为明显(p﹤0.05)。
结论:弓形虫速殖子培养上清对体外培养卵巢癌细胞SKOV-3细胞增殖有明显的抑制作用,其途径可能与调节周期蛋白cyclinB1、cdc2等蛋白表达或活性改变引起细胞株G2/M期阻滞有关;同时,弓形虫速殖子培养上清还可通过上调凋亡相关蛋白Bax表达和下调Bcl-2表达来促使卵巢癌细胞SKOV-3细胞凋亡。
Background and Objective: Ovarian cancer remains the most lethal gynaecological cancer. Ovarian cancer causes non-specific symptoms. Early diagnosis would result in better survival. However, lack of symptoms or vague symptoms in the early stages of ovarian cancer is why the illness is known as the 'silent killer'. The crude death rate of ovarian cancer was 4.8 per 100 000 female population. The treatment methods of this cancer including surgery,chemotherapy, Radiation therapy.But all the methods have side effects, long-term use of patients with immune system and high-dose hematopoietic system, cause a lot of damage.The medicine with less side effects and high selectivity seems to be could improve the clinical efficacy.
Toxoplasma gondii is a species of parasitic protozoa in the genus.The focus of recent reserch is on the factors of invasion of host cell by Toxoplasma and virulence. Now, it is proved that Toxoplasma gondii can inhibite host cell proliferation, affect the cell apoptosis and cell cycle.
In this study, we try to investigate the inhibitive effects of culture supernatants of T. gondii as a biological agent on the proliferation, apoptosis and cell cycle of human ovarian cancer cell line SKOV-3.
METHODS:
1. Growth inhibition rates of treated groups were measured with the MTT method.
2. FCM was used to test the rates of apoptosis of treated groups by culture supernatants of T. gondii with the fluorescent dye of PI.
3. FCM was used to test Cell cycle of treated groups by culture supernatants of T. gondii with the fluorescent dye of Annexin-v-FITC/PI.
4. After the treatment of SKOV-3 cells by culture supernatants of T. gondii, morphological change of apoptotic cells was investigated by Annexin-v-FITC/PI fluorescent staining under fluorescent microscope.
5. The agarose electrophoresis was used to detect the DNA changes of treated groups.
6.Western blot was used to detect the levels of cyclinB1、cdc2、bcl-2 and bax of different groups.
RESULTS:
1. The culture supernatants of T. gondii could inhibit the proliferation of SKOV-3 cells in a time-dose dependent manner.
2. In the flow cytometric analysis, cell cycle was significantly stopped at G2/M phase by the culture supernatants and a maximum apoptosis rate of 18.23% was detected at 48h of treatment with the supernatants, but the apoptosis rates decreased and dead rate increased at the treatment time beyond 48h.
3. The classic cellular morphology of apoptosis in the treated trophoblastic cells was observed under the fluorescence microscope.
4. The culture supernatant of T. gondii reduced the expressions of gene cyclinB1, cdc2 and bcl-2 of SKOV-3 cells and increased the expressions of gene bax. After 48 hours, contrast the levels of cyclinB1 in negative control group. The integrated density Value of the culture supernatants of T. gondii groups were 0.86+0.04, 0.73+0.06 and 0.60+0.05 respectively, the results of cdc2 proteins were 0.98+0.02, 0.84+0.05 and 0.65+0.06 and the results of cyclinB1 proteins were 0.97+0.05, 0.81+0.02 and 0.78+0.05, nd the results of cdc2 were 1.23+0.03, 1.54+0.04 and 1.75+0.02 respectively. The difference was statistically significant when compared with the negative control group (p<0.05).
CONCLUSION:
The culture supernatant of T. gondii may inhibit SKOV-3 cells and arrest the cell cycle mainly by regulating the expression of gene cyclinB1 and cdc2 ,While can promote apoptosis of SKOV-3 cells in vitro through up-regulating Bax expressions and down-regulating Bcl-2 expressions.
弓形虫是一种专性细胞内寄生的原虫,其寄生于宿主细胞过程中,虫体与细胞的生物学特性均会发生改变:研究二者间的联系,一直是国内外关注的焦点。业已证实的是弓形虫在寄生过程中可以抑制宿主细胞增值,引起细胞周期改变,并且诱导宿主细胞凋亡。本文利用液氮保种小鼠传代及CF-11纤维素纯化的方法提取制作弓形虫培养上清液,将其视为一种生物制剂在体外条件下人卵巢癌细胞SKOV-S细胞共培养,来观察弓形虫培养上清液对SKOV-S细胞生物学行为影响。
方法:
1.采用四甲基氮噻唑蓝(MTT)法检测空白对照组与刚地弓形虫培养上清处理组的吸光度(A490值)并按公式计算卵巢癌细胞SKOV-3细胞增殖抑制率。
2. PI染色后,上流式细胞仪技术检测空白对照组与刚地弓形虫培养上清处理组的细胞周期。
3. Annexin-v-FITC/PI染色后,上流式细胞仪技术检测空白对照组与刚地弓形虫培养上清处理组的细胞凋亡率。
4.荧光显微镜观察空白对照组与刚地弓形虫培养上清处理组的细胞在不同时项形态改变情况。
5.采用琼脂糖凝胶电泳观察培养上清处理组的凋亡DNA条带。
6.采用western blot,对培养上清处理前后,卵巢癌细胞SKOV-3细胞周期蛋白cyclinB1、cdc2及凋亡蛋白bcl-2及bax表达量进行检测。
结果:
1. MTT法检测结果显示卵巢癌细胞SKOV-3细胞增殖抑制率随着刚地弓形虫速殖子培养上清浓度和作用时间增加均明显增大,弓形虫速殖子培养试验组抑制率与对照组之间比较差异均有统计学意义(p﹤0.05)。
2.培养上清作用于卵巢癌细胞SKOV-3细胞48h后,对照组与3×107、6×107、12×107 / mL数量的弓形虫速殖子培养上清组的卵巢癌细胞SKOV-3细胞G2/M期细胞比例分别为(22.43±3.78)%、(35.56±5.22)%、(38.76±3.19)%、(44.95±4.74)%,以12×107 / mL数量组增加最为明显,差异具有显著性意义(n=5, p<0.05),提示培养上清具有诱导卵巢癌细胞SKOV-3细胞G2/M期停滞的作用,且有一定剂量依赖性。
3.以浓度为12×107 / mL弓形虫速殖子培养上清作用后24、36、48、72h后的卵巢癌细胞SKOV-3细胞用Annexin-v-FITC与PI双染后,FCM分析细胞凋亡,结果表明其凋亡率分别为15.23℅、16.12℅、18.23℅、17.64℅,与空白对照组0.64℅、0.83℅、1.56℅、2.67℅比较有显著差异(p﹤0.05)。
4.选择与数量为12×107 / ml弓形虫速殖子培养上清作用不同时间段的细胞置荧光显微镜下观察,可见2种细胞形态:早期凋亡细胞,细胞膜着绿色,核拒染;凋亡中晚期细胞与坏死细胞,核着红色,细胞膜着绿色,空白对照组的正常细胞不着色。
5.以浓度为12×107/ml弓形虫速殖子培养上清作用细胞不同时间后的琼脂糖凝胶电泳均出现了相差约180-200bp的典型的细胞凋亡的DNA梯形条带。
6.弓形虫速殖子培养上清作用前卵巢癌细胞SKOV-3细胞可见周期蛋白cyclinB1、cdc2与凋亡蛋白Bcl-2、Bax的表达的表达。上清作用48h后,与对照组相比,不同处理组的cyclinB1表达量(表达积分光密度值,下同)为0.86+0.04、0.73+0.06、0.60+0.05 ;cdc2蛋白表达量为0.98+0.02, 0.84+0.05、0.65+0.06;cyclinB1蛋白表达量为0.97+0.05, 0.81+0.02和0.78+0.05;cdc2蛋白表达量为1.23+0.03, 1.54+0.04、1.75+0.02,与对照组相比均有显著性差异(p<0.05),以12×107 / mL数量的弓形虫速殖子组最为明显(p﹤0.05)。
结论:弓形虫速殖子培养上清对体外培养卵巢癌细胞SKOV-3细胞增殖有明显的抑制作用,其途径可能与调节周期蛋白cyclinB1、cdc2等蛋白表达或活性改变引起细胞株G2/M期阻滞有关;同时,弓形虫速殖子培养上清还可通过上调凋亡相关蛋白Bax表达和下调Bcl-2表达来促使卵巢癌细胞SKOV-3细胞凋亡。
Background and Objective: Ovarian cancer remains the most lethal gynaecological cancer. Ovarian cancer causes non-specific symptoms. Early diagnosis would result in better survival. However, lack of symptoms or vague symptoms in the early stages of ovarian cancer is why the illness is known as the 'silent killer'. The crude death rate of ovarian cancer was 4.8 per 100 000 female population. The treatment methods of this cancer including surgery,chemotherapy, Radiation therapy.But all the methods have side effects, long-term use of patients with immune system and high-dose hematopoietic system, cause a lot of damage.The medicine with less side effects and high selectivity seems to be could improve the clinical efficacy.
Toxoplasma gondii is a species of parasitic protozoa in the genus.The focus of recent reserch is on the factors of invasion of host cell by Toxoplasma and virulence. Now, it is proved that Toxoplasma gondii can inhibite host cell proliferation, affect the cell apoptosis and cell cycle.
In this study, we try to investigate the inhibitive effects of culture supernatants of T. gondii as a biological agent on the proliferation, apoptosis and cell cycle of human ovarian cancer cell line SKOV-3.
METHODS:
1. Growth inhibition rates of treated groups were measured with the MTT method.
2. FCM was used to test the rates of apoptosis of treated groups by culture supernatants of T. gondii with the fluorescent dye of PI.
3. FCM was used to test Cell cycle of treated groups by culture supernatants of T. gondii with the fluorescent dye of Annexin-v-FITC/PI.
4. After the treatment of SKOV-3 cells by culture supernatants of T. gondii, morphological change of apoptotic cells was investigated by Annexin-v-FITC/PI fluorescent staining under fluorescent microscope.
5. The agarose electrophoresis was used to detect the DNA changes of treated groups.
6.Western blot was used to detect the levels of cyclinB1、cdc2、bcl-2 and bax of different groups.
RESULTS:
1. The culture supernatants of T. gondii could inhibit the proliferation of SKOV-3 cells in a time-dose dependent manner.
2. In the flow cytometric analysis, cell cycle was significantly stopped at G2/M phase by the culture supernatants and a maximum apoptosis rate of 18.23% was detected at 48h of treatment with the supernatants, but the apoptosis rates decreased and dead rate increased at the treatment time beyond 48h.
3. The classic cellular morphology of apoptosis in the treated trophoblastic cells was observed under the fluorescence microscope.
4. The culture supernatant of T. gondii reduced the expressions of gene cyclinB1, cdc2 and bcl-2 of SKOV-3 cells and increased the expressions of gene bax. After 48 hours, contrast the levels of cyclinB1 in negative control group. The integrated density Value of the culture supernatants of T. gondii groups were 0.86+0.04, 0.73+0.06 and 0.60+0.05 respectively, the results of cdc2 proteins were 0.98+0.02, 0.84+0.05 and 0.65+0.06 and the results of cyclinB1 proteins were 0.97+0.05, 0.81+0.02 and 0.78+0.05, nd the results of cdc2 were 1.23+0.03, 1.54+0.04 and 1.75+0.02 respectively. The difference was statistically significant when compared with the negative control group (p<0.05).
CONCLUSION:
The culture supernatant of T. gondii may inhibit SKOV-3 cells and arrest the cell cycle mainly by regulating the expression of gene cyclinB1 and cdc2 ,While can promote apoptosis of SKOV-3 cells in vitro through up-regulating Bax expressions and down-regulating Bcl-2 expressions.
引文
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[38]王璐.细胞周期蛋白B1在卵巢癌中的研究进展[J].国际复产科学杂志,2008,35(1):44-45.
[39] Friedrich N, Santos JM, Liu Y, et al. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectinsduring host cell invasion by apicomplexan parasites[J]. J Biol Chem. 2010.,285(3):2064-2075
[40]吴亮,陈盛霞,李琳婕等. [J].中国寄生虫学与寄生虫病杂志,2008,26(6):452-454.
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[42]饶华祥,侯玉英,岳慧萍等.弓形虫感染对妊娠中期小鼠胎盘组织中细胞凋亡相关蛋白表达的影响[J].中国人兽共患病学报,2010,26(1):57-61.
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[38]王璐.细胞周期蛋白B1在卵巢癌中的研究进展[J].国际复产科学杂志,2008,35(1):44-45.
[39] Friedrich N, Santos JM, Liu Y, et al. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectinsduring host cell invasion by apicomplexan parasites[J]. J Biol Chem. 2010.,285(3):2064-2075
[40]吴亮,陈盛霞,李琳婕等. [J].中国寄生虫学与寄生虫病杂志,2008,26(6):452-454.
[41] Angeloni M B , Silva N M, Castro A S, et al.Apoptosis and S Phase of the Cell Cycle in BeWo Trophoblastic and Hela are Differentially Modulate by Toxoplasma gondii Strain Types[J]. Placenta, 2009, 30: 785-791.
[42]饶华祥,侯玉英,岳慧萍等.弓形虫感染对妊娠中期小鼠胎盘组织中细胞凋亡相关蛋白表达的影响[J].中国人兽共患病学报,2010,26(1):57-61.
[43] enegas A, Villard O, Neuville A, et al. Toxoplasma gondii-induced foetal resoptin resorption in mice involves interferorn-gamma-induced apoptosis and spiral artery dilation at the maternofoetal interface[J].International Journal for Parasitology, 2009, 39(4):481-487.
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[48]张秀昌,蔡念光,孙黎,等.弓形虫诱导人白血病细胞K562凋亡的实验观察[J].中国寄生虫学与寄生虫病杂志,2007,3(25):185-188
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[51] Enegas A, Villard O, Neuville A, et al. Toxoplasma gondii-induced foetal resoptin resorption in mice involves interferorn-gamma-induced apoptosis and spiral artery dilation at the maternofoetal interface[J].International Journal for Parasitology, 2009, 39(4):481-487.
[52] Angeloni M B , Silva N M, Castro A S, et al.Apoptosis and S Phase of the Cell Cycle in BeWo Trophoblastic and Hela are Differentially Modulate by Toxoplasma gondii Strain Types[J]. Placenta, 2009, 30: 785-791.
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[1]牛小迎.弓形虫病的综述[J].青海畜牧兽医杂志, 2008, 38(3): 48-49.
[2]詹希美.人体寄生虫学北京[M].第五版.北京:人民卫生出版社,2001,138-140.
[3]彭碧文.钙离子与钙调蛋白在弓形虫侵染宿主细胞信号转导中的作用[J].国外医学寄生虫病分册,2001,28(6).
[4] Candolfi E,Hunter CA,Rcmington JS,et al.Roles of gamma interferon and other cytokines in suppression of spleen cell proliferative response to concanavalin A and Toxoplasma antigen during acute toplasmosis[J].Infect Iune, 1995,63(3):751-756.
[5] Haque S, Khan I, Haque A, et al.Impairment of the cellular immune response in acute murine toplasmosis:Regulation of interleukin 2 production and macrophage-mediated inhibitory effects[J].Infect Imune, 1994, 62(7):2908-2916.
[6]彭文伟.染性疾病与传染病学[M].北京,科学出版社,2000,1459-1476.
[7] Gross U. Toxoplasma gondii research in Europe[J].Parasitol Today,1996,12(1):1-3.
[8]奚琳琳,李威.弓形虫病机理、毒力及基因型研究进展[J].中国病原生物学杂志,2009,4(11):859-861
[9]张德林,李学瑞,王艳华等.弓形虫代谢分泌抗原在免疫中研究进展[J].中国人兽共患病学报,2007,23(12):1259-1261.
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