羟喜树碱对外源野生型p53转染宫颈癌细胞作用效果的研究
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摘要
目的探讨羟喜树碱(Hydroxycamptothecin,HCPT)对外源野生型p53转染宫颈癌细胞的作用效果。方法体外培养宫颈癌HeLa细胞,将其分为空白对照组、阴性对照组、HCPT组、p53组、HCPT+p53组。荧光显微镜下观察转染结果,qRT-PCR检测p53基因的表达,CCK-8法检测不同浓度HCPT作用于目的质粒转染后细胞的活力,流式细胞技术检测细胞凋亡率。结果荧光显微镜下可见细胞内有明显绿色荧光,说明质粒转染成功;HeLa细胞转染目的质粒后,p53基因mRNA表达上调,且随着转染质粒剂量的增加而逐渐升高;与空白组、阴性对照组相比,实验组细胞存活率降低,随着HCPT浓度的增加,对细胞活力的抑制作用明显增加,各浓度比较,差异有统计学意义(P<0.05);与空白组和溶剂组相比,HCPT组、p53组的凋亡率升高,差异具有统计学意义(P<0.05);p53+HCPT组凋亡率显著高于其他实验组,差异具有统计学意义(P<0.05)。结论外源野生型p53基因导入宫颈癌HeLa细胞后,可明显增强HCPT的作用,抑制HeLa细胞的增殖,促进其凋亡。HCPT和p53可协同抑制HeLa细胞的增殖并促进其凋亡。
Objective To investigate the effects of Hydroxycamptothecin(HCPT) on the cervical cancer cells transfected with wildtype p53. Methods He La cells were cultured in vitro, and some were transfected with the plasmid pcDNA3.1-HA-EGFP(negative control group) or the target plasmid pcDNA3.1-HA-TP53(experimental group). Then the cells were divided into 5 groups: blank control group, solvent group, HCPT group, p53 group and HCPT+p53 group. The expression of the p53 gene was detected by qRT-PCR, CCK-8 assay was used to detect the viability of cells treated with different concentrations of HCPT. The apoptosis rates of different groups were measured by flow cytometry. Results Under the fluorescent microscope, there was obvious green fluorescence in the cells, indicating that the plasmid was successfully transfected. After the HeLa cells were transfected with the target plasmid, the mRNA expression of p53 gene was up-regulated along with the increase of the transfection plasmid dose. Compared with the blank and negative control groups, the viability of cells in experimental groups was inhibited in a HCPT-dose dependent manner, and there were statistical differences when it treated with different doses of HCPT(P<0.05). Compared with the blank control group and solvent group, the apoptosis rates of HCPT group and p53 group were increased(P<0.05), and that of the p53+HCPT group was significantly higher than that of the other experimental groups(P<0.05). Conclusion The introduction of exogenous wild-type p53 gene into cervical cancer HeLa cells can significantly enhance the effect of HCPT in inhibiting the proliferation of cervical cancer cells and promoting its apoptosis.
Objective To investigate the effects of Hydroxycamptothecin(HCPT) on the cervical cancer cells transfected with wildtype p53. Methods He La cells were cultured in vitro, and some were transfected with the plasmid pcDNA3.1-HA-EGFP(negative control group) or the target plasmid pcDNA3.1-HA-TP53(experimental group). Then the cells were divided into 5 groups: blank control group, solvent group, HCPT group, p53 group and HCPT+p53 group. The expression of the p53 gene was detected by qRT-PCR, CCK-8 assay was used to detect the viability of cells treated with different concentrations of HCPT. The apoptosis rates of different groups were measured by flow cytometry. Results Under the fluorescent microscope, there was obvious green fluorescence in the cells, indicating that the plasmid was successfully transfected. After the HeLa cells were transfected with the target plasmid, the mRNA expression of p53 gene was up-regulated along with the increase of the transfection plasmid dose. Compared with the blank and negative control groups, the viability of cells in experimental groups was inhibited in a HCPT-dose dependent manner, and there were statistical differences when it treated with different doses of HCPT(P<0.05). Compared with the blank control group and solvent group, the apoptosis rates of HCPT group and p53 group were increased(P<0.05), and that of the p53+HCPT group was significantly higher than that of the other experimental groups(P<0.05). Conclusion The introduction of exogenous wild-type p53 gene into cervical cancer HeLa cells can significantly enhance the effect of HCPT in inhibiting the proliferation of cervical cancer cells and promoting its apoptosis.
引文
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[11]Tzafetas M, Mitra A, Kalliala I, et al. Fertility-sparing Surgery for Presumed Early-stage Invasive Cervical Cancer:A Survey of Practice in the United Kingdom[J]. Anticancer Res, 2018, 38(6):3641-3646. doi:10.21873/anticanres.12639.
[12]Tseng JH, Aloisi A, Sonoda Y, et al. Less versus more radical surgery in stage IB1 cervical cancer:A populationbased study of long-term survival[J]. Gynecol Oncol, 2018,150(1):44-49. doi:10.1016/j. ygyno.2018.04.571.
[13]Lu XY, Chen M, Chen DH, et al. Remifentanil on T lymphocytes, cognitive function and inflammatory cytokines of patients undergoing radical surgery for cervical cancer[J].Eur Rev Med Pharmacol Sci, 2018, 22(9):2854-2859. doi:10.26355/eurrev_201805_14987.
[14]Cibula D, P?tter R, Planchamp F, et al. The European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology guidelines for the management of patients with cervical cancer[J]. Radiother Oncol, 2018, 127(3):404-416. doi:10.1016/j. radonc.2018.03.003.
[15]Tomita N, Mizuno M, Kondo S, et al. Role of Extensive Lymphadenectomy in Early-Stage Cervical Cancer Patients With Radical Hysterectomy Followed by Adjuvant Radiotherapy[J]. Int J Gynecol Cancer, 2018, 28(6):1211-1217.doi:10.1097/IGC.0000000000001279.
[16]张晖辉,齐范,祖雄兵,等.膀胱癌BIU—87细胞羟基喜树碱耐药的蛋白质组学研究[J].肿瘤药学, 2011, 1(1):22-25. doi:10.3969/j. issn.2095-1264.2011.01.005
[17]Stiasny A, Freier CP, Kuhn C, et al. The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer[J]. Oncol Lett, 2017, 14(4):4467-4476. doi:10.3892/ol.2017.6752.
[18]Guo KY, Han L, Li X, et al. Novel proteasome inhibitor delanzomib sensitizes cervical cancer cells to doxorubicininduced apoptosis via stabilizing tumor suppressor proteins in the p53 pathway[J]. Oncotarget, 2017, 8(69):114123-114135. doi:10.18632/oncotarget.23166.
[19]de Almeida VH, de Melo AC, Meira DD, et al. Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer[J]. Braz J Med Biol Res, 2017, 51(1):e6822. doi:10.1590/1414-431X20176822.
[2]Cordeiro MN, De Lima RCP, Paolini F, et al. Current research into novel therapeutic vaccines against cervical cancer[J]. Expert Rev Anticancer Ther, 2018, 18(4):365-376.doi:10.1080/14737140.2018.1445527.
[3]Xu HH, Wang K, Feng XJ, et al. Prevalence of human papillomavirus genotypes and relative risk of cervical cancer in China:a systematic review and meta-analysis[J]. Oncotarget,2018, 9(20):15386-15397. doi:10.18632/oncotarget.24169.
[4]Bao H, Zhang L, Wang L, et al. Significant variations in the cervical cancer screening rate in China by individual-level and geographical measures of socioeconomic status:a multilevel model analysis of a nationally representative survey dataset[J].Cancer Med, 2018, 7(5):2089-2100. doi:10.1002/cam4.1321.
[5]Zhang Q, Zhao M, Cao D, et al. Assessment of the effectiveness of HPV16/18 infection referred for colposcopy in cervical cancer screening in Northwest of China[J]. J Med Virol, 2018, 90(1):165-171. doi:10.1002/jmv.24902.
[6]Yu L, Jiang M, Qu P, et al. Clinical evaluation of human papillomavirus 16/18 oncoprotein test for cervical cancer screening and HPV positive women triage[J]. Int J Cancer,2018, 143(4):813-822. doi:10.1002/ijc.31368.
[7]Wong JPH, Vahabi M, Miholjcic J, et al. Knowledge of HPV/cervical cancer and acceptability of HPV self-sampling among women living with HIV:A scoping review[J].Curr Oncol, 2018, 25(1):e73-e82. doi:10.3747/co.25.3855.
[8]Pedersen HN, Smith LW, Racey CS, et al. Implementation considerations using HPV self-collection to reach women under-screened for cervical cancer in high-income settings[J].Curr Oncol, 2018, 25(1):e4-e7. doi:10.3747/co.25.3827.
[9]Cook EE, Gershman ST, Kim JJ, et al. Trends of two HPVassociated cancers in Massachusetts:cervical and oropharyngeal cancer[J]. Cancer Causes Control, 2018, 29(4-5):435-443. doi:10.1007/s10552-018-1016-1.
[10]Das D, Sarkar B, Mukhopadhyay S, et al. An Altered Ratio of CD4+And CD8+T Lymphocytes in Cervical Cancer Tissues and Peripheral Blood-A Prognostic Clue?[J]. Asian Pac J Cancer Prev, 2018, 19(2):471-478. doi:10.22034/APJCP.2018.19.2.471.
[11]Tzafetas M, Mitra A, Kalliala I, et al. Fertility-sparing Surgery for Presumed Early-stage Invasive Cervical Cancer:A Survey of Practice in the United Kingdom[J]. Anticancer Res, 2018, 38(6):3641-3646. doi:10.21873/anticanres.12639.
[12]Tseng JH, Aloisi A, Sonoda Y, et al. Less versus more radical surgery in stage IB1 cervical cancer:A populationbased study of long-term survival[J]. Gynecol Oncol, 2018,150(1):44-49. doi:10.1016/j. ygyno.2018.04.571.
[13]Lu XY, Chen M, Chen DH, et al. Remifentanil on T lymphocytes, cognitive function and inflammatory cytokines of patients undergoing radical surgery for cervical cancer[J].Eur Rev Med Pharmacol Sci, 2018, 22(9):2854-2859. doi:10.26355/eurrev_201805_14987.
[14]Cibula D, P?tter R, Planchamp F, et al. The European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology guidelines for the management of patients with cervical cancer[J]. Radiother Oncol, 2018, 127(3):404-416. doi:10.1016/j. radonc.2018.03.003.
[15]Tomita N, Mizuno M, Kondo S, et al. Role of Extensive Lymphadenectomy in Early-Stage Cervical Cancer Patients With Radical Hysterectomy Followed by Adjuvant Radiotherapy[J]. Int J Gynecol Cancer, 2018, 28(6):1211-1217.doi:10.1097/IGC.0000000000001279.
[16]张晖辉,齐范,祖雄兵,等.膀胱癌BIU—87细胞羟基喜树碱耐药的蛋白质组学研究[J].肿瘤药学, 2011, 1(1):22-25. doi:10.3969/j. issn.2095-1264.2011.01.005
[17]Stiasny A, Freier CP, Kuhn C, et al. The involvement of E6, p53, p16, MDM2 and Gal-3 in the clinical outcome of patients with cervical cancer[J]. Oncol Lett, 2017, 14(4):4467-4476. doi:10.3892/ol.2017.6752.
[18]Guo KY, Han L, Li X, et al. Novel proteasome inhibitor delanzomib sensitizes cervical cancer cells to doxorubicininduced apoptosis via stabilizing tumor suppressor proteins in the p53 pathway[J]. Oncotarget, 2017, 8(69):114123-114135. doi:10.18632/oncotarget.23166.
[19]de Almeida VH, de Melo AC, Meira DD, et al. Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer[J]. Braz J Med Biol Res, 2017, 51(1):e6822. doi:10.1590/1414-431X20176822.