摘要
目的 探讨一氧化氮(NO)与HPV感染的关系及其对宫颈癌细胞增殖与凋亡的影响.方法 选择2009年12月1日至2010年8月5日在复旦大学附属妇产科医院宫颈疾病诊疗中心行阴道镜检查的患者115例,应用HPV分型检测试剂盒(可检测21种HPV亚型)进行HPV分型,同时应用Griss法间接检测宫颈局部NO含量.在体外培养的宫颈腺癌细胞株HeLa细胞和宫颈鳞癌细胞株Caski细胞中,加入不同浓度(终浓度分别为0.125、0.25、0.5 、1.0及2.0 mmol/L)NO供体--硝普钠(SNP)后培养24 h,采用四甲基偶氮唑蓝(MTT)比色法检测细胞生长情况,流式细胞仪检测细胞凋亡情况,并应用荧光定量逆转录PCR技术及蛋白印迹法检测SNP处理后细胞中HPVE6、E7 mRNA的表达及p53蛋白的表达.结果 (1)115例患者中,宫颈HPV感染93例,其中高危型50例,低危型43例;无HPV感染22例.115例患者的宫颈局部均检测到NO,其中高危型HPV感染患者的宫颈局部NO含量为(47.6±1.4)μmol/L,低危型HPV感染者为(24.1±1.2)μmol/L,无HPV感染者为(22.8±0.3)μmol/L,高危型HPV感染者高于低危型HPV感染者和无HPV感染者,差异有统计学意义(P<0.05);而低危型HPV感染者与无HPV感染者比较,差异无统计学意义(P>0.05).(2)不同浓度(0.125、0.25、0.5 1.0及2.0 mmol/L)SNP处理HeLa、Caski细胞24 h后,能抑制细胞生长、促进细胞凋亡,当SNP浓度≥1.0 mmol/L时,与SNP处理前比较,差异均有统计学意义(P<0.05).1.0 mmol/L的SNP处理24h后,HeLa细胞中HPV18 F6、E7 mRNA的表达水平(分别为19.181±0.360、17.571±0.010)明显低于SNP处理前(分别为27.362±0.191、22.962±0.053;P<0.05),p53蛋白的表达水平(1.17±0.03)明显高于SNP处理前(0.23±0.05;P<0.05);但Caski细胞中HPV16E6、E7mRNA和P53蛋白的表达在SNP处理前、后无明显变化(P>0.05).结论 宫颈局部微环境中NO含量增加与感染高危型HPV有一定的相关性;SNP能抑制宫颈癌细胞的生长,促进其凋亡,降低HPV18 E6、E7 mRNA的表达及活化p53蛋白,其机制可能是宫颈腺癌细胞株HeLa细胞对SNP更敏感.NO释放增加可能是宫颈局部微环境清除HPV的免疫机制之一.
Objective To study the relationship between nitric oxide within cervical microenvironment and different HPV types as well as the effect of sodium nitroprusside( SNP), a nitric oxide donor, on the proliferation and apoptosis of cervical cancer cell lines. Methods HPV typing test was assessed from 115 women by using high-risk HPV (HR-HPV) 21 typing test and the release of cervical nitric oxide(NO) was assessed as nitrate, nitrite in cervical fluid. Cervical NO was then compared between women showing different HPV types. Proliferation of Caski and HeLa cervical cells was determined by methyl thiazolyl tetrazolium (MTT) assay, cell apoptosis was detected by flow cytometry after 24 hours treated by different final concentration of SNP (0. 125,0. 25,0. 5,1.0 and 2. 0 mmol/L, respectively). The expressions of HPV E6,E7 gene mRNA and p53 protein were detected by SYBR Green Ⅰ quantitative real-time PCR and western blot. Results ( 1 ) The cervical NO release of women with HR-HPV was higher compared to that in HPV negative women [ (47. 6 ± 1.4) μmol/L vs ( 22. 8 ± 0. 3 ) μmol/L; P 〈 0. 05 ]; but there was no statistical difference between low-risk HPV (LR-HPV) group [ (24. 1 ± 1.2 ) μmol/L] and control group (P 〉0. 05 ). (2)After 24 hours treated by different final concentration of SNP, the results shown that SNP could inhibited the proliferation and increased apoptosis rate in Caski and HeLa cells, in which the concentration of SNP ≥ 1.0 mmol/L , there were significantly different ( P 〈 0. 05 ), while when SNP ≥2. 0mmol/L, the proliferation of cells inhibited seriously. Treated by SNP ( 1.0 mmol/L ) 24 hours, the expressions of HPV18 E6, E7 mRNA in HeLa cells were reduced from 27. 362 ±0. 191,22. 962 ±0. 053 to19. 181 ±0. 360, 17. 571 ±0. 010 and the protein expression of p53 increased from 1. 17 ±0. 03 to 0. 23 ±0. 05, there were statistically significant differences between adding SNP group and the control group ( P 〈0. 05); but there were no statistically significant differences in HPV16 E6, E7 mRNA and that of p53 in Caski cells( P 〉 0. 05 ). Conclusions The presence of HR-HPV is associated with an increased release of NO in the human uterine cervix; NO could inhibit the growth and proliferation and enhance the apoptosis of cervical cancer cells, inhibit the expression of HPV18 E6, E7 mRNA in HeLa cells and activate the expression of p53 protein, the mechanism may be due to higher sensitivity of HeLa cells (cervical adenocarcinoma cell) to SNP. The increasing release of NO may play a role in regulating the elimination of HPV in cervical microenvironment, which is a part of mucous membrane immunity.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2011年第4期260-265,共6页
Chinese Journal of Obstetrics and Gynecology
关键词
宫颈肿瘤
一氧化氮
细胞增殖
细胞凋亡
乳头状瘤病毒感染
Uterine cervical neoplasms
Nitric oxide
Cell proliferation
Apoptosis
Papillomavirus infections