·论著·

机体免疫应答与高级别宫颈上皮内瘤变术后HPV转归的关系

段小令 陆琦

【摘要】 目的 探讨机体免疫应答与高级别宫颈上皮内瘤变(HSIL)患者术后人乳头状瘤病毒(HPV)转归的关系。 方法 选择本院在2020年9月—2022年3月期间收治的HSIL患者213例作为研究对象,年龄为28～63岁,平均年龄(39.0±4.8)岁,对比手术前、术后3月、术后6月及术后12月HSIL患者各项血清免疫指标的变化。然后根据术后12月HSIL患者HPV转归情况分为转阴组(n=162)和未转阴组(n=51),对比两组一般资料和临床资料,利用多因素Logistic回归分析,筛选HSIL患者HPV转归的独立影响因素,比较各因素对HPV转归的预测效能,并分析各因素与HPV转归的关联性。 结果 HSIL患者术后12月复诊时CD3+细胞数、CD4+细胞数、Th/Ts、免疫球蛋白A(IgA)、免疫球蛋白G(IgG)、免疫球蛋白M(IgM)水平显著升高,CD8+细胞数则显著降低(P均<0.05);未转阴组患者年龄显著高于转阴组,且未转阴组患者有流产史的比例显著高于转阴组(P均<0.05);转阴组患者CD4+和Th/Ts水平显著高于未转阴组,且转阴组患者白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平显著低于未转阴组,两组唾液酸苷酶(SIA)阳性、H2O2阳性及阴道清洁度(CLE)异常的患者数差异具有统计学意义(P均<0.05);多因素Logistic回归分析结果显示,年龄大和TNF-α水平高是HSIL患者术后HPV未转阴的危险因素,CD4+细胞数多和Th/Ts水平高是HSIL患者术后HPV未转阴的保护因素(P<0.05),年龄+CD4++Th/Ts+TNF-α联合检测HSIL患者术后HPV转归情况AUC为0.881,约登指数为0.695、准确性为0.848,均高于其他预测模型;将年龄、CD4+、Th/Ts及TNF-α水平逐层划分,建立Logistic模型逐步排除混杂因素后,年龄、CD4+、Th/Ts及TNF-α水平仍为HSIL患者术后HPV转归的独立影响因素。随着年龄、TNF-α的升高,CD4+细胞数、Th/Ts水平的降低,其关联效应值也相应升高(P趋势<0.05)。 结论 与术前相比,术后12月HSIL患者细胞免疫应答和体液免疫应答均得到显著改善;年龄和TNF-α水平是HSIL患者术后HPV持续感染的危险因素,CD4+和Th/Ts是HSIL患者术后HPV持续感染的保护因素;年龄+CD4++Th/Ts+TNF-α联合检测HSIL患者术后HPV转归情况,其预测性能较好;消除混杂因素后,随着年龄、TNF-α的升高,CD4+细胞数、Th/Ts水平的降低,与HPV转归的关联效应值也相应升高。

【关键词】 机体免疫应答; 高级别宫颈上皮内瘤变; T淋巴细胞; HPV转归

宫颈癌(cervical cancer,CC)是常见的女性恶性肿瘤,全球每年有超过50万例新增病例[1]。宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)是CC进展过程中的必经阶段,CIN可分为低级别与高级别,其中宫颈高级别上皮内瘤变(high-grade squamous intraepithelial lesion,HSIL)较为多见,虽未到达癌症的程度,但已累及宫颈组织较深位置[2]。CIN的发展和最终导致侵袭性癌症的主要原因是人乳头状瘤病毒(human papilloma virus,HPV)的持续感染[3-4]。宫颈环形电切术(loop electrosurgical excision procedure,LEEP)是CIN临床治疗的常用手术方法,能切除病变组织并清除HPV,降低HSIL进一步癌变风险。但仍有部分CIN患者在LEEP手术后HPV持续感染,大幅增加了CC患病风险,严重危害患者生命健康,因此研究CIN患者LEEP术后HPV持续感染高危因素具有重要意义[5]。近期有研究表明,人体受到病毒感染会引起T淋巴细胞减少,损害细胞免疫功能呢,导致机体免疫应答水平降低,最终诱发持续感染和肿瘤的发生[6-7]。并且HSIL患者术后容易出现脱痂、出血的情况,难以完全清除病毒感染病灶,残留HPV感染的清除还需依靠患者自身免疫力[8]。因此本文将探讨机体免疫应答与HSIL患者术后HPV转归的关系,现报道如下。

对象与方法

一、研究对象

选择本院在2020年9月—2022年3月期间收治的213例HSIL患者为研究对象,年龄为28～63岁,平均年龄(39.0±4.8)岁,纳入标准:(1)明确诊断为HSIL[9];(2)至少有2年性生活史;(3)HPV感染者;(4)术前48 h内无性生活、阴道冲洗、放药;(5)近3个月内未进行阴道相关疾病治疗;(6)治疗方法为LEEP刀宫颈锥切术。排除标准:(1)严重肾功能异常者;(2)恶性肿瘤患者;(3)严重心功能异常者;(4)妊娠或哺乳期妇女。本研究治疗方案已通过本院医学伦理委员会批准。

二、研究方法

1.治疗方法:213例HSIL患者均接受LEEP刀宫颈锥切术治疗。术前进行常规检查,包括B超检查、白带常规、阴道镜检查等,告知患者术前24 h禁止性生活,术前排空膀胱。术中,指导患者取膀胱截石位,行常规消毒后进行宫颈碘试验,标记碘溶液在宫颈表面移行区范围外5 mm左右,明确病变范围,利用高频电波LEEP刀切割病变组织,将功率设定为50 W,切割深度5～10 mm,若病灶面积大,可进行分次切除,利用球形电极电凝止血,涂抹烧伤湿润膏,利用碘伏纱布压迫填塞。术后,采用抗生素处理预防感染。

2.一般资料:收集患者的基本资料用于本次研究,其中包括患者年龄、月经规律、经期同房、饮酒、主动或被动吸烟、孕次、产次、病程、首次性生活年龄、宫颈癌家族史及流产史等资料。

3.临床指标检测:分别在患者入院、术后3月、术后6月及术后12月时抽取肘静脉血两管各2 mL,一管抗凝处理后,利用流式细胞仪检测患者T淋巴细胞亚群分布情况,包括CD3+、CD4+、CD8+细胞绝对数,CD4+/CD8+计算值为辅助性T细胞(helper T cell,Th)与抑制性T细胞(suppressor T cell,Ts)比值(Th/Ts);另取一管采用免疫比浊法检测免疫球蛋白G(immunoglobulin G,IgG)、免疫球蛋白M(immunoglobulin M,IgM)及免疫球蛋白A(immunoglobulin A,IgA)水平。

患者入院次日清晨采集空腹血液样本5 mL,离心15 min(3 000 rpm/min,离心半径10 cm)分离血清,全自动生化分析仪以酶联免疫吸附法测定肿瘤坏死因子-α(tumor necrosis factor,TNF-α)和白介素-6(interleukin-6,IL-6)水平。

4.阴道微环境检测:患者入院时进行样本采集,于阴道上1/3段无菌棉签旋转10 s取材,并立即送检。镜检阴道乳酸菌、滴虫、线索细胞及其他病原微生物等。阴道微环境检测[10]:(1)采用五联检测试剂盒检测白细胞脂酶(leukocyte esterase,LE)、过氧化氢(H2O2)、唾液酸苷酶(Sialidase,SIA)及pH值。(2)乳酸杆菌:显微镜下计数乳酸杆菌,乳酸杆菌+或++++为阳性,++和+++为阴性。(3)阴道清洁度(Cleanliness,CLE):镜检分泌物以杆菌为主,并且可见大量上皮细胞为Ⅰ度;有部分杆菌、脓细胞及杂菌,并且可见上皮细胞为Ⅱ度;有大量脓细胞和杂菌,少量杆菌和上皮细胞为Ⅲ度;几乎全布是脓细胞和杂菌为Ⅳ度。Ⅰ～Ⅱ度为CLE正常,Ⅲ～Ⅳ度为CLE异常。(4)患者Nugent评分超过6分并且线索细胞比例超过20%诊断为细菌性阴道病(bacterial vaginosis,BV);镜检发现阴道毛滴虫诊断为滴虫性阴道炎(trichomonas vaginitis,TV);镜检发现芽生孢子或菌丝诊断为外阴阴道假丝酵母菌病(vulvovaginal candidiasis,VVC)。

5.HPV检测:患者入院和术后12月时进行HPV检测,使用阴道窥器充分暴露宫颈,宫颈刷采集宫颈脱落上皮细胞样本,将宫颈刷浸入细胞保存液中,检测时将该样本离心1 min(转速10 000 r/min,半径3 cm)弃上清液,加入裂解酶液,提取DNA。

溶液配置:PCRmix 23.25 μL+DNAtaq酶 0.75 μL+1 μL模板。

反应条件:95 ℃预变性5 min;95 ℃变性1 min、55 ℃退火30 s、72 ℃延伸30 s,共40个循环;72 ℃终延伸5 min。

导流杂交:(1)取PCR产物20 μL,95 ℃加热5 min,冰水浴至少2 min;(2)1 μL杂交液预热45 ℃,温育至少2 min后;(3)将DNA样品溶液加入0.5 ml预热至45 ℃的杂交液,温育10 min后,进行导流杂交;使用0.8 mL杂交液反复冲洗膜三次;(4)杂交仪设定25 ℃,加入0.5 mL酶标液温育4 min开泵,再设定温度36 ℃彻底洗膜4次,加入NBT/BCIP溶液0.5 mL,盖上盖板显色3～6 min;(5)阳性点为蓝紫色远点,根据HPV分型比对图(图1)判断HPV分型。

图1 HPV分型比对图

Figure 1 Comparison of HPV typing

三、统计学分析

应用SPSS 22.0软件进行数据分析,研究中计量资料和计数资料分别用均数±标准差和例数(%)表示,组间比较采用χ2检验、t检验。采用多因素Logistic回归分析HSIL患者术后HPV转归情况的影响因素,比较各因素对HPV转归的预测效能,绘制受试者工作特征(receiver operating characteristic,ROC)曲线,计算曲线下面积(area under curve,AUC)、灵敏度与特异度。Logistic模型调整混杂变量后,分析影响因素对HSIL患者术后HPV转归的关联性。

结 果

一、HSIL患者手术前后T淋巴细胞亚群水平比较

比较213例HSIL患者手术前后T淋巴细胞亚群水平发现,术后3月、6月及12月患者CD3+、CD4+细胞数和Th/Ts水平与术前比较显著升高,CD8+细胞数显著降低(P均<0.05),见表1。

表1 HSIL患者术前、术后3月、术后6月及术后12月T淋巴细胞亚群水平比较

Table 1 Comparison of T lymphocyte subsets in patients with HSIL before surgery, 3 months after surgery, 6 months after surgery and 12 months after surgery

Note:Compared with pre-operation,aP<0.05; Compared with 3 months after operation, bP<0.05; Compared with 6 months after operation, cP<0.05

ItemsPre-operation3 months after operation6 months after operation12 months after operationCD3+48.2±4.252.7±4.7a55.6±4.4ab57.2±4.4abCD4+20.3±3.022.6±2.5a24.8±2.6ab25.2±2.3abCD8+35.7±3.933.7±3.5a31.4±3.2ab28.6±3.4abcTh/Ts0.6±0.20.7±0.1a0.8±0.2ab0.9±0.2abc

二、HSIL患者手术前后免疫功能比较

术后HSIL患者IgA、IgG及IgM水平均显著降低,差异具有统计学意义(P均<0.05),随着术后时间的增加,IgA、IgG及IgM水平均呈下降趋势,见表2。

表2 HSIL患者术前、术后3月、术后6月及术后12月免疫功能比较

Table 2 Comparison of immune function of HSIL patients before surgery, 3 months after surgery, 6 months after surgery and 12 months after

Note:Compared with pre-operation,aP<0.05; Compared with 3 months after operation, bP<0.05; Compared with 6 months after operation, cP<0.05

ItemsPre-operation3 months after operation6 months after operation12 months after operationIgA (g/L)0.9±0.61.4±0.6a2.0±0.7ab2.3±0.7abIgG (g/L)7.2±1.49.8±2.5a10.8±2.2ab11.9±2.6abcIgM(g/L)0.6±0.20.8±0.5a1.0±0.6ab1.2±0.8ab

三、两组患者一般资料比较

对转阴组和未转阴组患者一般资料进行比较,发现饮酒、主动或被动吸烟、月经规律、经期同房、首次性生活年龄、孕次、产次、宫颈癌家族史、病程及是否HPV16/18感染对HSIL患者术后HPV转归无显著影响(P均>0.05),未转阴组患者年龄显著高于转阴组,且未转阴组患者有流产史的比例显著高于转阴组(P均<0.05),见表3。

表3 转阴组和未转阴组患者一般资料比较[例(%)]

Table 3 Comparison of general data of patients in the negative conversion group and the non-negative conversion group[n(%)]

Note:Comparison betweenthe two groups,*P<0.05

ItemsNegative group(n=162)Non-negative group(n=51)Age( x±s,years)∗38.4±4.641.3±4.4Tipple36(22.2)12(23.5)Active or passive smoking49(30.3)17(33.3)Menstrual regularity116(71.6)35(68.6)Regular sex20(12.4)7(13.6)Age of first sexual intercourse(years) ≤2046(28.4)14(27.5) >20112(71.6)37(72.5)Pregnancy times <285(52.5)27(52.9) ≥277(47.5)24(47.1)Childbirth times <2108(66.7)33(64.7) ≥254(33.3)18(35.3)Family history of cervical cancer19(11.7)7(13.7)History of abortion∗17(10.5)11(21.6)Course of disease( x±s, years)7.7±1.78.1±1.6HPV 16/18 Negative99(61.1)30(58.8) Positive63(38.9)21(41.2)

四、两组患者临床资料比较

两组HSIL患者各项临床资料对比结果显示,CD3+、CD8+、IgA、IgG、IgM、LE、乳酸杆菌、BV、TV、VVC及pH对HSIL患者术后HPV转归无显著影响(P均>0.05),转阴组患者CD4+和Th/Ts水平显著高于未转阴组,且转阴组患者IL-6和TNF-α水平显著低于未转阴组,两组SIA阳性、H2O2阳性及CLE异常的患者数差异具有统计学意义(P均<0.05),见表4。

表4 转阴组和未转阴组患者临床资料比较例(%)]

Table 4 Comparison of clinical data between the negative conversion group and the non-negative conversion group

Note:Comparison betweenthe two groups,*P<0.05

ItemsNegative group(n=162)Non-negative group(n=51)CD3+48.4±4.247.5±4.2CD4+∗20.7±2.219.0±2.4CD8+35.6±3.635.3±3.3Th/Ts∗0.6±0.20.5±0.1IgA(g/L)0.9±0.50.8±0.4IgG(g/L)7.2±2.17.1±2.2IgM(g/L)0.6±0.20.6±0.2IL-6(ng/L)∗9.1±2.511.8±2.3TNF-α(ng/L)∗39.7±5.245.2±6.2SIA(+)∗13(8.0)13(25.5)LE(+)24(14.8)9(17.7)H2O2(+)∗30(18.5)18(35.3)Lactobacillus(+)32(19.8)13(25.5) BV(+)22(13.6)11(21.6) TV(+)18(11.1)7(13.7) VVC(+)16(9.9)6(11.8)pH ≤4.875(46.3)19(37.3) >4.887(53.7)32(62.8)CLE∗ Normal128(79.0)30(58.8) Abnormal34(21.0)21(41.2)

五、T淋巴细胞亚群水平与HSIL患者术后HPV转归的关系

术后12月对比两组HSIL患者T淋巴细胞亚群水平,转阴组与未转阴组患者CD3+、CD8+细胞数无显著差异(P>0.05),转阴组患者CD4+细胞数及Th/Ts水平显著高于未转阴组(P<0.05),见图2。

图2 HSIL患者T淋巴细胞亚群水平与术后HPV转归情况对比

Figure 2 Comparison of the level of T lymphocyte subsets and postoperative HPV outcome in HSIL patients

六、多因素分析

将HSIL患者术后HPV转归情况(转阴=0,未转阴=1)作为因变量,将单因素分析中差异具有统计学意义的因素,年龄、流产史、CD4+、Th/Ts、IL-6、TNF-α、SIA、H2O2及CLE作为自变量,赋值见表5。多因素Logistic回归分析结果显示,年龄大和TNF-α水平高是HSIL患者术后HPV未转阴的危险因素,CD4+细胞数多和Th/Ts水平高是HSIL患者术后HPV未转阴的保护因素(P<0.05),见表6。

表5 各因素赋值情况

Table 5 Assignment of each factor

VariablesAssigned valueAge<40.00=0;≥40.00=1History of abortion否=0;是=1CD4+<20.14=0;≥20.14=1Th/Ts<0.52=0;≥0.52=1IL-6<10.09=0;≥10.09=1TNF-α<42.38=0;≥42.38=1SIA阴性=0;阳性=1H2O2阴性=0;阳性=1CLE正常=0;异常=1

表6 HSIL患者术后HPV转归多因素分析

Table 6 Multivariate analysis of postoperative HPV outcomes in HSIL patients

VariablesβSEWaldχ2OR95%CIPAge 1.0920.8271.7442.982.21-3.59 0.003History of abortion 0.2880.3050.8931.330.85-1.76 0.088CD4+-0.3230.8330.1500.720.50-0.92 0.026Th/Ts-0.5010.2683.4930.610.32-0.81<0.001IL-6 0.2320.4770.2361.260.95-1.54 0.257TNF-α 1.2501.4020.7943.491.87-5.07<0.001SIA 0.3520.2282.3841.420.83-1.76 0.094H2O2 0.1531.1970.0161.170.67-1.63 0.167CLE 0.3241.0210.1001.380.94-1.78 0.069

七、HSIL患者术后HPV转归预测效能比较

利用年龄、CD4+、Th/Ts及TNF-α水平单独检验预测HSIL患者术后HPV转归情况的ROC曲线AUC分别为0.714、0.676、0.778、0.730,见表7和图3。年龄+CD4++Th/Ts+TNF-α联合检测HSIL患者术后HPV转归情况AUC为0.881,高于任何单一因素预测模型,且约登指数和准确性也高于其他模型,见表7和图3。

表7 HSIL患者术后HPV转归预测效能对比

Table 7 Comparison of predictive efficacy of postoperative HPV outcomes in HSIL patients

VariablesAUC95%CISensitivitySpecificityAccuracyJorden indexAge0.7140.678-0.7520.7080.7170.7130.425CD4+0.6760.638-0.7110.9160.4190.6680.335Th/Ts0.7780.732-0.8140.8250.6710.7480.496TNF-α0.7300.689-0.7730.5180.9340.7260.452Age+CD4++Th/Ts+TNF-α0.8810.837-0.9260.9430.7520.8480.695

图3 年龄、CD4+、Th/Ts及TNF-α水平预测HSIL患者术后HPV转归的ROC曲线

Figure 3 ROC curve of age, CD4+, Th/Ts and TNF-α levels predicting HPV outcome in patients with HSIL

八、危险因素与HPV转归关联性分析

将年龄、CD4+、Th/Ts及TNF-α水平逐层划分(Q1-Q5),建立Logistic模型逐步排除存在共线性的混杂因素,最终校正饮酒、主动或被动吸烟、孕次、产次、病程、流产史、IL-6、SIA、H2O2、CLE,以消除混杂因素对HPV转归的影响,结果见表8。在未经调整的Logistic模型(未校正模型)中,年龄、CD4+、Th/Ts及TNF-α水平与ICP患者妊娠结局显著相关(P<0.001),调整后(模型5),年龄(OR=1.85,95% CI:1.57～2.13)、CD4+(OR=1.69,95% CI:1.41～1.95)、Th/Ts(OR=2.18,95% CI:1.94～2.43)及TNF-α(OR=1.86,95% CI:1.60～2.13)仍为HSIL患者术后HPV转归的独立影响因素。随着年龄、TNF-α的升高(Q2-Q5),CD4+、Th/Ts水平的降低(Q2-Q5),其关联效应值也相应升高,趋势性检验差异均有统计学意义(P趋势<0.05)。

表8 不同年龄、CD4+、Th/Ts及TNF-α对HSIL患者术后HPV转归的关联效应分析

Table 8 Analysis about the association effect of different levels of age, CD4+, Th/Ts and TNF-α on postoperative HPV outcome in patients with HSIL

Note:Model 1=Adjusted for history of abortion、levels of IL-6、SIA、H2O2 and CLE,Model 2=Model 1+Age,Model 3=Model 2+TNF-α,Model 4=Model 3+CD4+,Model 5=Model 4+Th/Ts

VariablesUnadjusted modelModel 1Model 2Model 3Model 4Model 5Age(years) Q1(<35)Reference Q2(35-40)1.28(1.00-1.53)1.33(1.07-1.60)1.27(1.02-1.53)1.28(1.00-1.53)1.23(0.97-1.47)1.31(1.06-1.59) Q3(40-45)1.44(1.18-1.70)1.48(1.23-1.75)1.41(1.15-1.969)1.46(1.19-1.73)1.47(1.20-1.71)1.42(1.15-1.69) Q4(45-50)1.63(1.39-1.89)1.60(1.34-1.86)1.66(1.40-1.91)1.68(1.43-1.93)1.52(1.33-1.78)1.59(1.35-1.84) Q5(>50)1.82(1.57-2.07)1.92(1.66-2.19)1.87(1.59-2.14)1.84(1.56-2.11)1.87(1.60-2.23)1.85(1.57-2.13) Ptendency<0.0010.002<0.0010.001<0.001<0.001CD4+ Q1(>24)Reference Q2(21-24)1.13(0.94-1.47)1.17(0.95-1.49)1.22(0.88-1.50)1.18(0.89-1.48)1.17(1.00-1.50)1.23(0.92-1.46) Q3(18-21)1.34(1.06-1.61)1.29(1.04-1.55)1.25(0.98-1.51)1.25(1.01-1.54)1.35(1.08-1.62)1.31(1.07-1.66) Q4(15-18)1.44(1.17-1.69)1.43(1.16-1.67)1.41(1.17-1.66)1.45(1.21-1.73)1.44(1.12-1.69)1.39(1.13-1.64) Q5(<15)1.56(1.31-1.84)1.57(1.30-1.82)1.59(1.34-1.84)1.55(1.37-1.89)1.52(1.36-1.90)1.69(1.41-1.95) Ptendency<0.001<0.0010.002<0.0010.001<0.001Th/Ts Q1(>0.7)Reference Q2(0.6-0.7)1.45(1.20-1.72)1.41(1.15-1.67)1.47(1.19-1.73)1.42(1.17-1.70)1.37(1.12-1.63)1.36(1.09-1.61) Q3(0.5-0.6)1.56(1.30-1.81)1.59(1.35-1.86)1.69(1.43-1.95)1.59(1.33-1.85)1.64(1.39-1.91)1.66(1.38-1.92) Q4(0.4-0.5)1.89(1.61-2.15)1.93(1.68-2.21)1.87(1.63-2.13)1.93(1.67-2.20)1.87(1.60-2.14)1.90(1.65-2.15) Q5(<0.4)2.16(1.90-2.43)2.15(1.91-2.42)2.14(1.88-2.38)2.26(1.98-2.53)2.29(2.08-2.57)2.18(1.94-2.43) Ptendency<0.001<0.001<0.001<0.001<0.001<0.001TNF-α Q1(<35)Reference Q2(35-40)1.29(1.03-1.57)1.34(1.08-1.61)1.25(1.01-1.49)1.34(1.05-1.61)1.26(1.03-1.53)1.29(1.03-1.55) Q3(40-45)1.38(1.11-1.64)1.40(1.14-1.67)1.37(1.11-1.65)1.38(1.12-1.69)1.40(1.14-1.68)1.48(1.27-1.81) Q4(45-50)1.55(1.28-1.83)1.52(1.27-1.78)1.68(1.40-1.95)1.58(1.33-1.85)1.61(1.34-1.88)1.62(1.35-1.91) Q5(>50)1.94(1.67-2.20)1.88(1.63-2.13)1.86(1.61-2.11)1.91(1.65-2.16)1.93(1.64-2.17)1.86(1.60-2.13) Ptendency<0.0010.003<0.0010.003<0.001<0.001

讨 论

CC是原发于子宫颈部位的恶性肿瘤,为女性生殖道最常见的恶性肿瘤,其发病早期缺乏典型临床表现,导致部分患者确诊时就已经是中晚期,从而错失最佳治疗时机,对生命安全造成威胁[11]。HSIL是宫颈癌前病变的典型表现,目前已明确的主要致病原因为HPV的持续感染[12]。LEEP刀宫颈锥切术能有效切除病变组织和清除HPV,降低CIN进一步癌变风险,但有些潜藏于皮肤深处的HPV无法完全消除,并且LEEP切口由于热损伤导致病变组织残留,可能会导致HPV持续感染甚至CIN复发[13-14]。HPV不仅存在于宫颈病变组织中,还能在阴道上皮组织等部位检测出,LEEP刀宫颈锥切术无法清除宫颈以外的HPV,只能依靠患者自身免疫力清除残留HPV,绝大多数HSIL患者术后HPV感染逐渐转阴,但少部分患者术后病变组织残留、免疫功能紊乱,导致HPV持续感染和病情复发[15]。因此,HSIL患者机体免疫应答与术后HPV转归关系的研究尤为重要,对临床治疗HSIL和清除HPV具有指导意义。

T细胞是细胞免疫功能中最重要的细胞群,而免疫球蛋白(immunoglobulin,Ig)是人体体液免疫功能中最重要的蛋白家族,当人体细胞和体液免疫应答产生变化时,T细胞和Ig水平也会随之变化[16]。本研究中HSIL患者术后12月复诊时CD3+、CD4+、Th/Ts、IgA、IgG、IgM水平显著升高,CD8+细胞数则显著降低,同时还发现CD4+细胞数及Th/Ts水平为HSIL患者术后HPV转归的独立影响因素。李世蓉[17]等研究也认为,CD4+百分比及CD4+/CD8+比值的消长对不同宫颈病变HPV感染的转归起着重要作用。HPV持续感染会对细胞免疫和体液免疫造成不同程度损伤,大量抗体和抗原被消耗,导致患者自身免疫无法完全清除HPV,进一步降低机体免疫力[18]。有研究表示CIN术后恢复情况与患者年龄和TNF-α水平有关[19-22],本研究也有类似发现,年龄大和TNF-α水平高均为影响HSIL患者术后HPV转归的危险因素,可能是由于患者雌激素的分泌随年龄增加而逐渐减少,阴道黏膜萎缩、变薄,导致阴道抵抗力降低,引起残留HPV持续感染[23]。而TNF-α生物学功能包括抗肿瘤、免疫调节、介导炎症等,TNF-α的过量产生,暗示了一些病理条件下所起的重要作用,包括疾病恶化、免疫紊乱等,影响了HSIL患者HPV的转归[24]。本研究利用年龄、CD4+、Th/Ts及TNF-α水平预测HSIL患者术后HPV转归情况发现,年龄+CD4++Th/Ts+TNF-α联合检测HSIL患者术后HPV转归情况AUC为0.881,高于任何单一因素预测模型,且约登指数和准确性分别为0.695、0.848,均高于其他预测模型。将年龄、CD4+、Th/Ts及TNF-α水平逐层划分分析其与HPV转归关联性,消除混杂因素后分析结果显示随着年龄、TNF-α的升高,和CD4+、Th/Ts水平的降低,其关联效应值也相应升高。

本研究尚存在一定的局限性,纳入的样本数据来自同一医疗中心,结果难免存在一定偏倚;研究缺少术后12月后相关数据分析,分析结果的深度和广度需进一步提升,可将远期预后相关数据分析作为未来研究重点。

综上所述,与术前相比,术后12月HSIL患者细胞免疫应答和体液免疫应答得到显著改善;年龄大和TNF-α水平高是HSIL患者术后HPV未转阴的危险因素,CD4+细胞数多及Th/Ts水平高是HSIL患者术后HPV未转阴的保护因素;年龄+CD4++Th/Ts+TNF-α联合检测HSIL患者术后HPV转归情况,其预测性能较好;消除混杂因素后,随着年龄、TNF-α的升高,CD4+细胞数、Th/Ts水平的降低,其与HPV转归的关联效应值也相应升高。

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Relationship between immune response and HPV outcome after high-grade cervical intraepithelial neoplasia

DUAN Xiaoling, LU Qi. Department of Obstetrics and Gynecology, Jinshan Hospital, Fudan University, Shanghai 201508, China

[Abstract] Objective To investigate the relationship between immune response and human papilloma virus (HPV) outcome in patients with high-grade cervical intraepithelial neoplasia (HSIL). Methods 213 patients with HSIL admitted to our hospital from September 2020 to March 2022 were selected as the research subjects. They aged 28-63 years with an average age of (39.0 ± 4.8) years. We compared the changes in various serum immune indicators of HSIL patients before surgery, 3 months after surgery, 6 months after surgery, and 12 months after surgery. According to the HPV conversion status of HSIL patients 12 months after surgery, they were divided into a negative conversion group (n=162) and a non-negative conversion group (n=51). We compared the general and clinical data of the two groups, and used multiple logistic regression analysis to screen independent influencing factors of HPV conversion in HSIL patients. We also compared the predictive efficacy of each factor for HPV conversion, and analyzed the correlation between each factor and HPV conversion. Results The levels of CD3+, CD4+, Th/Ts, immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) were significantly increased in patients with HSIL 12 months after surgery, while the levels of CD8+ were significantly decreased (P<0.05). The age of patients and the proportion of patients with abortion history in the non-negative conversion group were significantly higher than those in the negative conversion group (P<0.05). The levels of CD4+ and Th/Ts in patients with negative conversion were significantly higher than those in patients without negative conversion, and the levels of IL-6 and TNF-α in patients with negative conversion were significantly lower than those in patients without negative conversion. There were significant differences in the number of sialidase (SIA) positive, H2O2 positive and abnormal vaginal cleanliness (CLE) patients between the two groups (P<0.05). Multivariate logistic regression analysis showed that older age and higher TNF-α level were risk factors for postoperative HPV non-negative conversion in patients with HSIL, and more CD4+ cell counts and higher Th/Ts level were protective factors for postoperative HPV non-negative conversion in patients with HSIL (P<0.05). The area under curve (AUC) of postoperative HPV outcome in HSIL patients tested by age+CD4++Th/Ts+TNF-α was 0.881, the Yoden index was 0.695, and the accuracy was 0.848, which were all higher than other prediction models. After establishing logistic model through gradually excluding the confounding factors, we stratified by age, CD4+, Th/Ts and TNF-α levels, and found that age, CD4+, Th/Ts and TNF-α levels were still independent influencing factors for postoperative HPV outcome in patients with HSIL. The association effect values increased with the increment of age and TNF-α, and the decrease of CD4+ and Th/Ts levels (P trend <0.05). Conclusion The cellular and humoral immune responses of HSIL patients were significantly improved 12 months after surgery compared with those before surgery. Age and TNF-α level were risk factors for non-negative HPV conversion in HSIL patients, and CD4+ and Th/Ts were protective factors for non-negative HPV conversion in HSIL patients. The combined detection of postoperative HPV outcomes in HSIL patients by age+CD4++ Th/Ts+TNF-α showed good predictive performance. After the elimination of confounding factors, the associated effect sizes with HPV outcomes increased with the decreased levels of CD4+ and Th/Ts and the increment of age and TNF-α.

[Key words] body immune response; high-grade squamous intraepithelial lesion; T lymphocytes; HPV outcome

【中图分类号】 R71

作者单位：201508 上海,复旦大学附属金山医院妇产科

通信作者：陆琦(hathorl@163.com)

(收稿日期：2024-09-11)