宫颈癌患者血清可溶性B7-H4检测及临床意义

胡国艳,郑淑华,刘 伟,刘学军,吕 晶,王小梅,徐军发*

(1.广州市精神病医院 检 验科,广东广州 510370;2.广东医学院临床免疫学教研室,广东东莞 523808;3.广东医学院第一附属医院检验科,广东湛江 524001)

宫颈癌患者血清可溶性B7-H4检测及临床意义

胡国艳1,2,郑淑华2,刘 伟2,刘学军1,吕 晶2,王小梅3,徐军发2*

(1.广州市精神病医院 检 验科,广东广州 510370;2.广东医学院临床免疫学教研室,广东东莞 523808;3.广东医学院第一附属医院检验科,广东湛江 524001)

目的探讨宫颈癌患者血清可溶性B7-H4的水平及临床意义。方法在建立了检测可溶性B7-H4(sB7-H4)ELISA夹心法的检测50例宫颈癌患者、40例子宫肌瘤患者血清sB7-H4水平,以50例体检健康者血清sB7-H4水平为对照。结果正常体检者、子宫肌瘤患者和宫颈癌患者血清 sB7-H4浓度分别为(31.62±9.85)μ g/L、(34.49±8.17)μ g/L和(43.70±8.60)μ g/L。对照组者与子宫肌瘤患者血清sB7-H4水平差异无显著性(P＞0.05),宫颈癌患者血清sB7-H4水平显著高于对照组和子宫肌瘤患者(P均＜0.01)。手术前宫颈癌患者血清sB7-H4水平(46.88±8.89)μ g/L明显高于术后患者(39.29±5.99)μ g/L(P＜0.01)。宫颈癌患者血清sB7-H4水平与鳞状上皮癌肿瘤(SCC)水平呈正相关(P＜0.01)。结论sB7-H4与宫颈癌的发病有关,可能是宫颈癌的一种较好的肿瘤标志物,检测sB7-H4对宫颈癌的辅助诊断和疗效观察具有一定的临床意义。

宫颈癌;子宫肌瘤;sB7-H4

(Chin J Lab Diagn,2010,14:1425)

B7-H4是2003年发现的B7家族成员,在T细胞介导的免疫中起负调节作用,主要表现为抑制T细胞的增殖、G0/G1期的细胞周期进程和IL-2的产生[1]。B7-H4在正常组织中无表达或有极低水平表达,但已经发现在多种肿瘤组织中高表达,如卵巢癌[2]、肺癌[3]、肾细胞癌[4]、乳腺癌[5]、子宫内膜癌[6]、胰腺癌[7]和前列腺癌[8],并发现卵巢癌等患者血清中可溶性B7-H4(soluble B7-H4,sB7-H4)水平的增高[2],但有关可溶性B7-H4与宫颈癌的关系未见报道。本研究在建立了检测可溶性B7-H4 ELISA夹心法[9]的基础上对部分健康体检者和子宫肌瘤患者与宫颈癌患者血清B7-H4水平进行了检测。

1 材料与方法

1.1 实验对象 宫颈癌组50例,子宫肌瘤40例,均来自广东医学院附属医院肿瘤科,所有经病理检查确诊,50例宫颈癌中Ⅰ期患者17例,Ⅱ期患者25例,Ⅲ、Ⅳ期患者8例,手术前 29例,手术后21例,患者年龄31-77岁,平均年龄(50.18±10.50)岁。40例子宫肌瘤中,患者年龄 30-57岁,平均年龄(42.56±6.59)岁。健康对照组50例,来自广东医学院附属医院体检中心体检健康者标本,男29例、女21例,年龄18-67岁,平均年龄(45.65±11.38)岁,经体检无异常。

1.2 材料 酶联微量反应板,来源于Coning公司。抗人B7-H4单克隆抗体(mAb),购自eBioscience公司。重组小鼠B7-H4蛋白及兔抗小鼠B7-H4多克隆抗体由本室制备[9]。HRP标记羊抗兔IgG,购自北京博奥森生物技术有限公司。TMB底物及显色剂,沈阳惠民公司产品。BIOTEK Elx800型酶标仪等。SCC试剂盒为瑞典CanAg公司产品。

1.3 双抗体夹心间接ELISA检测方法 用0.05 mol/L pH9.6的碳酸盐缓冲液按1∶2 000稀释抗人B7-H4mAb,包被可拆酶标板,置4℃过夜,用PBST洗涤3次。采用100 ml/L FCS封闭,200μ L/孔,37℃作用2 h后用PBST洗涤3次。用PBST-BSA将200 mg/L重组小鼠B7-H4从1∶1 000做倍比稀释至1∶64 000作为标准品。在包被好的酶标板中加入标准品或待测血清标本100 μ L/孔。37℃作用 1 h,用PBST洗3次。加入1∶1 000稀释兔抗小鼠B7-H4多克隆抗体,100 μ L/孔,37℃作用1 h后用PBST洗3次,再加入1∶5 000稀释HRP标记羊抗兔 IgG,37℃作用1 h 后用 PBST洗 3 次,按100 μ L/孔加显色底物,作用15min,以2 mol/L硫酸终止,在酶标仪上测A450值。

1.4 样本收集与检测 被检者在早上空腹采血,静置1 h后,分离血清,小量分装,置-70℃保存备测。所有样本均在同一批次检测,每个样本设立两个平行检测,取平均A值计算样本中 sB7-H4的含量。SCC相关抗原检测由广州市精神病医院检验科完成。

2 结果

2.1 宫颈癌、子宫肌瘤患者及健康体检者血清sB7-H4水平

宫颈癌、子宫肌瘤患者及健康体检者血清sB7-H4水平见图 1,宫颈癌患者血清 sB7-H4水平[(43.70±8.60)μ g/L]显著高于子宫肌瘤患者[(34.50±8.17)μ g/L](t=4.106,P＜0.001)和健康体检者[(31.62±9.85)μ g/L](t=5.235,P＜0.001),而子宫肌瘤组与健康体检者对照组相比无统计学意义(t=1.881,P＞0.05)。

图1 宫颈癌、子宫肌瘤患者及健康体检者血清sB7-H4

2.2 术前和术后宫颈癌患者血清sB7-H4水平的比较

结果见表2。结果显示,癌术前宫颈患者血清sB7-H4水平显著高于后术患者,差异有显著性意义(t=3.390,P＜0.05)。

表2 术前和术后宫颈癌患者血清B7-H4水平

2.3 不同临床分期宫颈癌患者血清sB7-H4水平的变化

表3显示,不同临床分期宫颈癌患者随着病情加重血清sB7-H4水平有增高趋势,经方差分析,各组间比较差异无显著性(F=2.444,P＞0.05)。

表3 宫颈癌术前和术后血清B7-H4检测结果

2.4 宫颈癌患者血清B7-H4水平与SCC相关性

将50例宫颈癌患者血清sB7-H4水平与SCC水平进行单因素直线相关分析,结果显示sB7-H4水平与SCC水平呈正相关(r=0.632,P＜0.01)。

3 讨论

子宫颈癌是妇科最常见的恶性肿瘤之一。在全球范围内,每年约有20多万女性死于宫颈癌。在发展中国家,宫颈癌则属于常见多发的妇科肿瘤,排行榜首。宫颈癌早期没有任何症状,当宫颈癌的症状出现三个月后就诊者已有2/3为癌症晚期。

B7-H4于2003年被发现后,各种关于B7-H4与肿瘤关系的研究越来越多,研究发现多种肿瘤组织高表达B7-H4,如卵巢癌、肾细胞癌、非小细胞性肺癌、乳腺癌和前列腺癌[2-11]等,在前列腺癌[8]、乳腺癌[11]、肾癌[4]、非小细胞性肺癌[3]中 B7-H4的表达与肿瘤的分型、分期和病人的转归密切相关,而且在肺癌和前列腺癌中B7-H4的表达与肿瘤的转移相关,高表达B7-H4的前列腺癌更容易扩散、复发,患者死亡率也高[8],因此B7-H4被认为是一种较敏感的肿瘤标志物。但以上研究主要是基于肿瘤组织的免疫组化染色得到的结果,对于血清中可溶性B7-H4的检测少见有报道,我们用本室建立的检测可溶性B7-H4 ELISA夹心法对部分体检健康者、子宫肌瘤患者与宫颈癌患者血清B7-H4水平进行了检测,结果发现宫颈癌患者血清B7-H4水平与健康体检者和子宫肌瘤患者B7-H4水平的差异有统计学意义,而健康体检者与子宫肌瘤患者之间B7-H4水平差异无统计学意义,而且宫颈癌患者血清B7-H4水平与SCC水平呈正相关,提示B7-H4与宫颈癌的发病有关,对于鉴别子宫良、恶性肿瘤有一定的意义,可能是宫颈癌的一种较好的肿瘤标志物。我们的结果也表明术后宫颈癌患者血清sB7-H4水平显著低于术前患者,提示宫颈癌患者血清sB7-H4水平与治疗状况相关。宫颈癌患者血清sB7-H4水平与肿瘤的临床分期无显著相关性,可能与本研究观察病例偏少有关。本研究结果提示血清sB7-H4水平的检测对宫颈癌的辅助诊断和疗效观察具有一定的临床意义。

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Clinical significance of determination of serum B7-H4 in patients with uterine cervix cancer

HU Guo-yan1,2,ZHENGShuhua2,LIU Wei2,et al.(1.Department of clinical laboratory,Guangzhou psychiatric hospital,Guangzhou510370;2.Department of Clinical Immunology,Guangdong Medical College,Dongguan523808,China)

ObjectiveTo study the clinical significance of determination of serum B7-H4 in patientswith uterine cervix cancer.MethodsSerum B7-H4 levelswere determined in 50 patients with uterine cervix cancer and 40 patients with uterine fibroid as well as in 35 controls.ResultsThe serum B7-H4 levels in patients with uterine cervix cancer(43.70 ±8.60 μ g/L)were significantly higher than those in patientswith uterine fibroid(36.63±7.70μ g/L)and healthy controls(31.62±9.85μ g/L)(P＜0.01).But the B7-H4 in serum were not significant between patients with uterine fibroid and healthy controls(P＞0.05).The serum B7-H4 levels in patientswithuterine cervix cancer before surgical treatment(46.65±8.59μ g/L)washigher than that in patientswithuterine cervix cancer after surgical treatment(38.45±9.33μ g/L)(P＜0.01).There was correlation between the serum B7-H4 levels in patients with uterine cervix cancer and the serum SCC levels in patients with uterine cervix cancer(P＜0.05).ConclusionThese results suggest that the B7-H4 may correlationwith uterine cervix cancer.B7-H4 may be a new marker of diagnosis for uterine cervix cancer.Measurement of serum B7-H4,not only provide useful information for distinctive diagnosisof different kindsof uterine diseases,but also is a good target in evaluation of disease extent and effect of treatments in patients with uterine cervix cancer.

uterine cervix cancer;uterine fibroid;B7-H4

R730.3

A

1007-4287(2010)09-1425-03

国家自然科学基金(30922779),广东省社会发展计划项目(2006B36002021),东莞市科技计划项目(2008108101054)

*通讯作者

胡国艳(1976-),女,湖南石门人,主管检验师。

2009-11-11)