宮頸癌患者血清可溶性B7-H4檢測及臨床意義

胡國艷,鄭淑華,劉 偉,劉學軍,呂 晶,王小梅,徐軍發*

(1.廣州市精神病醫院 檢 驗科,廣東廣州 510370;2.廣東醫學院臨床免疫學教研室,廣東東莞 523808;3.廣東醫學院第一附屬醫院檢驗科,廣東湛江 524001)

宮頸癌患者血清可溶性B7-H4檢測及臨床意義

胡國艷1,2,鄭淑華2,劉 偉2,劉學軍1,呂 晶2,王小梅3,徐軍發2*

(1.廣州市精神病醫院 檢 驗科,廣東廣州 510370;2.廣東醫學院臨床免疫學教研室,廣東東莞 523808;3.廣東醫學院第一附屬醫院檢驗科,廣東湛江 524001)

目的探討宮頸癌患者血清可溶性B7-H4的水平及臨床意義。方法在建立了檢測可溶性B7-H4(sB7-H4)ELISA夾心法的檢測50例宮頸癌患者、40例子宮肌瘤患者血清sB7-H4水平,以50例體檢健康者血清sB7-H4水平為對照。結果正常體檢者、子宮肌瘤患者和宮頸癌患者血清 sB7-H4濃度分別為(31.62±9.85)μ g/L、(34.49±8.17)μ g/L和(43.70±8.60)μ g/L。對照組者與子宮肌瘤患者血清sB7-H4水平差異無顯著性(P＞0.05),宮頸癌患者血清sB7-H4水平顯著高于對照組和子宮肌瘤患者(P均＜0.01)。手術前宮頸癌患者血清sB7-H4水平(46.88±8.89)μ g/L明顯高于術后患者(39.29±5.99)μ g/L(P＜0.01)。宮頸癌患者血清sB7-H4水平與鱗狀上皮癌腫瘤(SCC)水平呈正相關(P＜0.01)。結論sB7-H4與宮頸癌的發病有關,可能是宮頸癌的一種較好的腫瘤標志物,檢測sB7-H4對宮頸癌的輔助診斷和療效觀察具有一定的臨床意義。

宮頸癌;子宮肌瘤;sB7-H4

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

B7-H4是2003年發現的B7家族成員,在T細胞介導的免疫中起負調節作用,主要表現為抑制T細胞的增殖、G0/G1期的細胞周期進程和IL-2的產生[1]。B7-H4在正常組織中無表達或有極低水平表達,但已經發現在多種腫瘤組織中高表達,如卵巢癌[2]、肺癌[3]、腎細胞癌[4]、乳腺癌[5]、子宮內膜癌[6]、胰腺癌[7]和前列腺癌[8],并發現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],但有關可溶性B7-H4與宮頸癌的關系未見報道。本研究在建立了檢測可溶性B7-H4 ELISA夾心法[9]的基礎上對部分健康體檢者和子宮肌瘤患者與宮頸癌患者血清B7-H4水平進行了檢測。

1 材料與方法

1.1 實驗對象 宮頸癌組50例,子宮肌瘤40例,均來自廣東醫學院附屬醫院腫瘤科,所有經病理檢查確診,50例宮頸癌中Ⅰ期患者17例,Ⅱ期患者25例,Ⅲ、Ⅳ期患者8例,手術前 29例,手術后21例,患者年齡31-77歲,平均年齡(50.18±10.50)歲。40例子宮肌瘤中,患者年齡 30-57歲,平均年齡(42.56±6.59)歲。健康對照組50例,來自廣東醫學院附屬醫院體檢中心體檢健康者標本,男29例、女21例,年齡18-67歲,平均年齡(45.65±11.38)歲,經體檢無異常。

1.2 材料 酶聯微量反應板,來源于Coning公司。抗人B7-H4單克隆抗體(mAb),購自eBioscience公司。重組小鼠B7-H4蛋白及兔抗小鼠B7-H4多克隆抗體由本室制備[9]。HRP標記羊抗兔IgG,購自北京博奧森生物技術有限公司。TMB底物及顯色劑,沈陽惠民公司產品。BIOTEK Elx800型酶標儀等。SCC試劑盒為瑞典CanAg公司產品。

1.3 雙抗體夾心間接ELISA檢測方法 用0.05 mol/L pH9.6的碳酸鹽緩沖液按1∶2 000稀釋抗人B7-H4mAb,包被可拆酶標板,置4℃過夜,用PBST洗滌3次。采用100 ml/L FCS封閉,200μ L/孔,37℃作用2 h后用PBST洗滌3次。用PBST-BSA將200 mg/L重組小鼠B7-H4從1∶1 000做倍比稀釋至1∶64 000作為標準品。在包被好的酶標板中加入標準品或待測血清標本100 μ L/孔。37℃作用 1 h,用PBST洗3次。加入1∶1 000稀釋兔抗小鼠B7-H4多克隆抗體,100 μ L/孔,37℃作用1 h后用PBST洗3次,再加入1∶5 000稀釋HRP標記羊抗兔 IgG,37℃作用1 h 后用 PBST洗 3 次,按100 μ L/孔加顯色底物,作用15min,以2 mol/L硫酸終止,在酶標儀上測A450值。

1.4 樣本收集與檢測 被檢者在早上空腹采血,靜置1 h后,分離血清,小量分裝,置-70℃保存備測。所有樣本均在同一批次檢測,每個樣本設立兩個平行檢測,取平均A值計算樣本中 sB7-H4的含量。SCC相關抗原檢測由廣州市精神病醫院檢驗科完成。

2 結果

2.1 宮頸癌、子宮肌瘤患者及健康體檢者血清sB7-H4水平

宮頸癌、子宮肌瘤患者及健康體檢者血清sB7-H4水平見圖 1,宮頸癌患者血清 sB7-H4水平[(43.70±8.60)μ g/L]顯著高于子宮肌瘤患者[(34.50±8.17)μ g/L](t=4.106,P＜0.001)和健康體檢者[(31.62±9.85)μ g/L](t=5.235,P＜0.001),而子宮肌瘤組與健康體檢者對照組相比無統計學意義(t=1.881,P＞0.05)。

圖1 宮頸癌、子宮肌瘤患者及健康體檢者血清sB7-H4

2.2 術前和術后宮頸癌患者血清sB7-H4水平的比較

結果見表2。結果顯示,癌術前宮頸患者血清sB7-H4水平顯著高于后術患者,差異有顯著性意義(t=3.390,P＜0.05)。

表2 術前和術后宮頸癌患者血清B7-H4水平

2.3 不同臨床分期宮頸癌患者血清sB7-H4水平的變化

表3顯示,不同臨床分期宮頸癌患者隨著病情加重血清sB7-H4水平有增高趨勢,經方差分析,各組間比較差異無顯著性(F=2.444,P＞0.05)。

表3 宮頸癌術前和術后血清B7-H4檢測結果

2.4 宮頸癌患者血清B7-H4水平與SCC相關性

將50例宮頸癌患者血清sB7-H4水平與SCC水平進行單因素直線相關分析,結果顯示sB7-H4水平與SCC水平呈正相關(r=0.632,P＜0.01)。

3 討論

子宮頸癌是婦科最常見的惡性腫瘤之一。在全球范圍內,每年約有20多萬女性死于宮頸癌。在發展中國家,宮頸癌則屬于常見多發的婦科腫瘤,排行榜首。宮頸癌早期沒有任何癥狀,當宮頸癌的癥狀出現三個月后就診者已有2/3為癌癥晚期。

B7-H4于2003年被發現后,各種關于B7-H4與腫瘤關系的研究越來越多,研究發現多種腫瘤組織高表達B7-H4,如卵巢癌、腎細胞癌、非小細胞性肺癌、乳腺癌和前列腺癌[2-11]等,在前列腺癌[8]、乳腺癌[11]、腎癌[4]、非小細胞性肺癌[3]中 B7-H4的表達與腫瘤的分型、分期和病人的轉歸密切相關,而且在肺癌和前列腺癌中B7-H4的表達與腫瘤的轉移相關,高表達B7-H4的前列腺癌更容易擴散、復發,患者死亡率也高[8],因此B7-H4被認為是一種較敏感的腫瘤標志物。但以上研究主要是基于腫瘤組織的免疫組化染色得到的結果,對于血清中可溶性B7-H4的檢測少見有報道,我們用本室建立的檢測可溶性B7-H4 ELISA夾心法對部分體檢健康者、子宮肌瘤患者與宮頸癌患者血清B7-H4水平進行了檢測,結果發現宮頸癌患者血清B7-H4水平與健康體檢者和子宮肌瘤患者B7-H4水平的差異有統計學意義,而健康體檢者與子宮肌瘤患者之間B7-H4水平差異無統計學意義,而且宮頸癌患者血清B7-H4水平與SCC水平呈正相關,提示B7-H4與宮頸癌的發病有關,對于鑒別子宮良、惡性腫瘤有一定的意義,可能是宮頸癌的一種較好的腫瘤標志物。我們的結果也表明術后宮頸癌患者血清sB7-H4水平顯著低于術前患者,提示宮頸癌患者血清sB7-H4水平與治療狀況相關。宮頸癌患者血清sB7-H4水平與腫瘤的臨床分期無顯著相關性,可能與本研究觀察病例偏少有關。本研究結果提示血清sB7-H4水平的檢測對宮頸癌的輔助診斷和療效觀察具有一定的臨床意義。

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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

國家自然科學基金(30922779),廣東省社會發展計劃項目(2006B36002021),東莞市科技計劃項目(2008108101054)

*通訊作者

胡國艷(1976-),女,湖南石門人,主管檢驗師。

2009-11-11)