細(xì)胞核內(nèi)特征值的改變可引起DNA倍體的變化

王景潔 孫全新 孫小蓉 牛曉泉

細(xì)胞核內(nèi)特征值的改變可引起DNA倍體的變化

王景潔1孫全新2孫小蓉3牛曉泉4＊

(1武漢市第五醫(yī)院保健科,武漢430050;2湖北中山醫(yī)院細(xì)胞室,武漢430033;3華中科技大學(xué)同濟(jì)醫(yī)學(xué)院中加腫瘤早期診斷研究中心,武漢 430022;4武漢市第五醫(yī)院科教科,武漢430050)

目的 通過(guò)測(cè)定不同DNA倍體細(xì)胞,研究細(xì)胞核內(nèi)特征值的改變。方法 用宮頸刷刷出宮頸細(xì)胞,經(jīng)固定后,用涂片離心機(jī)制成二張玻片,一張行巴氏染色作 TBS診斷,另一張行Feulgen染色做DNA定量測(cè)定。通過(guò)對(duì)宮頸細(xì)胞核圖像內(nèi)像素的統(tǒng)計(jì),計(jì)算出細(xì)胞核內(nèi)多種特征值,比較不同DNA倍體細(xì)胞內(nèi)特征值的不同。結(jié)果 161873例婦女行宮頸細(xì)胞學(xué)檢查,常規(guī)細(xì)胞學(xué)檢查發(fā)現(xiàn)2454例低級(jí)別鱗狀上皮內(nèi)病變(low-grade squamous intraepithelial lesion,LSIL)和523例高級(jí)別鱗狀上皮內(nèi)病變(high-grade squamous intraepithelial lesion,HSIL);而DNA倍體分析發(fā)現(xiàn)3412例有3個(gè)以上>5c細(xì)胞。84%以上的LSIL和 HSIL病例均可見(jiàn)倍體異常細(xì)胞。與2c細(xì)胞相比,4c、5c、7c及9c細(xì)胞核面積及核半徑明顯增大;7c、9c細(xì)胞核內(nèi)平均光學(xué)密度和緊實(shí)度均值也有明顯改變,而光密度方差和灰度熵?zé)o變化。結(jié)論 宮頸細(xì)胞DNA倍體改變往往伴有細(xì)胞形態(tài)和DNA核內(nèi)分布等特征值的改變。

DNA倍體; 圖像分析系統(tǒng); 宮頸癌; 非整倍體細(xì)胞

DNA圖像分析系統(tǒng)可對(duì)細(xì)胞核進(jìn)行定量分析從而測(cè)定出細(xì)胞核內(nèi)DNA含量,其主要過(guò)程是先將細(xì)胞圖像進(jìn)行分割,再計(jì)算細(xì)胞核的相關(guān)特征值,通過(guò)對(duì)這些特征值的統(tǒng)計(jì)和分析,最終將細(xì)胞分類(lèi)及計(jì)算細(xì)胞核DNA含量。DNA圖像分析已經(jīng)運(yùn)用于臨床檢測(cè),尤其是對(duì)宮頸細(xì)胞進(jìn)行定量測(cè)定[1-3]。宮頸癌是由人類(lèi)乳頭瘤病毒(human papilloma virus,HPV)持續(xù)感染宮頸細(xì)胞所致。HPV病毒可引起宮頸細(xì)胞DNA倍體發(fā)生改變[4-6]。因此在大多宮頸癌及癌前病變中,通過(guò)DNA圖像分析可發(fā)現(xiàn)宮頸細(xì)胞DNA含量異常,即DNA異倍體細(xì)胞[7]。雖然有很多對(duì)宮頸細(xì)胞進(jìn)行DNA定量測(cè)定的報(bào)道,但對(duì)其異倍體細(xì)胞的特征改變的研究較少。本文試圖對(duì)宮頸細(xì)胞的定量測(cè)量,研究異倍體細(xì)胞核內(nèi)特征值改變。

材料和方法

1.標(biāo)本來(lái)源

收集2008-2009年兩年內(nèi)武漢市第五醫(yī)院和湖北省中山醫(yī)院進(jìn)行子宮頸液基薄層細(xì)胞學(xué)檢查的病例161873例,所有病例的年齡均在25-54歲之間。宮頸細(xì)胞檢查試劑盒由武漢蘭丁醫(yī)學(xué)高科技有限公司提供,其中包括:宮頸刷、盛有固定液的標(biāo)本管、標(biāo)簽等。另有8例宮頸活檢證實(shí)為宮頸上皮內(nèi)瘤變(cervical intraepithelial neoplasm,CIN),其中 3 例CIN2、2例CIN3及3例宮頸癌。這8例來(lái)源于湖北省中山醫(yī)院細(xì)胞病理室。

2.取材

婦科醫(yī)生在陰道擴(kuò)張器直視下,手持宮頸刷的刷柄,通過(guò)擴(kuò)陰器,將刷頭中央較長(zhǎng)的刷絲從子宮頸外口插入子宮頸管內(nèi),周邊刷絲頂部抵觸在子宮頸黏膜面,順時(shí)針或逆時(shí)針旋轉(zhuǎn)3-5圈,取出宮頸刷后,用鑷子夾緊刷頭底座,拔脫刷頭,將刷尖向下垂直方向浸泡在樣本收集管固定液內(nèi),旋緊管蓋后震蕩搖晃,然后將貼上標(biāo)簽的標(biāo)本管送到細(xì)胞實(shí)驗(yàn)室制片。

3.液基薄層制片及染色

將裝有固定液和刷頭的標(biāo)本收集管,加入消化液放在振蕩器上振蕩2h,然后將消化后的細(xì)胞懸液離心5min(800轉(zhuǎn)/分),棄上清液后,加50%酒精分別清洗、離心2次(800轉(zhuǎn)/分×5min/每次)。然后將用固定液稀釋的細(xì)胞混懸液,放入細(xì)胞涂片離心機(jī)甩片5min,每例制成2張薄層細(xì)胞學(xué)片,其中1片進(jìn)行巴氏染色做常規(guī)細(xì)胞學(xué)診斷,另外1片進(jìn)行Feulgen染色做DNA定量測(cè)定。

4.常規(guī)細(xì)胞學(xué)診斷

所有巴氏染色片子經(jīng)兩位有經(jīng)驗(yàn)的細(xì)胞病理學(xué)醫(yī)生讀片。根據(jù) TBS診斷分為6組:(1)正?；蛄夹?(2)不明意義的非典型鱗狀上皮 (atypical squamous cells of undetermined significance,ASCUS);(3)不能除外高級(jí)別鱗狀上皮內(nèi)病變的非典型鱗狀上皮(atypical squamous cells,cannot exclude highgrade squamous intraepithelial lesion);(4)低級(jí)別鱗狀上皮內(nèi)病變(low-grade squamous intraepithelial lesion,LSIL);(5)高級(jí)別鱗狀上皮內(nèi)病變(highgrade squamous intraepithelial lesion,HSIL)或原位癌(carcinoma in situ,CIS);(6)不典型腺細(xì)胞(atypical glandular cell,A GC)。

5.DNA倍體分析

所有Feulgen染色片用蘭丁全自動(dòng)細(xì)胞圖像分析系統(tǒng)進(jìn)行掃描處理,該系統(tǒng)已達(dá)到歐洲定量細(xì)胞病理學(xué)會(huì)所制定的有關(guān)DNA定量分析圖像系統(tǒng)的各項(xiàng)標(biāo)準(zhǔn)[8]。細(xì)胞片染色質(zhì)量控制用大鼠肝細(xì)胞片子作對(duì)照。一般情況下,該系統(tǒng)對(duì)每張玻片上6000個(gè)以上的細(xì)胞核進(jìn)行掃描測(cè)定。經(jīng)掃描后的每個(gè)細(xì)胞核均有77個(gè)特征值,其中包括形態(tài)特征、吸光特征、具體結(jié)構(gòu)特征、Markovian和非Markovian結(jié)構(gòu)特征和長(zhǎng)度特征等。系統(tǒng)根據(jù)不同細(xì)胞成分所具有的不同特征參數(shù)來(lái)完成自動(dòng)細(xì)胞分類(lèi)和計(jì)數(shù)過(guò)程,如正常上皮細(xì)胞,增生或癌變細(xì)胞,淋巴細(xì)胞及未參與診斷的垃圾細(xì)胞(重疊細(xì)胞核,聚焦不良細(xì)胞核和核碎片等)。標(biāo)準(zhǔn)對(duì)照細(xì)胞為同張玻片上的100個(gè)正常上皮細(xì)胞,所測(cè)出的平均光密度 (integrated optical density,IOD)為2C的參考值,其CV(coefficient of variation)值小于5%。凡是有大于5C以上非整倍體細(xì)胞的玻片,都要通過(guò)細(xì)胞技術(shù)員在顯微鏡下逐一對(duì)大于5C細(xì)胞進(jìn)行核實(shí),以排除該系統(tǒng)將垃圾和重疊的細(xì)胞核誤認(rèn)為異常細(xì)胞。結(jié)合TBS診斷與DNA倍體分析結(jié)果,如有以下結(jié)果,建議作陰道鏡檢查及活檢:(1)LSIL、HSIL、鱗狀細(xì)胞癌;(2)ASCUS伴有DNA倍體異常者(>5c細(xì)胞);③常規(guī)細(xì)胞學(xué)正常,但有2個(gè)或3個(gè)以上5c細(xì)胞。

6.細(xì)胞核面積及DNA分布研究

8例病理證實(shí)為CIN2或CIN2以上級(jí)別的宮頸細(xì)胞樣本,經(jīng)DNA倍體分析后均發(fā)現(xiàn)有>5c、>7c、>9c的非整倍體細(xì)胞。每例宮頸樣本測(cè)定不同倍體細(xì)胞核面積及DNA分布。其8例被測(cè)細(xì)胞詳細(xì)情況見(jiàn)表1。

表1 8例不同倍體細(xì)胞Table 1 Cell number of different DNA ploid in 8 cases

細(xì)胞特征值定義及使用公式如下:

反映細(xì)胞核形態(tài)的特征值:

細(xì)胞核面積是指:核內(nèi)像素的總和,經(jīng)換算為平方微米。

平均半徑(單位:微米)的計(jì)算如下:

細(xì)胞的緊實(shí)度反映了細(xì)胞的圓度,細(xì)胞的緊實(shí)度值越低表明細(xì)胞越圓。其計(jì)算方法如下:

compat=p2/(4＊π＊S)

其中,p表示細(xì)胞的周長(zhǎng),S表示細(xì)胞的面積(平方微米)

反映細(xì)胞核內(nèi)DNA分布的特征值:

平均光學(xué)密度的計(jì)算方法為:

光學(xué)密度方差的計(jì)算方法為:

其中,f為積分光學(xué)密度,n為細(xì)胞在圖像中的像素個(gè)數(shù)。

灰度熵的計(jì)算方法為:

其中,i為圖像直方圖中柱狀的個(gè)數(shù),Ii為第i個(gè)柱狀對(duì)應(yīng)的值。

7.統(tǒng)計(jì)學(xué)方法

對(duì)細(xì)胞核面積和核內(nèi)DNA分布的統(tǒng)計(jì),每一例的各級(jí)倍體細(xì)胞的平均值代表這一例不同級(jí)別統(tǒng)計(jì)數(shù),例如2c細(xì)胞數(shù)據(jù)是指每例2c細(xì)胞的平均值,再將8例的2c細(xì)胞數(shù)據(jù)為一組,與另外倍體細(xì)胞組進(jìn)行比較統(tǒng)計(jì)。以2c細(xì)胞為標(biāo)準(zhǔn),用t配對(duì)檢驗(yàn),比較其他各不同倍體細(xì)胞組,當(dāng)其他組與2c組比較,P<0.05表示二組間差別為顯著性差別。

結(jié) 果

1.對(duì)161873例病例宮頸細(xì)胞進(jìn)行常規(guī)細(xì)胞學(xué)診斷和DNA定量分析

用常規(guī)細(xì)胞學(xué)診斷 161873例宮頸病例,有153146例診斷為正常和8727例異常,其中5675例為ASCUS,66例為ASC-H,2454例為L(zhǎng)SIL,523例為 HSIL,9例為A GC;而DNA定量測(cè)定發(fā)現(xiàn)有144207例正常、29例增生和17637例有異倍體細(xì)胞。在發(fā)現(xiàn)有異倍體細(xì)胞病例中,有1-2個(gè)>5c細(xì)胞的病例為14225例,3個(gè)以上>5c細(xì)胞的病例為3412例。其結(jié)果見(jiàn)表 2。從表2可見(jiàn)84.4%的LSIL和96%的 HSIL中可見(jiàn)有異倍體細(xì)胞,38.4%的ASCUS中可見(jiàn)有異倍體細(xì)胞。在常規(guī)細(xì)胞診斷為正常病例中有8.39%為異倍體細(xì)胞(見(jiàn)表2)。

表2 161873例婦女宮頸樣本常規(guī)細(xì)胞學(xué)和DNA倍體分析結(jié)果比較Table 2 Results of conventional cytology and DNA ploid analysis in 161873 cervical samples

2.對(duì)8例宮頸細(xì)胞核特征值測(cè)定

通過(guò)DNA倍體分析,發(fā)現(xiàn)宮頸異倍體細(xì)胞隨著DNA含量增多,細(xì)胞核的形態(tài)發(fā)生改變,例如:細(xì)胞核面積、核半徑逐漸增大,與二倍體細(xì)胞相比,均有顯著性的增大(P﹤0.05),核的緊實(shí)度有顯著性改變。細(xì)胞核內(nèi)DNA的分布也發(fā)生改變,例如:平均OD有顯著性改變?；叶褥睾凸饷芏确讲顭o(wú)顯著性差異(表3,表4)。

表中的C表示DNA含量的單位,例如2C表示2倍體,其中,面積的單位為平方微米,半徑的單位為微米。將以上數(shù)據(jù)其它倍體與2倍體進(jìn)行比較,進(jìn)行配對(duì)t檢驗(yàn),表中上標(biāo)＊表示與2C比較有統(tǒng)計(jì)學(xué)顯著性差異(P<0.05)。

討 論

DNA倍體分析用于宮頸細(xì)胞學(xué)檢查已在國(guó)內(nèi)外均有報(bào)道[3,7,9],在常規(guī)細(xì)胞學(xué)診斷為L(zhǎng)SIL和HSIL病例,大多均含有DNA非整倍體細(xì)胞,這與多個(gè)作者的結(jié)果一致[7,9,10]。而ASCUS病例中也有35%病例也可見(jiàn)DNA非整倍體細(xì)胞。在常規(guī)細(xì)胞學(xué)診斷為正常樣本中,有8%左右見(jiàn)有>5c非整倍體細(xì)胞,ASCUS病例伴有DNA倍體異?；虺Ｒ?guī)細(xì)胞學(xué)正常而伴有3個(gè)>5c以上的非整倍體細(xì)胞者多需進(jìn)一步做陰道鏡檢查或?qū)m頸病理活檢[2,7]。

隨著細(xì)胞DNA含量改變,細(xì)胞核在形態(tài)上,DNA含量及核內(nèi)DNA分布等特征值均發(fā)現(xiàn)了改變,即隨著DNA含量的增大,細(xì)胞核的面積、核半徑、積分光學(xué)密度等都逐漸增大,光學(xué)密度方差、灰度熵沒(méi)有發(fā)生顯著性增大或改變,DNA倍體或DNA含量的改變已作為判斷細(xì)胞癌變的一個(gè)很重要的生物特征,通過(guò)本文的研究,細(xì)胞核面積、半徑及平均光密度等也是隨細(xì)胞DNA含量而變化的,是否可將這些特征值作為判斷細(xì)胞是否有癌變,還有待進(jìn)一步研究。

總之,宮頸細(xì)胞DNA倍體改變常伴有細(xì)胞核內(nèi)面積、半徑及核內(nèi)DNA分布的改變。

[1]Bollmann R,Méhes G,Speich N,et al.Aberrant,highly hyperdiploid cells in human papillomavirus-positive,abnormal cytologic samples are associated with progressive lesions of the uterine cervix.Cancer,2005,105:96-100

[2]Lorenzato M,Caudroy S,Nou JM,et al.Contribution of DNA ploidy image cytometry to the management of ASC cervical.Cancer,2008,114:263-269

[3]Guillaud M,Benedet J,Cantor S,et al.DNA ploidy compared with human papilloma virus testing(hybrid capture II)and conventional cervical cytology as a primary screening test for cervical high-grade lesions and cancer in 1555 patients with biopsy confirmation.Cancer,2006,107:309-318

[4]Melsheimer P,Vinokurova S,Wentzensen N,et al.DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri.Clin Cancer Res,2004,10:3059-3063

[5]Graflund M,Sorbe B,Sigurdardóttir S,et al.Relation between HPV-DNA and expression of p53,bcl-2,p21WAF-1,MIB-1,HER-2/neu and DNA ploidy in early cervical carcinoma:correlation with clinical outcome.Oncol Rep,2004,12:169-176

[6]Heilman SA,Nordberg JJ,Liu Y,et al.Abrogation of the postmitotic checkpoint contributes to polyploidization in human papillomavirus E7-expressing cells.J Virol,2009,83:2756-2764

[7]Bocking A,Nguyen V.Diagnostic and prognostic use of DNA image cytometry in cervical squamous intraepithelial lesions and invasive carcinoma.Cancer Cytopath,2004,102:41-54

[8]Bocking A,Giroud F,Reith A.Consensus report of the ESACPtask force on standardization of diagnostic DNA image cytometry.Anal Cell Pathol,1995,867-874

[9]Sun XR,Wang J,Garner D,et al.Detection of cervical cancer and high grade neoplastic lesions by a combination of liquid-based sampling preparation and DNA measurement using automated image cytometry.Cell Oncology,2005,27:33-41

[10]Mehes G,Speich N,Bollmann M,et al.Chromosomal aberrations accumulate in polyploid cells of high-grade squamous intraepithelial lesions(HSIL).Pathol Oncol Res,2004,10:142-148

Changes of nuclear features with different DNA ploid

Wang Jingjie1,Sun Quanxin2,Sun Xiaorong3,Niu Xiaoquan4＊
(1Department of Health Care,Wuhan 5th Hospital,Wuhan 430050;2Department of Cytology,Hubei Zhongshan Hospital,Wuhan 430033;3China-Canada Early Cancer Detection Research Center,Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430022;4Department of Education,Wuhan 5th Hospital,Wuhan 430050,China)

Objective To study the nuclear features in different DNA ploids of cervical cells by DNA image cytometry.Methods Cervical cells were collected by a brush and fixed.Two slides per sample were made by a cytospin.One slide was stained by Pap solution for TBS diagnosis,whice the other one was stained by Feulgen for DNA ploidy analysis.Nuclear features,such as nuclear area and mean optical density(OD),were also measured.Results Cervical samples f rom 161,873 women were examined.2454 lowgrade squamous intraepithelial lesions(LSIL)and 523 high-grade squamous intraepithelial lesions(HSIL)were found by TBS diagnosis,while 3 and more than 3>5c aneuploid cells in 3412 cases were determined by a DNA image cytometer.Aneuploid cells werefound in the samplesof 84%LSIL and 96%HSIL cases.Compared to those of 2c cells,the nuclear area,radius,mean OD and compactness were significantly different in 7c and 9c cells,while variance and gray entropy were found the some change.Conclusion Nuclear features of cervical cells are changed with different DNA ploids.

DNA ploid; Image cytometer; Cervical cancer; Aneuploid cell

R361.3

A

10.3870/zgzzhx.2011.04.016

2011-04-10

2011-06-10

王景潔,女(1963年),漢族,副主任醫(yī)師。

＊通訊作者(To whom correspondence should be addrssed)