中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
3期
218-223
,共6页
任传利%韩崇旭%王大新%汪步海%徐兴祥%章佳新%周林%吴志峰
任傳利%韓崇旭%王大新%汪步海%徐興祥%章佳新%週林%吳誌峰
임전리%한숭욱%왕대신%왕보해%서흥상%장가신%주림%오지봉
抗原,肿瘤%细胞黏附分子%纳米粒子%肿瘤循环细胞%细胞系,肿瘤
抗原,腫瘤%細胞黏附分子%納米粒子%腫瘤循環細胞%細胞繫,腫瘤
항원,종류%세포점부분자%납미입자%종류순배세포%세포계,종류
Antigens,neoplasm%Cell adhesion molecules%Nanoparticles%Neoplasm circulating cells%Cell line,tumor
目的 建立EpCAM抗体偶联纳米磁珠阳性分离上皮来源的实体瘤外周血CTC的方法(简称"磁珠分离法"),探讨该方法分离CTC的灵敏度和应用意义.方法 用7.5 ml健康志愿者外周血裂解红细胞分别倍比稀释加入5、10、20、50、100个乳腺癌细胞系MCF7、食管癌细胞系KYSE70、胰腺癌细胞系BxPC-3及胃癌细胞系9811P,通过磁珠分离法富集癌细胞,荧光显微镜下检测体外加入癌细胞的回收率.然后,用磁珠分离法富集未治疗的30例食管癌(Ⅰ期+Ⅱ期6例、Ⅲ期+Ⅳ期24例)、35例乳腺癌(Ⅰ期+Ⅱ期15例、Ⅲ期+Ⅳ期20例)、30例胰腺癌(Ⅰ期+Ⅱ期5例、Ⅲ期+Ⅳ期25例)、33例胃癌患者(Ⅰ期+Ⅱ期13例、Ⅲ期+Ⅳ期20例)和30例慢性胃炎及30名健康志愿者的CTC,同时结合IF和HE染色鉴定各组的CTC.另外,用FISH分别检测分析食管癌、乳腺癌、胰腺癌、胃癌患者200个CTC中8号和20号染色体拷贝数水平.结果 用4,6-二眯-2苯吲哚(DAPI)染色食管癌、乳腺癌、胰腺癌、胃癌等4种癌细胞系并加入健康志愿者7.5 ml外周血的组内试验中,荧光显微镜下计数4种癌细胞的组内的平均回收率分别为87%、87%、86%和88%;5种稀释梯度的细胞数在4种癌细胞系的组间的平均回收率分别是88%、85%、87%、88%和87%.在107的外周血白细胞中能富集到1个癌细胞.采用磁珠分离法从食管癌、乳腺癌、胰腺癌及胃癌患者中检出≥2个CTC的阳性率分别为50%(15/30)、63%(22/35)、70%(21/30)及61%(20/33),健康志愿者和炎症患者均未检出CTC.食管癌、乳腺癌、胰腺癌组分别与健康对照组和炎症组比较,经精确概率法分析,差异均有统计学意义(P值均为0.000).80%的食道癌、75%的乳腺癌、65%的胰腺癌和59%的胃癌患者CTC中均发生8号和20号染色体非整倍体改变.结论 建立的磁珠分离法对富集分离实体瘤外周血CTC具有较好的分选灵敏度和准确性.食道癌、乳腺癌、胰腺癌和胃癌患者中的CTC多发生8号和20号染色体非整倍体改变.
目的 建立EpCAM抗體偶聯納米磁珠暘性分離上皮來源的實體瘤外週血CTC的方法(簡稱"磁珠分離法"),探討該方法分離CTC的靈敏度和應用意義.方法 用7.5 ml健康誌願者外週血裂解紅細胞分彆倍比稀釋加入5、10、20、50、100箇乳腺癌細胞繫MCF7、食管癌細胞繫KYSE70、胰腺癌細胞繫BxPC-3及胃癌細胞繫9811P,通過磁珠分離法富集癌細胞,熒光顯微鏡下檢測體外加入癌細胞的迴收率.然後,用磁珠分離法富集未治療的30例食管癌(Ⅰ期+Ⅱ期6例、Ⅲ期+Ⅳ期24例)、35例乳腺癌(Ⅰ期+Ⅱ期15例、Ⅲ期+Ⅳ期20例)、30例胰腺癌(Ⅰ期+Ⅱ期5例、Ⅲ期+Ⅳ期25例)、33例胃癌患者(Ⅰ期+Ⅱ期13例、Ⅲ期+Ⅳ期20例)和30例慢性胃炎及30名健康誌願者的CTC,同時結閤IF和HE染色鑒定各組的CTC.另外,用FISH分彆檢測分析食管癌、乳腺癌、胰腺癌、胃癌患者200箇CTC中8號和20號染色體拷貝數水平.結果 用4,6-二瞇-2苯吲哚(DAPI)染色食管癌、乳腺癌、胰腺癌、胃癌等4種癌細胞繫併加入健康誌願者7.5 ml外週血的組內試驗中,熒光顯微鏡下計數4種癌細胞的組內的平均迴收率分彆為87%、87%、86%和88%;5種稀釋梯度的細胞數在4種癌細胞繫的組間的平均迴收率分彆是88%、85%、87%、88%和87%.在107的外週血白細胞中能富集到1箇癌細胞.採用磁珠分離法從食管癌、乳腺癌、胰腺癌及胃癌患者中檢齣≥2箇CTC的暘性率分彆為50%(15/30)、63%(22/35)、70%(21/30)及61%(20/33),健康誌願者和炎癥患者均未檢齣CTC.食管癌、乳腺癌、胰腺癌組分彆與健康對照組和炎癥組比較,經精確概率法分析,差異均有統計學意義(P值均為0.000).80%的食道癌、75%的乳腺癌、65%的胰腺癌和59%的胃癌患者CTC中均髮生8號和20號染色體非整倍體改變.結論 建立的磁珠分離法對富集分離實體瘤外週血CTC具有較好的分選靈敏度和準確性.食道癌、乳腺癌、胰腺癌和胃癌患者中的CTC多髮生8號和20號染色體非整倍體改變.
목적 건립EpCAM항체우련납미자주양성분리상피래원적실체류외주혈CTC적방법(간칭"자주분리법"),탐토해방법분리CTC적령민도화응용의의.방법 용7.5 ml건강지원자외주혈렬해홍세포분별배비희석가입5、10、20、50、100개유선암세포계MCF7、식관암세포계KYSE70、이선암세포계BxPC-3급위암세포계9811P,통과자주분리법부집암세포,형광현미경하검측체외가입암세포적회수솔.연후,용자주분리법부집미치료적30례식관암(Ⅰ기+Ⅱ기6례、Ⅲ기+Ⅳ기24례)、35례유선암(Ⅰ기+Ⅱ기15례、Ⅲ기+Ⅳ기20례)、30례이선암(Ⅰ기+Ⅱ기5례、Ⅲ기+Ⅳ기25례)、33례위암환자(Ⅰ기+Ⅱ기13례、Ⅲ기+Ⅳ기20례)화30례만성위염급30명건강지원자적CTC,동시결합IF화HE염색감정각조적CTC.령외,용FISH분별검측분석식관암、유선암、이선암、위암환자200개CTC중8호화20호염색체고패수수평.결과 용4,6-이미-2분신타(DAPI)염색식관암、유선암、이선암、위암등4충암세포계병가입건강지원자7.5 ml외주혈적조내시험중,형광현미경하계수4충암세포적조내적평균회수솔분별위87%、87%、86%화88%;5충희석제도적세포수재4충암세포계적조간적평균회수솔분별시88%、85%、87%、88%화87%.재107적외주혈백세포중능부집도1개암세포.채용자주분리법종식관암、유선암、이선암급위암환자중검출≥2개CTC적양성솔분별위50%(15/30)、63%(22/35)、70%(21/30)급61%(20/33),건강지원자화염증환자균미검출CTC.식관암、유선암、이선암조분별여건강대조조화염증조비교,경정학개솔법분석,차이균유통계학의의(P치균위0.000).80%적식도암、75%적유선암、65%적이선암화59%적위암환자CTC중균발생8호화20호염색체비정배체개변.결론 건립적자주분리법대부집분리실체류외주혈CTC구유교호적분선령민도화준학성.식도암、유선암、이선암화위암환자중적CTC다발생8호화20호염색체비정배체개변.
Objective To establish an isolation method for solid GTC in peripheral blood using EpCAM antibody-linked nanobeads and evaluate the sensitivity of the method and its application significance. Methods Five, ten, twenty, fifty and one hundred MCF7 (breast cancer), KYSE70 (esophageal cancer), BxPC-3 (pancreatic cancer) and 9811P (stomach cancer) cells were added into 7. 5 ml erythrocyte lysed peripheral blood obtained from healthy volunteers respectively. EpCAM antibodylinked nanobeads were used to enrich cancer cells. The recovery rates of the in vitro added cancer cells were evaluated by fluorescence microscopy. Then, the untreated thirty cases of esophageal cancer (six cases at stage Ⅰ and Ⅱ, twenty-four cases at stage Ⅲ and Ⅳ), thirty-five cases of breast cancer (fifteen cases at stage Ⅰ and Ⅱ , twenty cases at stage Ⅲ and Ⅳ), thirty cases of pancreatic cancer (five cases at stage Ⅰ and Ⅱ , twenty-five cases at stage Ⅲ and Ⅳ), thirty-three gastric cancer (thirteen cases for stage Ⅰ and Ⅱ ,twenty cases at stage Ⅲ and Ⅳ) were enrolled to enrich the peripheral blood CTC. Thirty healthy volunteers and thirty gastritis patients served as two groups of control. Meanwhile the enriched CTC was identified by IF and HE staining. FISH was used to analyze the copy number of chromosome 8 and chromosome 20 in two hundred esophageal cancer, breast cancer, pancreatic caner and gastric cancer CTC. Results After DAPI staining and mixing with 7.5 ml peripheral blood from healthy donors, the average cell recovery rates of KYSE70, MCF7, BxPC-3 and 9811P cells evaluated under fluorescence microscope were 87%, 87%, 86% and 88% (within group), and the recovery rates of 5 gradient dilution levels were 88%, 85%, 87%, 88% and 87% (intergroup). With a high sensitivity, this method was able to isolate one cancer cell in 107 white blood cells of peripheral blood. The positive rates of more than 2 CTC in the peripheral blood detected by this method were 50% (15/30) of esophageal cancer, 63% (22/35) of breast cancer, 70% (21/30) of pancreatic cancer and 61% (20/33) gastric cancer patients respectively,but no CTC was detected in the peripheral blood of healthy volunteers and gastritis patients (P = 0. 000).The aneusomy of chromosome 8 and chromosome 20 were found in 80% esophageal cancer, 75% breast cancer, 65% pancreatic cancer and 59% gastric cancer. Conclusions The CTC isolation technique with EpCAM antibody-linked nanobeads is sensitive and accurate. The aneusomy of chromosome 8 and 20 is frequent in CTC from esophageal cancer, breast cancer, pancreatic cancer and gastric cancer.
