中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2015年
10期
762-767
,共6页
吕建美%何彦津%谢连凤%刘勋%朱利民
呂建美%何彥津%謝連鳳%劉勛%硃利民
려건미%하언진%사련봉%류훈%주이민
癌,腺样囊性%泪器%眼肿瘤%肿瘤干细胞%肿瘤细胞,培养的
癌,腺樣囊性%淚器%眼腫瘤%腫瘤榦細胞%腫瘤細胞,培養的
암,선양낭성%루기%안종류%종류간세포%종류세포,배양적
Carcinoma,adenoid cystic%Lacrimal apparatus%Eye neoplasms%Neoplastic stem cells%Tumor cells,cultured
目的 分离、培养人泪腺腺样囊性癌(LACC)细胞系并研究其肿瘤干细胞(CSC)特性.方法 实验研究.应用无血清悬浮培养法分离培养人LACC-CSC,显微镜下观察细胞形态学改变.实验分为两组,即LACC-CSC实验组和LACC对照组,应用流式细胞仪检测干细胞标记CD133、ATP结合转运蛋白G超家族成员2(ABCG2)的表达,Tanswell小室检测CSC侵袭力,并诱导CSC向血管内皮细胞分化,体外异种移植检测CSC致瘤力.实验组与对照组组间比较采用t检验.结果 LACC接种于无血清培养基后悬浮成球生长,10～ 12 d可形成形态规则的肿瘤微球体.流式细胞仪检测示LACC-CSC表达CD133比率为(35.67±6.86)％,LACC为(0.46±0.48)％,二者比较差异具有统计学意义(t=-8.867,P＜0.05);LACC-CSC表达ABCG2为(39.99±4.54)％,LACC为(6.75±1.34)％,二者比较差异具有统计学意义(t=-9.932, P＜0.05).Transwell体外侵袭实验示,培养24 h后,LACC-CSC穿过Matrigel基底膜平均为(32.60±8.79)个/高倍镜视野,LACC为(10.20±2.77)个/高倍镜视野,两组比较差异有统计学意义(t=5.433,P＜0.05);培养48 h后LACC-CSC穿过Matrigel基底膜平均为(62.60±4.83)个/HP,LACC为(44.00±5.34)个/HP,两组比较差异有统计学意义(t=5.779,P＜0.05).应用含VEGF和bFGF等的血清培养基低氧诱导,LACC-CSC呈贴壁生长,细胞形态改变,连续诱导后三维基质胶中可形成血管腔样结构,表达血管内皮标记CD31、CD34.体外移植瘤实验LACC-CSC组9d时全部出瘤,LACC组12d全部出瘤,两组成瘤率均为100％.结论 通过无血清培养法获得的LACC-CSC悬浮成球生长,表达经典干细胞标记CD133及ABCG2,具有更强的迁移侵袭力以及体内致瘤力,并具有多向分化潜能,具有干细胞的一般特性.
目的 分離、培養人淚腺腺樣囊性癌(LACC)細胞繫併研究其腫瘤榦細胞(CSC)特性.方法 實驗研究.應用無血清懸浮培養法分離培養人LACC-CSC,顯微鏡下觀察細胞形態學改變.實驗分為兩組,即LACC-CSC實驗組和LACC對照組,應用流式細胞儀檢測榦細胞標記CD133、ATP結閤轉運蛋白G超傢族成員2(ABCG2)的錶達,Tanswell小室檢測CSC侵襲力,併誘導CSC嚮血管內皮細胞分化,體外異種移植檢測CSC緻瘤力.實驗組與對照組組間比較採用t檢驗.結果 LACC接種于無血清培養基後懸浮成毬生長,10～ 12 d可形成形態規則的腫瘤微毬體.流式細胞儀檢測示LACC-CSC錶達CD133比率為(35.67±6.86)％,LACC為(0.46±0.48)％,二者比較差異具有統計學意義(t=-8.867,P＜0.05);LACC-CSC錶達ABCG2為(39.99±4.54)％,LACC為(6.75±1.34)％,二者比較差異具有統計學意義(t=-9.932, P＜0.05).Transwell體外侵襲實驗示,培養24 h後,LACC-CSC穿過Matrigel基底膜平均為(32.60±8.79)箇/高倍鏡視野,LACC為(10.20±2.77)箇/高倍鏡視野,兩組比較差異有統計學意義(t=5.433,P＜0.05);培養48 h後LACC-CSC穿過Matrigel基底膜平均為(62.60±4.83)箇/HP,LACC為(44.00±5.34)箇/HP,兩組比較差異有統計學意義(t=5.779,P＜0.05).應用含VEGF和bFGF等的血清培養基低氧誘導,LACC-CSC呈貼壁生長,細胞形態改變,連續誘導後三維基質膠中可形成血管腔樣結構,錶達血管內皮標記CD31、CD34.體外移植瘤實驗LACC-CSC組9d時全部齣瘤,LACC組12d全部齣瘤,兩組成瘤率均為100％.結論 通過無血清培養法穫得的LACC-CSC懸浮成毬生長,錶達經典榦細胞標記CD133及ABCG2,具有更彊的遷移侵襲力以及體內緻瘤力,併具有多嚮分化潛能,具有榦細胞的一般特性.
목적 분리、배양인루선선양낭성암(LACC)세포계병연구기종류간세포(CSC)특성.방법 실험연구.응용무혈청현부배양법분리배양인LACC-CSC,현미경하관찰세포형태학개변.실험분위량조,즉LACC-CSC실험조화LACC대조조,응용류식세포의검측간세포표기CD133、ATP결합전운단백G초가족성원2(ABCG2)적표체,Tanswell소실검측CSC침습력,병유도CSC향혈관내피세포분화,체외이충이식검측CSC치류력.실험조여대조조조간비교채용t검험.결과 LACC접충우무혈청배양기후현부성구생장,10～ 12 d가형성형태규칙적종류미구체.류식세포의검측시LACC-CSC표체CD133비솔위(35.67±6.86)％,LACC위(0.46±0.48)％,이자비교차이구유통계학의의(t=-8.867,P＜0.05);LACC-CSC표체ABCG2위(39.99±4.54)％,LACC위(6.75±1.34)％,이자비교차이구유통계학의의(t=-9.932, P＜0.05).Transwell체외침습실험시,배양24 h후,LACC-CSC천과Matrigel기저막평균위(32.60±8.79)개/고배경시야,LACC위(10.20±2.77)개/고배경시야,량조비교차이유통계학의의(t=5.433,P＜0.05);배양48 h후LACC-CSC천과Matrigel기저막평균위(62.60±4.83)개/HP,LACC위(44.00±5.34)개/HP,량조비교차이유통계학의의(t=5.779,P＜0.05).응용함VEGF화bFGF등적혈청배양기저양유도,LACC-CSC정첩벽생장,세포형태개변,련속유도후삼유기질효중가형성혈관강양결구,표체혈관내피표기CD31、CD34.체외이식류실험LACC-CSC조9d시전부출류,LACC조12d전부출류,량조성류솔균위100％.결론 통과무혈청배양법획득적LACC-CSC현부성구생장,표체경전간세포표기CD133급ABCG2,구유경강적천이침습력이급체내치류력,병구유다향분화잠능,구유간세포적일반특성.
Objective To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties.Method Experimental study.Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed.Cells were divided into two groups,the LACC-CSC experimental group and the LACC control group.The flow cytometry instrument was used to detect the expression of classical stem cell markers CD 133 and ABCG2.Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells.The tumorigenic force in vitro xenotransplantation were applied.Result LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days.Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67± 6.86)％, significantly different to LACC with (0.46 ±0.48)％, (t=8.867, P＜0.05).Similarly ,the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99±4.54)％, significantly different to LACC with (6.75 ± 1.34)％,(t=-9.932, P＜0.05).In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60± 8.79)/HP contrary to LACC with average (10.20±2.77)/HP after 24 hours, showing statistically significance(t=5.433, P＜0.05) between the two groups.After training for 48 hours, the difference between two groups was still obvious (t=5.779, P＜0.05) with LACC-CSC average(62.60±4.83)/HP to LACC (44.00±5.34)/HP.When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells.When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34.Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100％ tumorigenic rate, whereas LACC group 12 days with 100％ tumorigenic rate.Conclusions LACC-CSC can be obtained through serum-free culture method.LACC-CSC grew suspensively and expressed classical stem cell markers.LACC-CSC were identified as cancer stem cells with stronger migration and invasion.LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.
