CAJ | 학술논문

目的建立人肝癌血管内皮细胞(TEC)分离培养方法并鉴定比较.方法采用CD105、CD31抗体交联免疫磁珠方法优化分选TEC并传代培养;通过形态观察、功能实验、流式检测及实时聚合酶链反应(real-time PCR)等验证比较.结果 CD105+TEC呈细长"纤维条索状",CD31+TEC则呈"梭形";流式检测TEC表达CD68和α-平滑肌肌动蛋白(α-SMA)低于5%,排除巨噬细胞和成纤维细胞污染,PCR检测CD105+TEC、CD31+TEC甲胎蛋白mRNA显著低于肿瘤细胞HepG2(P＜0.01),表达水平低于HepG2的1/8,排除肿瘤细胞污染;95%以上阳性分选细胞呈乙酰化低密度脂蛋白摄取实验阳性;增殖实验检测CD105+TEC和CD31+TEC生长48 h的吸光度值分别为0.45±0.04和0.35±0.02(P＜0.05),CD105+TEC增殖能力强于CD31+TEC;TEC在Matrigel胶中可形成毛细管网状结构,CD31+TEC强于CD105+TEC.结论 CD105、CD31抗体交联免疫磁珠分选法均可分离得到高纯度人肝癌TEC.
목적 건립인간암혈관내피세포(TEC)분리배양방법병감정비교.방법 채용CD105、CD31항체교련면역자주방법우화분선TEC병전대배양;통과형태관찰、공능실험、류식검측급실시취합매련반응(real-time PCR)등험증비교.결과 CD105+TEC정세장"섬유조색상",CD31+TEC칙정"사형";류식검측TEC표체CD68화α-평활기기동단백(α-SMA)저우5%,배제거서세포화성섬유세포오염,PCR검측CD105+TEC、CD31+TEC갑태단백mRNA현저저우종류세포HepG2(P＜0.01),표체수평저우HepG2적1/8,배제종류세포오염;95%이상양성분선세포정을선화저밀도지단백섭취실험양성;증식실험검측CD105+TEC화CD31+TEC생장48 h적흡광도치분별위0.45±0.04화0.35±0.02(P＜0.05),CD105+TEC증식능력강우CD31+TEC;TEC재Matrigel효중가형성모세관망상결구,CD31+TEC강우CD105+TEC.결론 CD105、CD31항체교련면역자주분선법균가분리득도고순도인간암TEC.
Objective To isolate, identify and compare vascular endothelial cells derived from human hepatocellular carcinoma (HCC). Methods Modified immunomagnefic methods using magnetic beads conjugated with anti-CD105 and anti-CD31 antibody were used to isolate tumor-derived endothelial cells (TEC), namely CD31 +TEC and CD105 +TEC. CD31 +TEC and CD105 +TEC were cultured in vitro,identified and compared by morphological appearance, functional characterization, flow cytometry and realtime polymerase chain reaction (PCR) analyses. Results The isolated CD105 + TEC presented "fibrous bands" appearance while CD31 + TEC presented "fusiformis" appearance. Macrophages, fibrocytes, and tumor cells contamination was excluded. Surface CD68 and α-SMA expression was less than 5%. Alpha fetoprotein expression was significantly lower in CD31 + TEC and CD105 + TEC than that in HepG2 cells ( P ＜0. 01 ), and the expression level was less than 1/8 folds of HepG2 cells. Internalization of acetylated lowdensity lipoprotein was positive in more than 95% of isolated cells. The absorbance (A) values of CD105 +TEC and CD31 + TEC grown for 48 h were 0. 45 ± 0. 04 and 0. 35 ± 0. 02 respectively ( P ＜ 0. 05 ). Proliferation of CD105 + TEC was significantly more active than CD31 + TEC when cultured in the serum supplemented with medium for 24, 48, and 72 h. The isolated cells could form capillary-like tubes on Matrigel matrix, stronger capability of CD31 + TEC than CD105 + TEC. Conclusion CD31 + TEC and CD105 + TEC can be conveniently purified by modified immunomagnetic methods.