【作者】 宋宗昌；

【导师】 胡义德；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2008， 硕士

【摘要】 【目的】本课题通过分析人肺腺癌细胞系经化疗药物作用后侧群(side population,SP)细胞的比例变化,以及利用免疫磁珠分选出耐药人肺腺癌细胞系中ABCG2+细胞并进行裸鼠移植瘤实验,初步探讨人肺腺癌干细胞样细胞的分选方法及其基本生物学特性。【方法】以小剂量阿霉素(Adriamycin,ADM)诱导人肺腺癌A549细胞系和SPC-A-1细胞系,比较亲代细胞和诱导后细胞的形态;绘制两系细胞诱导前后的增殖曲线,计算倍增时间;用MTT比色法检测多种化疗药物对肿瘤细胞增殖的杀伤效应;显微镜下测定诱导前后两系细胞中SP细胞比例。以ADM诱导后的人肺腺癌SPC-A-1细胞系为研究对象,利用免疫磁珠分选出SPC-A-1/ADM细胞系中ABCG2+细胞和ABCG2-细胞,分别进行流式细胞分析及裸鼠体内移植,分析ABCG2+细胞和ABCG2-细胞的体内成瘤性。【结果】1.ADM诱导前后人肺腺癌细胞系的群体体外倍增时间和ADM诱导后人肺腺癌细胞系的耐药指数ADM诱导前后A549细胞的群体体外倍增时间分别为37.6h和36.1h,并无显著差异(P=0.989,t=0.013)。A549/ADM细胞对ADM、顺铂、长春新碱、足叶乙甙、紫杉醇五种药物的耐药指数分别为9.588、6.348、1.736、11.213、4.333。ADM诱导前后SPC-A-1细胞的群体体外倍增时间分别为35.9h和35.6h,并无显著差异(P=0.956,t=0.056)。SPC-A-1/ADM细胞对ADM、顺铂、长春新碱、足叶乙甙、紫杉醇五种药物的耐药指数分别为4.283、3.672、6.308、9.112、2.716。2.ADM诱导前后人肺腺癌细胞系中SP细胞比例变化ADM诱导前后,A549细胞中SP细胞的比例分别为4%和45%,差别显著(X2=307.210,p=0.000);SPC-A-1细胞中SP细胞的比例分别为1%和10%,差别显著(X2=102.201,p=0.000)。3.培养SPC-A-1和SPC-A-1/ADM细胞中ABCG2+细胞的比例变化ABCG2染色荧光观察显示,在SPC-A-1/ADM细胞中,每个视野均可见被Mouse Anti-Human ABCG2/BCRP-FITC标记的细胞,ABCG2+细胞数量较SPC-A-1细胞中明显增多。流式细胞分析结果显示,SPC-A-1细胞空白对照组中ABCG2+细胞比例为0.047% ,同型对照组中ABCG2 +细胞比例为0.087 % , Mouse Anti-Human ABCG2/BCRP-FITC标记的SPC-A-1细胞中ABCG2+细胞比例为0.247%,而Mouse Anti-Human ABCG2/BCRP-FITC标记的SPC-A-1/ADM细胞中ABCG2+细胞比例达到9.9%,是SPC-A-1细胞中ABCG2+细胞比例的40.08倍。4.从SPC-A-1/ADM细胞中免疫磁珠分选ABCG2+细胞的得率及ABCG2+细胞的比例变化结果显示,从SPC-A-1/ADM细胞中,每1×107个细胞可分离出2×104个ABCG2+细胞,ABCG2+细胞得率为0.2%。A群细胞中Mouse Anti-Human ABCG2/BCRP-FITC标记阳性率为0.127%,B群细胞中Mouse Anti-Human ABCG2/BCRP-FITC标记阳性率为84.71%,是A群细胞中Mouse Anti-Human ABCG2/BCRP-FITC标记阳性率的667t倍。5.A群和B群人肺腺癌细胞的裸鼠体内成瘤能力比较接种SPC-A-1细胞和A群细胞的裸鼠未见成瘤,成瘤率分别为0/4、0/4。接种B群细胞的裸鼠3周后开始成瘤,第8周时,共有3只裸鼠成瘤,成瘤率为3/4。【结论】1.人肺腺癌A549和SPC-A-1细胞系经过ADM诱导后对多种化疗药物的耐药性明显增加,耐药细胞中SP细胞的比例显著升高,提示SP细胞比例增加与肺腺癌的耐药性形成密切相关。2.SPC-A-1/ADM细胞群中ABCG2表达阳性率显著高于SPC-A-1细胞,这为基于ABCG2来分离干细胞创造了条件。3.SPC-A-1/ADM细胞群中的ABCG2+细胞裸鼠体内移植成瘤率较高,而ABCG2-细胞和SPC-A-1细胞在裸鼠体内未见移植瘤形成,表明基于ABCG2所分离的肺腺癌细胞具有干细胞特性。更多还原

【Abstract】 Objective:The proportion of SP cells was analyzed in human lung adenocarcinoma cell lines induced by chemotherapeutic drug,and ABCG2+cells were isolated from multidrug resistant cell lines by magnetic activated cell sorting(MACS). Tumorigenicity was detected in nude mice. We studied primarily on the isolation method and biological characteristic of human lung adenocarcinoma stem-like cells.Method:Human lung adenocarcinoma cell lines A549 and SPC-A-1 were induced by low dose adriamycin(ADM).The morphology of the cells was compared before and after induction.The growth curves in vitro were drew and their doubling times(DT) were calculated.The lethal effects were detected by MTT method and the proportion of the SP cells was assayed in the two cell lines under fluorescence microscope.ABCG2+cells were isolated from drug resistance cell line SPC-A-1/ADM by MACS, and analyzed by flow cytometry.Then transplanted them into nude mice. The tumorigenicity of ABCG2+cells and ABCG2-cells was analyzed.Result:1. DT and resistance factor (RF) of human lung adenocarcinoma lines in vitro before and after induction by ADM.The DT of A549 cell line in vitro before and after induction by ADM was 37.6h and 36.1h, respectively. The RF of A549/ADM cell line to ADM, DDP, VP-16, VCR, TAX was 9.588、6.348、1.736、11.213、4.333, respectively.The DT of SPC-A-1 cell line in vitro before and after induction by ADM was 37.6h and 36.1h ,respectively. The RF of SPC-A-1/ADM cell line to ADM, DDP, VP-16, VCR, TAX was 4.283、3.672、6.308、9.112、2.716, respectively.2. Proportion of SP cell in human lung adenocarcinoma cell line before and after induction by ADM.The proportion of SP cell was 4% and 45% in A549 cell line before and after induction by ADM. The proportion of SP cell was 1% and 10% in SPC-A-1 cell line before and after induction by ADM.3. Proportion of ABCG2+ cells in SPC-A-1 and SPC-A-1/ADM cells.Under fluorescence microscope, cells labeled by Mouse Anti-Human ABCG2/BCRP-FITC were observed in each field of the SPC-A-1/ADM cells. The number of ABCG2+ cells in SPC-A-1/ADM cell line was more than SPC-A-1.Flow cytometry analysis showed that the proportion of ABCG2+ cells in the blank group of SPC-A-1 cell line was 0.047%,in the isotype control group of SPC-A-1 cell line was 0.087%, in SPC-A-1/ADM cell line was 9.9%. It was 40.08 times higher than that of SPC-A-1cell line.4. ABCG2+ cells isolated from SPC-A-1/ADM cell line by MACS.Result revealed that 2×104 ABCG2 + cells could be isolated from 1×107 SPC-A-1/ADM cells. the separating proportion of the ABCG2+ cells was 0.2%. The positive rate of the cell labeled by Mouse Anti-Human ABCG2/BCRP-FITC in ABCG2- cells (group A) was 0.127%, the positive rate of the cell labeled by Mouse Anti-Human ABCG2/BCRP-FITC in ABCG2+ cells (group B) was 84.71% and it was 667 times higher than that of group A.5. Tumorigenicity of group A and group B cells in nude mice.The transplanted tumors were not found in SPC-A-1 cell group (0/4) and group A (0/4) at the 8th weekend. The transplanted tumors were found in group B at the 3 th weekend, at the 8th weekend, the tumorigenicity ratio was 3/4.Conclusion:1. The multidrug resistance increased obviously in A549 cell line and SPC-A-1cell line induced by ADM. The proportion of SP cell increased obviously in multidrug resistance cells. All of these implied that increasing of SP cells was related with multidrug resistance in human lung adenocarcinoma.2. The proportion of ABCG2+ cells was higher in SPC-A-1/ADM cell line than that in SPC-A-1,and it would benefit for stem cell isolating base on SPC-A-1/ADM cells.3. The tumorigenicity of ABCG2+ cells coming from SPC-A-1/ADM cell line was higher than that from ABCG2- cells and SPC-A-1 cells in nude mice. It indicated that the cells isolated from the chemotherapeutic drug resistant lung adenocacinoma based on ABCG2+ possess stem cell characteristics.更多还原