| 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. |
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