| Lung cancer has become the leading cause of cancer mortality,the morbidity is increasing year by year.Even though some progresses in diagnosis and therapy have been maded in the last twenty years,combined therapy become the common method used in clinical,but the effect of treat is not significant.The 5 year survival rate is less than 15%.It is an problem urgently to be solved and a large challenge for people to explore the molecular mechanism in tumorigenesis and development in lung cancer,and to search for the new effective targets to attack lung cancer.In the past few years,increasing evidence suggest that some cell subsets in tumor possess the ability of self-renew and differentiation potentiality,although they are a fraction of the tumor cells.They are probably the begaining of the tumorigenesis,develoment,drug resistance,metastasis and recurring of the tumor.These type of cells have been called tumor stem cells because they have the characters of stem cells.Tumor stem cells hypothesis presume that malignant tumor consist of three cells:①Tumor stem cells:They possess the infinite reproductive activity and could form new tumor clone.②The cells that have the finite reproductive activity but could not form new tumor clone.③The cells that have neither the reproductive activity nor the ability that form new tumor clone.Now the therapy could made the tumor to contract, some can even obtain complete remission,but most of the tumor would relapse or metastasize.That might because the existing therapy could not eliminate the critical cells in tumor—tumor stem cells.Tumor stem cells has become a popular in resent years.It has gained some progression and shed light on the possibility of treatment of malignant tumor.We try to develop a method of identification lung cancer stem cells,to provide experience and evidence to find more tumor stem cells,to support tumor stem cell therapy.In this study,we intend to demonstrate some of the biological charaters of SP cells(the cell subsets related to stem cells)in human lung cancer cell line A549,to provide some new therapeutic mechanism for further exploring the molecular distinction, the prognosis monitoring and target therapy.The clinical significance of ABCG2 expression in non-small lung cancerObjective:To investigate the expression of ABCG2 in normal lung tissues and non-small lung cancer(NSCLC).Methods:The mRNA expression of ABCG2 in 8 normal lung tissue and 48 NSCLC tissues from resected specimens of patients were detected by RT-PCR. The relationship between the ABCG2 and the clinicopathologic characteristics in NSCLC was analysed,and the role of ABCG2 in prognosis of NSCLC was discussed.Results:The positive rate of the expression of ABCG2 was 100%(8/8)in normal lung tissues and 45.8%(22/48)in 48 NSCLC tissues,the expression of ABCG2 was significantly higher than the NSCLC(p＜0.01);the expression of ABCG2 in various clinicopathologic characteristics of NSCLC was not significantly different(p＞0.05),the survival time of the patients with ABCG2(+) was shorter than ABCG2(-)patients(p＜0.05),and it shows the trend that shorter than the overall survival time,the survival time of the patients with ABCG2(-) has the trend that longer than the overall survival time.Conclusion:The expression of ABCG2 was an independent factor for prognosis in NSCLC,the prognosis was worse in ABCG2(+)patients.Isolation and analysis of side population cells in human lung cancer cell line A549Objective:To set up one simple but efficient method of isolation lung cancer stem cells.Methods:Both the proteins expression of CD34 and ABCG2 in cell line A549, and the CD34 protein in 15 lung cancer tissues from resected specimens of patients were detected by immunohistochemistry.SP and NSP cells in cell line A549 were isolated by MACS and FACS respectively,and their differentiation was analysed.ABCG2 expression in the tow cell subsets was detected by RT-PCR.Results:CD34 and ABCG2 protein in cell line A549 and 15 lung cancer tissues were undetectable by immunohistochemistry,but ABCG2 was detectable in cell line A549,the positive rate was about 2%.SP and NSP cells could not isolated by MACS,but could be isolated by FACS,the percentage of SP cells was about 1.09%in all of the alive cell A549,and decreased to 0.20%after treated by Verapamil.SP cells could produce both SP and NSP cells,but NSP cells could only produce NSP cells.Conclusion:FACS is an effective way to isolate SP cells in cell line A549,and the SP cells in cell line A549 may enrich tumor stem cells. The biological behavioral trait of SP cells in in cell line A549 in vitroObjective:To explore the mechanism of the biological distinction,and the mechanism of invasion and metastasis of SP cells in cell line A549 in vitro.Methods:SP and NSP cells were cultured in vitro,and the cell growth curves, the cell divisional index,the cell cycle and the cell proliferation index were measured;the cloning efficiency was test by plate clone formation test;the ability of the two subsets were examed by migration and invasion assay;the chromosome nuclear type of the tow cell subsets were also analysed.Results: There ere no difference in the cell growth curves,the cell divisional index,the cell cycle,the cell proliferation index of the tow cell subsets(p＞0.05),and the cells which cross the membrane in migration test were not significantly different either(p＞0.05);but the SP cells which imigrate the filter membrane in invasion assay was significantly more than the NSP cells(p＜0.05),the cloning efficiency of SP cells in vitro was significantly higher than NSP cells(p＜0.05).The chromosome nuclear type analysis showed that they were not significantly different.Conclusion:The ability of the vitro clone formation and invasion of SP cells was surpassed than the NSP cells.The biological distinction of SP cells in cell line A549 in vivoObjective:To explore the biological character of SP cells in A549 cell line in vivo.Methods:SP and NSP cells were prepared in different order of magnitude, and than were injected into both of the armpit subcutaneouly of the BALB/c nude mices,and then observed the development of the transplanted tumors.The tissues type of the transplanted tumors was confirmed by HE staining;the expression of ABCG2 in transplanted tumor was detected by RT-PCR and immunohistochemistry.Results:After 3 to 5 weeks of injection,new tumors were observed in SP cells at 1x 10~5 in 5 of 5 situs,and at 1x 10~4 in 3 of 5 situs,whereas NSP cells at any of the order of magnitude(included 1x10~6 and less than 1x10~6)failed to form new tumors,suggesting the efficiency of tumor formation of SP cells was much higher than NSP cells.The transplanted tumors were confirmed adenocacinoma by HE staining;the expression of ABCG2 in transplanted tumors was detectable by RT-PCR and immunohistochemistry,the expression rate of ABCG2 was about 10%by immunohistochemistry.Conclusion:SP cells in cell line A549 have self-renew and high tumorigenesis ability.The chemotherapeutics susceptibility of SP cells in cell line A549 in vitroObjective:To explore the chemotherapeutics susceptibility of SP cells in cell line A549 and the possible mechanism of multidrug resistance.Methods: The susceptibility of SP and NSP cells to DDP,5-FU,VP16,NVB and GEM were detected by drug susceptibility test,and IC50s were calculated;after 2 hours treated by the five chemotherapeutics(the dose≈IC50s of NSP cells to the drugs),the intracellular drug levels were test by high performance liquid chromatograph.Results:The difference of the IC50s to DDP,and of the intracellular drug levels between the tow subsets was not significantly different (p＞0.05),whereas the IC50s to the other four drugs of SP cells were significantly higher than that of the NSP cells(p＜0.01);and the intracellular drug levels of the other four drugs in SP cells were significantly lower than that of the NSP cells(p＜0.01).Conclusion:Compares to NSP cells,SP cells in cell line A549 are more drug resistant to the chemotherapeutics,and the multidrug resistance of SP cells is closely related to the function of SP cells discharging the chemotherapeutics medicine.
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