Isolation And Identification Of Cd133 Positive And Cd133 Negative Cells In Human Lung Cancer, And Screening Of Their Differential Genes

ObjectiveDrug resistance , metastasis and recrudescence of cancers lead to the failure of clinical treatment and death of patients with malignant tumors. Searching for genes associated with drug resistance and metastasis of tumor is very important for solving problems of clinical treatment, and play a key role in clarifying the mechanism of tumor metastasis and drug resistance. According to the background,we plan to isolate CD133+ cells and CD133- cells by using magnetic activated cell corting method.Then evaluate their bionomics by different methods . On this basis, we will screen the differential genes of cancer metastasis and drug resistance by DNA arrays.Methods1.Immunohistochemical staining was used to detect the expression and distribution of CD133 in 81 cases of lung cancer and 10 cases of normal lung tissue. Then find out the relationships of CD133 expression and tumor histological type ,tumor grading , tumor staging by statistical analysis.CD133 was detected by immunocytochemistry in human lung cancer cell line A549.2.Isolated CD133+ cells and CD133- cells from human lung cancer cell line A549 by MACS; Detected the purity of sorting cells by immunofluorescence.3.To observe the growth status and the formation of sphere,CD133+cells and CD133- cells were cultured in serum-free DMEM-F12 medium (containing EGF, bFGF) in vitro.4.Tested the colony formatting efficiency of CD133+ cells ,CD133- cells and cells without sorting by colony-forming assay.5.The differentiation of sphere was induced in vitro .And immunohistochemical staining was used to detect the expression of CD133 and CK7 of differentiated and undifferentiated cells.6.Detected the metastasis-related genes(84 genes) and the resistance-related genes(84 genes) of CD133+ cells and CD133- cells by using DNA microarray. Analyze the differences by technology of bioinformatics.Results1.The expression of CD133 in lung cancer.Among 81 lung cancer examples ,there were 43 expressing CD133(53.1%). The positive rate of squamous cell carcinoma and adenocarcinoma were 48.3%(14/29)and 52.9%(18/34), respectively.And the positive rate of. other types of lung cancer was 61.1%(11/18).There were nocorrelation between CD133 expression and histology type, tumor grading and TMN classification (P >0.05).2.A small number of CD133 positive cells could be found in human lung cancer cell line A549.3.The percentage of CD133+ cells ,isolated from human lung cancer cell line A549 by using the MS column,was 0.2%.4.Almost all CD133+ cells showed fluorescence,while fluorescence were difficult to be seen in CD133- cells.It means that the sorting cells ,isolated by MACS ,were with high purity.5.For 24 hours, adherent cells were found in CD133-negative group and CD133-positive group , but the number of adherent cells in CD133-positive group is significantly less than in CD133-negative cell group.CD133+ cells formed sphere in the ninth day ,while the CD133- cells failed to form sphere.6.The colony formation rates of CD133+ cells, CD133- cells and cells without sorting were 57.1%, 3.3%, 8.7%, the rate of colony formation among the three groups was statistically significant difference (P <0.05).7.Immunocytochemistry results showed that CD133 expressed in undifferentiated cells while the expression of CK7 could not be found ; The case in differentiated cells was different ,CK7 instead of CD133 expressed in them .8.Compared with the CD133- cells,the expressions of 30 resistance-related genes and 19 metastasis-related genes drop in CD133+cells.And only one resistance-related genes was highly expressed in CD133+ cells . In addition, the difference of the expression levels reached or exceed 2 times.Conclusion1.Lung cancer contained rare CD133 + cells. The expression of CD133 has nothing to do with tumor histological type, tumor grading and TMN classification..2.CD133+ cells could be isolated from human lung cancer A549 cell lines by MACS successfully.3.It showed that CD133+ cells with differentiation potential can form "tumor sphere" and their proliferation potential is significantly higher than CD133- cells.CD133+ cells have stem cell characteristics.4.The result of drug resistance DNA microarray prompted that RARG gene may play an important role in drug resistance mechanism.5.The result of tumor metastasis DNA microarray prompted that high proliferative potential , adhesive attraction and immune escape property of CD133 + cells may play a major role in the metastasis process of tumors.CD82 gene may play an important role in mechanism of tumor metastasis.