Radiobiology Characteristics Of CD133Positive Cells In Human Lung Cancer

Background and Purpose:Lung cancer is the leading cause of cancer deaths in the world annually，The5-year survival rateis very low,Current by means of surgery, chemotherapy, radiation therapy, and comprehensive treatmentcan not control the death rate of lung cancer,recently with deeply study of tumor and stem cell,thinkly thetumor may be a stem cell disease, At the present time, one of the most exciting concepts being explored incancer research is the idea of CSCs, suggesting that small populations of undifferentiated cells withpotential for self-renewal and differentiation initiate and have been displayed to be the source of many solidtumours including lung carcinoma,CSCs play an important role in tumor maintenance, develop, metastasisand recurrence, CSCs are radio-resistant and chemotherapy resistance.Radiotherapy has a curative potential in many solid human tumors in modern medicine, and thatradiation therapy to kill tumor cells mainly by causing DNA serious damage, Including single-strand breaks(SSB), double-strand breaks (DSB), base damage, DNA-DNA cross-linking, and DNA-proteincross-linking various forms, DNA Double-Strand Break is the most serious DNA damage, the ability ofDSBs repair is one of the important factors influencing radiosensitivity, Radiation therapy induced DSBactivation of DNA-PK, ATM and ATR, etc, can quickly repair the DSB,can also activate the DNA damagesignal pathway to promote repair the transcription of the relevant factors, caused cell cycle arrest orinduction of apoptosis. Although radiation therapy can eradicating a large number of tumor cells, butsurviving tumor stem cells may become a cause of treatment failure. Therefore accurate separation ofidentification and eradication of cancer stem cells is very important. CD133is a transmembraneglycoprotein,and the first is found in human hematopoietic stem cells,which is thought as a surface markerof several stem cells.This article through a wide range of tumor markers CD133the application magneticactivated cell sorting method to isolated CD133+lung cancer cells, and then to do a study of radiobiologyproperties.Materials and Methods:1. Application magnetic activated cell sorting method from lung cancer H2228cells isolated CD133+cells.2. The cell counting depicts the growth curve of the CD133+lung cancer cells and lung cancerH2228cells.3. Clonogenic survival assay was performed to determine survival fraction of the CD133+lungcancer cells and lung cancer H2228cells under different doses．The parameters DO and Dq for thesingle-hit multitarget model and the parameters α, β and SF2for the linear quadratic model were calculatedto evaluate radiosensitivity of these two cells．4. Cell cycle and cell apoptosis percentage were detected with flow cytometry.Results:1. The percentage of CD133+cells,isolated from human lung adenocarcinoma cell line H2228by using the magnetic activated cell sorting method，was1.7%.2. Almost all CD133+cells after immunofluorescence staining emits a red fluorescence.It meansthat the sorting cells,isolated by MACS,were with relatively high purity.3. by growth curve,we found that CD133+lung cancer cells rapid growth, culture soon enter thelogarithmic phase, the total number of cells rendering index upward trend. By contrast, the H2228thegrowth of lung cancer cells is slower, which may be attributed to the unlimited proliferation and self-renewof CD133+lung cancer cells ability.4. In this study, using colony formation assay further compare of CD133+lung cancer cells andH2228radiation sensitivity differences.The survival curve looked smoother in the CD133+lung cancercells than in the H2228．Also．the survival fraction at every doses was higher in the CD133+lung cancercells than in the H2228． In the single-hit multitarget model, The parameters D0are：2.445and2.308，Dq are2.610and1.121, D0is larger, the cells to radiation more resistant. Dq value is smaller, the weakercells sublethal damage repair capacity, small doses can make the cells into the fatal damage death stageCD133+lung cancer cells experimental results with radiation resistance than H2228. otherwise，in thelinear quadratic model，The parameters α are0.038and0.185,β are0.036and0.04,SF2are79.89%and61.03%（P<0，05）, the greater the value of α, the greater the extent of the damage, and β represents the highdose portion of the biological effects described in the portion of the low-dose,the CD133+lung cancer cells more radiation resistance than H2228.SF2was higher in the CD133+lung cancer cells than in theH2228，indication that the cells survive for more.All these results suggested that CD133+lung cancercells is much more radiosensitive than H2228．5.The result of the flow cytometry displays the irradiation dose2Gy24h CD133+lung cancercells cell cycle distribution changed little, the H2228cell cycle after irradiation distribution for the increasein S phase and G2/M phase to reduce, which indicates that the lung cancer stem cells in the cell cycleredistribution is not obviously.Therefore, CD133+lung cancer cells have a radiation resistance. The resultsalso show the before and after CD133+lung cancer cells irradiation compared to the rate of apoptosis wasno significant difference (P>0.05), the H2228before and after irradiation, compared to the rate ofapoptosis there are significant differences (P <0.05), CD133+lung cancer cells are more radiation resistantthan H2228.Conclusion：1. CD133+cells could be isolated from human lung cancer H2228cell lines by MACSsuccessfully, the CD133+cells were in a very small percentage of the totall cell population.2. CD133+lung cancer cells have unlimited proliferation and self-renewal capacity, also has aradiation resistance.3. CD133+lung cancer cells radiation resistance may be related to cell cycle redistribution andapoptosis associated with cell cycle redistribution is not obvious and the apoptosis rate was not changedsignificantly,the stronger the radiation resistance.