The Mechanism On The Ionizing Radiation-induced Adaptive Response In Fibroblasts Under2-dimensional And3-dimensional Conditions

Adaptive response induced by low doses of ionizing radiation refers to theresistance to the subsequent high-dose ionizing radiation caused by the small doses ofionizing radiation in advance, which can reduce the injury or the subsequence causedby high dose irradiation. The related mechanism mainly involves the immuneregulation, activities of reactive oxygen species (Reactive oxygen species, ROS),DNA repair activation, the effect of cell signal transduction and cell cycle blockrelated to p53protein, etc. The tumor microenvironment composed of fibroblastsplays an important role in tumor occurrence and development. Related studies showthat some biological effects of fibroblast induced by low doses of ionizing radiationplays a role in such aspects as the tumor proliferation that cannot be ignored. Itsmechanism is not very clear, and remains to be further researched.3-D cell culture is a manner in which cells are cultured in a simulation of aformicro environment.Compared with flat(two-dimensionl) cell culture, itscharacteristics is the cell polarization and adhesion complex formation and thecharacteristic appearance of cytoskeleton and cell volume and leads to the regulationof cell phenotype, function and signal conditioning to make it closerto thePhysiological and pathological characterization.3-D cell culture offers uniquetechnical support to the simulating of the physiological and pathological environment,the research of molecular mechanism, the vitroproduction and the tissue used fortransplant.Radiation induces the break of DNA Double-strand, which can serve as a means of monitoring biological effects of ionizing radiation. Phosphorylation ofH2AX genes(gamma-H2AX) are involved in DNA damage repair and accumulatesIn DNA Double-strand breaks, and are often used as a DNA Double-strand breaksmonitoring indicators.Objective:To explore adaptive response of various fibroblast cell lines cultured under2-Dand3-D induced by ionizing radiation of different doses and dose rates and its relatedmechanisms.Method:1. Culture various fibroblasts （RMP-4、IMR-90and MEF）under the conditionof2-D and3-D.2. Radiation doses range between0~100mGy as low dose (D1) to induceadaptive response, range between1~5Gy as the attacking dose to induce the ruptureof double-stranded DNA. The time interval of6hours.3. Observe the γ-H2AX focus and fluorescence intensity at different time anddifferent dose of radiation of RMP-4cells cultured under2-D to determine the besttime and dose.RMP-4cells under the two-dimensional culture condition, different doses anddifferent time detection observation of gamma–H2AX focus.4. Detect γ-H2AX focus in various fibroblasts through immunofluorescenceanalysis, evaluate double-stranded DNA breakage and adaptive response induced bylow dose ionizing radiation.5. Monitor the expression of related gene protein (p53, p21and p53(ser15)expression with Western blot and verify the related mechanism.6. under the2-D and3-D condition with the same dose and different dose rateirradiation of RMP-4cells by immunofluorescence gamma-H2AX focus, evaluatedthe DNA double strand breaks of of different dose rate ionizing radiation induced.7. Observe the cell morphology in2-D and3-D culture condition， after pholloidin staining under the Fluorescence microscopy and confocal microscopyobservation after different dose radiation.8. Do the gray analysis for protein electrophoresis banding with ImageJ software.9. The statistical results are expressed in the form of mean+standard deviation,and analyzed the obtained results with variance analysis. P<0.05was consideredstatistically significant. All of the data is analyzed with SPSS.Origin6drawingsoftware.Results:1. The analysis of damage schedule/concentration-response of the DNA offibroblasts RMP-4induced by ionizing radiation induced into fiber cell lines ofRMP-4Under the condition of2-D culture, expose2Gy to ionizing radiation, and thencontinue to culture, respectively,0.5,1,2,4hours later, observe γ-H2AX focus andfluorescence intensity.0.5hours after irradiation, the number of γ-H2AX focus in thecell increases obviously, to its peak in1hour, and in the flowing4hours is stillincreasing significantly. The number of positive cells (more than10cells werepositive for focus) increased from15%in controls to1hours of87%, intermediate0.5,2, and4hours respectively45%,85%and81%, and statistically significantdifferences were obvious（P<0.05）.RMP-4cells cultured under the same condition were given0,0.1,0.5,1,2and5Gy (0.8676Gy/min),1hour after exposed to ionizing radiation, found low dose0.1Gy can induce the increase of fibroblasts gamma-H2AX focus, the number of positivecells (cells positive for more than10focus)21%of0Gy group increased to nearly100%at5Gy group（P<0.05）.Ionizing radiation induced rupture of Double-strandedDNA of fibroblast cell lines RMP-4partly depend on time and does within a certainrange dose and time.2. The adaptive response of fibroblast cell lines RMP-4induced by low dose ofionizing radiation and its mechanisms RMP-4cells exposed to low dose irradiation of50mGy or100mGy, and wereexposed to attack dose of2Gy, and detected the gamma-H2AX focus andfluorescence intensity in cells by immunofluorescence staining. Through statisticalanalysis and counting, gamma-H2AX positive cells of2Gy irradiation group reducedto65%and45%respectively from87%（P<0.05）, which shows that low doseirradiation in advance can obviously increase the resistance of RMP-4cells to theattack dose2Gy of ionizing radiation.RMP-4cells were exposed to D1low doseirradiation, and then to joint D2(2Gy) attack dose of ionizing radiation in six hours,and Western blot was used to detect related protein expression. Stripe gray analysisshows that p53, p21and gamma-H2AX irradiated by direct D2dose increasedsignificantly（P<0.05）, while p21and gamma-H2AX protein expression levels of thegroups were exposed in advance to25,50,75and100mGy of D1dose2Gy followedby D2doses significantly decreased（P<0.05）, especially the group of75mGy+D2reduced significantly. But the changes of p53protein in D1+D2in various treatmentgroup were not obvious. So in the subsequent experiments phosphorylation p53(ser15)protein expression will be detected.3. The adaptive response of fibroblast cell lines IMR-90induced by low dose ofionizing radiation and its mechanismsFibroblast cell lines IMR-90were exposed to D2(2Gy) or low dose of D1+D2(6hours) of ionizing radiation, and cell immunofluorescence tests were used todetect gamma-H2AX and p53(ser15) expression and aggregation, by counting thenumber of focus of cells or positive cells to statistical analyzing differences betweendifferent treatment groups. After exposed to D2attack dose, gamma-H2AX focus ofIMR-90cells increased to40/cells from the average number of10/cells（P<0.05）.However, IMR-90cells were exposed to D1(25,50,75and100mGy),and then to attack dose irradiation D2on6hours, and the average number of γ-H2AXfocus are significantly lower than that of single D2dose group （P<0.05）, and thenumber of the focus of the75mGy+D2group is least and about24/cell.The number of the focus of the100mGy+D2group is on average30/cell, but is stillsignificantly lower than that of the attack alone dose D2group. According to thenumber of positive cells to analyse the results of all groups and found that the rates ofgamma-H2AX positive cells of the example group, D2,25mGy+D2,50mGy+D2,75mGy+D2and100mGy+D2are respectively20%,98%,97%,60%,50%and80%.The difference between single D2group and the D1+D2groups was statisticallysignificant （P<0.05）.The γ-H2AX positive cells of75mGy+D2group account fornearly50%, which is the lowest proportion of positive cells in all treatment groups.Western blot was used to detect the expression of γ-H2AX protein and gray analysisof protein bands were analyzed to calculate relative levels of the protein expression.Each protein stripe grey value is0,2.9,0.90,0.85,0.90and0.85. The differencebetween each D1+D2group and the single D2group was statistically significant（P<0.05）. The results above show that low doses of ionizing radiation can inducethe resistance of fibroblasts cell lines IMR-90to the attack irradiation dose D2,namely, adaptive response. Analysing the p53(ser15) positive cells number throughthe cell immunofluorescence, and got the value of each group, which was respectively4%,40%,39%,29%,20%and30%. The difference between group50mGy+D2,group75mGy+D2, group100mGy+D2and single D2group, was statisticallysignificant （P<0.05）. Analysing the grey value of p21protein bands of each Westernblot group, the gray value of each group was respectively2.0、2.75、2.25、2.25、2.50and2.65. The results show that compared with single D2group,the25mGy+D2,50mGy+D2,75mGy+D2the difference was statistically significant （P<0.05）.Relative grey value of p21protein bands of each group were1.80,2.85,2.10,1.65,1.75and1.25. The difference between group50mGy+D2,75mGy+D2,100mGy+D2and single D2group, was statistically significant （P<0.05）.4. The adaptive response of fibroblast cell lines MEF induced by low dose ofionizing radiation and its mechanismsFibroblast cell lines MEF were exposed to D2(2Gy) or low dose of D1+D2(6 hours) of ionizing radiation, and cell immunofluorescence tests were used to detectgamma-H2AX and p53(ser15) expression and aggregation, by counting the numberof focus of cells or positive cells to statistical analyzing differences between differenttreatment groups. After exposed to D2attack dose, γ-H2AX focus of MEF cellsincreased to35/cells from the average number of8/cells（P<0.05）.However, MEFcells were exposed to D1(25,50,75and100mGy), and then to attack doseirradiation D2on6hours, and the average number of γ-H2AX focus are significantlylower than that of single D2dose group （P<0.05）, and the number of the focus issignificantly lower than that of the single D2group（P<0.05）). The numbers of thefocus of the75mGy+D2are the lowest, which is about16/cell. The numbers of thefocus of the25mGy+D2group and100mGy+D2are about20/cell. According to thenumber of positive cells to analyse the results of all groups and found that the rates ofgamma-H2AX positive cells of the example group, D2,25mGy+D2，50mGy+D2,75mGy+D2and100mGy+D2are respectively17.5%，84%，69%，49%，45%and70%.The difference between single D2group and the D1+D2groups was statisticallysignificant （P<0.05）.The proportion of γ-H2AX positive cells of75mGy+D2group is the lowest among all treatment groups. Western blot was used to detect theexpression of γ-H2AX protein and gray analysis of protein bands were analyzed tocalculate relative levels of the protein expression. Each protein stripe grey value is0、1.75、0.50、0.32、0.35and0.45. The difference between each D1+D2group and thesingle D2group was statistically significant（P<0.05）. Analysing the p53(ser15)positive cells number through the cell immunofluorescence, and got the value of eachgroup, which was respectively0、20%、15%、15%、5%and5%. Analysing the greyvalue of p53protein bands of each Western blot group, the gray value of each groupwas respectively0.29、0.65、0.45、0.40、0.40and0.40. the grey value of p21proteinbands of each group, was respectively0.49、0.78、0.60、0.50、0.40and0.40.Theanalysis results of gene bands of p53and p21indicate that the difference betweeneach D1+D2group and single D2group is statistical significant（P<0.05）.The results show that compared with single D2group,the25mGy+D2,50mGy+D2,75mGy+D2the difference was statistically significant（P<0.05）. Relative grey value of p21protein bands of each group were1.80,2.85,2.10,1.65,1.75and1.25. The differencebetween group50mGy+D2,75mGy+D2,100mGy+D2group and single D2group,was statistically significant（P<0.05）.The results above show that low doses ofionizing radiation can induce the resistance of fibroblasts cell lines MEF to the attackirradiation dose D2, namely, adaptive response. And genes p53and p21aresignificantly related to the adaptive response induced by low dose of ionizingradiation.5. The comparison of adaptive response induced by ionizing radiation of cellscultured under2-D and that of3-DRMP-4are cultured under the condition of2-D and3-D, and were exposed to0.5Gy of ionizing radiation and the dose rate is respectively: low (LDR)0.0184Gy/min,(MDR) in0.2Gy/min and high dose rate (HDR)0.8676Gy/min. The resultsshowed that after being exposed to ionizing radiation, under the condition of twokinds of culture the number of.γ-H2AX focus positive cells of RMP-4cells culturedunder the two different conditions increased along with the increase of the dose rateThis shows that under the same condition of ionizing radiation, different dose ratemakes a significant difference to the damage of the DNA double chain fracture, andespecially on the cells cultured under the three-dimensional cultivation condition. Theproportion of2-D positive cells in group γ-H2AX were4.9%,5.2%,11%and46%（P<0.05）; the proportion of3-D positive cells in group γ-H2AX were6%,9%,17%and72%（P<0.05）.This indicates that the damage of DNA of cells cultured under3-Dculture are more sensitive to the increase of dose rates. In order to further compare theeffect of culture conditions of2-D and3-D on the adaptive response induced byionizing radiation, observe the cell morphological changes under electron microscope.Under the condition of2-D cultivation, cell morphology under the electronmicroscope observation are the growth thick, stretch together with each other.RMP-4 cells cultured under3-D culture were observed under electron microscope,characterized by clear individual cells, stretch, and full cell bodies. After beingexposed to ionizing radiation of0.1,0.5or2Gy, cell morphology change obviously,there is an obvious abnormal morphology, and part of the cell performancespseudopod disappeared, cell curl, etc. Another part of the cells are between normaland abnormal shape, which we define as the intermediate state.2-D culture condition,RMP-4cells treated with2Gy or75mGy+2Gy irradiation, electron microscopeobservation, RMP-4cell morphology and statistics. The results showed that:2-Dculture conditions, cell growth and stretch tight, intertwined,2Gy after irradiation cellshrinkage crimping, but the texture is still clear, after75mGy+2Gy irradiation, thereis no obvious morphological change of the cells;Under3-D culture condition, the cells were exposed to ionizing radiation of2Gyor75mGy+2Gy, and were observed under electron microscope to do the cellmorphology statistics.The results show that the cell number of abnormal formincreased obviously, and increased from the underlying rate28%to80%（P<0.05）,and the intermediate state of cells increased slightly (about5%).Theabnormal cells of the75mGy+2Gy group account for34%and are obvious lowerthan that of2Gy group, but are more than the control group（P<0.05）.The cells ofintermediate state increased to20%, and are much more than2Gy group（P<0.05）.These results showed that:3-D culture conditions and culture conditionsof the2-D more easily observed into adaptive response of fiber cells, which may becloser to the cell growth in vivo of the real environment and3-D culture conditions,relatively easier to activate other signaling pathway, thus activating the adaptiveresponse of cells.Conclusion1.Low doses of ionizing radiation can induce the adaptive response of fibroblastscultured under the condition of2-D and3-D;2.The adaptive response can be easily induced under the conditions of3-D culture than under the2-D culture conditions, and this may indicate that there May be apotential lethal damage repair mechanism;3.p53and p21may be involved in the adaptive response of low dose induced byionizing radiation, and the mechanism of adaptive response of low doses induced byionizing radiation requires further research;4.The adaptive response induced by low dose ionizing radiation of cells culturedunder the condition of3-D is much easier to be observed in morphology.In conclusion,this study of project established a three-dimensional cell culturemodel which is used to simulate in vivo conditions, in the experiment the cellmorphology of a variety of fibroblast were compared under the condition of cellculture in2-D and3-D, and we also observe whether the ionizing radiation withdifferent dose and dose rate can adaptive response induced in3-D culture conditions,to provide the basis for the study of3-D radiation characteristics of culture under thecondition of fibroblasts and its effect on tumor cells, and lay the foundation for futherresearch that the ionizing radiation of microenvironment of tumor cells in thetreatment of the role and mechanism of tumor.