The Efefct Of MSCs On Inhibition Of Radiation Induced Thymoma And Its Correlation With P53Methylation

Purpose:Clear and confirmed the inhibition of MSCs in radiation induced thymoma process ontumor formation, observe that the p53promoter methylation is the one of the mainmechanisms in radiation induced thymoma formation, MSCs protecting p53promotermethylation which effect radiation induced thymoma formation.Methods:1. Preparation of radiation induced murine thymoma C57BL/6modelDeep X ray therapeutic instrument is used to carry out four times total body irradiationwith single dose rate of1.75Gy continued for336second, once per week, total dose of7Gy.The applied voltage and current are200kV,10mA, respectively, under1.0mm Al and0.5mmCu Filter Plate. In the last radiation, all rats were sacrificed after six months radiation andtheir thymus tissues as well as those of normal rats were taken for pathological observation.2. Inhibitory effect of MSCs on radiation induced murine thymomaIsolation and culture of MSCs: The rats were killed by cervical dislocation and soak in75%alcohol solution for5min. After aseptic dislodgment of thighbones and humerus,wiping out metaphyseal and washing medullary cavity repeatedly, they were made intosingle cells for cultivation.Model establishment of MSCs injection treatment through tail vein: we collected theMSCs, adjusted the concentration to2.0×106/ml and took0.5ml for injection in model ratswith radiation induced thymoma through tail vein once a week for four times. The rats incontrol group were given0.5ml normal saline through tail vein for comparison. Then, wecarried out pathological observation on thymoma tissue in the treatment group and analysison the thymoma incidence in both control group and treatment group.3. Gene sequence analysis and detection of methylation in p16gene promoterAccording to the published sequence of p53gene promoter in C57BL/6rats (the GenBankaccession number: AF287146.1)，designed for specific PCR methylation primer (MSP) andmethylation sequencing PCR primer (BMP), genome extraction, sulphites have processing,methylation PCR amplification, agarose gel electrophoresis, methylation primer detectionand methylation sequence analysis. Result:1. Control group: All of the phenomenon including no clear boundary betweenthymus and cortical areas, diffuse distribution of lymphoid neoplastic cells, degeneration andnecrosis of normal lymphocyte along with largen and deeply strained cell nucleusdemonstrate the formation of radiation induced thymic lymphoma. In MSCs treatment group,normal thymus tissue, little larger lymphocyte and anachromasis of cell nucleus can befound.According to the results of pathological analysis, we concluded that the incidence ofMurine thymoma was72.73%(8/11) in control group and20%(2/10) MSCs treatmentgroup.2. After PCR amplification on the condition of MSP primer5carried out on GenomicDNA by Sulfite treatment, no methylated amplified bands were found in normal group,control group and MSCs treatment group. The sequence where MSP primer5PCR amplifiedfragments were located was further amplified by PCR technique on the condition of sodiumbisufite genomic sequencing (BSP) primer7. Target genes were shown in agarose gelelectrophoresis for normal group, control group and MSCs treatment group.3The PCR products were subjected to TA cloning and those positive clones wereselect for sequencing. The measured sequence of PK7, PM7and PZ7with were consistentwith the predictions, implying that they were p53promoter methylation. Comparisonbetween the predicted p53promoter methylation sequence and measured sequence of PK7,PM7and PZ7by ClustalW2-Multiple Sequence Alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/) indicated that there were no the predicted methylated loci in p53promotermethylation sequence for control group and model group, while cytosine (C) methylationappeared in1724loci in model group and no methylation were found in MSCs treatmentgroup.ConclusionFurther confirmed that MSCs has a protective effect of radiation damage, inhibition ofradiation induced thymoma occurred; the p53promoter of the radiation induced thymomaoccur abnormal methylation; thymoma tissue of MSCs treatment group occur DNAmethylation, possibly through aberrant methylation protect thymus tissues from radiationinjury...