| p53 is an essential factors which maintains the stability of genome and involves in cell emergency response. p53 was activated in response to DNA damage when the exogenous and endogenous damage as well as the oncogene activated, and determined cells fate according to the severity of the damage. p53 could repair the damage by arresting cell cycle progression and induce apoptosis for the unrepairable damage. p53 is a critical anti-oncogene. More than 50% of human tumors carry TP53 mutation.MSCs was widely used in the treatment of injuries such as human bone, cartilage, cardiovascular, nervous system,and skin because of its low immunogenicity and multi-differentiation potential. Our previous studies have evidenced the ability of MSCs in reducing the incidence of irradiation-induced thymoma in mice. Although numerous researches have been made to investigate the mechanisms of damage recovery involved in MSCs, the detailed mechanism remains unclear. In this research, p53 was used as an entry point to investigate the recovery effect and its mechanism of irradiation-induced damage of MSCs from epigenetics, p53 mutation and signal transmission.Method: 1. Preparation of thymoma model of mice C57BL/6J mice were selected, divided into normal group and irradiation group. The X?ray was used to irradiate the whole mice body at dosage rate of 300 c Gy/s. The dosage was set up 1.75 Gy/time according to the description by Kaplan. The irradiation was given once per week for 4 weeks. The total dosage was 7 Gy. Then, the radiation group was divided into irradiation alone group and MSCs-treated irradiation group. Mice was observed every day, thymoma was identified after 6months. 2. MSCs isolation, cultured and identification The method of bone marrow adherent was applied to isolate and purify MSCs. C57BL/6J suckling mice were selected and sacrificed by dislocation. The femurs were taken out and metaphyses were cut off in a sterile condition. The medullary cavity was repeatedly washed with L-DMEM culture medium. Single cell suspension was prepared, cell concentration was adjusted to 2.0×105/ml and transferred to 25cm2 culture flask containing L-DMEM complete medium including 10% fetal bovine serum and cultured at 37°C in an atmosphere of 5% CO2.The medium was replaced 48 h later to remove the non-adherent cells. Cells were observed everyday and the medium was changed twice per week. When up to 80–90% full of the flask bottom,the cells were passaged at 1:2 and MSCs were identified by induced differentiation. The third passage of cells were collected and adjusted to 5.0×105/ml and seeded in 6-well cell culture plate covered with cover glasses, 2ml/well. Osteogenic inducing culture medium(L-DMEM medium, 20% fetal bovine serum, 10 mmol β- glycerophosphate, 10 mol dexamethasone, 50μg / m L ascorbic acid) and adipogenic inducing culture medium(10mg/L of recombinant human insulin, 0.5mmol/L IBMX, 1μmol/L dexamethasone, 100μmol/L indomethacin) were added into plate,cells were observed everyday. Then the alkaline phosphatase staining and oil red O staining were processed after 2 weeks. 3. MSCs were injected into irradiated mice 0.2ml of 2×106 cells / ml of MSCs were injected into mice through caudal vein on the day or the next day after irradiated 4 times, the second injection was performed one week later with a total injection of 2 times. 4. Measurement of p53 expression with immunohistochemistry The thymus tissues were embedded in paraffin and sectioned.The sections were deparaffinized followed by antigen retrieval.1:300 dilution of rabbit anti-mouse p53 antibody were dropped and then incubated at 4°C overnight. Biotin-labeled goat anti-rabbit antibody were dropped and incubated for 20 min at room temperaturefollowed by dropping streptomyces avidin- peroxidase and incubated for 20 min at room temperature. The sections were developed with DAB and contrast stained,dehydrated with graded ethanol, cleaned with xylene, and covered. 5. RT-PCR detected the change of gene expression RNA from mouse thymus tissue was isolated using tissue / cell RNA rapid extraction kit(EASYspin) and RT-PCR was performed using mouse p53 and related gene-specific primers. The reaction conditions were: 94 °C for 1 min,94 °C for 30 s, 55 ~ 65 °C for 30 s, and 72 °C for 30 s.Amplification was performed for 30 cycles, followed by extension at 72°C for 5 min. The PCR products were electrophoresed in 1%~2% agarose gel. 6. detection and analysis of p53 promoter methylation The thymus genomic DNA was extracted using tissue / cell genomic DNA extraction kit and bisulfite treatment was performed using EZ DNA methylation-Gold Kit, p53 promoter methylation was detected using methylation specific primers, and sequencing primers were used to amplify methylated p53 promoter and sequencing. 7. Target gene sequencing and analysis The correct PCR products were identified using agarose gel electrophoresis, and agarose gel purification extraction kit was used for recovery and purification. The purified PCR products were ligated with the TA vector p MD18-T, transformed into E. coli DH5α competent cells. Monoclonal were picked by ampicillin resistance screening to amplify. The plasmid was extracted using plasmid small amount of fast extraction kit,and restriction enzyme Bam HⅠ(or Eco RⅠ)and Hind Ⅲ were used for identification, then the positive recombinant plasmid were selected for sequencing. The sequences were analyzed by BLAST to determine whether the sequence is the target gene, and Clustal W software was used for comparative analyze.Results: 1. MSCs cultured and identification C57 BL / 6J suckling mouse bone marrow MSCs were cultured by bone marrow all-adherent culture method and inoculated, the cells began to adhere 48 h later withsmall volume and mainly rounded observed by microscope;72h later, adherent cells began to appear shuttle type showing fibroblast-like growth. 10 days or so cell densitie was up to 80%~90% with mainly fibroblast-like morphology. Round cells were gradually disappeared after passage and cells showed a shuttle type and grew like shoal of fish or arrangement growth like paving stones. MSCs were induced into osteogenesis and adipogenic differentiation. The results showed that osteogenesis-induced MSCs have a deep staining of alkaline phosphatase and cells formed aggregates while non-induced group did not. Lipid droplets were appeared in Cytoplasm of adipogenic-induced MSCs and lipid droplets gradually increased with time,indicating that MSCs can be induced to differentiate into bone and fat. 2. Pathologically analysis of thymic structure in mice C57BL/6J mice after 6 months X-ray irradiation were dissected. Thymus in irradiation alone group visible to the naked eye was significantly larger compared with normal control group with no normal thymus structure and showed pale yellow. The size of thymus in MSCs-treated irradiation group was close to the normal control group with no normal thymus leaf structure, and with the color between the normal control group and irradiation alone group. After the thymus tissue biopsy, thymus tissue structure of mice was normal in the control group observed in light microscopy with clear structure of cortex and medulla, mainly lymphocytes, regular shape, uniform size and were round or oval. The normal thymic structure was destroyed in irradiated group with no normal lymphocytes, and lymphoma cell diffuse distribution, forming a typical characteristic of lymphoma cells with necrosis debris,indicating the formation of irradiation-induced thymoma. The structure of thymus could be seen in MSCs-treated irradiation group and the lymphocytes became normal with no characteristic lymphoma cells. 3. MSCs up-regulated the expression of p53 protein and m RNA in irradiated mouse thymus tissue. Immunohistochemistry and RT-PCR results showed that the expression of p53protein and m RNA in thymoma tissues in irradiation alone group was higher than in normal control group(t=3.73,p=0.02 and t=18.768,p<0.001), and the expression of p53 protein and m RNA was higher in nontumor thymus tissues in MSCs‐treated irradiation group than in irradiation alone group( t=3.065,p=0.037 and t=5.801,p=0.004). 4. Analysis the influence of p53 promoter methylation in thymus tissue from irradiated mice response to MSCs. The agarose gel electrophoresis showed no amplified methylation bands in normal control group, irradiation alone group and MSCs-treated irradiation group. Sulfite sequencing analysis showed that p53 promoter methylation was not observed in all three thymus tissues in normal control group. Only one thymus tissues was found methylation in irradiation alone group.The methylation located at +143 of the first exon and-1190 of the promoter distal end. These two methylation loci were not included in the known transcription factors binding motif. However, Matlnspector Professional Database(www.genomatix.de/) analysis showed-1190 located in binding motif of transcription factor E2 A. It showed that in three nontumor thymus tissues from MSCs-treated irradiated mice, only one thymus tissues exhibited methylation. The methylation located at +198 of the first exon and-1043,-1090, and-1158 of the promoter distal end.+198 located in the binding motif of transcription factor Pax(+195/+201).-1043 and-1090 located at the either side of the negative regulation region(- 1065/-1084). 5. MSCs affect p53 gene mutation in thymus from irradiated mice. Primers were designed according to the p53 m RNA CDS region of C57 BL / 6J mice, mouse thymus p53 gene was obtained by amplification followed by clone sequencing. Ten recombinant plasmids selected from each mouse were sequenced. Sequences were analyzed by BLAST and converted into amino acid sequences after non-mice p53 genes were removed. Then the amino acid sequences were analyzed by Clustal W software. The results showed that p53 amino acid mutations enriched in irradiation alone group and p53 mutations in different clones of the same mouse weredifferent as well as the mutations from different mice. In addition,the mutations were more concentrated in exons 4,5,6,7,8 with a mutation rate of 78%. However, p53 amino acid mutations were significantly reduced in MSCs-treated irradiation group, the mutation rate was 48%. 6. MSCs influence expression of gene involved in p53 signal pathway.(1)The effect of MSCs on p53 upstream factors The m RNA levels of p53 upstream factors were detected by RT-PCR.Results showed the expression of Chk2 decreased(t=0.317,p=0.767)and the expression of Mdm2(p53 negative feedback regulation factor) as well as p19 increased(t=5.514, p=0.005 and t=2.424, p=0.072)in irradiation alone group compared to the normal control group.In MSCs-treated irradiation group, the expression of Chk2 significantly decreased(t=4.386,p=0.012) and the expression of Mdm2(inhibitor of p53) and p19 decreased(t=13.833,p<0.001 and t=1.352,p=0.248)compared to the normal control group.In addition, the expresion of the other regulatory factors, such as DNMT1, that significantly reduced after irradiation(t=19.356, p<0.001), while the expression was increased in MSCs-treated irradiation group(t=3.302,p=0.030).(2)The effect of MSCs on p53 target factors RT-PCR showed that p21(a major target gene of p53) and CDC25c(cell cycle regulation gene) m RNA increased(t=0.227, p=0.831 and t=10.505, p<0.001)in irradiation alone group compared to the normal control group, though a decrease was seen in Bax, Fas(apoptosis gene) and DDB2(repair gene)(t=5.101, p=0.007; t=0.330, p=0.758 and t=4.296, p=0.013), however, the DDIT4 and GADD45α(repair gene) m RNA increased(t=1.912, p=0.128 and t=3.631, p=0.022). In irradiation alone group, the p21 and CDC25 c m RNA reduced(t=1.833, p=0.141 and t=2.995, p=0.040). Bax and Fas m RNA increased(t=3.648, p=0.022 and t=0.988, p=0.379)in MSCs-treated irradiation group compared to the irradiation alone group, though a decrease was seen in DDB2, DDIT4 and GADD45α(t=6.137, p=0.004; t=2.617, p=0.059 and t=3.687, p=0.021).Conclusion: 1. MSCs can upregulate p53 protein and m RNA expression in thymus tissues from mice. 2. MSCs can promote p53 promoter methylation in thymus tissue from irradiated mice and effect the binding of negative regulation factors to increase the p53 expression. 3. MSCs can protect or repair the p53 amino acid mutations caused by radiation, so the mutation rate dropped from 78% in irradiation alone group to 48% to restore the functions of p53 which have lost due to mutation. 4. MSCs can downregulate Mdm2 expression to increase p53 expression by reducing the degradation of p53 caused by Mdm2. 5. MSCs can relieve cell cycle arrest by reducing p53 target gene p21 and CDC25 c m RNA expression. 6.MSCs can induce apoptosis in cells which can not repair the DNA damage by decreasing the p53 target gene DDB2,DDIT4 and GADD45α and increasing the expression of Bax and Fas gene. |
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