| Hydroquinone(HQ) is the intermediate metabolite of benzene in vivo.Epidemiology and animal experiment discovered that the target organ of HQ wasbone marrow. The bone marrow includes haematogenesis and matrix sisytems. Bonemarrow mesenchymal stem cells (BMSCs) as the precursor cell of many marrowstroma cells are the important part of hemopoietic microenvironment, and areessential of sustaining the characteristic and homing of haemopoietic stem cell.Poly(ADP-ribose)polymerase-1(PARP-1) as an earlier stess molecule, plays animportant role in apoptosis, transcription, DNA reparation, cell death, chromatosomefunction, genome integrity, DNA methylation and so on. RNA interference is aprimary process of living. It makes the objective gene silence after specificitytranscription through expressing or inducing double strands RNA. It avoids the sideeffect of chemistry inhibitor. We planed to research the toxic effect of HQ on BMSCsat the practic contact level, elucidate the role of PARP-1in the toxic effect of HQ andthe possible relationship between PAPR-1and DNA Methyltransferase (DNMTs).Objective:To construct and screen the RNAi expression vector for rat poly(ADP-ribose) polymerase-1(PARP-1) gene. To research the toxic effect of HQ onBMSCs at the practic contact level, elucidate the role of PARP-1in the toxic effect ofHQ and the possible relationship between PAPR-1and DNMT.Methods:The cells were expanded by subculture and the threeth passage cell,proven to be capable of differentiation into osteoblasts and lipocyte.To design andsynthsis oligonucleoside according to the gene order and the principle of short hairpinRNA, and construct the RNAi expression vector for rat PARP-1gene. After enzymecutting and sequencing correctly, constructed vector was transfected into rat BMSCsby LipofectamineTM2000, and the transcriptional level of PARP-1mRNA wasdetected by RT-PCR, and the effective RNAi expression vector of PARP-1wasscreened. the biological character of PARP-1defective BMSCs after transfecting for 24h was observed.The defective cells and normal cells exposed HQ with the dose ofPBS,2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L,80.0μmol/L,160.0μmol/L,320.0μmol/L for24h, the eligible toxic dose was found by MTTmethos.The rates of apoptotic and necrosis of defective and normal cells was detected24h after treated with PBS,2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L HQ. Therates of micronucleus and dyskaryosis of defective and normal cells was determined24h after treated with PBS,5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L HQ.The level of PARP-1, DNMT1, DNMT3a, DNMT3b, MBD2and MeCP2mRNA ofdefective and normal cells was detected24h after treated with PBS,2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L HQ.Results:1. BMSCs in high purity and with character of adherence were obtained.BMSCs submited hypodispersion, colony-like growth and fusiform shape in primaryculture. BMSCs obviously improve homogeneity in serial subcultivation and passagewas very stable.2. The oligonucleotides were designed and synthesized according to the PARP-1gene reported in GenBank and the general principle of shRNA design, based on which4recombinant RNAi expression vectors targeting PARP-1gene were constructed,identified by restriction analysis and sequencing, then transfected to rat BMSCs. Thevalid RNAi expression vector for rat PARP-1gene was screened and the inhibitionratio was above75%.3. The biological character of PARP-1defective BMSCs had no obviouslychange within the state of normal growth, growth velocity and cell vigor. The cellscould be used to subsequent research.4. The dose of HQ between2.5μmol/L and40.0μmol/L was more compatiblebecause the level was according with the practical occupational expose level.5. The rates of early apoptotosis and the rates of apoptotosis and necrosis ofdefective and normal cells24h after treated with PBS,2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L HQ increased at first and then decreased.The rates of earlyapoptotosis and the rates of apoptotosis and necrosis of defective cells had significantdifference compared with normal cells at the same dose.It elucidated that the ability ofrepairing DNA damage of PARP-1defective cells was more weaken. They were more sensitive to environmental adverse factors.6. HQ induced the appearance of micronuclei and dyskaryosis.The PARP-1defective cells occurred more. It elucidated that DNA of PARP-1defective cells wasbroken more easily than normal cells.Therefore, PARP-1defective cells could beregarded as sensitive cells to detect environmental adverse factors.7. The express level of PARP-1of defective and normal cells24h after treatedwith PBS,2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L,40.0μmol/L HQ increasedat first and then decreased. Increasing of PARP-1express level was benefit torepairing DNA damage. But overexpression of PAPR-1would induce exhaust ofNAD+, which could down-regulate the express level of PARP-1.8. HQ increased the express level of DNMT1and MBD2, but decreased theexpress level of DNMT3a. The changes were more obvious in defective cells. Itdemonstrated that abnomal level of PARP-1would influenc the expression level ofDNMTs and methylation.Conclusions:1.A method for isolation and purification in vitro of BMSCs frombone marrow had been established. It was observed by morphous and bionomics. Itsuggested that the method of isolation, high purification and active proliferation ofBMSCs by adherent culture and tissue blot culture in special culture system was veryconvenient and effective.2. The inhibited effect of PARP-1expression by RNAi was stable.3. HQ at low dose had heredity toxic action. PAPR-1participated in the toxityresponder.4. PARP-1participated in the regulation of DNA methylation. The branching andchain structure of PARP-1could prevent the effect of DNMTs.
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