Study Of Adenovirus-mediated HIL-1Ra Gene Transfer To Rat Bone Marrow MSC And Modulation Effects On Its Immunoregulation Factors Expression

Background and Objective Interleukin-1 receptor antagonist (IL-1Ra) is a natural inhibitor of the biologic actions of IL-1, which is known to be a pathogenic mediator in numerous inflammatory and degenerative conditions of rheumatoid arthritis (RA). The anti-inflammatory effect of IL-1Ra is realized by competitively binding to the IL-1 receptorⅠand then blocking the biologic activities of IL-1. The recombinant human IL-1Ra has been approved as a kind of clinical biological agent (Anakinra) for RA treatment, and it has been shown to effectively reduce joint inflammation and cartilage destruction in RA. However, Anakinra has major shortcomings, which include its lack of oral availability and short biologic half-life. These limitations are the reason why a new therapeutic approach such as gene therapy is needed. Theoretically, gene therapy can provide a long-lasting therapeutic effect and local gene delivery using various vectors can reduce systemic side effects in RA., Therefore, the gene therapy of IL-1Ra may probably be the more promising approach in clinical application.Mesenchymal stem cell (MSC), a special cell population derived from mesoderm, possesses a high reproduction property and multilineage differentiation potential. Under some given conditions, MSC could be induced differentiate to adipogenic, chondrogenic and osteogenic lineages, and moreover, to endoderm and ectoderm organism. Recent advanced studies show that this cell population exhibits an usefulness in field of gene delivery. Firstly, they are easily isolated, cultivated and expanded to large numbers in vitro without changing their phenotype and multilineage potential. Secondly, it could be feasibly modified by foreign gene in vitro with efficiently long-term expression in vivo. Thirdly, grafted MSC could evade the attacks from the host immune system because their surface phenotype mirroring a poorer immunogenicity recognized by T cells. Moreover it can directly inhibit the activity of T cells and indirectly influence antigen presentation of APC. Lastly, MSC also have the unusual tropism towards matrix components and could target the pathologic organs which could make them ideal vehicles used to express therapeutic reagents such as cytokines gradually on targeted sites. These intrinsic properties of MSC make itself become an attractive candidate for a cellular gene vehicle.The purpose of our study is to construct the recombinant adenovirus vector carrying hIL-1Ra gene by the method of homologous recombination in bacteria; and after isolate, culture and identify of rat bone marrow derived mesenchymal stem cells (BM-MSC) in vitro, to transfect hIL-1Ra gene into rat BM-MSC. By detecting foreign hIL-1Ra expression in rat BM-MSC, and investigating its modulation effects on Immunoregulation factors expression in rat BM-MSC, to estimate the feasibility of MSC modified by foreign hIL-1Ra gene and lay a foundation for further study on using it as an immunosuppressant.Methods 1. The primary culture of MSC was isolated from the bone marrow of SD rat by density gradient centrifugation plus adhesion separation. After expended in LG-DMEM supplemented with 10 % FBS and by digestion-controlled passage, the subculture were expanded and purified. The morphology of cells was observed with phase-contrast microscope, and the growth curve was draw. The surface molecule expressions of cells were examined by immunocytochemistry and flow cytometric analyses. The multilineage differentiation capability of MSC was examined by culturing cells under conditions favorable for adipogenic, chondrogenic and osteogenic differentiation. 2. With total RNA was extracted from PHA-stimulated health human peripheral blood mononuclear cells, the cDNA of human IL-1Ra was amplified by RT-PCR and then was inserted into eukaryotic expression vector pcDNA3 by digested with HindⅢand EcoRⅠ. After confirmed by restriction analysis, PCR amplification and DNA sequencing, the gene of hIL-Ra was subcloned into the plasmid of pShuttle-CMV with EcoRⅤand HindⅢdigestion. After linearization by PmeⅠand dephosphorylation by calf intestinal alkaline phosphatase (CIP), the plasmids of pShuttle-CMV/hIL-1Ra and the superhelix plasmids of pAdEasy-1 were co-transformed into E.coli BJ5183. The positive clones of homologous recombinant plasmid, pAd/hIL-1Ra, were selected by kanamycin resistance and then were expanded in E.coli DH5α. After digested by PacⅠ, the linearized pAd/hIL-1Ra plasmids were transfected, mediated by LipofectamineTM Reagent, into HEK293 cells, and the particles of recombinant adenovirus AdhIL-1Ra were packaged in the cells later. After further infections into HEK293 cells, the AdhIL-1Ra were amplified. After then, the recombinant adenovirus sample was titrated with TCID50 and the number of viral particles per milliliter was measured by A260nm. 3. By diluted with LG-DMEM to a relative smaller volume, the AdhIL-1Ra virus, according MOI 100, were add onto rat BM-MSC which were passage 3 subculture at 90% cell-fusion. After 2-hour absorption, the cells were supplied with complete medium and continued culture for 48 hours. The expression of foreign hIL-1Ra gene in the MSC was tested by RT-PCR, west-blotting and immunofluorescence. 4. According sequences recorded in GENBANK, design the primer pairs of Rat IL-1β, Rat IL-Ra, Rat IL-10, Rat TGF-β3, Rat TNF-α, Rat IDO, Rat Col-2 and RT1 class II (Rat MHC II) genes. After adjusted cell concentration to 2×104 cells/ml, the passage-2 BM-MSC from three rat were subcultured in 6-well clusters at 2ml/well and divided into 4 groups: The group of MSC with normal culture; The group of MSC with LPS (5ug/ml) treatment; The group of MSC with foreign hIL-1Ra gene modification; The group of MSC with foreign hIL-1Ra gene modification plus LPS (5ug/ml) treatment. The culture of MSC in all groups started at same time and ended at the sixth day. The foreign hIL-1Ra gene modification of MSC was mediated by adenoviral infection at the third day, and the methods of infection were as mentioned above. The LPS supplement was performed at the fifth day. At the end of culture, the total RNA of the cells were extracted and the expression of the rat genes in BM-MSC was detected by RT-PCR. The mean levels (X±S) of gray scale were compared within groups, and the correlation coefficient within the factors was determined.Results 1. The primary cultured MSC adhered to plastic surface within 48 hours and exhibited oval, asteroid and fusiform shape. After cultured in LG-DMEM medium containing 10% FBS for12 days, the cells reached 90 % confluence in single layer. By digestion-controlled passages combined with adhesion separation,the morph of subculture uniformly trended to be the fusiform shape and the cells arranged tightly in a swirl-like fashion. From passage 1 to passage 3, the cells showed a high proliferation property with a population doubling time of 21.3±2.8 and 22.1±3.3 hours and maintained the uniform fibroblast-like morphology. Compare to passage 1 and passage 3, the proliferation rate of passage 5 cells was decreased with a population doubling time of 24.1±4.1 hours (P<0.05). Immunocytochemistry showed passage 2 cells were positive for CD44 and fibronectin (FN), while negative for CD14 and CD34. The results of FMC analysis indicated the positive rate of CD44 was 92.09 % and of FN was 90.55 %. By culturing in adipogenic, chondrogenic and osteogenic medium respectively for 2 weeks, and then by identified by Oil Red O, Alcian Blue and Alizarin Red S staining, the cells of passage 3 were successfully induced to differentiate into these three lineages. 2. The size of RT-PCR products was 570bp as expected, and after restriction analysis, PCR amplification and DNA sequencing, the pcDNA3-hIL-1Ra was found to be well constructed. The correct sequence of hIL-1Ra which had been cloned was 100% identified with 0.0 E-value of BLAST to GenBank. By subcloned hIL-1Ra gene into Shuttle vector and homologous recombinant with backbone plasmid in bacteria, we obtained the pAd/hIL-1Ra plasmids. After transfected it into HEK293 cells, and by continued culture for 10 days, the cells showed a set of cytopathic effects. Through repeat freezen and thawed these cells at -70℃～37℃, and used its supernatant as template in PCR, the recombinant adenovirus AdhIL-1Ra was confirmed to be packaged. The number of viral articles of AdhIL-1Ra was 1.19×1011OPU/ml, and titer was 3.6×109TCID50 /ml. 3. With compared to control group, the expression of hIL-1Ra in rat BM-MSC was detected by RT-PCR, west-blotting and immunofluorescence after 48-hour transfection of AdhIL-1Ra. 4. The results of RT-PCR indicated: Firstly, the RT1 class II gene was not expressed in rat BM-MSC, no matter in which group. Secondly, in AdhIL-1Ra-modified group, compare with normal group, the levels of expression of Rat-IL-1β, Rat-IL-Ra, Rat-IL-10 and Rat-TGF-β3 were up-regulated (p<0.01), but of Rat-TNF-α, Rat-IDO and Rat-Col-2 were decreased (p<0.01). Seemly, the Rat-IL-1β, Rat-IL-Ra, Rat-IL-10, Rat-TNF-αand Rat-IDO gene expressions of BM-MSC were higher but the Rat-TGF-β3 and Rat-Col-2 gene expressions of BM-MSC were lower in LPS-treated group than normal group (p<0.01). Thirdly, the variance degrees (%) of Rat-IL-1β, Rat-IL-Ra, Rat-IL-10 and Rat-Col-2 between the group of AdhIL-1Ra-modified and the group of AdhIL-1Ra-modified plus LPS treatment was lower than its between the group of normal and the group of LPS treatment. Lastly, within Rat-IL-1β, Rat-IL-Ra and Rat-IL-10, and within Rat-TNF-αand Rat-IDO, the correlation coefficient of them were positive (p<0.05). However, the correlation coefficient of Rat-TNF-αto Rat-TGF-β3, Rat-IL-10 to Rat-IL-1βand Rat-IL-Ra were negative (p<0.05). The correlation coefficient between Rat-TNF-αand Rat-IDO was negative too (p<0.001).Conclusion 1. By the methods of density gradient centrifugation, and with the capability of adhesion to culture plastic, the cells isolated from rat bone marrow could greatly proliferate in vitro and maintain the phenotypic and functional properties of mesenchymal stem cells. 2. The recombinant adenovirus AdhIL-1Ra was successfully constructed by the method of homologous recombination in bacteria, which lay a foundation for further study on adenoviral vector-mediated hIL-1Ra gene modification of BM-MSC. 3. Rat BM-MSC could successfully express the foreign hIL-1Ra gene after AdhIL-1Ra infection. 4. The results of our study indicate: The BM-MSC has a low- immunogenicity property and could express various immune-associated factors including IL-1β, IL-Ra, IL-10, TGF-β3, TNF-αand IDO. High correlations within these factors indicate there is a immunoregulation network in BM-MSC. Foreign hIL-1Ra gene modification of MSC leads to significantly effects on expression of immune-associated factors in rat BM-MSC. LPS stimulation could induce BM-MSC produce more inflammatory cytokines and it could be inhibited by foreign hIL-1Ra gene modification. The results indicate also that the chondrogenic differentiation property of BM-MSC is depended on its immune condition. When normal-cultured MSC was stimulated with some inflammatory signal, no matter which came from environment or cytokine network, the potential tendency of MSC to differentiate to chondrogenic lineage would be blocked.