| Background:Massive bone defect,caused by severe trauma,infection,tumor and such diseases like these,is a very common but difficult clinical work,and now engineering bone tissue is considered to be a promising method to treat this problem,which will be of great value in future clinic research.As one of the most crucial part of engineering bone tissue,seed cells have been widely studied in recent years.Generally,proper seed cells for engineering bone tissue should meet following demands:First,having reliable and convenient cell source and facilitating expansion in vitro;Second,bearing specific differentiation phenotype or oriented differentiation potential;Third,having favorable histocompatibility with recipient and not inducing transplantation rejection.Up to now,osteoblasts,embryonic stem cells and bone marrow-derived mesenchymal stem cells(MSCs) are three available candidates for engineering bone tissue,in which osteoblasts and embryonic stem cells are not very satisfying.Osteoblasts,as adult terminal cells have limited proliferation potential and scarce sources.Due to some ethnical reasons and complicated regulatory mechanism of osteogenesis,embryonic stem cells are excluded as well.Compared with the other two cells, MSCs,which own multi-oitented differentiative potential and self-renewal capability,have convenient sources and can expand in vitro cell culture,is regarded as the optimal seed cell of engineering bone tissue.What's more,in both normal animal models of autotransplantation and immunodeficient animal models of allotransplantation,MSCs were proved to be effective to repair bone defects.However,further researches indicate that,to be applied in clinic as the seed cell, hMSCs still have a long way to go.For example,there are problems such as how to control their multi-oriented differentation,the interaction between hMSCs and recipients, phenotype identification and the insufficient cell number of autotransplantation,among which,the insufficient cell number is the main problem which limits their application in clinic.This is because that massive bone defects require massive seed cells,while hMSCs only account for 0.01—0.001%of mononuclear cells in bone marrow,and what's worse, they will decreases with age,expansion in vitro culture is only 38PD(population doublings)on average and they are hard to expand in a short time.The feasible solution comprises of two aspects:one is to enhance the proliferation activity of MSCs,the other is to apply allogeneic or xenogeneic MSCs.As we know,telomere is a clustered and repeated TTAGGG sequence in chromosome ends,which will gradually shorten during cell division.When telomere in one or more chromosomes is shorten to its extreme,cell senescence and growth stop will occur.The length of telomere is sustained by telomerase and human telomerase reverse transcriptase (hTERT) is contact reaction of subunit of telomerase.Some researchers reported that ectopic expression of hTERT in MSCs could extend their life-span and maintain their osteogenic potential.Simonsen JLetc.transferred hTERT into hMSCs and obtained hMSCs-T cell clone which showed no obvious senescent phenomena,maintained multipotential differentiation,had no tendency to cause tumor after more than 200PD in vitro culture,which indicated hMSCs with hTERT transgene could prolong their life span and maintain multi-differential potential.Although recent studys indicate that MSCs are low hypoimmunogenic,they are not completely immune privileged cells,So allogenic MSCs transplantation still faces immune rejection problem.Tissue and organ transplantations have made great progress with the application of immunosuppressive drugs,but immunosuppressive drugs would decrease the immune function and lead to tumor and severe infection.As we know,there is no method that can induce donor specific,complete and permanent immune tolerance.Among numerous studies of immune tolerance,CTLA4 is one of the most important focus points. CTLA4 can bond B7 molecule on APC competitively with CD28 and block the B7-CD28 costimulatory pathway,which inhibits T cell full-activation and induces T cell anergy, apoptosis or clone deletion,resulting in immune tolerance.CTLA4Ig,a fusion protein of CTLA4 extracellular domain and partial segment of IgG- Fc,has the same function with CTLA4.Our previous study has modified MSCs with CTLA4Ig gene and screend out transfected cells.The modified MSCs can secrete CTLA4Ig protein to induce immune tolerance,which has been demonstrated in rat model.Thingking about security and graft requisition,adenovirus vector is safe and has efficient infectivity,we design to transfer CTLA4Ig gene by adenovirus vector in this study.Objective1.To construct the retroviral hTERT expression vector.2.To prepare the retroviral hTERT expression vector transferred hMSCs (hTERT-hMSCs).3.To prepare CTLA4Ig gene transferred hTERT-hMSCs(CTLA4Ig-hTERT-hMSCs).4.To study the biological characters of CTLA4Ig-hTERT-hMSCs both in vitro and in vivo.Materials and methods1.MSCs were isolated from bone marrow of human iliac crest by a modificative procedure of Pittenger and density gradient centrifugation of Percoll and were cultured by routine methods.The symbols CD105/CD34 at passage 3 were detected by FACS.2.Construction recombination plasmid pLXSN-hTERT by using molecular biological technique.3.Package of hTERT-retroviral vector and determination of titer:pLXSN-hTERT was transfected into PT67 cells mediated by lipoplast2000.The positive clones were screened by G418,clone line and limiting dilution assay.TCID50 was used to determine the virus titer.4.hTERT-retroviral vector infection human MSCs:PT67 cells involved hTERT-retroviral vector and passage2 hMSCs were cocultured.The hTERT—hMSCs were screened with G418.5.The expression of hTERT protein in hTERT—hMSCs was immunocytologically demonstrated.6.The telomerase activity was detected with TRAPEZE Telomerase Detection Kit.7.Both hMSCs and hTERT—hMSCs were infected by Adv-CTLA4Ig-EGFP.The expression of EGFP was observed under the inverse fluorescent microscope and the infection rate was detected by FACS.8.The expression of CTLA4IG protein in both CTLA4Ig- hMSCs and hTERT—hMSCs was detected by Immunocytochemistry,and CTLA4IG protein in cell culture fluid was detected by Western Blot.9.It was observed with Inverted microscope that morphous and growth state of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs,and CTLA4Ig-hTERT-hMSCs.10.The cells cycles of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs,and CTLA4Ig-hTERT-hMSCs had been studied by FACS method.11.Osteogenesis of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs were detected by calcium salts dying,alkaline phosphatase dying and osteocalcin immunocytochemistry.12.Engineering bone tissue was prepared by DBH seeded by CTLA4Ig -hMSCs.13.Osteogenesis of engineering bone tissue in radius defect of rabbits was observed by X-ray on designed time points(i.e.,2-days,2,4,8,12weeks post-surgery).Results:1.Like fibrocyte,cultured primary hMSCs showed an atractoid or triangular shape and arrayed in whirpool.FACS detection indicated that 90.8%of isolated cells expressed CD105 and 96.2%were CD34 negetive.2.Plasmid pGRN145 was cut into a 11.5kb vecter fragment and a 3.5kb hTERT target fragment,Plasmid pLXSN was cut into a 5.9kb linear fragment by EcoR I resPectively. Then we gained recombinant plasmid pLXSN—hTERT by ligating hTERT and pLXSN. DNA sequencing illustrated that 3402 bases of recombinant plasmid pLXSN—hTERT matched with 3402 bases of hTERT sequence of gene bank.The matching rate was 100%.3.We used recombinant plasmid pLXSN—hTERT to transfect PT67 cells and obtained hTERT—PT67 package cells after G418 resistant screening.The virus tilter of cell culture supernatant was 1.2×104.4.After 5 days coculture of pLXSN—hTERT—PT67 package cells and hMSCs and 30 days G418 resistant screening,the survival cells were named hTERT-hMSCs.The cells didn't show obvious cell shape change when they were cultured to 46th passage.5.Immunocytochemical stain of hTERT showed that both hTERT-hMSCs and hMSCs expressed hTERT protein in cell nucleus.The whole nucleus of hTERT-hMSCs was deeply stained,and the positive staining of hTERT-hMSCs was 99%while hMSCs was 65%under 10times highpower field.6.Telomerase activity detection indicated that compared with untransfected hMSCs, hMSCs with hTERT transgene had stronger telomerase activity.7.48hours after AdvCTLA4Ig-EGFP infecting hMSCs and 24 hours after AdvCTLA4Ig-EGFP infecting hTERT—hMSCs,we observed that both a few hMSCs and hTERT—hMSCs expressed green fluorescence,and the cell number gradually increased. The expression peak of green fluorescent protein in both cells was at the fifth day,and the green fluorescence began to decrease at the 28th day.Flow cytometry indicated that the infection rate of hMSCs was 81.14%,while hTERT-hMSCs was 83.75%.8.Immunocytochemistry detection demonstrated that most of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs positively expressed CTLA4Ig in cytoplasm,especially around nucleus,hMSCs that expressed hTERT were small,long spindleshape while hMSCs that negatively expressed hTERT were mainly large,flat shape.Western blotting indicated that CTLA4Ig protein existed in culture solution of both CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs.9.We observed that most of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs were long spindle—shaped and a few were triangular,hMSCs gradually became wide and short along with passage,and the growth velocity slowed down, while hTERT-hMSCs remained long spindle—shaped and the growth velocity changed little.The shape of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs showed little change before and after CTLA4Ig transfection,however,the growth velocity slowed down.10.Detection of cell cycle1-12 passage hMSCs in G0/G1 phase increased while in G2/M and S phase decreased gradually,and statistical analysis showed significant difference,which indicated that proliferative capability of hMSCs weakened gradually.1-18 passage hTERT—hMSCs showed similar change tendency but statistical analysis showed insignificant difference, which suggested that proliferative capability of hTERT-hMSCs changed little accompanying passage.Statistical analysis demonstrated that hTERT—hMSCs had higher proliferative capability than hMSCs did.Cell cycle comparison between hMSCs and CTLA4Ig-hMSCs,and comparison hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs indicated that CTLA4 gene transfection could slow down cell proliferationAll of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs were diploid cells.11.During the course of osteogenesis in vito,the shape and growth speed of CTLA4Ig-hMSCs and CTLA4Ig -hTERT-hMSCs changed little in the first 2 week;then the cells became wide and short,cells with polygonal shape increased,and the cells had the tendency to cluster together.During 3 to 4 weeks,most of cells were polygonal and clustered.Calcareous infarct could be detected by tetracycline dying in cell clump;AKP dying was strong positive;the expression of osteocalcin could be detected by immunocytochemistry.12.In our experiment,there were about 5×105 cells attached to per gram DBM.13.CTLA4Ig-hMSCs xenotransplantationLow density vague shadow in bone defect could be seen 2 weeks and 4 weeks post-surgery in group treated with DBM+CTLA4Ig-hMSCs under X-ray;at 8 weeks,the shadow was high-density,similar with bone tissue;at 12 weeks,the bone defect was completely ossified,and medullary cavity was recanalized.Compared with group treated with DBM+CTLA4Ig-hMSCs,in group treated with DBM+MSCs,similar shadow appeared in the first 4 weeks,but at 8 weeks,the shadow density was apparently lower than the former;the bone defect didn't heal completely at 12 weeks.Group treated with DMB showed vague shadow early,but the shadow disappeared gradually.During the observation, bone defects diminished in both group treated with DBM and blank group,but obvious bone defects still remained 12 weeks post-surgery.Conclusions:1.We successfully constructed recombinant retroviral expression vector-pLXSN—hTERT.2.We obtained package cell lineage hTERT—PT67 which has the ability to produce hTERT recombinant retrovirus.3.Compared with hMSCs,hTERT—hMSCs show stronger telomerase activity,higher proliferative capability and longer life-span..4.Expression and secretion of CTLA4Ig protein could be detected in both CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs,which last more than 4 weeks.5.The times of population doublings could be prolonged and growth speed slowed after hMSCs and hTERT—hMSCs had been modified by adenovirus mediated-CTLA4 gene transfection6.Both hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs could be induced to differentiate into osteoblast in vitro,which indicates that adenovirus mediated-CTLA4 gene transfection don't alter hMSCs' osteogenesis potential.7.CTLA4Ig-hMSCs have better ostogenesis effects than hMSCs as seed cells for engineering bone tissue on xenotransplantation animals.The ostogenesis effects of CTLA4Ig -hTERT-hMSCs on xenotransplantation animals need further studying. |
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