| Obiectives:Mesenchymal stem cells are multipotent cells derived from mesoderm and ectoderm,which have capabilities for differentiation into various lineages of mesenchymal tissues and hematopoiesis supportion and enhancing the engraftment of hematopoietic stem cells.Following with deeply realization about MSC,people pay more attention to the feature of its immunoregulationã€1,2】.Nowdays, MSCs are generally researched as a desirable therapeutic methods in the field of treating immunomediated diseasesã€26,27,28,29】.In our previous studys,hMSCs with pretreated can powerfully protect the ConA mice,and the mechanism may be related with high IL-6 expression.There are not relative studies after our literature retrieve. thus,in this experiment we determine to use vaious strategy which can regulate MSC IL-6 expression,and identify whether IL-6 is a key molecule in protection function of MSC.Methods: Umbilical cords were obtained and then wharton jelly was isolated. we incubate wharton jelly with collagenase typeâ…£and then collect cells after centrifugation . After 3 passage,we studied basic bilolgical characteristics by morphology,flow cytometry,differentiational potential of stable state cells.Kinds of regulation mythods,such as: lentiviral expressing vector and lentiviral interfere vector of IL-6,the supernatant of activated lymphocytes,combinations of TNF-αand IFN-γ,TNF-αor IFN-γrespectively,are applied to regulate IL-6 expression which detected by RT-PCR and real-time PCR.Result:1 Isolation of umbilical cord mensenchymal stem cells and evaluation of its biological characteristics:1.1 UCMSCs were isolated from adhering cell clones consisting of two different morphologies:the most of cells had a fibroblastoid morphology,while the little had endothelioid morphology.With passaging,the endothelioid cells gradually disappeared from culture.By passage 4,the UCMSCs show a homogeneous fibroblast-like morphology.1.2 Flow cytometry analysis revealed that these cells expressed signs ofCD29,CD90,CD105,but not CD34,which is a sign of HLA-DR and Lympocytes.1.3 After adipogenic differentiation,lipid droplet can be observed in the cytoplasm of isolated cells,which were stained using oil red solution.The UCMSCs become quadrate after osteogenic differentiaton and calcium deposition can be detected using von Kossa procedure.It is obvious that the isolated cells have a potential of osteogenic and adipogenic differentiation.Above suggest that the isolationed cells were mesenchymal stem cells and can be used in the next study.2 Construction of IL-6 expression vectors2.1 Using plasmid of pLXSV/IL-6 as a templates,IL-6 gene fragment are amplificated by PCR and then connected with pGEM-T vector.After conversion and amplification,gene order is evaluated.The results display the sequence is coincident,which suggest that the gene of IL-6 has been successfully connected with pGEM-T vector.2.2 IL-6 is connected with lentivirus pBPLV vectors,and then gene order is evaluated after conversion and amplification.The results display the sequence is coincident,which suggest that the IL-6 expressing vectors (pBPLV/IL-6)are constructed.3 IL-6 expressing vectors infected UCMSCs. The result of RT-PCR display that the IL-6 expression levels in MSCs infected by pBPLV/IL-6 vectors are significantly high than the normal MSCs and MSCs infected by pBPLV control vectors(p<0.05).Real-time PCR also displays a identical result in IL-6 expression levels(p<0.05).The above result suggest pBPLV/IL-6 transfection can apparently up-regulate IL-6 expression. 4 IL-6 interference vectors infected UCMSCs.The result of RT-PCR display that the IL-6 expression levels in MSCs infected by pSicoR/IL-6 vectors are significantly descendent than the normal MSCs and MSCs infected by pSicoR control vectors(p<0.05).Real-time PCR also displays a identical result in IL-6 expression levels(p<0.05).The above result suggest pSicoR/IL-6 transfection can apparently down-regulate IL-6 expression.5 The effect of the supernatant of activated lymphocytes on MSC IL-6 expression.the result of RT-PCR and real-time PCR display the expression of IL-6 in different concentration are high than the control(p<0.05);the result of RT-PCR and real-time PCR display the expression ofIL-6 in different time are high than the control(p<0.05);The above result display that the supernatant of activated lymphocytes can up-regulate IL-6 expression.6 The effect of the cytokine of TNF-αand/or IFN-γon IL-6 expression.the result of RT-PCR display the expression of IL-6 at the combinaton group in different concentration are high than the control(p<0.05);the result of RT-PCR and real-time PCR display the expression ofIL-6 at the combination and TNF-αgroup in different time are high than the control,but the IFN-γgroup is lower than the control(p<0.05).The above result display that the combination of TNF-αand IFN-γor individual application of the TNF-αcan up-regulate IL-6 expression,but the factor of IFN-γcan down-regulate IL-6 expression.Conclusions:1 Mesenchymal stem cells can be isolated from Wharton jelly using collagenase digestion.2 Construction of IL-6 expression vectors3 Some factors as lentiviral expressing vector of IL-6,the supernatant of activated lymphocytes,combinations of TNF-αand IFN-γor merely TNF-αup-regulate IL-6 expression.4 lentiviral interfere vector of IL-6 and merely IFN-γcan prominently down-rugulate IL-6 expression. |
