| Objective:Human amniotic mesenchymal stem cells(hAMSCs)were transfected with lentivirus carrying C57BL/6 mouse IL-10 gene to construct IL-10 gene modified hAMSCs(IL-10-hAMSCs).Preliminarily explored the synergistic enhancement of hAMSCs and IL-10 in regulating factors related to skin wound healing in mice.Method:1.The amniotic membrane of the placenta of healthy parturients under 30 years old was extracted under aseptic conditions.The hAMSCs were isolated and extracted in vitro by trypsin and collagenase two-step method.The hAMSCs were cultured in a sterile clean incubator and purified for 3-5 generations and then frozen for further experiment.2.Utilized the lentivirus which carried the gene of IL-10 of mice to transfect the good condition cultured hAMSCs then cloned the IL-10 gene into hAMSCs and get IL-10-hAMSCs,made a assessment of transfection efficiency with Inversed Fluorescent Microscope.3.Sixty C57B6/L mice aged 8 weeks were randomly divided into control group,hAMSCs group,IL-10-hAMSCs group and 40 mice in each group.After anesthesia with 4%chloral hydrate solution,a full-thickness skin defect wound model was made symmetrically along the back spine.After the wound was modeled,the control mice were injected with 200 ml PBS buffer around the wound.The hAMSCs group and IL-10-hAMSCs group were injected with 200 uL of hAMSCs and IL-10-hAMSCs suspension(5×10~5/100μl)at multiple points around the wound,respectively.4.Observed the healing status after the wound model were established at 1st day,3rd day,7th day and 14th day.At each time took the photograph and excuted 10 mice of each group and cut off the wound tissue in the back.Some of wound tissue would be used to make paraffin section via HE hemotoxylin and eosin staining to observed the healing and inflammatory infiltration status.Calcuate the wound healing rate and ditected the expresssion of IL-10,TGF-β3,MIP-1α,MIP-2 by qPCR.Results:1.The well cultured hAMSCs were seen under the observation of optical microscope like the shape of polygonal and spindle,they were proliferated fast and grown follow the appearance like a nest or vortex shape,under the high power view there were no taint in the field.2.Utilized the lentivirus with IL-10 gene to transfection the well cultured hAMSCs,then observed the transfection effect by Fluorescence Inversion Microscope System.Saw 90 percent of cells showed green fluorescence indicated the transfection were suscessful.3.The result of wound healing rate shoued that at the 7th day IL-10-hAMSCs group had better healing effection than hAMSCs group,the control group showed worst,the 14th day the IL-10-hAMSCs remained hardly no obvious scar tissue,the hAMSCs group’s scar tissue was smaller than the control group.3.After the HE-staining paraffin sections of wound tissue were done,they showed all the paraffin sections which cut off at the 1st day were obviously infiltrated by inflammatory cells.The sections which cut off at the 3rd day showed all tissue’s inflammation were receded than the 1st day,The IL-10-hAMSCs group were at the best situation with less inflammation,better than the control group and hAMSCs group,and the fresh granulation tissue had already come out.And at the 7th day,the sections showed inflammation of 10-hAMSCs group and hAMSCs group was significently receded.Yet meanwhile the control group still hadinflammatory infiltration.The collagen of all three group were disorganized,had no significent difference.Then the 14th day saw all wound had healed up.Sections showed almost no inflammatory cells in three groups,no significent difference in three groups.4.Used qPCR to detect the relative expression of IL-10、TGF-β3、MIP-1α、MIP-2’s mRNA:At the first day,third day,seventh day and fourteenth day,the relative expression of IL-10 in control group was:1.0261±0.4656,4.5871±0.3184,6.8933±0.1584/0.4824±0.1414;the hAMSCs group was:1.4333±0.2681,6.3306±0.5022,16.1559±0.1403,8.8166±0.2333;theIL-10-hAMSCsgroup was:3.6190±0.2166,12.4598±0.9519,22.1927±0.1370,10.5074±0.0283.The relative expression of TGF-β3 in control group was:1.0040±0.1826,1.4060±0.4699,1.1949±0.5001,0.2185±0.0777;the hAMSCs group was:1.2094±0.2123,1.1950±0.1331,1.9377±0.2215,0.4363±0.0586;the IL-10-hAMSCs group was:1.1922±0.1665,1.1426±0.2257,2.8269±0.2616,2.0054±0.9263;The relative expression of MIP-1αin control group was:3.8526±0.3464,6.9142±0.1626、3.6482±0.1131、2.2501±0.0461;the hAMSCs group was:0.3407±0.1979,0.6484±0.0353,0.6114±0.1060,0.7629±0.0494;the IL-10-hAMSCs group was:1.7952±0.0282,0.4116±0.0141,0.2517±0.0353,0.2118±0.1626.The relative expression of MIP-2 in control group was:11.1194±0.0494,8.6342±0.0919,5.7217±0.0212,4.6824±0.0355;the hAMSCs group was:1.3771±0.0989.1.8412±0.0424,1.1814±0.0353,0.5532±0.0282;the IL-10-hAMSCs group was:1.0003±0.0070,1.0682±0.0636,1.3757±0.1272,0.7875±0.0565;Conclusion:The local transplantion of IL-10-hAMSCs shows the IL-10 and hAMSCs had synergistic effect of elevanting the expression of the cruclial anti-inflammation cytokines IL-10 to facilitate wound healing,can also elevant the expression of TGF-β3 towards the same way and restrict the scarring formation.Meantime,the expression of pro-inflammatory cytokines:MIP-1αand MIP-2,can be downregulated.Thus lead to the scarless wound healing. |
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