| Objective:Evaluate IL-10-h AMSCs about whether changes in h AMSCs phenotype,growth characteristics and multilineage differentiation ability of biological characteristics,and detection and analysis the effect of IL-10-h AMSCs local transplantation on the expression of vascular related factors VEGF and b FGF in the full-thickness skin defect of mice,provide experimental basis for clinical application of IL-10 and IL-10-h AMSCs.Methods:1.By flow cytometry(FCM)were cultured h AMSCs,IL-10 modified h AMSCs and h AMSCs transfected with empty plasmid by CD73,CD90,CDl05 and CD44 molecular identification,draw the growth curve of h AMSCs were MTT,and osteogenic and adipogenic induction medium were detected h AMSCs differentiation ability.2.Use cell culture nick test to detect the migration ability of IL-10-hAMSCs.3.With 80 healthy 7 weeks age male C57 BL / 6 wild mice,the experiments were divided into PBS transplant group,h AMSCs transplant group,IL-10-h AMSCs transplant group and h AMSCs transfected with empty plasmid transplant group.20 in each group,after anesthesia to establish a full-thickness skin defect model with 1cm× 1cm size was cut on both sides of the back of the mouse.Different according to grouping,at the time of modeling,subcutaneous multiple points around each wound(the midpoint of the wound margin from the wound 1mm)were injected with 100μl PBS suspensed different groups of h AMSCs(the total number of cells was 1 x 106).PBS transplantation group injected equal amount of PBS.4.Each 5 mice were killed by 1 day,3 days,7 days and 14 days after modeling respectively,and the whole layer of wound was cut along the edge of the wound.HE staining was used to observe the inflammatory cell infiltration and collagen arrangement.The expression of VEGF and b FGF in the wound tissue homogenate of mice by ELISA.Immunofluorescence staining was used to observe the expression of VEGF and b FGF in the full-thickness skin defect of mice.Results:1.FCM results showed that: the IL-10-h AMSCs were highly expressed CD73,CD90,CD105,CD44,the positive rate was 98.6%,93.8%,99.8%,97.7%,empty plasmid-h AMSCs were highly expressed CD73,CD90,CD105,CD44,the positive rate was 94.1%,98.1%,96.6%,94.8%,Neither of the two expresses CD34,CD45,CD11 b,CD19,HLA-DR.Phenotype conforms to the requirements of the International Association for cell therapy for the labeling of mesenchymal stem cells.The growth curve showed that the growth curve of the third generation IL-10-h AMSCs cells was "S",When the cells were cultured in 21 d,alizarin red S staining was induced by bone formation,The oil red O stain was induced by lipid,and the red color was displayed at the lipid droplets.There was no significant difference between IL-10-h AMSCs and h AMSCs and empty plasmid-h AMSCs in inducing ability.2.Scratch experiment show,three groups migration is basically same in 0 hour,IL-10-h AMSCs group migrate to the blank area is slightly larger than the h AMSCs group and empty plasmid group in 6 hours,12 hours,18 hours.showed that IL-10 can enhance the migration ability of h AMSCs.3.HE results show: Under the microscope,wound tissues of mice in 1day have a large number of inflammatory cell infiltration and necrosis tissue;in 3 days wound tissue inflammatory cell infiltration less than the 1day,the emergence of a large number of new capillaries,fibroblasts gradually appeared,the h AMSCs group,IL-10-h AMSCs group and empty plasmid group the inflammatory cells in wound tissue was less than that of group PBS,the number of fibroblasts and new capillaries generated more than PBS group,the IL-10-h AMSCs group of optimal,forming a typical fresh granulation tissue;the 7days inflammatory cells in wound tissue were significantly reduced compared with the 3days,fibroblast cell becomed maturation,increase the number compared with 3days,the number of capillaries compared with 3days was decrease,the number of fibroblasts was least in IL-10-h AMSCs group compared with the other three groups,most in the PBS group;the14d mice were no obvious inflammatory cells,The number of capillary decreased with7 days,and the number of fibroblasts was more than 7days.The number of fibroblasts inIL-10-h AMSCs was less than other three groups,and the PBS group was the most.4.Full-thickness skin defect model tissue detected the content of VEGF and b FGF by ELISA,after modeling 1 day,3 days,7 days and 14 days,the VEGF contents in PBS group were:96.78±4.87、100.84±4.69、103.08±4.21、99.70±5.05ng/L;the VEGF contents in h AMSCs group were:101.77±4.05、115.2±4.03、117.48±6.1、112.61±2.23ng/L;the VEGF contents in IL-10-h AMSCs group were:132.71±3.09、136.86±4.75、142.17±7.39、138.93±5.84ng/L;the VEGF contents in empty plasmid-h AMSCs group were:105.31±4.70、111.89±4.41、115.02±6.96、110.79±3.97ng/L.The b FGF contents in PBS group were:8.78±0.70、11.61±0.22、13.11±0.36、11.20±0.49ng/L;the b FGF contents in h AMSCs group were:9.90±0.44、15.73±1.43、19.50±1.11、16.20±1.96ng/L;the b FGF contents in IL-10-h AMSCs group were:16.70±1.78、21.73±1.85、23.13±1.67、22.19±1.55ng/L;the b FGF contents in empty plasmid-h AMSCs group were:9.10±0.64、15.93±1.51、18.90±1.49、16.40±1.66ng/L。the content of VEGF and b FGF in each groups increased gradually from 1day to 7days,the content of 14 days is decrease with 7days,The contents of VEGF and b FGF in h AMSCs group,IL-10-h AMSCs group and empty plasmid group were significantly higher than the control group(PBS group)from 1day to 7days,and the IL-10-h AMSCs group increased more significantly,the difference was statistically significant(P < 0.05),h AMSCs group and empty plasmid group had no obvious difference.5.Immunofluorescence staining was used to observe the wound tissue specimens and the expression of b FGF and VEGF in each group,after modeling 1 day,3 days,7 days and 14 days,the positive rate of VEGF in PBS group were: 33.24±0.85、35.65±1.75、38.57±1.32、34.94±1.25;the positive rate of VEGF in h AMSCs group were:37.25±1.05、42.87±0.79、48.92±1.46、43.93±1.68;the positive rate of VEGF in IL-10-h AMSCs group were:54.57±1.38、67.10±0.94、71.11±1.53、68.35±1.09;the positive rate of VEGF in empty plasmid-h AMSCs group were:35.29±0.57、41.34±0.95、48.22±1.44、43.61±1.56.The positive rate of b FGF in PBS group were: 16.99±1.04、22.00±0.46、25.97±0.70、21.85±1.15;the positive rate of b FGF in h AMSCs group were: 20.13±1.06、26.20±1.15、31.17±1.21、28.18±0.76;the positive rate of b FGF in IL-10-h AMSCs group were:26.41±0.86、34.42±1.85、38.57±1.33、33.97±0.38;the positive rate of b FGF in empty plasmid-h AMSCs group were:19.23±1.02、25.16±1.11、30.27±0.93、27.22±1.17.from 1day to 7days,VEGF and b FGF positive rate increased gradually,the positive expression of 14 d was slightly decreased compared with 7d,the positive rate in IL-10-h AMSCs group,h AMSCs group and empty plasmid group was higher than the control group(PBS group)at each time point,and the positive expression of IL-10-h AMSCs group was highest,the difference was statistically significant(P < 0.05).Conclusion:(1)Using lentiviral vector carrying IL-10 gene modified h AMSCs,in addition to inhibiting the proliferation of cells to a certain extent,it would not affect the basic biological characteristics of h AMSCs phenotypic characteristics and multilineage differentiation ability;(2)IL-10 can enhance the migration ability of h AMSCs,Transplantation of IL-10-h AMSCs can increase the expression of VEGF and b FGF,promote angiogenesis,and promote wound healing. |
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