Effects Of Hyperuricemia On Biological Characteristics Of Rat Adipose Mesenchymal Stem Cells

Objective:1.To establish hyperuricemia model in rats.2.To explore the effects of hyperuricemia internal environment on the related biological characteristics of rat adipose mesenchymal stem cells（ADSCs）.The differences in morphological characteristics,expression of cell surface markers,proliferation ability,senescence related characteristics,adipogenic differentiation ability,osteogenic differentiation ability and migration ability of ADSCs between normal group and hyperuricemia rats were compared.Methods:1.Twenty SPF grade 8-week-old male Wistar rats with bodyweight in the range of 200±20g were selected.After one week of adaptive feeding,they were divided into 2 groups with 10 animals in each group according to random number table method.Model group（M group）:yeast extract feed（yeast extract:ordinary rat maintenance feed is 1:4）feeding combined with potassium oxazinate intragastric administration（750mg/kg）,Control group（C group）:ordinary feed combined with equal volume of solvent intragastric administration（750mg/kg）;Each group was modeled for 4 weeks according to the above feeding methods.During modeling,orbital blood samples were taken to detect uric acid level,which was more than 2 times of the base value,and the modeling was successful.2.After successful modeling,the rats were dissected under aseptic conditions to obtain subcutaneous fat in the groin,extract ADSCs from adipose tissue and culture,and then subcultured to P₂.The morphology of ADSCs in the two groups was observed under inverted phase contrast microscope.CCk-8 kit was used to detect the short-term proliferation of both cells.The differentiation potential of ADSCs in the two groups were determined after14 days and 21 days of adipogenic and osteogenic induction systems,respectively.Flow cytometry was used to detect the expression of cell surface markers,and SPSS25.0 was used for statistical analysis.The migration ability of ADSCs in the two groups was detected by scratch test,and the migration ability of ADSCs was measured by Image J-win64.In vitro culture,the degree of replicative senescence of ADSCs in both groups was measured.Results:After 4 weeks of feeding with potassium oxazinate and yeastextract,the UA level was 3 times or more than the base value,and the hyperuricemia model was induced successfully.The cell morphology of ADSCs was observed under microscope:the cells were evenly distributed,the cell morphology was uniform,and the cells grew in vortex shape.Cell refraction in group C was stronger,with smaller and round cells and smaller nucleus;cell refraction in group M was weaker,with flat cell morphology,spindle shape and larger nucleus.The expression rates of CD29,CD90 and CD44 of positive ADSCs in the two groups were above 98%,and the expression rates of CD45 of negative ADSCs were lower than 0.4%,which was consistent with the expression levels of rat ADSCs cell surface markers.There was no significant difference in the expression of ADSCs surface markers between the two groups（ns,P≥0.05）,and the short-term proliferation ability of ADSCs in M group was significantly decreased compared with that in C group（P<0.001）.In terms of the induced differentiation potential of ADSCs in vitro,the adipogenic and osteogenic differentiation abilities of ADSCs in group M were lower than those in group C（P<0.001）.In the cell scratch experiment simulating the wound healing model,ADSCs in group C healed faster in the percentage of scratch healing within 12h,while the scratches in both groups healed completely after 36h culture.The number of stained cells inβ-galactosidase staining of ADSCs in M group was more than that in C group,indicating that cells in M group were more prone to replicative senescence under the same culture system,and there was a significant difference compared with C group（P<0.01）.Conclusion:The model of hyperuricemia in rats can be successfullyestablished by yeast extract feed combined with potassium oxazinate ingastric administration.Hyperuricemia internal environment has a significant negative effect on the proliferation and differentiation potential of ADSCs in rats,and the hyperuricemia internal environment also has a certain negative effect on the migration ability of ADSCs in the early stage of wound healing.