Isolation, Culture, Purification And Identification Of Mouse Adipose Derived Mesenchymal Stem Cells And Repairment Of Acute Kidney Injury Caused By Cyclophosphamide

Objective:1 Isolate and culture mouse adipose derived mesenchymal stem cell(ADSCs)which can passaged stably,description and identification of its biological characteristics;2 to obtain high purity ADSCs in the method of differential attachment culture method;3 explore the repair effect of ADSCs in the acute renal injury caused by cyclophosphamide.Method:1 In aseptic environment,separate and extract the inguinal adipose tissues of mice,using ophthalmology scissors to cut adipose tissues to 1mm3 group tissue mass,applicate type I collagenase and trypsin to digest them,made them into single cell suspension,divide them into several culture flasks according to certain cell concentration,adding suitable amount containing10% fetal bovine serum DMEM / F12 culture medium,culture in the standard environment,regularly change the fluid,observe and take photo of the primary cells.2 After the primary cells covered the bottom of culture bottle above80%,then subculture it,take the third generation cells cryopreserved and resuscitated,explore its biological activity.3 Verify the biological characteristics of cells of the third generation,including drawing the growth curve by MTT method,cultured in osteogenic induction medium of the presence of dexamethasone 0.1umol/L,vitamin C50umol/L,sodium glycerophosphate 10mmol/L,10% fetal bovine serum,induced osteoblastic differentiation,culture in adipogenic induction culture medium of adipogenic induction culture medium,induced the differentiation into adipocytes,explore the potential ability of multi differentiation,verity the expression of CD29,CD34,Cd44,CD45 in the application of flow cytometrymethod.4 To obtain high purity ADSCs in the method of differential attachment culture method;5.Divide randomly 27 BALB/c mice into 3groups,named as blank control group A,model control group B and model treatment group C,in d-1,intraperitoneal injection B and C group with cyclophosphamide,which concentration is 10mg/ml,each mouse cyclophosphamide dosage is200mg/Kg,in d0,B group received tail intravenous injection of normal saline0.2ml,C group tail intravenous injection of ADSCs 0.2ml,the cell number is about 1 * 10~6,in d1,d3 and d7 taking venous blood of the mice,test the level of BUN,SCR and NGAL,the kidney tissue specimens stained by HE,according to the scores of international general method of acute renal tubular necrosis degree,score of acute kidney injury caused by CTX,the statistical analysis of the above data.Results: 1 Successfully cultured ADSCs from the mice inguinal fat cells,and the cells were spindle shaped,aggregation,swirling growth,in the second and third generation has strong growth ability,suitable for scientific research,after 4 generations still passage cells,but cell morphology compared with the previous differences cell reticular or paving stone like,in the culture conditions,most cultured 10 passages,cell proliferation activity decreased significantly;2 Successfully induced ADSCs differentiation into osteoblast and fat cells,by alizarin red staining,oil red O staining confirmed,cells were successfully differentiated into osteoblast and fat cells.ADSCs were cultured with multi differentiation potential;3 Mouse ADSCs were positive express CD29 and CD44,negative express CD34 and CD45;4 The application of differential adhesion culture method purified cells the expression rate of CD44 was 72%,the common culture group,the expression rate of CD44 was 55%,the difference between the two kinds of training methods was significant(P<0.05).Application of differential attachment method can produce high purity ADSCs;5 Compare the B group and C group with A in the first day and the third day,no significant difference exist between pathological morphology,under the microscope were visible glomeruli,renal tubular cells arranged indense arrangement rules,but in the 7th day,we can see slightly damage glomerular structure in B group,glomerular cells arranged in disorder,the renal tubular deformed,cells arranged loosely,compared group C with group B the kindney structure is slightly better,statistically analyzed the kidney pathological scores,there was no statistical significance,but the NGAL level of group C in 3 time were significantly lower than those in B group(P<0.05),in the 3rd and the 7th days,the level of BUN and SCR were decreased significantly compared with B group(P<0.05).Conclusion:1 Successfully isolated and cultured the mouse ADSCs,under the condition of this experiment can be stablely culture at least 7 generations,in accordance with the ADSCs morphological characteristics,and have the multiple differentiation potential,positively express CD29 and CD44,negative express CD34 and CD45.2 The application of differential adherent method can culture high purity ADSCs.3 ADSCs played a role in repairing acute kidney injury caused by cyclophosphamide.