| Mesenchymal stem cells(MSCs)are adult stem cells with the ability of self-renewal and multi-lineage differentiation,which exist in the connective tissue of the body.MSCs have become preferred seed cell for clinical treatment of stem cells due to its advantage of wide range of sources,low immunogenicity and strong proliferation ability.With the improvement of people’s living standards,Cat has become an important companion animal for humans and pet welfare has got great attentions from people,which one breeding has increased dramatically.Feline acute renal injury(AKI)is a common clinical disease,which is caused by acute renal ischemia,nephrotoxic drugs,urinary tract obstruction and so on.Although medical technology has been greatly developed in recent years,the therapeutic effect of AKI has not been fundamentally improved and its mortality has increased year by year.Stem cell therapy has been widely recognized for its small side effects and remarkable curative effects,which provides a new way for the treatment of many diseases.In order to select the seed cells for the treatment of cat diseases and to study the therapeutic effects and mechanism of MSCs on cat AKI,the following studies were carried out in this experiments:1.Comparisons of biological characteristics of MSCs from four sources of adipose,bone marrow,amniotic membrane and umbilical cord.To compare the biological characteristics of Mesenchymal Stem Cells(MSC)derived from adipose(AD),bone marrow(BM),amniotic membrane(AM)and umbilical cord(UC),the method of whole blood culture was used to isolate BM-MSCs and digestive method was used to isolate others-derived MSCs.Then the morphology,growth characteristics,differentiation capacity and cell surface markers were compared and analyzed.The results showed that feline MSC derived from 4 different tissues had common characteristics of adherent growth,spindle-shaped and polygonal-shaped.They had the ability of adipogenic and osteogenic differentiation.The cell proliferations of AD-MSCs and BM-MSCs were faster than AM-MSCs and UC-MSCs,and the doubling time on AD-MSCs,BM-MSCs,AM-MSCs and UC-MSCs were 30.46 hours,36.51hours,55.85 hours and 58.06hours respectively,which AD-MSCs and BM-MSCs were shorter.The AD-MSCs demonstrated the best growth activity and potential application prospect at clinic,because it maintained fine proliferative activity when they passed to the 9th generation,which can be used as a therapeutic seed cell.Cell surface markers of CD44,CD90 and CD105 were higher expressed,and CD34 were less expressed in four kinds of MSC.2.Regulations of AD-MSCs and its conditioned medium on proliferation and apoptosis of Crandell Rees Feline Kidney(CRFK)injuryed by Gentamicin(GM).In this experiment,the toxic effects of gentamicin on CRFK was studied firstly,and then the effects of AD-MSCs and its conditioned medium on proliferation and apoptosis of CRFK damaged by GM were determined by CCK-8 method,Annexin V flow assay and fluorescent quantitative PCR.The results showed that the cell activity of each group was significantly lower than that of the normal group when the different concentrations of GM acting on CRFK after 12 hours,and with the increase of GM injury time and concentration,the activity of CRFK had became lower,with a significant time and dose-dependent.The results of Q-PCR showed that the expression of proliferation-related genes CCND1,PCNA and apoptosis-related genes Bcl-2 was significantly increased and Bax gene was decreased in AD-MSC group.The results suggest that AD-MSCs promotes the repair of GM-induced crfk damage by increasing cell proliferation activity and reducing apoptosis of CRFK.The activity of CRFK cells in AD-MSCs and AD-MSCs-CM group was significantly higher than that of GM group,and the apoptosis rate in AD-MSCs and AD-MSCs-CM group was significantly lower than that of GM group.The results of Q-PCR showed that the expression of proliferation-related genes CCND1 and PCNA in AD-MSCs group was significantly higher than that in GM group,while that in AD-MSCs-CM group had an increasing trend but not significant.The ratio of Bcl-2/Bax in AD-MSCs group was significantly higher than other groups,and there was no significant difference between other groups.3.Observation on the effect of AD-MSCs in the treatment of AKI in cats.In this study,18 domestic cats of 4-5-months-old weighing 1.31kg were used for the experiments.Cats were respectively divided into 3 groups:control group(n=6),model group(n=6)and treatment group(n=6).Each cat in the treatment group and the model group received a dose of 200 mg/kg GM via Subcutaneous injection.In the control group,each animal received an equal volume of physiological saline.The injections were administered twice a day for 7 days consecutively.AD-MSCs(2×10~6/kg)was transplanted intravenously in the treatment group which was injected once every 5 days for a total of 5 times,and the same amount of physiological saline was injected in the control group and the model group.Blood samples and kidney tissues were collected for histopathological,serological,and immunohistochemical detection at the 7th,14th,21th and 36th days after treatment.The results showed that the average survival time of the treatment group was longer than that of the model group,and the mortality rate was lower than that of the model group.At the 7th day,the blood creatinine values of the treatment group and the model group were were higher or significantly higher than those of the control group,While the model group was significantly higher than the treatment group.The blood urea of the model group was higher than the treatment group and control group,but there was no significant between the treatment group and the control group;The values of blood phosphorus in the treatment group and the model group were no significantly different from those in the control group.The levels of blood creatinine and blood urea nitrogen in the treatment group were significantly lower than those in the model group(P﹤0.05),while the blood phosphorus was no significant.On the 14th day,the blood creatinine,urea nitrogen and phosphorus in the model group were significantly higher than those in the treatment group(P<0.05),and the urea nitrogen were no significantly.At 21d and 36d,the serum creatinine,urea nitrogen and phosphorus values of the model group were still higher than those of the treatment group and the control group.Histopathological results show:At 14th day most of the renal tubules in the model group were necrotic,exfoliated and the bare basement membrane,while in the treatment group,most of the renal tubular epithelial cells were swollen and only a few were necrotic and exfoliated.The results of immunohistochemical staining of ki67showed that the renal tubules and renal interstitial cells of the model group were almost stained without brown cells,while only a few renal tubular epithelial cells were stained with brown in the treatment group.At the 21st day,some of the renal tubules in the model group were necrotic and exfoliated,the bare basement membrane and the interstitium has thickened,while in the treatment group,most of the renal tubular cells are neatly arranged with brush border structure,and only a few renal tubular epithelial cells were swollen,necrotic and exfoliated.The results of immunohistochemical staining of ki67 showed that most of the renal interstitial cells in the model group and the renal tubular epithelial cells in the treatment group were stained brown.On the 36th day,swelling and exfoliation of renal tubular epithelial cells and glomerular Mesangial and interstitial hyperplasia were still observed in the model group,while brush border structure could be seen in most of the renal tubular cells in the treatment group.The results of immunohistochemical staining of ki67showed that a large number of renal interstitial cells were stained brown in the model group and most of the renal tubular epithelial cells in the treatment group were stained brown.Frozen section observation showed that PKH26 labeled MSCs could be homed to kidney tissue,but not in model group and blank group.Above results suggest that AD-MSCs transplantation can significantly reduce the contents of creatinine,urea nitrogen and phosphorus in blood of GM-AKI cats.PKH26 labeled MSCs could homed to kidney tissue to reduce the swelling,necrosis,exfoliation and interstitial hyperplasia of renal tubular epithelial cells,and promote the proliferation of renal tubular epithelial cells. |
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