Inhibition Of Canine Mammary Gland Tumors By Canine Amniotic Fluid-derived Mesenchymal Stem Cells

As people work and change their lifestyles,pets have become a part of the family.At present,the incidence of canine mammary tumor in dogs is getting higher and higher,butthere are no effective and side-effect-free drugs for the treatment of canine mammary tumor.Therefore,it's necessary to study cell therapy and cell-drug therapy.Amniotic fluid mesenchymal stem cells(AF-MSCs)are pluripotent stromal cells isolated from perinatal amniotic fluid tissue.They have immunosuppressive function and tissue repair capability,which can be widely used in clinical practice.Moreover,AF-MSCs do not form teratomas in allogeneic transplantation.Besides,the biological characteristics,culture characteristics of canine amniotic fluid-derived mesenchymal stem cells(c AF-MSCs)in vitro and their roles in cancer therapy have not been systematically studied.Therefore,c AF-MSCs were selected for in vitro proliferation,multi-directional differentiation,and the research on anti-cancer activitis,which provided a preliminary research basis for the treatment of canine breast cancer.1.Cells from perinatal amniotic fluid were conformed to the culture characteristics in vitro of mesenchymal stem cells.in,Amniotic fluid was collected in the canine caesarean section and cells were obtained by centrifugation,and the isolated cells were cultured and identified in vitro.The results showed that the cells obtained from the canine amniotic fluid were spindle-shaped adherent growth,arranged in a vortex or reticular order,and the growth curve was "S" type.The cells could be stained by Oil red O and alizarin red and alexin blue after they've been differentiated into adipocytes,osteoblasts and chondroblasts respectively.The cells induced by the neurogenic differentiation fluid could express NESTIN and GFAP,suggesting that the isolated cells could differentiate into neural cells.These cells expressed CD44,CD73,CD90,CD105,HLA-ABC,but did not express CD45,HLA-DR.The above results demonstrated that the cells isolated and cultured in thisexperiment were AF-MSCs which conformed to the basic characteristics of mesenchymal stem cells that have the ability of multi-directional differentiation and low immunogenicity.,2.Inhibition of canine mammary tumor cells(CTM1211)by c AF-MSCs in vitro and in vivo.The activity of CTM1211 cells was detected after co-culture with c AF-MSCs for 24 h,48 h,72 h and 96 h.It was found that c AF-MSCs had the best inhibit effect on CTM1211 after co-culturing for 72 h.Cell migration was calculated after co-culturing for 0 h,12 h,24 h,and 36 h.The rusults found that c AF-MSCs inhibited the migration of CTM1211,and the difference was significant between 24 h and 36 h.Apoptosis rate of cells after co-culturing 72 h was showed by Annexin V-FITC/PI double staining assay that the c AF-MSCs significantly promoted early apoptosis of CTM1211.q PCR detection of apoptosis-related factors revealed that the expression of p53 and Bcl-2 in CTM1211 was increased,and the expression of survivin and Bax in CTM1211 was decreased,which suggesting that c AF-MSCs increased the expression of apoptotic genes in CTM1211.CTM1211 were used subcutaneously injection to established the model on nude mice(BALB/c),and then the c AF-MSCs were injected into the tail vein of the model for the treatment.After three treatments,the mice were dissected and the tumors were taken out for histological analysis.The proportion of apoptotic cells in tumor tissues was calculated.It was found that the apoptotic cells in the experimental group accounted for 7.43%,which was higher than the control group.The heart,liver,spleen,lung and kidney were detected by histology,and no metastasis was found in the tumor cells.The expression of genes and proteins in tumor tissues was detected.The expressions of apoptosis-related genes p53,protein Bax,Bax in the treatment group were significantly higher than those in the control group,and the expression of survivin and Bcl-2was significantly lower than that in the control group.The results suggested that c AF-MSCs could promote apoptosis and inhibit migration of canine breast tumors.