Effects Of Umbilical Cord Mesenchymal Stem Cells And Their Exocrine Factors On Reproductive Damage Induced By Busulfan In Male Mice

Objective:To explore the therapeutic effect of human umbilical cord mesenchymal stem cell secretory factor injection on azoospermia model mice caused by busulfan.Methods:1.The isolation of human umbilical cord mesenchymal stem cells(HUMSCs)is mainly obtained by tissue adherence method.The isolated human umbilical cord mesenchymal stem cells are subjected to osteogenic,adipogenic differentiation and identification of cell surface markers;2.Collect the fourth-generation HUMSCs in vitro serum-free F12/DMEM medium supernatant,and pass through 0.22μm filter ultrafiltration to obtain the concentrated HUMSCs secretion factor(conditioned medium,CM)and record it as HUMSCs-CM;3.40 mg/kg of Busulfan was injected into the abdominal cavity of male BALB/C mice,and the control group was the saline group.At the 4th weekend of busulfan injection,hematoxylin-eosin(HE)staining was used to observe the structure of seminiferous tubules;the relative expression of meiotic genes STRA8,TNP2,SCP3,DDX4,OCT4 mRNA was detected by fluorescence quantitative PCR;Detect the expression of adhesion proteins P-cadherin,N-cadherin and intercellular adhesion molecule(ICAM-1)between testicular germ cells by western-blot;4.The 40 mg/kg dose of busulfan was injected into the abdominal cavity of male BALB/C mice for 4 weeks,and they were randomly divided into 4 groups:normal saline group,busulfan group,HUMSCs group,HUMSCs-CM group.HUMSCs group and HUMSCs-CM group were injected with HUMSCs suspension 200μl(1×10~7cells)and HUMSCs-CM 200μl through the tail vein,respectively,and the tail vein injection was performed once every 3 days for 4 weeks.At the end of the 8th week,the testicular tissue was stained with hematoxylin-eosin(HE)to observe the structure of seminiferous tubules;the relative expression of STRA8,TNP2,and SCP3 meiotic gene mRNA was detected by real-time fluorescent quantitative polymerase chain reaction(q PCR);Detect the expression of testicular cell adhesion proteins P-cadherin,N-cadherin and intercellular adhesion molecule(ICAM-1)by western-blot。Results:1.The primary HUMSCs were isolated and cultured using the tissue adherence method,and the primary HUMSCs were passaged to the 4th generation by trypsinization.Under the microscope,the cells are tightly arranged,and the cell morphology is diverse,mainly fusiform and oval.The positive rates of HUMSCs surface markers CD90,CD44,CD45,and CD29 were 99.66%,97.40%,0.10%,and 94.47%,respectively.After the cells are induced by osteogenic and adipogenic,stained with Alizarin Red and Oil Red O,HUMSCs can successfully differentiate into calcium salt granules and adipocytes;2.HUMSCs were cultured in serum-free F12/DMEM medium in vitro,and the supernatant culture medium HUMSCs-CM was collected,and the protein concentration was determined to be 0.60 mg/ml;3.The 40 mg/kg dose of busulfan was injected into the abdominal cavity of mice.At the end of the 4th week,the structure of the seminiferous tubules in the mouse testes was observed by HE staining.The spermatogenic tubules in the mouse seminiferous tubules were reduced severely,and the sperm motility was reduced.The basement membrane and spermatogenic cells are broken and separated,the spermatogonia and supporting cells in the seminiferous tubules are severely reduced,and the spermatogonia and spermatocytes are vacuolated;4.At the end of the fourth week,compared with the control group,the expression of STRA8,TNP2,SCP3,DDX4 and OCT4 genes of spermatogenic cells in the testis of the busulfan group was significantly reduced(all P<0.01);the busulfan group The expression levels of cell adhesion-related proteins N-cadherin,P-cadherin,and ICAM-1 were significantly lower than those of the control group(P<0.01);5.At the end of the 8th week,by HE staining of testicular tissue,HUMSCs-CM group compared with Busulfan group and HUMSCs group,the separation of basement membrane and spermatogenic cells and cell vacuolation were improved,and there were a large number of germ cells at all levels in the seminiferous tubules exist;6.Compared with the Busulfan group,the expression levels of STRA8,TNP2,and SCP3 in spermatogenic cells of the HUMSCs-CM group increased(all P<0.01),while the expression levels of the meiotic genes in the HUMSCs group did not increase significantly;Compared with the HUMSCs group,the expression levels of STRA8,TNP2,and SCP3 in spermatogenic cells of the HUMSCs-CM group increased(all P<0.05);7.Compared with the Busulfan group,the HUMSCs-CM group showed an increase in the expression levels of adhesion-related proteins N-cadherin,P-cadherin,and ICAM-1between germ cells in the testis of mice(all P<0.01).Compared with the busulfan group,the HUMSCs group had no significant increase in the expression levels of cell adhesion-related proteins N-cadherin and P-cadherin,but the HUMSCs group had an increase in the expression of ICAM-1 protein compared with the busulfan group(P<0.05).Conclusions:1.HUMSCs can be isolated successfully by using tissue adhesion method and trypsin digestion method;2.HUMSCs were cultured in serum-free F12/DMEM medium,and the supernatant was collected and ultrafiltered with a 0.22μm filter to obtain concentrated HUMSCs secretory factors;3.Intraperitoneal injection of busulfan inhibited the expression of meiotic genes STRA8,TNP2,SCP3,DDX4,OCT4 and the expression of cell adhesion related proteins N-cadherin,P-cadherin,and ICAM-1,resulting in spermatogenesis damage in mice;4.HUMSCs secretion factors protect mice with spermatogenic damage caused by busulfan,and promote the expression of spermatogenic cell meiosis genes STRA8,TNP2,SCP3 and cell adhesion related proteins N-cadherin,P-cadherin,ICAM-1.