| Objectives:1.To evaluate of C57BL/6 mouse aging model;2.To evaluate the effect of human umbilical cord mesenchymal stem cells(hUCMSC)on the degeneration of senile organ function in aged mice by transplanting into aging model;The isolation and identifica-tion of hUCMSC-derived exosomes and establish HUVEC aging oxidative stress cell model,then to observe and analysis the mechanism of hUCMSC-exo in delaying or reversing aging by antioxidative Stress in cell model,to provide a theoretical basis for hUCMSC treatment of senile degenerative diseases.Methods:1.The preparation of hUCMSC The hUCMSCwas cultured in vitro by the tissue culture method and observe the morphology of hUCMSC.The cell surface markers of hUCMSC was detected via flow cytometry,and the property of pluripotency was induced for adipogenesis,osteogenesis and cartilage differentiation and detected the positive rate of hUCMSC by Lv-GFP-labeled in vitro.2.The evaluation of aging mouse models C57BL/6 mice were fed to 72weeks age by conventional feed(n=60),According to the mouse coat color,physical activity,mental state,stool observation and body weight detection method to filter.The aged mice,follow the coat color dark,gloss reduction,white hair,poor mental state,the reduced activity,stool dry or thin frequency,the weight of the standard in the range of(20 ± 5)g,which meet the standard would be put in this experiment as natural aging mouse model(n=40)and 20 for female and male both.The young mice were randomly selected as young group by observing the average condition;the hair color was black,shiny,smooth;spirit actively;full of energy,;normal diet,stool and weight(15 ± 2.5)g.Randomly selected 8 mice from the young mice(8w)and the aging mouse models(72w)as the young group and the model group.The general condition of the natural aging mice in the model group was evaluated by comparing the young group.The structural changes of vital organs(heart,liver,spleen,lung,kidney and brain)in aging group were observed by HE staining;The expression of senescence-related proteins such as p16,p53,SOD2 and Catalase in the important organs(heart,liver,spleen,lung,kidney and brain)in the model group were detected by immunohistochemistry,and the transcriptional levels of p16,p53,SOD2 and Catalase in the important organs(heart,liver,spleen,lung,kidney and brain)of the model group were tested by qPCR and the total SOD activity was detected by the SOD kit.3.Groups and hUCMSC transplantation The model mice were randomly divided into experimental and control group(n=16),In the experimental group,hUCMSC was injected into the tail vein,following 2×105,200ul volume a mouse,once a week for 4 times continuously.The control group received the same volume of normal saline.After 4weeks transplantation and routine feeding,the effect of the hUCMSC was observed.4.The evaluation of hUCMSC transplantation To compare the differences in general station,the structure of important organs was observed by HE staining and the level of transcription and expression of senescence-related genes(p16,p53,SOD2 and Catalase)in vital organs between the two groups of mice was detected by qPCR and immunohistochemistry.The difference of the total SOD activity was tested by SOD kit.The level of autophagy related genes(becline 1,LC3b,sirtl,sirt5)in the main organs between the two groups were found by qPCR.5.The preparation and identification of exosomes The exosome was isolated from purified hUCMSC by ultra-speed centrifugation,and identified exosomes positive markers HSP90,HSP70,CD63 and negative markers TAPA by Western blot,and detected the distribution of exosomes by NTA and observed the morphology of exosomes by electron microscopy.6.The Mechanism The construction of HUVEC cell senescence model was cultured in the medium including the H2O2(400nmol/L).And in the transwell co-culture system with hUCMSC,LC3a/b,mTOR and p-mTOR protein levels were detected by Western blot,and the autophagy of the cell model was observed by Confocal microscopy and the formation of autophagy was found by transmission electron microscopy.The effect of exosomes on the proliferation of HUVEC cells was detected by CCK8 method and apoptosis was found by Flow cytometry.The activation of LC3a/b,p62 autophagy-related proteins and the activation of important signal transduction pathways of exosomes on HUVEC cells were observed by Western Blot.Results:1.The preparation of hUCMSC hUCMSC dispersed from the cultured tissue blocks 5 days later,and the third generation cells still had the typical swirling spiral growth shapes.The expression rates of CD29 and CD44 were 97.1%and 99.6%,and the expression rates of CD34,CD45 and CD105 were 3.18%,0.04%and 0%in the third generation of hUCMSC.Adenal-induced differentiation was observed after 10 days,and alizarin red staining was positive after 3 weeks of osteogenesis induction.Alcian Blue staining was positive after 4 weeks of cartilage induction,The GFP-labeled cells were found in all organs by immunohistochemistry.2.The evaluation of aging mouse models Compared to the young mice(8 weeks),the aging model(72weeks)is with physical activity decreased,hair color gloss with white hairs,poor mental state,dryness,or dilution increased.HE staining showed different degrees of inflammatory,cell edema,necrosis,fibrous tissue hyperplasia and other degeneration;The immunohistochemistry and qPCR test results were consistent with each other like p16,p53 in high levels,while SOD2 and catalase in the low levels of the important organs of aging mice,the difference was statistically significant(p<0.05).Serum total SOD activity in aging group was significantly lower than that in young group,the difference was statistically significant(p<0.05).3.The evaluation of transplantation In the experimental group,after hUCMSC transplantation,the hair color gloss and the body activity of the mice increased,the mental state was better,the abnormal rate of feces decreased;Significant organ structure improved in different degrees;The levels of p16 and p53 were decreased while the levels of SOD2 and Catalase were increased by immunohistochemistry and qPCR,the difference was statistically significant(p<0.05),serum total SOD activity increased,the levels of autophagy-related genes(becline 1,LC3b,)in the main organs were increased,the difference was statistically significant(p<0.05).4.Preparation of exosomes The exosome,isolated from hUCMSC source(hUCMSC-exo)by Ultracentrifuge,was a three-dimensional structure of the cup,with a about 136nm diameter,expressed exosome protein marker CD63,HSP90 and HSP70,not expressed the negative sign exosomes TAPA1.5.The Mechanism After the HUVEC cells growed in the H2O2 culture medium,the cell proliferation ability decreased and apoptosis increased.and after co-culture with hUCMSC,the expression of autophagic marker LC3a/b increased,and the electron microscopy showed autophagy and autophagy.Then after co-culture with hUCMSC-exo,the proliferation of HUVEC increased and the HUVEC cell apoptosis decreased.At the same time,the expression autophagy marker LC3a/b increased,while p62 decreased,the cell autophagy bubble increased significantly,the expression of p38,mTOR,hsp27,stat3 and other autophagy-related signal transduction were suppressed.Conclusions:1.We could get the aging mouse models with the general characteristics of aging and degeneration of tissue structure by nomal feed in 72weeks.2.In the C57BL/6 aging mouse model,hUCMSC could improve the physiological characterization of aging mice from from hair,spirit,activity,diet and body weight,and hUCMSC could reduce the pathological changes of the aged mice,delay or reverse the body oxidative stress state.The hUCMSC could inhibit the senescence gene expression to promote the expression of antioxidant-related genes.Therefore,this study suggests that hUCMSC have a certain anti-aging effect.3.The intervention of hUCMSC may promote the autophagy of the senescent cells by secreting exosomes through hUCMSC,cleaning ROS in aging cells,and reducing the oxidative damage induced by ROS. |
PDF Full Text Request