| In the condition of serum-free co-culture,to research the effect of UC-MSCs on proliferation,apoptosis,milk fat and protein synthesis in BMECs,and to reveal the specific mechanism of exogenous cytokines on the regulation of BMECs lactation.This experiment is divided into three parts:The first part,the UC-MSCs and BMECs were invoked as the research object.Those two kinds of cell with the best growth performance were purified by P5 UC-MSCs and P8 BMECs and selected for co-culturing in the way of direct or indirect contact.In direct contact groups,UC-MSCs and BMECs were co-cultured in the concentration ratios of 2:1,1:1,1:2,1:3,1:4,1:5,and 1:10.In an indirect contact group,the supernatant of UC-MSCs was used as the conditioned medium to re-suspend BMECs.In control groups,UC-MSCs and BMECs were cultured alone.The cell growth situation in each group was observed at 0,4,8,12,24,36,48,60,and 72 h,and cell proliferation was detected using cell counting kit-8(CCK-8)assay,the optimal co-culture conditions of UC-MSCs and BMECs were screened out combined with the cells morphological observation.The second part,based on the best ratio and time of co-culture,UC-MSCs and BMECs were co-cultured directly on the concentration ratios of 1:2,in control groups,UC-MSCs and BMECs were cultured alone.Using ELISA method to detect the IGF-?levels in each group supernatant at 48 h,and two kinds of cells were separated by Transwell Chambers.The IGF-?and IGF-?R mRNA expression values of each group were estimated by Real-time PCR.The third part,in the basis of IGF-I optimal secretion conditions was verificated by Transwell,BMEC was cultured alone as control group,IGF-I R inhibitor AG1024,JAK2 signal blocking agent AG490,and PI-3K signaling inhibitor LY294002 treated cells,observed cell growth of each groups when before treatment and after treatment,flow cytometry combined with Annexin V-FITC/PI double parameter method were adopted to detect apoptosis changes of cells,the IGF-I,CSN2,CSN3,and TG content of supernatant were determined,RT-PCR was used to determine the relative expression of signaling pathway related genes PI-3K,AKT,mTOR,JAK2,STAT5,ELF5,and the relative quantitative value of cell apoptosis genes Bcl-2,Bax,Caspase-3 mRNA,and the expression abundance of milk fat,milk protein synthesis key genes ACACA,FASN,SREBP1,and CSN2,CSN3 mRNA.Results:In the first part,the optimal co culture conditions of UC-MSCs and BMECs were optimized,the results showed that at 48 h,the optical density of the conditioned medium-BMECs group was significantly higher compared with the control groups(P<0.05).Meanwhile,the optical density in the direct contact group with the concentration ratio of 1:2 reached the peak,which was extremely significantly higher compared with the control groups(P<0.01)and significantly higher compared with the other direct contact groups and the conditioned medium-BMECs group(P<0.05).;The second part,Transwell was clearly the interaction between UC-MSCs and BMECs,results showed that at 48 h,the IGF-I,IGF-I R m RNA expression was significantly or extremely significantly higher than the pure culture group through two co culture programs(P<0.05,P<0.01),the IGF-?concentration value of UC-MSCs and BMECs mixed co-culture was significantly higher than the UC-MSCs group(P<0.05),and extremely significantly higher than the BMECs group(P<0.01),BMECs/UC-MSCs group was significant difference compared with UC-MSCs/BMECs group(P<0.05);The third part,confirmed the possible pathway of UC-MSCs on BMECs about apoptosis,milk fat and milk protein,results showed,the apoptosis rate of experimental group was extremely significantly lower than other groups(P<0.01),the expression of Bcl-2 in the experimental group was significantly increased than control group(P<0.01),and the expression of Caspase-3,Bax mRNA were significantly down regulated(P<0.05),after treatment with AG1024,LY294002 and AG490,the apoptosis rate of the expression of Bax,Caspase-3mRNA from each group were raised,the expression of Bcl-2 mRNA was reduced,they were statistically significant;milk fat and milk protein synthesis results showed,the indexes of the experimental group were significantly higher than the control group(P<0.05,P<0.01),AG1024 had significant inhibition effect on IGF-I(P<0.01),the contents of TG,CSN2,CSN3 and relative abundance of each gene were significantly or extremely reduced(P<0.05,P<0.01),the relative abundance of CSN2 and CSN3 mRNA were significantly reduced after AG490 treatment(P<0.05),but the expression of ACACA,FASN,SREBP1 mRNA had no significant change(P>0.05),the expression of PI-3K(P<0.01),AKT,mTOR(P<0.05)mRNA were significantly inhibited when LY294002 treatment,the expression of TG,CSN2,CSN3 and the content of each genes were significantly decreased(P<0.05),when AG1024 and AG490treatment together,the expression of milk fat synthesis key gene had no significant difference compared with AG1024 treatment alone(P>0.05),when AG1024 and LY294002 treatment together,the expression of CSN2,CSN3 mRNA were significantly down regulated(P<0.05),and the other indicators were extremely significantly reduced(P<0.01).In summary,the optimal co culture conditions of UC-MSCs and BMECs are established in this study,and the effect between UC-MSCs and BMECs is defined by Transwell~?chamber,and found that UC-MSCs could promote the expression of IGF-?and it's receptor related genes in BMECs,IGF-I have a hand in UC-MSCs regulation of apoptosis,milk fat and milk protein synthesis in BMECs,PI-3K/AKT/mTOR and JAK2/STAT5 signaling pathway are the common pathway of IGF-I regulating BMECs apoptosis and lactation,but JAK2/STAT5 signaling pathway is not involved in UC-MSCs regulation of milk fat synthesis in BMECs,so,UC-MSCs mediate PI-3K/AKT/mTOR and JAK2/STAT5signaling pathway to inhibit BMECs apoptosis by IGF-I,and increase the survival rate,thereby,promote BMECs milk fat and protein synthesis,regulation of BMECs lactation performance,not only make up for the deficiency of IGF-I secretion in BMECs,it also provides a theoretical basis for the further study on the regulation of endogenous IGF-I on the biological function of BMECs. |
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