| In order to study the role of the optimal concentration ratio and the best time to its mechanism about UC-MSCs co-cultured with BMECs. In this study, it will put the P3generation of UC-MSCs and P3generation of BMECs to the concentration in proportion (1:1,1:2,1:3,1:4,1:5,1:10,1:50,1:100,1:1000,2:1etc.) and cultivate them randomly, while the establishment of UC-MSCs and BMECs pure culture was the control group, and morphological changes were observed in each group of cells in Oh,24h,48h,72h,96h,120h and144h when by MTT cell proliferation assay to measure the rate of adherent cells and detect EGF, bFGF, TGF-β, HGF, TNF-α, TNF-β and LD expression levels simultaneously detect CcO I, HK, LDH and PK activity to explore the mechanism of UC-MSCs co-cultured with BMECs, thereby providing new ideas and theoretical basis for the UC-MSCs research applications in livestock production.The results showed:UC-MSCs cultured in vitro cell morphology uniform,with the appearance of fibroblast-like cells, tightly packed, there is some directionality; through alkaline phosphatase staining cells were seen after dark brown red; it can be induced to become fat, osteoblasts; the cell growth in good condition, no inhibition when the UC-MSCs and BMECs co-cultured with different concentrations according to the mixing ratio; MTT proliferation detection OD values found highest concentrations of1:2, the fastest cell proliferation was significantly higher than UC-MSCs group and simple BMECs groups, each group OD values was highest at72h, then decreased; adherent cells after co-culture with time is proportional to the rate in which the concentration of cells adherent1:2highest rate was significantly higher than BMECs; UC-MSCs and BMECs with different concentrations by co-cultured mixed1:2concentrations of EGF in the culture supernatant was significantly higher than1:1000, simple UC-MSCs group and simply BMECs (P<0.05), and at72h,96h,120h significantly higher than Oh (P<0.05);1:2group of TGF-a secretion lowest level,1:1group,1:2and2:1group was significantly lower than that BMECs group (P<0.05),1:1group,1:2group,1:3group,2:1group TGF-p levels were significantly higher than1:1000group and pure BMECs group (P<0.05), and72h, TGF-a and TGF-P secretion levels were significantly lower than Oh and144h (P<0.05);1:1group,1:2and2:1group bFGF secretion group were significantly higher than1:50,1:100,1:1000, and BMECs alone group (P<0.05), and48h,72h secretion was significantly higher than Oh,24h,144h (P <0.05); HGF secretion level difference between the groups was not significant,but one:secretion two largest groups, each group HGF secretion highest at72h, significantly higher than Oh,24h,48h (P<0.05);1:1,1:2,1:3,1:4concentrations HK’s activity was significantly higher than1:1000, BMECs cultured alone group (P<0.05), and at72h,96h,120h when the activity was significantly higher than Oh,24h,48h,144h (P<0.05); expression of LD in1:2concentrations reach the highest1:1,1:2,2:1concentration was significantly higher than BMECs pure culture group (P <0.05), and, when compared with Oh72h significant difference (P<0.05); PK activity in the highest concentrations of1:2,1:1,1:2, and the concentration of the active group was significantly higher than1:1000, BMECs pure culture group (P<0.05), and increased its activity gradually increased over time, while at72h the highest, significantly higher than the Oh,120h,144h (P <0.05); LDH activity1:1,1:2,1:3concentration was significantly higher than1:1000and BMECs pure culture group (P<0.05) and LDH activity increased decreased gradually with time, Oh group when compared with the other groups were significantly different (P<0.05),72h group and Oh,24h,144h group were significantly different compared (P<0.05);1:2group showed the lowest expression of TNF-a, was significantly lower than1:10,1:50,1:1000, UC-MSCs cultured alone group and BMECs pure culture group (P<0.05), and the concentration at0h-72h between showing a decreasing trend, and reaches a minimum at the time of72h, significantly lower than Oh,96h,120h and144h time (P<0.05);1:1group,1:2group, TNF-β expression of2:1group significantly lower than1:10,1:50,1:100,1:1000, UC-MSCs cultured alone group and BMECs pure culture group (P<0.05), and the expression level of the lowest in72h, and then began to rise,48h,72h expression was significantly lower than Oh,120h and144h time (P<0.05); co-culture group CcO I activity changed little, except1:100,1:1000,2:1group, the other co-culture of each group CcO I activity was significantly lower than BMECs pure culture group (P<0.05), and the lowest activity at72h, it is lower than the group except it at48h (P<0.05).The experiment reached the following conclusions:①The experiment separated the cows umbilical cord mesenchymal stem cells successfully.②The experiment established a UC-MSCs and BMECs co-culture system successfully, and found that UC-MSCs and BMECs concentration ratio of1:2is the best at72h through the test.③The co-cultured between UC-MSCs and BMECs can promote the proliferation system, and improve the cell adhesion rate.④co-culture between UC-MSCs and BMECs can improve the co-culture system EGF, HGF, bFGF secretion, inhibition of TGF-α and TGF-β secretion levels.⑤The co-culture system of UC-MSCs and BMECs can improve glucose metabolism.⑥The co-cultured between UC-MSCs and BMECs can control the liveness of CcO I and reduce the secretion TNF-α and TNF-β. |
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