| Objective:To explore the dose-response relationship of chondrogenic differentiation of PBMSCs separately promoted by TGF-β 3 and BMP-2,and to further study the dose-response relationship of chondrogenic differentiation of PBMSCs co-promoted by TGF-β 3 and BMP-2.Methods:(1)The peripheral blood of Diannan mini pig was mobilized by G-CSF and AMD3100 and collected,density gradient centrifugation and adherent culture were used to culture PBMSCs,the morphology of PBMSCs was observed under inverted phase contrast microscope.The expression of CD44,CD90,CD34 and CD45 of P2 generation PBMSCs was detected by flow cytometry.The PBMSCs of P2 generation was cultured and the osteogenic and adipogenic ability of multidirectional differentiation was detected by alizarin red and oil red O staining respectively.(2)The P2 generation PBMSCs were amplified and cultured in vitro,and different concentrations of TGF-β3(2.5ng/ml-500ng/ml)and BMP-2(25ng/ml-1600ng/ml)were separately added to promote it’s chondrogenic differentiation,and the blank control group was set up.On the 2nd,4th,6th,8th,10th,12th and 14th day of culture,the proliferation of cells in each group was detected.On the 3rd,7th,14th and 21st day of culture,the supernatant of cells in each group was collected and the level of Collagen Ⅱ in the supernatant was detected by ELISA.On the 21st day of culture,immunocytochemical staining was used to observe the expression of Collagen Ⅱ,and toluidine blue staining was used to observe the expression of Aggrecan.The growth and Collagen Ⅱ changes of PBMSCs at different time points were analyzed by repeated measurement ANOVA,and the best dosage of TGF-β 3 or BMP-2 single factor to induce chondrogenic differentiation of PBMSCs was found.(3)Based on the optimal dosage of single factor BMP-2 in 400ng/ml and TGF-β 3 in 40ng/ml obtained in the second part of the experiment,9 experimental groups were set up in cross combination,and the blank control group was set up to compare with the common dose combination from the literature.Different concentrations of chondrogenic inducers were added to P2 generation PBMSCs to induce cartilage differentiation.Cell proliferation was detected by CCK8 on the 2nd,4th,6th,8th,10th,12th and 14th day of culture.The expression of mRNA(SOX-9,Collagen Ⅱ,Collagen X,Aggrecan,MMP-13)was detected by RT-PCR after 21 days of culture.The protein expression levels of Aggrecan and Collagen Ⅱ in the supernatant were detected by Elisa kit at the 3rd,7th,14th,21th day of culture.After 21 days of culture,the expression of Collagen Ⅲ and Aggrecan was detected by immunofluorescence double labeling.Results:(1)The peripheral blood PBMSCs samples obtained after G-CSF+AMD3100 mobilization showed colony-like growth after culture,and the results of P2 generation PBMSCs flow cytometry showed that CD34 and CD45 were negative,but CD44 and CD90 were positive.After osteogenic induction for 21 days,alizarin red staining showed calcium nodule formation,oil red O staining was positive for 14 days after adipogenic induction,and lipid droplets could be seen in the cytoplasm.(2)The growth curve of PBMSCs induced by each single factor was similar,which was significantly different from that of the control group at each time point(P<0.05).With the extension of culture time,the morphology of PBMSCs in each group was basically similar,roughly in the shape of "S".With the increase of TGF-β 3 concentration in 2.5ng/ml-40ng/ml,the growth and proliferation ability of PBMSCs was enhanced,and the growth and proliferation ability of PBMSCs induced by 40ng/ml was decreased.With the increase of BMP-2 concentration,the growth and proliferation ability of PBMSCs increased with the increase in 25ng/ml-400ng/ml concentration,and then decreased when it exceeded 400ng/mlPBMSCs concentration.The cell growth activity decreased significantly at 1600ng/ml concentration,which cause cytotoxicity.The results of ELISA showed that the Collagen Ⅱ content of TGF-β3 was the highest at each time point of 40ng/ml and 80ng/ml,and there was no significant difference between the two concentration groups(P>0.05).At the same time,there was significant difference among the two groups and the other group(P<0.05).The content of Collagen Ⅱ in BMP-2 was the highest at each time point of 400ng/ml,and there was significant difference compared with other groups at the same time point(P<0.05).The results of toluidine blue staining and immunohistochemistry showed that after 21 days of induction,the staining intensity of BMP-2 in 400ng/ml,TGF-β3 in 40ng/ml and 80ng/ml was significantly higher than that in other concentration groups.(3)The results of CCK-8 cell proliferation induced by two factors showed that there was no significant difference in cell viability among the two concentration groups,but there was significant difference between the two concentration groups and the control group(P<0.05),and the cell proliferation activity in each concentration group was significantly better than that in the control group.The results of cartilage-related gene expression detected by RT-qPCR showed that on the 21st day,the expression level of cartilage-related genes(SOX-9,Aggrecan)in control group and A-E group was significantly lower than that in F,G,H,I and J groups,and that in F,G,H,I groups was significantly lower than that in J group.The expression level of cartilage-related gene Collagen Ⅱ in control group,A-E,H and I groups was significantly lower than that in F,G and J groups,and that in F and G groups was significantly lower than that in J group(P<0.05).The expression levels of cartilage hypertrophy related genes(MMP-13,Collagen X))in control group and A-F group were significantly lower than those in G,H,I,J groups,and those in G,H,I groups were significantly lower than those in J group.The relative expressions of F,G cartilage and bone related genes(S OX-9,Aggrecan,Collagen Ⅱ,MMP-13 and Collagen X)in the cycle were significantly lower than those in H,I and J groups.ELISA results showed that higher than TGF-β3 40ng/ml+BMP-2 400ng/ml concentration group could promote the differentiation of PBMSCs to produce more Collagen Ⅱ and Aggrecan,but this trend did not increase with the increase of concentration.Although the average value of Aggrecan and Collagen Ⅱ increased with the increase of factor concentration,there was no significant difference.Immunofluorescence showed that after chondrogenic induction for 21 days,the expression of Collagen Ⅱ in control group was negative,there was weak expression of Collagen Ⅱ in group A,B,C,D and E,but it was significantly less than that in group F,G,H,I and J,and a large amount of Collagen Ⅱ expression was found in group I,J and K,which was significantly more than that in group G and H.The expression of Aggrecan was consistent with that of Collagen Ⅱ.Conclusion:TGF-β3 in 40ng/ml and BMP-2 in 400ng/ml could effectively promote the proliferation and chondrogenic differentiation of PBMSCs from Diannan mini pig.The combined application of the optimal concentration of TGF-β3 and BMP-2 can maximize the chondrogenic differentiation of PBMSCs,maintain the cartilage phenotype,and avoid its hypertrophic differentiation. |
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