| Objective:In this study,Rabbit adipose stem cells(RADSCs)were extracted from groin fat of large rabbits and cultured in vitro.After several generations of culture,the proliferation and osteogenic differentiation ability of RADSCs were detected,and the characteristics of adipose stem cells were proved.On the basis of the previous cell extraction experiment,the rabbit adipocytes were isolated and cultured to prepare the drug-containing serum of Zhitong Jiangu recipe,which were divided into control group and experimental group to induce their osteogenic differentiation,and the effects of Zhitong Jiangu Recipe on BMP-7,alkaline phosphatase(ALP)and the proliferation ability of adipocytes were observed.It provides scientific experimental basis for treating osteonecrosis of femoral head with acetanilide and bone strengthening prescription,and has important application prospect and scientific value.Methods:An appropriate amount of fat tissue from the groin of rabbits was cut and washed repeatedly with PBS and the excess tissue was removed.After digestion with type I collagenase,the cells were separated and suspended,and then cultured to the second generation.Cell differentiation was induced by osteogenesis and lipid-forming induction solution,and the cells were identified as RADSCs by alizarin red staining and oil-red O staining.The rabbits were given intragastric administration of analgesic and bone-building formula for 7 consecutive days,and then anesthetized after intragastric administration on the last day.Blood was taken from abdominal aorta to prepare drug-containing serum at 0%,5%,10%,15%and 20%concentrations,and blank serum with the same concentration was set.Cells were divided into control group,blank serum group and drug-containing serum group.After adding corresponding serum,cell proliferation was detected by CCK-8 method.After the addition of osteogenic induction solution,the expression concentrations of ALP and BMP-7 in the three groups were detected by ELISA,and the graphs were drawn to interpret the effects of different concentrations of serum and drug-containing serum on cell osteogenic differentiation,and to confirm the optimal induction concentration.Results:The cells were digested,isolated and inoculated with type I collagenase,and then isolated to the second generation.CD44 and CD90 antibodies were added,and positive expression of CD44 and CD90 cell surface antigens was detected by flow cytometry.After cell differentiation was induced by osteogenic and lipogenic induction solution,alizarin red and oil red O staining were performed,and the re suits were positive,indicating that the cells were RADSCs.After the cells were cultured with different concentrations of serum and drug-containing serum,the value-added rate of ADSCs was detected by CCK-8 method.It was observed that there was no significant difference in each group until 24 hours after the culture(P>0.05),but the relative value-added rate of ADSCs was the highest in the group containing 10%drug-containing serum after 48 and 72 hours.Compared with the control group,the difference was statistically significant(P<0.05),so the optimal concentration of ADSCs proliferation in drug-containing serum was 10%.After 2 1 days of ADSCs cell culture,compared with blank control group,the bMP-7 content and ALP activity of ADSCs cells in the serum containing Zhitong Jiangu Formula were increased(P<0.01);Compared with blank serum-5%group,the bMP-7 content(P<0.01)and ALP activity of ADSCs cells in tzhitong Jiangu Formula containing serum were increased in different degrees(P<0.05);Compared with-10%group,bMP-7 content(P<0.01)and ALP activity of ADSCs cells in-20%group were increased(P<0.05).The bMP-7 content and ALP activity of ADSCs cells in the-5%group were decreased(P<0.05).It is suggested that tzhitong Jiangu prescription can significantly increase the content of BMP-7 and activity of ALP in ADSCs cells,and the effect is more significant when the concentration of tzhitong Jiangu prescription is 10%and 20%.Conclusion:In this experiment,RADSCs were successfully extracted,isolated and cultured from the fat in the groin of rabbits,which had the ability to proliferate and differentiate into osteogenesis and lipids in vitro.At 20%concentration,the drug serum of Zhitong Jiangu Prescription can significantly promote the osteogenic differentiation of RADSCs and increase the protein expression of BMP-7 and ALP.Therefore,the drug serum of Zhitong Jiangu Prescription can be used as an inducing factor of adipose stem cells to osteogenic differentiation and has certain experimental basis for application in new bone tissue engineering.It provides some ideas for further study of zhitong jiangu recipe to promote adipose stem cells to osteogenic differentiation. |
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