Experimental Reasearch On Effect Of Chinese Kidney-tonifying Drugs Combined With BMP-2on Proliferation And Osteogenic Activity Of The Human Umbilical Cord Blood Mesenchymal Stem Cells In Vitro

Objective:To establish culture system for the human umbilical cord blood mesenchymal stem cells (UCB-MSCs), observe the biological characteristics, proliferation, alkaline phosphatase (ALP)and osteocalcin (BGP) expression of UCB-MSCs induced by Chinese kidney-tonifying drugs serum combined with BMP-2in vitro, and explore its effect on transformation of UCB-MSCs into osteoblasts.Methods:18SD rats were given with Chinese kidney-tonifying drugs by gavage for7days, taking carotid arterial blood to make Chinese kidney-tonifying drugs serum, collecting Human umbilical cord blood samples under sterile conditions and separating UCB-MSCs by density gradient centrifugation. Observed the biological characteristics of Primary culture of UCB-MSCs under inverted phase contrast microscope, and cells were identified by flow cytometry. The3rd generation of UCB-MSCs Were divided into four groups:group A (control group), group B (5%Chinese kidney-tonifying drugs serum group), group C (50ng/mlrhBMP-2group), group D (5%Chinese kidney-tonifying drugs serum combined with50ng/mlrhBMP-2group). Observing the influence of proliferation each group on UCB-MSCs on the induction of1st,3rd and5th by MTT assay, the effect of Chinese Kidney-tonifying Drugs on osteogenic activity of UCB-MSCs by ALP staining, ALP activity and osteocalcin (BGP) content assay on the induction of3rd,6th,9th and12th.Results:(1)The primary culture cells of UCB-MSCs were round.72hours after primary culture,only a small amount of adherent cells were round or oval. The medium was changed for the first time on the7th, in which can be seen scattered MSCs radial growth. After two weeks the cells morphology were fusiform, being colony fusion with each other after three weeks. Most of Passage cells were adherented in24h.Generally, they can reach80%to90%confluence in3days, packed tightly, and the form is relatively uniform,mostly spindle.(2)Compared with the control group,Chinese kidney-tonifying drugs serum combined with BMP-2group, BMP-2group was able to promote proliferation of the UCB-MSCs on the induction of the3rd,5th day (P<0.05), and especially the Chinese kidney-tonifying drugs combined with BMP-2group was best (P<0.01). There was significant difference between Chinese kidney-tonifying drugs serum combined with BMP-2group and BMP-2group on the first5day (P=0.041<0.05).However, the difference was not statistically significant between Chinese kidney-tonifying drugs serum group and the control group (P>0.05).(3)The induction group has weak expression of the ALP staining on the7th day. On the14th the ALP staining was enhanced, but the intensity was different from each group,of which the control group had weak positive expression,Chinese kidney-tonifying drugs combined with BMP-2and BMP-2more significant, while the combination group was the optimal.The positive expression of the ALP staining was weakened on the21st. However,for control group,the ALP staining was negative throughout the induction process.(4)Compared with the control group, BMP-2group, Chinese Kidney-tonifying Drugs serum combined with BMP-2group could improve ALP activity on the3rd,6th,9th, and12th day after the induction (P<0.05), of which Chinese kidney-tonifying drugs serum combined with BMP-2group after the induction of the3rd,6th,9th day was statistically significant different (P<0.01).There was significant difference between Chinese kidney-tonifying drugs serum combined with BMP-2group and BMP-2group on the induction of the first6d(P=0.011<0.05).(5)Both BMP-2group and Chinese kidney-tonifying drugs serum combined with BMP-2group on the induction of the6th,9th, and12th day can improve the content of the osteocalcin (BGP), compared with control group, there was significant difference (P<0.05), of which Chinese kidney-tonifying drugs serum combined with BMP-2group on the the first9day after the induction was statistically significant different (P=0.004<0.01),compared with control group.Conclusion:The method that Chinese kidney-tonifying drugs serum based on the theory of the kidney generating marrow and dominating bone combined with BMP-2induced UCB-MSCs into osteoblasts in vitro is feasible and effective. Chinese kidney-tonifying drugs serum combined with BMP-2can promote the proliferation and directional osteoblastic differentiation of UCB-MSCs. However, for Chinese kidney-tonifying drugs serum alone, this effect is not significant.