Experimental Study Of Induction Of Granula Of Penetrating Bone And Removing Pain On Chondrogenic Phenotype Differentiation Of Bone Marrow-Derived Mesenchymal Stem Cells In Vitro

Objective:To observe the experiments of the Granula of Penetrating Bone and Removing Pain on extraction,separation,purification,cultivation,identification and the Cbfal,Sox9 gene on chondrogenic phenotype differentiation of bone marrow-derived mesenchymal stem cells (BMSCs)in vitro.We explored the treatment mechanism of osteoarthritis in TCM,to provide the substantial clinical foundations for treating osteoarthritis in TCM,and then to achieve the objective of preventing and treating osteoarthritisMethod:(1) The BMSCs were obtained from SD rats' bone marrow by the bone marrow adherence cultivation through separation,purification,cultivation in vitro.The growth conditions of the BMSCs were observed by inverted phase contrast microscope for us to investigate them in vitro.The third generation BMSCs were tested cell phenotype by flow cytometerCD90,CD45(2) The 48 SD rats aged 4 weeks were randomly divided into 2 groups, i.e.the Penetrating Bone and Removing Pain group(32 rats),the rats were enforced intragastric administration of equivalent dose for 3 days.1 hour later after the last intragastric administration,the serums of the Granula of Penetrating Bone and Removing Pain were separated;The blank group(16 rats),those were enforced intragastric administration of equivalent dose of normal saline and then the serums were prepared by the same way.The second generation BMSCs were randomly divided into 3 groups,i.e.the blank group,the pure chondrogenic inductor group and the group of the Granula of Penetrating Bone and Removing Pain mixed with chondrogenic inductor.We adopted pro-culture solution,pure chondrogenic induced culture solution(TGF-β₃10ug/L,Dex10^-7mol/L,VitC50mg/L) and the chondrogenic induced culture solution which included the serum of the Granula.All groups were cultivated in 50ml cell culture bottles.The effects of the Granula of Penetrating Bone and Removing Pain on chondrogenic phenotype differentiation of BMSCs were investigated after being cultivated for 1,2weeks,then cells observed by inverted phase contrast microscope, electron microscope and RT-PCR(3) The statistics and analysis of datas:The datas were expressed in the form of Mean and Standard Deviation,then statistically analyzed by statistics software named SPSS 14.0.Result:(1) The cultivated BMSCs grow well in vitro.We can obtain better homo geneous BMSCs.The experiment proves that the bone marrow adherence cultivation is a more successful method of separating BMSCs.It proves the success of cultivating B MSCs in vitro.The third generation BMSCs were tested cell phenotype by flow cytome ter:CD90:97.25%,CD45:3.26%.It proved that the purity of the third generation BMSCs was above 97%,which could provide purification cells for the later experiments.(2) Inverted phase contrast microscope:The Granula of Penetrating Bone and Removing Pain group BMSCs mixed together,growed intensively,changed to the form of triangle or polygon.The pure chondrogenic inductor group changed to the form of rotundity or ellipse.The blank group held on the single form of fusiform.Electron microscope:The BMSCs of chondrogenic phenotype differentiation were mostly ellipse.The protuberances on its surface were more,the nucleolus were larger,the rough surfaced endoplasmic reticulums and Golgi apparatus were more abundant,the endoplasmic reticulum cisternas dilated obviously.The BMSCs,not chondrogenic phenotype differentiation,were fusiform or ellipse.The protuberances on its surface were less, the nucleolus were less and irregular,the rough surfaced endoplasmic reticulums and Golgi apparatus were less abundant,the endoplasmic reticulum cistemas had dilatation.RT-PCR:1 week later: The blank group had no gene expression on Sox9.The Penetrating Bone and Removing Pain group and the inducer one all had gene expression on Sox9,but had no difference(P＞0.05).There was no gene expression on Cbfal though the three groups.2 weeks later:The blank group had no gene expression on Sox9 as well.The Penetrating Bone and Removing Pain group and the inducer one all had gene expression on Sox9 and they had highly significant difference(P＜0.01).The blank group had no gene expression on Cbfal.The Penetrating Bone and Removing Pain group and the inducer one all had gene expression on Cbfal and they had significant difference(P＜0.05).Conclusion:(1) It providided pure basic sell after the BMSCs of SD rats were extracted, separated,purified,cultivated and identification in vitro.(2) The Granula of Penetrating Bone and Removing Pain can significantly promote the gene expression on Sox9 but inhibit the gene expression on Cbfal,which proved that the Granula of Penetrating Bone and Removing Pain promote the BMSCs the chondrogenic phenotype differentiation and inhibit its final differentiation.It optimize the seeding cells in cartilage tissue engineering system and improve the quality of cartilaginous tissue and postpone the changes of retrogression in cartilage.