| Part One Regulation of Calcium Ion Flow in Ratbit Aortic Smooth Muscle Cells Induced by High Phosphorus.Objective: To investigate the effect of wenyang active blood method on calcium ion flow in rabbits aortic smooth muscle cells induced by high phosphorus.Materials and methods: The cells were randomly divided into 6 groups,namely: beta-GP(-)group,beta-GP(+)group,blank serum group,verapamil group,the JM No.1 Wenyang Decomposed Recipes group(hereinafter referred to as the Decomposed Recipes group)and the JM No.1 complete Recipes(hereinafter referred to as the Complete Recipes group).10 mmol/Lbeta glycerol phosphate add to 10% FBS culture medium cultured cells to seted up calcification model for 14 days.Beta-GP(-)group with 10%FBS culture medium for cells cultivated.After building model 14 days,Alizarin red S(1%,PH4.2)was uesd to dye VSMCs to judged the calcification degree.Eight healthy male New Zealand white rabbits were selected to prepare the serum containing drug for the blank serum group,the verapamil group,the Decomposed Recipes group and the Complete Recipes group.MTT assay was used to detect the cytotoxic effect of each group serum containing drug,and to calculate the half inhibition rate(IC50).The cells which inoculated to 12 orifice plates used the corresponding culture medium to interferring with cell for 6 days respectively.Flou-4 AM probe was added to observe the fluorescence intensity of calcium ions in calcified cells under fluorescence microscope.The concentration of calcium ions in each group was determined by colorimetric method.ELISA was used to determine the activity of cell CAN after intervention.Results : MTT results showed that the inhibitory effect of beta glycerol phosphate on cell viability was outstanding(P < 0.05),and the inhibition rate was 19.65%.Rabbit blank serum has a promoting effect to CCC-SMCs(P <0.05).with the increased concentration of serum,the cells vitality was strengthen.5%,10%,15% and 15% of the blank serum cell activity were one to one correspondence to 134.2%,158.7%,161.5%,169.3%.Compared with the blank serum group,verapamil group had no significant inhibitory effect on cell activity(P=0.675).The inhibition rate of cell was increased with the increase concentration of serum.The IC50 of the Complete Recipes group was19.67%,and the IC50 of the Decomposed Recipes group was 25.98%.The results of alizarin red S stain(1%,PH4.2)showed that there were many orange-red calcium nodules and cytoplasmic red stains in the CCC-SMCs calcification group induced by the beta glycerol phosphate.The control group also showed a small amount of calcified nodules and cytoplasmic red dye.The results showed that CCC-SMC cells spontaneously formed calcified nodules after 14 days of normal continuous culture,and the formation of calcification was promoted by the CCC-SMCs14 induced by he beta glycerol phosphate.Fluo4 AM fluorescent probe detection of intracellular calcium ion shows that compared with beta GP(-)group,beta GP(+)group average fluorescence intensity increased obviously,P < 0.05.It was suggesting that the beta GP increased intracellular calcium ion concentration significantly after intervention;The fluorescence intensity of the blank serum group was significantly higher than that of the beta-gp(-)group,and P < 0.05.It was indicating that rabbit serum could promote the flow of intracellular calcium ion.With blank serum group as the negative control group,the Complete Recipes group,the Decomposed Recipes group and the verapamil group cell fluorescence intensity were significantly weaker,P < 0.05.It is indicated that the medicated serums of the Complete Recipes group,the Decomposed Recipes group and the verapamil group can inhibit the flow of intracellular calcium ion.The calcium ion concentration in the CCC-SMCs was shown to be significantly higher than that of the beta-gp(-)group,and P<0.05.As the negative control group was the blank serum group,the concentration of intracellular calcium ion of the Complete Recipes group,the Decomposed Recipes group and the verapamil group were significantly lower than the blank serum group,P < 0.05.It is indicates that the medicated serum of the Complete Recipes group,the Decomposed Recipes group and the verapamil group can inhibite the flow of intracellular calcium ion.After the intervention in each group for six days,ELISA determined the CAN activity of the rabbit aortic smooth muscle cells.The standard curve was established,the R2 value was the correlation coefficient,R2=0.9859,indicating that the curve linear relationship was good and feasible.Used beta-GP(-)group as the negative control group,there was a significant increase of CAN activity in the cells after the intervention of the beta-GP(+)group,P < 0.05.Used blank serum group as the negative control group,it was shown that there was a significant inhibitory effect on the intracellular CAN activity after the intervention of the verapamil group,the Decomposed Recipes group and the Complete Recipes group,P < 0.05.Conclusion:1.The Complete Recipes group,the Decomposed Recipes group and the verapamil group can inhibit the flow of intracellular calcium ion. 2.Both the drug contained serum of the Complete Recipes group,the Decomposed Recipes group and the verapamil group,can inhibited the activity of the cell CAN.3.Compared with the Decomposed Recipes group and the verapamil group,the inhibition effect on the flow of intracellular calcium ion was obvious by the Decomposed Recipes group.4.It is possible to prevent and treat atherosclerotic occlusion by inhibiting intracellular CAN activity and the flow of intracellular calcium ion on Warming yang and Promoting Blood Circulation Treatment.Part Two Warming Yang and Promoting Blood Circulation Treatment Inhibits VSMCs Transdifferentiate into Osteoblast-like Cells.Objective: To investigate the effect of Warming yang and Promoting Blood Circulation Treatment on osteoblast-like cells transdifferentiation of rabbit vescular smooth muscle cells.Materials and Methods: The cells were divided into six groups: beta-GP(-)group,beta-GP(+)group,blank serum group,verapamil group,the JM No.1Wenyang Decomposed Recipes group(hereinafter referred to as the Decomposed Recipes group)and the JM No.1 complete Recipes(hereinafter referred to as the Complete Recipes group).After the intervention for six days,the cells were collected,and the ALP activity was determined by microenzyme labeling.The relative expressions of RUNX2 and SMAD1 m RNA were detected by fluorescence quantitative PCR.Results: Standard method to determine trace enzyme ALP activity in rabbit aortic smooth muscle cells,according to data in beta-GP(-)group as a negative control,beta glycerol phosphate group of intracellular ALP activity significantly increased after the intervention,P < 0.05;Used blank serum group as the negative control group,it was shown that there was a significant inhibitory effect on the ALP activity in the cells after the intervention of the verapamil group,the Decomposed Recipes group and the Complete Recipes group,P < 0.05.The each groups `s total RNA purity and concentration extracted for Real-time fluorescent quantitative PCR were conform to the requirements of the experiments.The dissolution curve of the gene GAPDH RUNX2,SMAD1 were unimodal.The specificity of primers is strong.There were no product of non-specific amplification and no purpose gene.The results of fluorescence quantitative PCR amplification products were identified by 2% agarose gel electrophoresis.The results showed that the gene was expressed in the cells of the various groups.The amplification products did not show a number of bands or tails,indicating that the primers were specific and specific amplification.The undirected DNA was produced without the primer dimer.The length of PCR product is the same as that of the corresponding target gene.The 2-△△CT method was used for the relative quantitative analysis of RUNX2 and SMAD1 genes in each group.Real-time fluorescent quantitative PCR results showed: with beta GP(-)group as the negative control group comparison,The relative expression levels of RUNX2 and SMAD1 m RNA were significantly increased after intervention by beta GP(+)group in 3d,6d and 9d,and P<0.05.Used blank serum group as the negative control group,the verapamil group,the Decomposed Recipes group and the Complete Recipes group compared with each group of RUNX2 m RNA expression of relative quantity had no obvious difference for 3 days(P > 0.05),the relative expression of SMAD1 m RNA in the whole party group,the party group,verapamil group 3 d SMAD1 m RNA expression were significantly increased,P < 0.05).The expression levels of RUNX2 and SMAD1 m RNA in the verapamil group,the Decomposed Recipes group and the Complete Recipes group were significantly decreased after 6 days,and P<0.05.After intervented for 9 days,there was no significant difference in the relative expression of RUNX2 m RNA in the verapamil group,the Decomposed Recipes group and the Complete Recipes group.The expression levels of SMAD1 m RNA in the blank serum group were significantly decreased after 9d in the verapamil group,the Decomposed Recipes group and the Complete Recipes group,and P<0.05.Conclusion:1.The verapamil group,the Decomposed Recipes group and the Complete Recipes group have inhibitory effects on the activity of ALP in cells.2.The expression of RUNX2 and SMAD1 m RNA was inhibited by the verapamil group,the Decomposed Recipes group and the Complete Recipes group.3.The inhibition effect of the expression of RUNX2 and SMAD1 m RNA in the verapamil group,the Decomposed Recipes group and the Complete Recipes group changed while the time increasing.4.Warming yang and Promoting Blood Circulation Treatment may inhibited the expression of intracellular ALP activity and the expression of RUNX2 and SMAD1 m RNA which was used to inhibit VSMCs transdifferentiate into osteoblast-like cells in the high phosphorus environment. |
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