The Experimental Study Of The Effects Of Tetramethylpyrazine On Activity Of Cartilage Cells And CDK4, CyclinD1

Objective:With the construction of vitro culture system of cartilage cells and the intervention of tetramethylpyrazine(TMP), the experimental results of proliferative activity of cartilage cells and the differential expression of CDK4,CyclinDl help probe the effects of tetramethylpyrazine on growth cycle of cartilage cells, and provide the theoretical evidence for the influence of tetramethylpyrazine on prevention and treatment of osteoarthritis with its possible mechanism.Methods:1. Acquire cartilage cells which were identified by toluidine blue staining from collagenase to construct the vitro culture system of cartilage cells.2. The fluorescent morphological features of the cells were observed under inverted microscope. The growth curve of cartilage cells was detected by MTT approach.3. Randomly divided the well-grown 2nd generation cartilage cells into four groups which included blank control group and three exprimental groups, then divided each group into 24 h,48 h and 72 h groups. The influence that tetramethylpyrazine had exerted upon proliferative activity of cartilage cells can be detected by MTT approach, and the cell cycle can be showed by flow cytometer.4. The differential expression of CDK4,CyclinD1 in target cells was detected with reverse transcriptase-polymerase chain reaction(RT-PCR). Results:1. The vitro culture system of cartilage cells was successfully constructed. Cells began to adhere, enlarge, extend and form processes after 24 hours'cell inoculation. The formation of cells cluster can be seen after 4 days, which was presented in spindle or oval shape. Moreover, the overlapping growth appeared. Right after about 8 days, proliferation occurred. Cells gradually fused and formed monolayer, which was presented in the pattern of irregular solid "paving-stone"2. The growth curve of cartilage cells:active proliferation happened from the 2nd day to the 4th day to the 2nd generation cartilage cells; in the period of the 5th day to the 7th day, the proliferation curve gradually appeared flat; After eight days, the growth curve began to drop down.3. The influence that tetramethylpyrazine had exerted upon proliferative activity of cartilage cells was as follows. There has been a gradual increase in OD value of cartilage cells of each group with 24 hours',48 hours'and 72 hours'intervention. There has no significant difference among OD value of each group with 24 hours' intervention. The OD value of the 2nd group and the 3rd group with 48 hours' intervention was obviously higher than that in control groups(P=0.036, P=0.023). The OD value of the 2nd group and the 3rd group with 72 hours'intervention was noticeably higher than that in control groups (P=0.008, P=0.004). The OD value of the 2nd group and the 3rd group with 72 hours'intervention was distinctly higher than that in group one.4. The effects that tetramethylpyrazine had worked upon proliferating cycle of cartilage cells were listed below. There has no significant difference in cell cycle among all groups with 24 hours'intervention. The G0/G1 Period of the2nd group and the 3rd group with 48 hours'intervention was obviously less than that in synchronous control groups(P=0.026, P=0.024) while S Period (P=0.040, P=0.025) and PI (P=0.026, P=0.024) were both noticeably higher. In the case of 72 hours'intervention, G0/G1 Period of the2nd group and the 3rd group was obviously less than that in synchronous control groups (P=0.003, P=0.002); G2/M Period was considerably higher than that of synchronous control groups(P=0.022, P=0.014) and that of 1st group (P=0.025, P=0.016). PI was also markedly higher than that of synchronous control groups (P=0.003, P=0.002)5. The differences in the expression of CDK4 mRNA,CyclinDl of each group:There has been the expression of CyclinDl, CDK4 mRNA in the 2nd generation cartilage cells with 24 hours'intervention,48 hours'and 72 hours'. There has no significant difference in those groups with 24 hours'intervention. The expression of CyclinDl mRNA of group two and group three with 48 hours'intervention and 72 hours'intervention was obviously higher than that in control groups (P=0.038, P=0.021; P=0.013, P=0.018); The expression of CDK4 mRNA of group two and group three with 48 hours'intervention and 72 hours'intervention was noticeably higher than that in control groups (P=0.023, P=0.020; P=0.034, P=0.017).The expression of CDK4 mRNA of group two and group three with 72 hours' intervention was distinctly higher than that in group one (P=0.043, P=0.022)Conclusion:1. Tetramethylpyrazine had some stimulative effects on the proliferation of cartilage cells.50 ug/L was the preferable drug concentration for intervention. The promotive effects were comparatively remarkable on 48 h and 72 h groups.2. The underlying mechanism may worked in the way as following. The cyclic protein complexes were activated by increasing the expression of cyclinDl, CDK4 in cartilage cells, which furthered the cycle of cartilage cells over G1/S checkpoint, and then marched in the proliferation period.