| Objective:We probe the protection and impossible mechanism of danshensu(DSS) on atherosclerosis and base the application of Chinese materia medica on experiment of modern medicine and propagate pharmaceutical sciences of fatherland by studying the effects and mechanism of danshensu on rat vascular smooth musle cell proliferation and apoptosis induced by ox-LDL.Method:1. The primary culture and identification of rat arterial smooth muscle cell : The VSMCs of Wistar rat thoracic aorta were obtained by a repeatedly blood vessel patch-attaching method.The cells were identified by inverted microscope and immunohistochemistry.The 5-10 generation cells were used in experiment.2. The preparation of ox-LDL: The LDL was purchased and the protein level was assayed by Lowry method. LDL was oxidizd by CU2+.The MDA level of LDL and the treated LDL were detected to comfirm the oxidation of LDL.3.The preparation of DSS: The 10mg DSS was dissolved in 5ml PBS and filtered by 0.22μm micropore film then store in 4℃.4.The effects of different concentration of ox-LDL on the growth of VSMCs: Exponential growing cells were harvested in 96-well plates with 2x103 cells every hole.All cells were divided into 10 groups , 6 bore each group.The cells was cultured with 10% fetal bovine serum (FBS) DMEM ,2 days later with DMEM,the next day with 5%FBS DMEM and each group was handled as follow: each group deal s with 0,20,30,40,50,60,70,80,90,100mg/l of ox-LDL.24 hours later,the OD value were detected by MTT method.The experiment was repeated 3 times.The growth curve was drawn with average OD value of every hole of each group.5. The effects of different concentration of danshensu(DSS) on the proliferation of VSMCs induced by low concentration ox-LDL:The cells were harvested in 96-well plates with 2 x103 cells every hole, 4 groups ,6 bore each group. The cells were cultured as same as the above for 3 days then handled as follow:①5%FBS DMEM;②5%FBS DMEM + 50mg/l ox-LDL;③5%FBS DMEM + 50mg/l ox-LDL+50 mg/L DSS;④5%FBS DMEM + 50mg/l ox-LDL+100 mg/L DSS. 20 hours later,the cells proliferation were detected by MTT method.The experiment was repeated 3 times.The cells was harvested in 4 bore of 6-well plates with 6x104 cells every bore.The cells were cultured as same as the above for 3 days then handled as same as the above.20 hours later,the cells m-RNA of of each bore were extracted and the expression of PDGF-BB m RNA of each bore was detected by RT-PCR method.The experiment was repeated 3 times.6. The effects of different concentration of danshensu(DSS) on the apoptosis of VSMC induced by high concentration ox-LDL: The cells were harvested in 4 bore of 6-well plates with 6x104 cells every hole, the cells was cultured for 3days then each bore was handled as follow:①5%FBS DMEM;②5%FBS DMEM + 100mg/l ox-LDL;③5%FBS DMEM + 100mg/l ox-LDL+50 mg/L DSS;④5%FBS DMEM + 100mg/l ox-LDL+100 mg/L DSS.The morphous of cells of each bore were observed and apoptosis index of each bore was detected by flow cytometry.The experiment was repeated 3 times.7. Statistical analysis: The SPSS statistical package (version 11.5)was employed to test the difference using univariate analysis of S-N-K and q test.Results:1. The primary culture and identification of rat arterial VSMCs : The VSMCs were cultured by a repeatedly blood vessel patch-attaching method,large number of VSMCs were obtained in a short time.3 days later,the cells radiatively and diversified grew around the blood vessel patches that were well attched.4 to 10 days later, the cells grew in number and parallelly converded into fasciculation or radiation.The specificpeak or valley feature of VSMCs was abvious.Cells grew condensely and passaged. Immunohistochemistry stain:myofilament appeared in cytopiasm which spread parallelly and longitudinally with dense area.Positive VSMCs (brown granule)stained by immunohistochemistry were found.2. The growth characteristics of the primary rat arterial smooth muscle cells :The local cells were so dense that can be passeged by 1:1 or 1:2.Several hours after passage, the adherence of some cells were observed.After 3-4 days the cells overgrow and could be passeged by 1:3 or 1:4.3. The preparation of Oxidative Low Density Lipoprotein:The content of ox-LDL detected by lowry method was 6.0mg/ml which has volum of 330μl.The MDA of LDL and treated LDL was 1.5μmol/L and 17.2μmol/L which verified that LDL was oxidized into ox-LDL4. The effects of different concentration of ox-LDL on the growth of VSMCs :MTT test showed that OD value reached to the peak at 50mg/l ox-LDL group then decreased with the increasing concentration of ox-LDL.There was apoptosis being observed in 100mg/l ox-LDL. so intervention concentration of ox-LDLon prolifertion was 50mg/l and intervention concentration of ox-LDL on apoptosis was 100mg/l.5. The effects of different concentration danshensu(DSS) on the proliferation of VSMC induced by ox-LDL: MTT test : OD value of the second group cells increased.DSS decreased the OD value with dose dependent.The OD value of each group had significant deviation.RT-PCR: The expression of PDGF-BB m RNA in each group had significant deviation,DSS decreased the expression of PDGF-BB m RNA increased by ox-LDL with dose dependent.6. The effects of different concentration DSS on the apoptosis of VSMCs induced by ox-LDL: The cells morphology was observed as follow:①Tipical growth characteristics of smooth muscle cells observed. Cells were fusiform and ranked in monolayer and fasciculation;②Cells membrane were vague, the shape of cells were round , the cells bubble increased;③some cells were small and round and detach from wall ;④the exfoliation and necrosis of cells were observed,many of the cells fall off wall.The apoptotic index of each group had significant deviation. DSS increased the apoptotic index induced by 100 mg/l ox-LDL with dose dependent. Conclusions:①Repeatedly blood vessel patch-attaching method for primary culture of rat aortic VSMCs is simple ,low-cost way to obtain cells in a shorter cycle time.②DSS can dose dependently inhibit the proliferation of VSMC induced by low concentration of ox-LDL through decreasing the expression of PDGF-BB m RNA of VSMCs.③DSS can dose dependently promote the apoptosis of VSMC induced by high density ox-LDL.
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