| Extremity arteriosclerosis obliterans(ASO),a intractable peripheral vascular disease,is a severitydisease with extremity functional disturbance or even gangrene because of extremity ischemiaresult from arthrosclerosis(AS). Percutaneous transluminal angioplasty(PTA) is one of the effectivetreatments for curing ASO.However,restenosis will take place to about 30~50% patients 6 monthesafter PTA, which influences the long-term effect of this therapy enormously and has hinderedgeneralization and apply of PTA.So preventing and curing restenosis after PTA is the key of boostingthe long-term effect of PTA and is one of the problems focused by domestic and overseas researcherswho are engagedin angionosis study. Tong Mai Ning, prescribed by professor Chen Shu Chang, has the effect of supplyingQi,promoting bloodflow and circulation and alleviating pain,which is aeffective prescription to cureASO and prevent and cure RS after PTA. Animal experiment of prophase has showed that it hasbetter effect of anti- RS after PTA.The forming of RS is a very complex patho-process, which relateto multiple element and factor,and proliferation of vascular smooth muscle cell(VSMC)is one of thekey pathological component elements. This study took VSMC as target cell and made use of serumpharmacological method to observe the effect of whole prescription , supply Qi part and promotingblood flow part of Tong Mai Ning on proliferation of VSMC induced by angiotensin (Ang ) orlysophosphatidyl choline(LPC) and related factors so as to elucidate cellular and molecularmechanism of Tong Mai Ning preventing and curing RS after PTA.Serum containing medicine of whole or partial prescription of Tong Mai Ning was obtained fromrabbit 3 days after intragastric administration everyday.VSMC was isolated and culturedfrom aorta ofsuckling rabbit by stickingpieces of tissue. The 4th to5th generation of VSMCwas divided randomlyinto groups as follows: blank, AngII, LPC, whole prescription, supply Qi part and promoting bloodflow part.VSMC of every group was induced by AngII or LPC except for blank.1. Applying MTT colorimetric method to observe the effect of medicine on proliferation of VSMC.2. Observing the change regularity of α-actin in VSMC endochylema by immunohistochemical stain(DAB).3. To detect the quantitative alteration of cell cycle phase by flow cytometry.4. Using biochemical method to determinate total SOD activeness and MDA content of VSMC supernatefluid.5. Applying nitrate reductase method to observe the regulate effect of medicine on NO content and NOS enzyme activity of VSMC supernate fluid6. Intracellular calcium ion was evaluated by flow cytometry with Fluo-3 fluorescent probe marked.7. To detect the changes of PDGF-BB in in VSMC endochylema by immunohistochemical stain(DAB).8. Making use of RT-PCR to examine the expression of c-fos and c-myc semiquantitatively which is controlling gene of VSMC. The experimental results show Ang and LPC can promote the proliferation of VSMC throughpromoting cells going from G0/G1 to S phases in the cell cycle,regulating NO and MDA secretedbyVSMC attenuating NOS and SOD activity,degrading the express of α-actin in VSMC endochylema,increasing the concentration of intracellular calcium ion, augmenting the express of PDGF-BB inVSMC endochylema, up-regulating the expression of c-fos and c-myc. Tong Mai Ning can inhibitthe proliferation of VSMC through inhibiting cells going from G0/G1 to S phases in the cell cycle,decrease MDA secreted by VSMC increasing NOS and SOD activity and NO production,strengthening the express of α-actin in VSMC endochylema, decreasing the concentration ofintracellular calcium ion, promoting the express of PDGF-BB in VSMC endochylema,down-regulating the expression of c-fos and c-myc.So Tong Mai Ning can counteract the effection ofAng and LPC. supply Qi part and promoting blood flow part also have the effect of inhibitingproliferation of VSMC in some aspect, but through aggregate... |
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