The Research On Vascular Endothelial Cell (VEC) Injury Model Of Blood Stasis Syndrome (BSS) Associated With Hypertension Disease

ObjectiveTo establish the vascular endothelial cell(VEC) injury model of blood stasis syndrome(BSS) associated with hypertension disease, to observe the model cells' change of activeness, shape, ultrastructure and function, and to provide reference in the methodology for the establishment of traditional Chinese medicine(TCM) cell model based on integration of disease and syndrome.Methods1. The activeness of cultivated human umbilicus vein endothelial cells(ECV-304) were assayed by MTT chromatometry, which were incubated by the different concentrations' (20%, 15%, 10%, 5%) serum of patients with BSS associated with hypertension disease for different time(6h, 12h, 24h, 48h), and finally we select that the serum concentration is 10% and the time is 24h to establish the model; The effect of tanshinoneâ…¡A(Tanâ…¡A) of different concentrations on the activeness of ECV-304 injured by 10% concentration serum of patients with BSS associated with hypertension disease was investigated.2. The change of model cells' shape was observed by the inverse phase contrast microscope and that of model cells' ultrastructure was observed by the transmission electron microscope(TEM) and the scanning electron microscope(SEM); The effect of Tan IIA of 10μg/ml on the shape and ultrastructure of model cells.3. Model cells' intracellular free calcium ([Ca²⁺]i) was assayed by laser scanning confocal microscope(LSCM); The effect of Tanâ…¡A of different concentrations on [Ca²⁺]i of model cells was investigated.4. The injury symbols of VEC: vWF, TM and EPCR excreted by model cells were assayed by enzyme-linked immunosorbent assay (ELISA); The effect of Tanâ…¡A of different concentrations on vWF, TM and EPCR excreted by model cells was investigated.5. The nitric oxide(NO) excreted by model cells was assayed by nitric acid deoxidizing enzyme method, and the endothelin(ET) was assayed by non-balance method; The effect of Tanâ…¡A of different concentrations on NO and ET excreted by model cells was investigated.Results1. After the serum acted on cells for 24h, the cells' activeness was lower in the blood stasis group and the non-blood stasis group than in the health group and the control group, the difference is distinct(P＜0.05 or P＜0.01); when the serum concentration is 10% or 5%, the cells' activeness was lower in the blood stasis group than in the health group, the difference is distinct(P＜0.05 or P＜0.01); Tanâ…¡A had protective function to cells' activeness when 10% serum of patients with BSS and it acted on cells at the same time, and when its' concentration is 10μg/ml, the effect is obvious.2. By the inverse phase contrast microscope, we discovered that: cells in the model group shrank, the intercellular space increased, dark granules existed in the cytoplasm; by the TEM we discovered that: in the model group, there were only slight tiny flosses in cells' surface, the amount of vacuoles increased, the amount of rough endoplasmic reticula(RER) decreased, RERs expanded, ribsomes lost partly, smooth endoplasmic reticula(SER) expanded obviously and assumed vacuole shape, mitochondria were thin and small, ridges were unclear or disappeared; by the scanning electron microscope(SEM) we discovered that model cells shrank, separated and become radiate star body.3. Fluorecence intensity (FI) was higher in the model group than in the health group, the difference is distinct (P＜0.05); FI was higher in the model group than in the control group, the difference is distinct (P＜0.01); there was no distinct difference between the model group and the non-blood stasis group (P＞0.05); FI was lower in the Tanâ…¡A group than the model group, but there was no distinct difference (P＞0.05).4. vWF, TM and EPCR secreted were higher in the model group, the non-blood stasis group and the health group than in the control group; vWF, TM and EPCR secreted were higher in the model group, the non-blood stasis group than in the health group; thereinto, the difference is distinct between the model group and the health group(P＜0.05 or P＜0.01); Tanâ…¡A of some concentrations can decrease the amount of vWF, TM and EPCR secreted by the model cells, there was distinct difference between the Tanâ…¡A group and the model group(P＜0.05 or P＜0.01).5. NO secreted was lower in the model group than in the control group and in the health group, the difference was distinct(P＜0.05 or P＜0.01); ET secreted was higher in the model group than in the control group , the health group and the non-blood stasis group, the difference was distinct(P＜0.05 or P＜0.01); NO secreted was lower in the Tanâ…¡A groups of 40, 20μg/ml and higher in the Tanâ…¡A groups of 10, 5μg/ml than in the model group, but no distinct difference was shown; ET secreted was higher in the Tanâ…¡A group of 40μg/ml and lower in the Tanâ…¡A groups of 20, 10, 5μg/ml than in the model group, but no distinct difference was shown too.Conclusions1. According to MTT chromatometry, we can filtrate the condition of duplication for cell model of BSS associated with hypertension disease. The patients' serum concentration is 10% and the acting time is 24h;2. Model cells have activeness, shape, structure and function injury;3. The injury is more serious in the model group than in the health group;4. Tanâ…¡A which is Chinese traditional medicine monomer with activating blood function can lighten this kind of injury;5. The injury which the serum of patients with BSS bring on the cultivated VECs (change out of body) has uniformity to the VECs' injury existed in the patients or animals with BSS(change in body);6. The VEC injury model of BSS associated with hypertension disease can be established successfully.