| Objective:First,To study the stability and bioactivity of 125I-AS in vitro. Second,To investigate the AS receptor internalizing, capability of ECV304 cell line, and the lethal effect of 125I-AS in ECV304 cell line. Finally,To investigate the effect factor of ECV304 cell AS receptor internalizing.Methods:The extracted AS was validated, and then labeled with 125I; The labeling efficiency of the production was measured by paper chromatography. It was supported by Xinhua filter paper in double developing solution to detect the Rf value; The stability of 125I-AS was observed in bovine serum albumin (BSA),saline and cysteine(Cys); The bioactivity of 125I-AS was detected by inhibition experiment of vessel endothelial in ECV304 cell line.ECV304 cells were incubated together with 125I-AS at 37℃for different periods of time(0.25 h, 0.5 h, 1 h,4 h,8 h,16 h,20 h,24h), the amounts of internalized 125I-AS were detected withγcounter. By using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra -zolium bromide(MTT) assay, the lethal effect of various dosage of 125I-AS, free 125I and AS on ECV304 cells after 24h,48h,72h,96h incubation were measured. ECV304 cells were incubated together with 125I-AS at different temperature(4℃, 24℃a nd 37℃) for different periods of time(0.5h,1h,4h,8h,24h), the amount of membrane receptor internalized 125I-AS were detected withγcounter, respectively. ECV304 cells were incubated together with different concentration 125I-AS at 37℃for different periods of time(0.5h,1h,4h,8h,24h), the amount of membrane receptor internalized 125I-AS were detected respectively.Results:Measured by paper chromatography, the labeling efficiency of the production can achieve 85%;The radioactivity remained associated with the 125I-AS respectively declined by 2.3%,5.2%,8.0% after 1h,4h,24h in 0.9% saline solution, declined by4.6%,9.1%,11.0% in 10g/L BSA . The radioactivity can be decreased in different molar ration of Cys. It decreased by no more than 11% when the molar ration was 5 within 4 hours. It indicates that the production is stable in vitro. Treated ECV304 cells with 125I-AS and AS in various dosage and took 0.2 M 6-aminocaproic acid as control, the both had inhibition effects on ECV304 cell line but the effect of 125I-AS was stronger than AS. That indicates 125I-AS had better biological activity than AS.125I-AS was rapidly internalized mediated via the membrane receptor of ECV304 cells at 37℃. The receptor internalization ratio, membrane receptor binding ratio and total binding ratio of ECV304 cells showed time-dependent manner in 0.5h.The receptor internaliza -tion ratio(12.6±1.1)% of total radioactivity amount, membrane receptor binding ratio and total binding ratio of ECV304 cells(19.1±2.2)%, (31.7±1.6)% respectively at 0.5h(r=0.814, P<0.05). The receptor internalization ratio, membrane receptor binding ratio and total binding ratio of ECV304 cells (28.0±3.0)%(,10.8±1.4)%,(38.8±2.3)% respectively at 24h(r=-0.681, P<0.05). The internalization showed time-dependent manner with concomitant decrease of membrane binding. The supreme lethal effect was found at 96h with 370KBq/ml 125I-AS, 125I-AS has stronger growth-inhibit effect on ECV304 cells than Na125I or AS (F=312.9013 ,P<0.01). The ratio of internalized 125I-AS: at 4℃is lower, at 24℃is increased, at 37℃is higher. As the concentration increasing, the internalization ratio become lower at 37℃. The ratio of AS receptor internalized increasing as the time delays at any temperature and concentration.Conclusions:125I labeling AS by the method of Ch-T is simple and feasible; 125I-AS can inhibit the growth of ECV304 cell line; 125I-AS is stable in vitro and had better biological activity. 125I-AS can be internalized into ASR positive cells mediated by ASR. The ECV304 cells can be killed by Auger electron emitting from 125I-AS. The lethal effect showed does-dependent and time-dependent. The internalization of ECV304 cell AS receptor has be effected by the factor of temperature, time and 125I-AS concentration. At the range 4℃～37℃,the ratio of AS receptor internalized in time- and temperature-dependent manner. |
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