| Objects : To investigate the influence of danloutablets on apoptosis induced byrapamycin in human aortic endothelial cells, and try to exploreitspossible mechanism..Methods: 1. In different dose of rapamycin, in turn, effect on HAEC s, tetramethyl methyl thiazolyl tetrazolium method(MTT method) was developed for the determinat ion of HAEC cell proliferation inhibit ion rate; By calculat ing cell proliferation inhib it ion rate and proliferation inhib it ion curve drawing, comprehensive analysis of rapamycin affect HAEC s proliferation activity. 2. By different concentration of rabbit serum medicated and rapamycin comb ined action in HAEC s, tetramethyl methyl thiazolyl tetrazolium method(MTT method) was developed for the determinat ion of HAEC cell inhibit ion rate of change, for appropriate drug-containing serum concentration into the next phase of the experiment.. 3. using the flow cytometry, detection of cell apoptosis rate, to investigate the changes ofapoptosis rate and the common effect of serum containing rapamycin under. 4. The real- time fluorescent quantitative PC R technology and detection of intracellular protein immunoblot technology related to apoptosis(C aspase 3, Bax, Bcl- 2) gene and protein expression, the effect of using protein immunoblot technology to detectp- Akt protein expression changes in PI3 K /Akt pathway. Results : 1. rapamycin has marked inhibit ion to cell growth in HAEC s display, compared with thenegative control group, the difference was statistically significant(P<0.05), and he doseand time dependence.. 2. Each group after treatment with different concentrations of serum medicated, in 12 h, 24 h, 48 h point time, the blank rabbit serum containing 5%, 5%, 15% concentration of rapamycin group compared with the corresponding bla nk group, the cell surviva l rate decreased, with statistical significance(P< 0.05), 5%, 15% concentration of Danlou tablets gro up compared with rapamyc in group, the cell surviva l rate rise, and statistically significant( P<0.05), but no statistical difference was found between cell surviva l wit h Rosuvastatin group(P>0.05), 5%, 15%, Mixed group compared with the Rosuvastatin and Da nlou tablets group cell surviva l rate increases, and were statistically s ignificant( P<0.05). 15% medicated serum concentration under the action of each cell surviva l rate is highest, medicated serum concentration and dose not exist between the cell survival rate.. 3. Each group chooses 1 ng/ml concentrations under the action of rapamycin, 15% medicated serum concentration process cells for 24 h, flow cytometry instrument to detect apoptosis rate, Danlou tablets group compared with rapamyc in apoptosis rate(18.23±1.36 vs23.23±0.76, p<0.05), Danlou tablets group and can be no significant difference between Rosuvastatin group(18.23±1.36vs17.48±0.66, p>0.05), Mixed group compared with Danlou tablets group and Rosuvastatin group, can be set, reduceapoptosis rate( 14.87±0.61vs18.23±1.36, p>0.05; 14.87±0.61 vs 17.48±0.66, p< 0.05). 4. Rt-PC R detection 1 ng/ml concentration under the action of rapamycin 15% medicated serum concentration processing cell apoptosis related gene expression after 24 h, Danlou tablets group compared with rapamycin group Bax(4.46 ±0.45 vs 5.24±0.56), Caspase-3(2.37±0.25vs3.96±0.22,) reduced the volume of expression(p<0.05), the Bcl- 2 expression increase(0.58 ±0.06 vs 0.47±0.08, p<0.05), Danlou tablets group compared with Rosuvastatin group,the Bax,Bcl- 2, C aspase-3 expression had no statistical difference(p>0.05); Mixed group compared with Danlou tablets group Bax(2.88 ±0.47vs4.46±0.45), C aspase-3(1.63±0.28vs2.37± 0.25,) expression quantity reduced( p<0.05), the volume of the Bcl- 2 expression increased(0.83 ±0.08 vs 0.58±0.06, p< 0.05). 5. Western blot detection 1 ng/ml concentrations under the action of rapamycin 15% medicated serum concentration process cells apoptosis protein expression and p- Akt protein expressionafter 24 h, Danlou tablets group compared with rapamycin group Bax(63.26 ±5.06vs77.92±5.28), Caspase-3(112.24±4.81vs147.14±7.94) reduced the volume of expression( p<0.05), the Bcl-2 expression increase(34.13±13.23vs11.66±10.40, p<0.05), Danlou tablets group campared witn Rosuvastatin group,the Bax Bcl- 2, C aspase- 3 expression had no statistical difference(p>0.05). Mixed group compared with Danlo u tablets group Bax(31.76±5.75vs63.26±5.06), C aspase- 3(76.25±3.88vs112.24±4.81,) reduced the volume of expression( p<0.05), the Bcl- 2 expression increase(63.32 ±11.68 vs 34.13±13.23, p<0.05).Danlou tablets group compared with rapamyc in group, reduced expression of p- Akt(97.90±15.71vs134.56±20.21, p<0.05), Mixed group campared with Danlou tablets groupp- Akt expression increase(58.83 ±6.09vs97.90 ±15.71, p<0.05), Danlou tablets group and Rosuvastatin group c an be compared with no significant difference(97.90 ±15.71vs83.15±9.99, p> 0.05). Conclus ion: 1. rapamycin has pro apoptotic effect on HAECs, and the dose and time dependent,the greater the dose, the longer the time, the greater the inhibitory rate of cell.Alphaasarone inhib its growth of huma n esophageal carcinoma cells by proliferation inhibition,apoptosis promotion and direct killing. 2. Danlou tablets can suppress the HAEC s apoptosis induced by rapamyc in, when rapamycin is 1 ng/ml, 15% concentration of medicated serum inhib it apoptosis effect is best. 3. Dan fructus piece can increase antiapoptotic genes are expressed the Bcl- 2, by promoting apoptosis gene Bax, caspase 3 expression, suppress the HAECs apoptosis induced by rapamycin. 4. Danlou tablets can regulate the expression of p- Akt, that its HAEC s apoptosis of rapamycin induced under protection may through PI3 K /Akt pathway. |
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