| Pancreatic carcinoma is highly malignant tumor. Because of rapid progression, late diagnose and low rectal rate et al, presently people are searching for other method to treat the disease. Recent 20 years, the correlation between estrogen and pancreatic tumor is paid atten-tion. A series of study demonstrate the estrogen receptor not only high express in tumor of hormone target organ such as breast uterus ova-ry prostate et al . A lot of study proved that estrogen was relative with biological behavior of pancreatic tumor.Bel - 2 gene was discovered in human follicular lymphoma t frag-mentation in 1985 by Tsujimoto. It is protooncogene, and prevent cell from apoptosis, so that promote occurrence, development of tumor. Which express the Bel -2 proteins. Bax is Bel -2 homology protein. Now it was proved that Bax has been agitation protein to promote apop-tosis. Recent study show that Bel-2 expression not only He in B andT lymphocyte but also in other cell. In normal pancreatic tissue, Bel ?2 protein mainly express in pancreatic ductal epithelia cell, which is pancreatic carcinoma original cell. And a lot of study show Bel - 2 correlate to pancreatic carcinoma.Estrogen is related with pancreatic carcinoma , so is Bel - 2. It is unclear Whether estrogen is related with Bel - 2 or not. Our study object investigate antiestrogen drug TAM affect on the pancreatic car-cinoma Bxpc - 3 cell of high expressed estrogen receptor. In order to reveal that TAM inhibit the growth of pancreatic carcinoma cell, in-duce cell apoptosis by controlling the balance of Bel - 2 and Bax. I-dentify that estrogen correlate with Bel - 2 with the development of pancreatic carcinoma.Method1. Cell Culture :The Bxpc - 3 cell was maintaine inl0% fetal bovine serum ( FBS), at 37t: in a 95% room air and 5% CO2 humidity incubator.2. MTT Analysis of TAM inhibiting affect on Bxpc -3 cellThe cell (10 per sample) planted in 96 - well flatbottom was given different concentrative TAM, and incubated separetedly for 3, 6, 9 days, added into 10 ul MTT(5mg/ml) , then incubated4 hours , added into 200#l DMSO to solubilize the formazan, fifteen minutes later , the plates were read onmicroplate reader. Hie MTT results were used to calculate the inhibiting rate.3. Flow cytometric analysis of apoptosisThe cell (106 per sample) was added into 10-5 mol/L TAM until it was 24, 48, 72, 144 hours. The cells were detached from the plas-tic by trysinization and then washed in PBS. The cell were fixed in 70% ethanol and stored at 4?C until the analysis. The cells were trea-ted with RNase for 40 minites. Next, the cells were stained with pro-pidium iodide for 1 hour. FAC Scan flow cytometric assay the intensity of fluorecence. the content of DNA in cell cycle analysis were per-formed by cellquest software to achieve the apoptosis rate, then plot the curve of growth inhibition rate.4. Immunohistochemistry Analysis of Bel - 2, BaxThe Bxpc - 3 cell pasting on carry sheet glass were cultured for 24 hours , then added into different concentration TAM for 72 hours. Next, prepared sample. The samples wered stained by immunohisto-chemistry to detect Bel - 2 and Bax. Using hyperploidy microscope to observe 200 cells from different sight, calculate positive cell rate.5. The results -were showed by mean ?SD , t check - out and analysized by SPSS software.Result1. After MTT spected the TAM effected on the Bxpc - 3 cell, the cell growth apparently was inhibited. The curve of growth inhibition rate show the IC50 value of 3, 6, 9 days is separately (5. 03 ?. 08)x!04mol/L,(2. 18 ?.04) xl04mol/L, (1.82 ?.09) x 104mol/ L. Our study demonstrate the affect of TAM on Bxpc - 3 cell is de-pend - dose, depend - time.2. After TAM effected on Bxpc - 3 cell, the apoptosis rate in-creased with drug time extending. Comparing to the control, through t check - out difference is significant (P < 0. 01) . In addition, among the different group the difference is significant.3. After TAM affect on the cell for 72 hou...
|
PDF Full Text Request