| Objectives: To study the relationship between BCL-2 gene and clinical stage of bladder cancer by detect BCL-2mRNA in low stage (Tis-1)and high stage(T2-4) bladder cancer, and to test the effect and mechanisms of BCL-2antisense oligonucleotides on bladder cancer EJ cell line in vitro and in vivo , and investigate the possibility and the pathway and the approach of bladder cancer therapy with antisense oligonucleotides of BCL-2 gene.Methods: In the first part of this experiment, BCL-2mRNA have been detected in 8 low stage (Tis-1) and 10 high stage(T2-4) bladder cancer samples using RT-PCR methods, optical density was scanned after electrophoresis and an area curve of optical density was drawn to analyze its levels of BCL-2mRNA.In the second part of this study, Fully phosphorothioated BCL-2 antisense oligonucleotides (BCL-2PS-ASON) with sequence 5/-tctcccagcgtgcgccat-3/ which can recognize the first exon of BCL-2 (this oligonucleotides sequence was complementary to the first six codons of mRNA of BCL-2 gene ) were added to bladder cancer EJ cell line served as experiment group and adding fully phosphorothioated BCL-2 sense oligonucleotides (BCL-2PS-SON)with sequence 5/-taccgcgtgcgaccctct-3/ served as the negative control group ,without any drug in cells served as blank control group；Before experiment and when 12th,24th,48th,72nd hours of experiment,we used microscope to observe morphologic changes and the growth condition of cell, and performed MTT analysis to measure inhibiting rate,and analyzed the apoptotic rate by flow cytometry and used electron microscope to observe the changing of microstructure. Colony formed and nude mouse transplantation neoplasia experiment was performed with the cells of after 72hs experiment mention above to evaluate the　effect of drug on the ability of　cells neoplasia; Another experiment groups of adriamycin and BCL-2PS-SON plus adriamycin and BCL-2PS-ASON plus adriamycin were built ,MTT assay was performed to measure inhibiting rate and flow cytometry was used to examine apoptotic rate to compare inhabitation rate of cell proliferation and apoptosis rate 12h after experiment between these groups；In the third part of this experiment, nude mice which beared tumors were injected with BCL-2PS-SON and BCL-2PS-ASON respectively ,Put to death after two weeks ,tumors were dissected and weighted,then pathological examination was performed to compared the inhibited effect。Results: The results of first part demonstrated that expression levels of BCL-2mRNA in low stage (Tis-1)and high stage(T2-4) bladder cancer were 1.173±0.779 and 0.462±0.304 respectively, and there was significantly difference between low stage and high stage bladder cancer .The results of second part indicated that 1,at all time points analyzed, the inhibitory rate of cell proliferation in experiment group and negative control group were 2.97%, 4.29%, 6.00%,8.45% and 2.00%,2.86%,3.50%,4.58% respectively ,there was no significantly difference between negative control group and experiment group(P>0.05),this results indicated that BCL-2PS-ASON had no effect on the rate of cell growth.2,There were no significant peak of apoptosis between monoploid and diploid both in negative control group and blank control group; there was not significantly different from negative control group and blank control group(P>0.05). There were significant peak of apoptosis between monoploid and diploid in experiment group; The apoptosis rate of experiment group were 5.82%±1.23% and 14.50%±2.56% and 24.52%±3.585 and 31.90%±4.66% respectively ,there were significantly difference between experiment group and two control groups. 3,The apoptosis rate and the inhibitory rate in adriamycin and BCL-2PS-SON plus adriamycin and BCL-2PS-ASON plus adriamycin groups were 9.22±2.13%,9.31±2.01%,23.45±4.26% and 8.84%, 9.68%,17.87% respectively,BCL-2PS-ASON plus adriamycin groups were significantly different from other groups(P>0.05);12 hours after drugs added, some cells of the...
|
PDF Full Text Request