Examination Of Survivin And VEGF In Bladder Cancer And The Treatment Function Of Their ASODN On Bladder Cancer

Bladder cancer is the most common cancer of urology in China.Its incidence increases year by year.About 70% of bladder cancer is superficial cancer.Exairesis is the main treatment method.But recurrence rate is high.About 60% will recur in 5 years and 88% will recur in 15 years.10%~30% of recurring cancer will be infiltrating type and this kind of patients need total resection of bladder.The traditional treatment method of bladder cancer is operation, chemotherapy,radiotherapy and biotherapy.Combined therapy is the main method to increase therapeutic effect in clinic.Combined therapy can reduce relapse rate and increase quality of life.Adjunctive therapy after operations is the key to treatment and the hot spot of research.Adjunctive therapy of bladder cancer mainly includes chemotherapy and biotherapy. Chemotherapeutics can only relieve relapse of tumor and has few effect on tumor development.Biotherapy have showed enormous potentiality in animal experiment and clinical experiment.Biotherapy is the only hope of inhibiting tumor progression now.It includes immunostimulation of extrinsic source,cytokine,tumor vaccine,monoclonal antibody and gene therapy.The technique of antisensenucleic acids is a totally new technique in molecular biology since gene cloning and recombination.Antisense oligonucleotide(ASODN) technique,in other words,antisense DNA technique is to use an artificial section of special oligonucleotide which can be complementation fixation with DNA or RNA.ASODN can specially inhibit gene expression.The mechanism of action of ASODN mainly includes 2 ways. Oligodeoxyri- bonucleotide is designed and synthesized. Oligodeoxyribon- ucleotide is incorporated in special area of genome.Triplex DNA,D loop or oese function to transcription factor forms to inhibit or close expression of target gene.On the other hand,ASODN can combine with mRNA and form duplex structure of DNA and RNA to stimulate internal ribonuclease H(RnaseH).RnaseH can specially degrdn mRNA of crossbred and stop translation of mRNA. Zammeenik was the first scientist to use ASODN.Since then,a lot of studies have showed that ASODN can specially inhibit expression of gene in vitro and in vivo.Combination of ASODN with traditional chemotherapeutic medicines is the main method in tumor study now.Now there are few domestic and overseas studies about treatment effect of ASODN of survivin and vascular endothelial growth factor(VEGF) on bladder cancer.Patients with bladder cancer and T24 of bladder cancer cell line were selected in our experiments.Expression of survivin and VEGF were examined and the function of their ASODN on bladder cancer was observed.Initial study about the treatment function of their ASODN on bladder cancer was done.1 The examination of survivin and VEGF in bladder cancer82 samples of bladder cancer were got by exairesis.Examination of survivin and VEGF was done by immunohistochemistry of SP and Western blot.Microvessel density(MVD) of sampls of bladder cancer was measured.Hybridization in situ was done by oligonucleotide probe of survivin.Tissues of bladder cancer were detected by SABC.Apoptosis was examined by TUNEL.The results have demonstrated:(1)There was significant difference in expression of survivin in tissues of bladder cancer and normal bladder(P<0.01).The expression of survivin and VEGF had dependablity (P<0.05).Positive signals of survivin mRNA were mainly in cytoplasm.(2)There was strong positive expression of survivin and VEGF in bladder cancer.There was no expression of survivin and there was weakly positive expression of VEGF in control.(3)MVD of bladder cancer was significant different from that of control(P<0.01).(4)Positive staining of caspase-3 was mainly in cytoplasm.There was positive expression of caspase-3 in 56 cases of bladder cancer.Apoptosis cells could be seen in 51 cases of bladder cancer.The distribution of apoptosis cells was diffused.Fragmented or pyknosis nuclei were stained with brown or yellow brown.Cytoplasm was stained with no color or light color.Inspissate cytoplasm sticked to nuclear membrane.There was chromosome condensation or aggregation around the nuclei.There was positive expression of caspase-3 and survivin mRNA at the same time in 56 cases of bladder cancer.Apoptosis index of negative survivin group was significantly higher than that of positive survivin group(P<0.01).In conclusion,there was high expression of survivin in bladder cancer and the expression of survivin had intimate correlation with VEGF.2 Effect of survivin targeting on cell proliferation and apoptosis in bladder cancer cellsThe T24 cell line of transitional cell carcinoma of bladder was cultured conventionally.Oligonucleotide was transported with liposome.Different concentrations of compound of oligonucleotide with liposome(ODNS-Lip) were made.Expression of survivin was done by Western blot.Cell inhibition rate was detected by MTT.Changing of cell shape was observed by transmission electron microscope.Apoptosis was detected by flow cytometry. Apoptosis morphology was observed by fluorescein stain.Expression of caspase-3 was detected by SP immunohistochemistry. The results have demonstrated:(1)Expression level of survivin decreased with increasing concentration of ODNS-Lip.Comparing with sense oligonucleotide and control group,difference was significant(P<0.01).(2)Growth inhibiting rate increased with increasing concentration of ODNS-Lip and time.Comparing with sense oligonucleotide and control group,difference was significant (P<0.01).(3)Recovery of cytoplasm could be seen under inverted microscope after T24 cells was cultured with different ODNS-Lip.Cell body change round.Some cells became suspension cells.Cell volume contracted under transmission microscope.There were karyopyknosis, chromatin condensation and swelling of mitochondria.Vacuolus increased in cytoplasm.Some nuclei broke into pieces and apoptotic body formed.(4)Typical hypodiloid could be detected by flow cytometry and peak value increased with time.Comparing with sense oligonucleotide and control group,difference was significant (P<0.01).(5)Apoptosis cells increased with increasing of ODNS-Lip concentration after 48 hours.Comparing with sense oligonucleotide and control group,difference was significant(P<0.01).(6)Expression of caspase-3 increased with increasing of ODNS-Lip concentration after 48 hours.The increasing was concentration dependent.Comparing with sense oligonucleotide and control group,difference was significant(P<0.01).In conclusion,ASODN of survivin could regulate down expression of survivin.The expression of survivin was concentration dependent.ASODN of survivin which was transfected with liposome could induce apoptosis of bladder cancer cells.Apoptosis rate was partly time dependent.3 The effect of VEGF antisense oligonucleotide combined with low molecular weight heparin on the growth and metastasis of mice bladder cancerThe T24 cell line was cultured conventionally.Mouse models of bladder cancer were made.Each group was randomization.Histopathologic slides of tissues were made and observed after treatment.Microvessel density of tumor was measured.Expression of VEGF was detected by Western blot. The results have demonstrated:(1)The tolerance of mice for treatment was good.There was no case of bleeding and adverse side effect.Only 1 mouse died before operation.Mice weight of ASODN group,LMWH group and therapeutic alliance group was more than those before experiment.All the mice had tumors in 9 days.Tumors in control group and MSODN group appeared earlier than those in ASODN group and therapeutic alliance group.(2)Tumor cells in each group was inequality size after HE staining.There was abundant cytoplasm.Nuclei were big and deeply stained.There were abundant necrosis foci in ASODN group,LMWH group and therapeutic alliance group.Borderline effect was obvious.There was karyolysis.Intercellular substance was less than that of control group and MSODN group.(3)There was metastasis to lymph node of pelvic cavity under light microscope after HE staining.(4)MVD of ASODN group and therapeutic alliance group was lower than those of control group and MSODN group(P<0.01).MVD of LMWH group was lower than those of control group and MSODN group(P<0.05).(5)There was high level expression of VEGF in control group and MSODN group,which was examined by Western blot.Expression of VEGF was down-regulated in ASODN group and therapeutic alliance group.In conclusion,tumor growth was obviously inhibited in ASODN group and therapeutic alliance group.The inhibition was more evident in therapeutic alliance group.There was no inhibition of tumor growth in MSODN group.There was partly inhibiton of tumor growth in LMWH group.There was no adverse side effect of VEGF ASODN and LMWH.Metastasis rate decreased in ASODN group,LMWH group and therapeutic alliance group.