| ObjectiveTumor is a kind of disease that is characterd by the maladjustment of cell division,cell death;cell and cell with extra cellular matrix.The occurrence of tumor is associated with cell apoptosis and proliferation.. Survivin,a novel inhibitor of apoptosis,is a unique member of inhibitor of apoptosis protein. Survivin is attentioned abroad by tumor researcher for it's uniqure structure, special tissure distribution and anti-apoptosis.Bladder cancer is the most common tumor in urinary system. 50 — 70% patients will be recurrence after operation, and 10~20% become more malignant. The pathological stages and cell classification are very important to the diagnosis , treatment and prognosis clinically. There is no true medical marker , So it's important to find a bladder tumor marker in clinic.There are few tumor marker for the early dignosis of cancer now, so it's more significance to early dignosis , especially no wound dignosis. The gold standard to the dignosis of bladder cancer are endoscope and urine cytology on clinic. There have invasion , painness and more expensive with endoscopy examination. There has high specify with urine cytology ,but the sensibility is only 40~60%. Apoptosis plays important role on normal cell conformation and inner environment. The weak of apoptosiscan enhance tumor invision and metastasis . Singular enhancing cell ex-sistance can enduce gene mutation and the resistivity of cell toxicity based on immunology. The early reseach was focus on Bel — 2 family. Bel — 2 was the first discovered anti apoptosis gene which can promote cell survival cycle. Besides, the wide tissure distribution of the family of the inhibitor of apoptosis proteins, IAPs , and the antiapoptosis cause more recognition, play important role on celll apoptosis.Survivin, a novel inhibitor of apoptosis , is a member of the inhibitor of apoptosis protein (IAP) family expressiong on many cancers. Survivin is a 16. 5kDa member of the inhibitors of apoptosis. Its chromosomal location is at 17q25 ,approximately 3% of the distance from the telomere,and it is comprised of 3 introns and 4exons, encoding 142 amino acids. Its' specity of expression is in the first yourth of the gene expression over 100 tumors. Survivin both inhibit cell apoptosis and regulate cell proliferation in tumor forming and expressed in cancer cells, being the poor prognosis marker of some tumor.Our experiment is to study the expression of suvivin in bladder cancer and in urine and the early dignosis marker of bladder cancer , predict the prognosis of bladder cancer, and also to find the apoptosis way of MMC , HCPT to bladder tumor and the influence to the expression of suvivin.MethodsThere are 40 cases of bladder cancer. All cases were diagnosed by en-doscope and pathology. No treatment of chemical, radiology cure or immunology were done before operation. There are 32men and 8women, aging form 48~60 years old, mean 65. 33 cases is original tumor and 7 cases is recurrence . The clinical stages is divided according to UICC. The superficial tumor (Tis~Tl) is 22 cases, and invasive tumor (T2—T4) is 18 cases. The cell classification is divided according to WHO . grade I is 16, grade II is 18, grade III is 6. The normal bladder tissue was obtainedduring operation of benign prostate hyperplasia. Immunohistochemistry was used to examinate the expression of survivin;RT—PCR was used to exmaninate the expression of survivin mRNA with the consultation of |3— actin . GIS—700D gel image management system was used to scan the result of electrophoresis. Western blot was used to survey the expression of survivin protein , and the results was scaned by gel image management system.Urine from 30 subjects in normal people,30 in patients with bladder cancer,30 with other urinary diseases,20 after treatment of bladder cancer were collected. All patients were diagnosed with cytology, pathology. All urine were centrifuged and cell exfoliated cell were collected and total RNA were obtained. Proliferation of PCR was carried out to detect the expression of survivin mRNA . GIS—700D gel image management system was used to scan the result of electrophoresis.Cell cultureHuman bladder cancer cell line(EJ) were incubated (37°C,5//oC02, 100% humidity) with 10% nonfat dry milk and RPMI1640 in penicillin (lOOU/ml) , streptomycin( lOOmg/ml).Cell suspension was added into slide, and immunohistochemistry was carried out. Another immnohistochemistry was done again using the cell suspension treated with MMC,HCPT(0. 20 mg/ml,48h) ,and compared the positive rate of survivin. 1000 cells was counted randomly under microscopy (10X40) in a slide. The cell positive rate is the number of positive cell /total cell X100%. If the number of positive cell is over 5% ,the result is positive, 5 %~15 %( + ) ,15 %~30 %(2 + ) ,30 %~45 % (3 + ) , > 45 %(A + ) .Results1. Immunohistochemistry results, the superficial tumor of expressionof survivin (Tis - Tl) was 66. 6%i and the invasive tumor (T2 —T4) was 77. 2%. cell classification are: G1S57. 1%,G2:56. 3%,G3:60%. There were no expression of survivin on normal bladder tissure.2. Western blot result;a 16. 5 Kd protein was detected with Western blot, and there was no detection on normal bladder tissure.3. RT—PCR of bladder cancer : a 279bp product was detected with RT—PCR on all bladder cancer, and there was no products on control group.4. RT — PCR products of urine were detected in group of bladder cancer corresponding to an expected 279bp product. There were no products in group of normal control and other urinary diseases.5. The cell apoptosis rate of MMC was similar to that of HCPT . There were significance change of the positive rate of survivin about Im-munohistochemistry before and after using MMC(P0.05).Conclusions1. The over expression of survivin was associated with the occurrence and development of bladder tumor.2. The expression of survivin was not associated with pathology stage and cell grade.3. The expression of suvivin in urine is along with that in bladder cancer4. Survivin may become the early diagnosis marker of bladder cancer.5. Both MMC and HCPT can cause cell apoptosis , and MMC may inhibite caspase—3 through survivin, but HCPT cann't, it may cause cell apoptosis in another way.
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