膀胱移行细胞癌患者尿脱落细胞Survivin mRNA表达的研究
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摘要
目的：探讨Survivin mRNA在膀胱移行细胞癌患者尿脱落细胞中的表达及其意义。
方法：采用巢式逆转录聚合酶链反应（巢式RT-PCR）检测：1.膀胱癌细胞株T24 Survivin mRNA表达。2.56例TCCB患者、20例非膀胱的泌尿系肿瘤患者、10例非泌尿系肿瘤疾病患者及10例健康志愿者尿脱落细胞Survivin mRNA表达,β-actin作为内参照。
结果：1.膀胱癌细胞株T24 Survivin mRNA表达为阳性。2.56例 TCCB患者中51例尿脱落细胞Survivin mRNA阳性表达，阳性率为91.1%，对照组40例中有1例血尿待查病人Survivin mRNA阳性表达，阳性率为2.5 %，TCCB组与对照组Survivin mRNA阳性率有统计学差异（p<0.01）。检测尿脱落细胞Survivin mRNA表达诊断TCCB，敏感性为91.1%，特异性为97.5%。按临床分期尿脱落细胞Survivin mRNA表达率T1期为90.5%（19/21），T_2-4期 91.4%（32/35），尽管T_2-4期表达率稍高于T1期，但两者差异无统计学意义(p>0.05)。按病理分级尿脱落细胞Survivin mRNA表达率G_1为90.6%（29/32），G_2为90.9 %（10/11），G_3为92.3 %（12/13），尽管随分级增加Survivin mRNA表达率增加，但三者之间Survivin mRNA表达率差异无统计学意义(p>0.05)。对49例行保留膀胱手术的TCCB患者复查尿脱落细胞Survivin mRNA表达，其中46例术前尿脱落细胞Survivin mRNA
阳性表达患者术后阳性表达5例，阳性表达率为10.9%（5/46），3例术前尿脱落细胞Survivin mRNA阴性表达者术后均阴性表达。本组有11例患者获得随访6个月，其中术后尿脱落细胞Survivin mRNA阳性表达3例，阴性表达8例，均无复发，对照组中1例血尿患者尿脱落细胞Survivin mRNA阳性表达，随访6个月后经膀胱镜检发现可疑病灶，取活检和手术后病理证实为T1期TCCB。
结论：1.巢式RT-PCR检测尿脱落细胞Survivin mRNA表达诊断TCCB敏感性和特异性高，无创伤性，Survivin可作为TCCB诊断的一个敏感指标。2.尿脱落细胞Survivin mRNA表达与TCCB的临床分期、病理分级无关。
Objective: To determine if detection of Survivin mRNA expression in urine by using nested RT-PCR can provide a novel noninvasive tool for the diagnosis of transitional cell carcinoma of the bladder .
Methods: Survivin mRNA in T24 bladder cancer cell line, exfoliated urinary cells of 56 patients with TCCB, 20 patients with urinary cancers except bladder, 10 nonneoplastic urinary diseases patients and 10 healthy volunteers were detected. Survivin and β-actin were amplified by using nested RT-PCR.
Results: 1. T24 bladder cancer cell line showed positive Survivin mRNA signal. 2. Only 1 of 40 (2.5%) showed positive Survivin mRNA signal in urine from control group. 51 of 56 (91.1%) patients with TCCB showed positive Survivin mRNA signals in urine. The Survivin mRNA expression difference between experimental group and control group was significant (p<0.01). The positive rate of Survivin mRNA in T1 was 90.5%（19/21）,T_2-4 91.4%（32/35）；G_1 90.6%（29/32）,G_2 90.9%（10/11），G_3 92.3%（12/13）.5 of 46
preoperative Survivin mRNA positive patients showed positive Survivin mRNA signals in urine after tumor removal. 3 of 3 preoperative Survivin mRNA negative patients showed negative Survivin mRNA signals in urine after tumor removal. No postoperative patients, including 3 Survivin mRNA positive and 8 Survivin mRNA negative patients suffered from recurrence during a 6-month follow-up period. One patient with positive Survivin mRNA signal in hematuria had a positive cystoscopy and operation result for TCCB (T1) during a 6-month follow-up period.
Conclusions: 1.Highly sensitive and specific determination of urine survivin mRNA appears to provide a simple, noninvasive diagnostic test to identify patients with TCCB. 2.There is no relationship between survivin mRNA and the stages or grades of the TCCB.
方法：采用巢式逆转录聚合酶链反应（巢式RT-PCR）检测：1.膀胱癌细胞株T24 Survivin mRNA表达。2.56例TCCB患者、20例非膀胱的泌尿系肿瘤患者、10例非泌尿系肿瘤疾病患者及10例健康志愿者尿脱落细胞Survivin mRNA表达,β-actin作为内参照。
结果：1.膀胱癌细胞株T24 Survivin mRNA表达为阳性。2.56例 TCCB患者中51例尿脱落细胞Survivin mRNA阳性表达，阳性率为91.1%，对照组40例中有1例血尿待查病人Survivin mRNA阳性表达，阳性率为2.5 %，TCCB组与对照组Survivin mRNA阳性率有统计学差异（p<0.01）。检测尿脱落细胞Survivin mRNA表达诊断TCCB，敏感性为91.1%，特异性为97.5%。按临床分期尿脱落细胞Survivin mRNA表达率T1期为90.5%（19/21），T_2-4期 91.4%（32/35），尽管T_2-4期表达率稍高于T1期，但两者差异无统计学意义(p>0.05)。按病理分级尿脱落细胞Survivin mRNA表达率G_1为90.6%（29/32），G_2为90.9 %（10/11），G_3为92.3 %（12/13），尽管随分级增加Survivin mRNA表达率增加，但三者之间Survivin mRNA表达率差异无统计学意义(p>0.05)。对49例行保留膀胱手术的TCCB患者复查尿脱落细胞Survivin mRNA表达，其中46例术前尿脱落细胞Survivin mRNA
阳性表达患者术后阳性表达5例，阳性表达率为10.9%（5/46），3例术前尿脱落细胞Survivin mRNA阴性表达者术后均阴性表达。本组有11例患者获得随访6个月，其中术后尿脱落细胞Survivin mRNA阳性表达3例，阴性表达8例，均无复发，对照组中1例血尿患者尿脱落细胞Survivin mRNA阳性表达，随访6个月后经膀胱镜检发现可疑病灶，取活检和手术后病理证实为T1期TCCB。
结论：1.巢式RT-PCR检测尿脱落细胞Survivin mRNA表达诊断TCCB敏感性和特异性高，无创伤性，Survivin可作为TCCB诊断的一个敏感指标。2.尿脱落细胞Survivin mRNA表达与TCCB的临床分期、病理分级无关。
Objective: To determine if detection of Survivin mRNA expression in urine by using nested RT-PCR can provide a novel noninvasive tool for the diagnosis of transitional cell carcinoma of the bladder .
Methods: Survivin mRNA in T24 bladder cancer cell line, exfoliated urinary cells of 56 patients with TCCB, 20 patients with urinary cancers except bladder, 10 nonneoplastic urinary diseases patients and 10 healthy volunteers were detected. Survivin and β-actin were amplified by using nested RT-PCR.
Results: 1. T24 bladder cancer cell line showed positive Survivin mRNA signal. 2. Only 1 of 40 (2.5%) showed positive Survivin mRNA signal in urine from control group. 51 of 56 (91.1%) patients with TCCB showed positive Survivin mRNA signals in urine. The Survivin mRNA expression difference between experimental group and control group was significant (p<0.01). The positive rate of Survivin mRNA in T1 was 90.5%（19/21）,T_2-4 91.4%（32/35）；G_1 90.6%（29/32）,G_2 90.9%（10/11），G_3 92.3%（12/13）.5 of 46
preoperative Survivin mRNA positive patients showed positive Survivin mRNA signals in urine after tumor removal. 3 of 3 preoperative Survivin mRNA negative patients showed negative Survivin mRNA signals in urine after tumor removal. No postoperative patients, including 3 Survivin mRNA positive and 8 Survivin mRNA negative patients suffered from recurrence during a 6-month follow-up period. One patient with positive Survivin mRNA signal in hematuria had a positive cystoscopy and operation result for TCCB (T1) during a 6-month follow-up period.
Conclusions: 1.Highly sensitive and specific determination of urine survivin mRNA appears to provide a simple, noninvasive diagnostic test to identify patients with TCCB. 2.There is no relationship between survivin mRNA and the stages or grades of the TCCB.
引文
1 Droller MJ. Bladder cancer: state of the art care[J]. CA Cancer J Clin,1998,48(5):269-284.
2 Grossman HB. New methods for detection of bladder cancer[J]. Semin Urol oncol,1998,16(1):17-22.
3 Ramakumar S,Bhuiyan J,Besse JA,et al. Comparison of screening methods in the detection of bladder cancer[J]. J Urol.,1999;161(2):388-394.
4 Brown FM,Urine cytology:it is still the gold standard for screening?[J].Urol Clin North Am,2000,27:25-37..
5 Ambrosini G,Adida C,Altieri DC,et al. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J].Nat Med,1997,3(8):917-921.
6 Li F, Ambrosini G, Chu EY,et al. Control of apoptosis and mitotic spindle checkpoint by survivin [J].Nature,1998,396(6711):580-584
7 Suzuki A,Ito T,Kawano H,et al.Survivin initiates procaspase-3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death[J].Oncogene,2000.19(10):1346-1353
8 Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7[J].Biochemistry,2001,40(4):1117-1123.
9 O'Connor DS, Grossman D, Plescia J,et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin[J]. Proc Natl Acad Sci USA,2000,97(24):13103-13107.
10 Mesri M, Morales-Ruiz M, Ackermann EJ,et al. Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting[J].Am J Pathol,2001,158(5): 1757-1765
11 O'Connor DS,Schechner JS,Adida C,et al.Control of apoptosis during angiogenesis by survivin express in endothelial cell[J].Am J Pathol,2000,156(2):393-398.
Papapetropoulos A,Fulton D,Mahboubi K,et al.Angiopioetin-1 inhibits
12 endothelial cell[J].J Biol chem. 2000,275(13):9102-9105.
13 Tran J,Bak J,Sheehan C,et al.Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells[J].Biochem Biophys Res Commmun.1999,264(3):781-189.
14 Olie RA,Simoes-Wust AP,Baumann B,et al.A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy ,Cancer Res,2000,60:2805-2809.
15 Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspase,and anticancer drugs.Cancer Res,1998,58:5315-5320.
16 Adida C，Crotty PL，McGrath J，et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation[J].Am J Pathol,1998,152(1): 43-49.
17 Velculescu VE,Madden SL,Zhang L,et al.Analysis of human transciptomes[J].Nat Genet.1999,23:387-388.
18 Grossman D,McNiff JM,Li F,et al.Expression of the apoptosis inhibitor,survivin ,in nonmelanoma skin cancer and gene targeting in a keratinocyte cell[J].Lab Invest 1999,79:1121-1126.
19 Grossman D,McNiff JM,Li F,et al.Expresson and targeting of the aoptosis inhibitor,survivin,in human melanoma[J].J Invest Dermatol,1999,113:1076-1081.
20 Takamizawa S, Scott D, Wen J,et al. The survivin:fas ratio in pediatric renal tumors [J].J Pediatr Surg ,2001 ,36(1):37-42.
21 Swana HS, Grossman D, Anthony JN,et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer[J]. N Engl J Med,1999,341(6):452-453.
Smith SD, Wheeler MA, Plescia J,et al. Urine detection of survivin and
22 diagnosis of bladder cancer[J]. JAMA,2001,285(3):324-328.
23 Stein JP,Grossfeld GD,Ginsberg DA,et al.Prognostic markers in bladder cancer:a contemporary review of the literature[J].J Urol,1998,160:645-659.
24 金冬雁，黎孟枫，译.分子克隆实验指南[M].第二版,北京：科技出版社，1995:343.
25 Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biolchem,1998,273(14):7787-7798.
26 刘秉文，陈俊杰，医学分子生物学[M]，第1版，中国协和医科大学出版社，2000:566-580.
27 Matsumura M, Niwa Y, Hikiba Y, et al.Sensitive assay for detection of hepatocellular carcinoma associated gene transcription (AFP mRNA) in blood.Biochem Biophys Res Commun,1995,207(2):813-818.
28 Huben RP. Tumor markers in bladder cancer[J].Urolgy,1984,23(3 Suppl):10-14.
29 陈必成，吴秀玲，张方毅,等.Survivin在膀胱移行细胞癌中的表达及意义[J].浙江医学，2002,24(6):324-325.
2 Grossman HB. New methods for detection of bladder cancer[J]. Semin Urol oncol,1998,16(1):17-22.
3 Ramakumar S,Bhuiyan J,Besse JA,et al. Comparison of screening methods in the detection of bladder cancer[J]. J Urol.,1999;161(2):388-394.
4 Brown FM,Urine cytology:it is still the gold standard for screening?[J].Urol Clin North Am,2000,27:25-37..
5 Ambrosini G,Adida C,Altieri DC,et al. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J].Nat Med,1997,3(8):917-921.
6 Li F, Ambrosini G, Chu EY,et al. Control of apoptosis and mitotic spindle checkpoint by survivin [J].Nature,1998,396(6711):580-584
7 Suzuki A,Ito T,Kawano H,et al.Survivin initiates procaspase-3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death[J].Oncogene,2000.19(10):1346-1353
8 Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7[J].Biochemistry,2001,40(4):1117-1123.
9 O'Connor DS, Grossman D, Plescia J,et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin[J]. Proc Natl Acad Sci USA,2000,97(24):13103-13107.
10 Mesri M, Morales-Ruiz M, Ackermann EJ,et al. Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting[J].Am J Pathol,2001,158(5): 1757-1765
11 O'Connor DS,Schechner JS,Adida C,et al.Control of apoptosis during angiogenesis by survivin express in endothelial cell[J].Am J Pathol,2000,156(2):393-398.
Papapetropoulos A,Fulton D,Mahboubi K,et al.Angiopioetin-1 inhibits
12 endothelial cell[J].J Biol chem. 2000,275(13):9102-9105.
13 Tran J,Bak J,Sheehan C,et al.Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells[J].Biochem Biophys Res Commmun.1999,264(3):781-189.
14 Olie RA,Simoes-Wust AP,Baumann B,et al.A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy ,Cancer Res,2000,60:2805-2809.
15 Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspase,and anticancer drugs.Cancer Res,1998,58:5315-5320.
16 Adida C，Crotty PL，McGrath J，et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation[J].Am J Pathol,1998,152(1): 43-49.
17 Velculescu VE,Madden SL,Zhang L,et al.Analysis of human transciptomes[J].Nat Genet.1999,23:387-388.
18 Grossman D,McNiff JM,Li F,et al.Expression of the apoptosis inhibitor,survivin ,in nonmelanoma skin cancer and gene targeting in a keratinocyte cell[J].Lab Invest 1999,79:1121-1126.
19 Grossman D,McNiff JM,Li F,et al.Expresson and targeting of the aoptosis inhibitor,survivin,in human melanoma[J].J Invest Dermatol,1999,113:1076-1081.
20 Takamizawa S, Scott D, Wen J,et al. The survivin:fas ratio in pediatric renal tumors [J].J Pediatr Surg ,2001 ,36(1):37-42.
21 Swana HS, Grossman D, Anthony JN,et al. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer[J]. N Engl J Med,1999,341(6):452-453.
Smith SD, Wheeler MA, Plescia J,et al. Urine detection of survivin and
22 diagnosis of bladder cancer[J]. JAMA,2001,285(3):324-328.
23 Stein JP,Grossfeld GD,Ginsberg DA,et al.Prognostic markers in bladder cancer:a contemporary review of the literature[J].J Urol,1998,160:645-659.
24 金冬雁，黎孟枫，译.分子克隆实验指南[M].第二版,北京：科技出版社，1995:343.
25 Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biolchem,1998,273(14):7787-7798.
26 刘秉文，陈俊杰，医学分子生物学[M]，第1版，中国协和医科大学出版社，2000:566-580.
27 Matsumura M, Niwa Y, Hikiba Y, et al.Sensitive assay for detection of hepatocellular carcinoma associated gene transcription (AFP mRNA) in blood.Biochem Biophys Res Commun,1995,207(2):813-818.
28 Huben RP. Tumor markers in bladder cancer[J].Urolgy,1984,23(3 Suppl):10-14.
29 陈必成，吴秀玲，张方毅,等.Survivin在膀胱移行细胞癌中的表达及意义[J].浙江医学，2002,24(6):324-325.