膀胱移行细胞癌尿液中MMP_2及MMP_9检测的临床意义
摘要
前言
膀胱移行细胞癌是泌尿系统最常见的恶性肿瘤，复发及转移为其显著的生物学特征，基底膜(base-ment membrane,BM)是肿瘤浸润转移的一道天然屏障，基底膜的主要成分是Ⅳ型胶原。Ⅳ型胶原酶MMP_2(明胶酶A分子量72KD)和MMP_9(明胶酶B分子量92KD)与膀胱移行细胞癌的关系最为密切，能够特异性地降解基底膜(BM)的主要成份Ⅳ型胶原，而促进肿瘤的浸润转移，以往研究MMP_2和MMP_9与膀胱移行细胞癌的关系多采用免疫组化和原位杂交的方法，膀胱移行细胞癌尿液MMP_2及MMP_9的检测报道较少，本实验采用明胶酶谱法(Zymograpky 法)检测不同分期、分级膀胱移行细胞癌患者尿液中MMP_2和MMP_9的表达，并与尿脱落细胞学检查进行对比，探讨膀胱移行细胞癌患者尿液中MMP_2及MMP_9与膀胱移行细胞癌分期分级的关系，以明确MMP_2及MMP_9是否具有早期诊断价值，以及对膀胱移行细胞癌的术式选择是否有价值，报告如下。
材料与方法
一、一般资料
2001.09～11月住院的膀胱移行细胞癌患者40例，其中男30例，女10例，年龄36～77岁，平均62岁，40例膀胱移行细胞癌患者均经术后病理证实，均为移行细胞癌，其中按WHO标准Ⅰ级11例，Ⅱ级23例，Ⅲ级6例，按UICC标准T_1 21例，T_2 12例，T_(3-4) 7例。
二、实验所需仪器及试剂
仪器：DYY-Ⅲ型电泳仪
DYY一皿28C型电泳槽
QUEVE恒温箱
LKB2219型恒温水浴箱
超速低温高心机
试剂：丙烯酸胺
甲叉双丙烯酸胺
明胶
10助 SDS－PA防胶（含 1．omg／Inl明胶）
2．5％Titon X－100
考马斯亮兰R250
三、方法
二．标本收集
留取膀就移行细胞癌患者晨尿50nil，4℃条件下300r／min离
心10分钟，收集沉淀，－20℃保存备用，同时常规行尿脱落细胞检
查。
2．明胶酶活性分析（Zymoonho）
①常温下标本解冻，加裂解液，匀浆。
欧℃条件下15000r／min，离心60分钟。
③收集上清，酚试剂法进行蛋白定量，在相同蛋白浓度条件下
取 25 gi样本上样于含 1．omg／rrd明胶的 10％SDS－PAGE胶（聚
丙烯酸胺凝胶X上层覆以 5％的浓缩胶作为分离胶，以 30毗恒
流电流电泳3小时。
④电泳后凝胶于 2．5％Triton X－100中浸泡 3 ／J’时。
⑤然后在 50Mm Tis—Hcf，200rnmVL NaCI，10nunol／L
CaC12＊H值 7．5溶液中孵育 30h。
⑤孵育结束后以0．5％考马斯亮兰R25030％甲醇上0％乙酸
染色4小时。
①脱色液（甲醇：乙酸：水一3：l：6）脱色4小时。
·2·
3．图像分析
利用UVP凝胶成像分析系统对ZymograPhy电泳结果进行灰
度扫描，以灰度面积代表酶活性，对各细胞的酶活性进行定量比
较。
四、统计学分析
两组间均数比较采用 t检验，两组间率的比较采用X‘检验。
结 果
一、膀脱移行细胞癌尿液 MMPZ及 MMPg活性比较
MMPz及MMPg阳性结果为蓝色背景下的透亮带。40例膀fi
移行细胞癌中 MMPz表达者 32例，MMPO表达者 38例，MMPz和
MMPO同时未表达者2例，同时表达者32例。MMPz及 MMPO的
活性随着膀航移行细胞癌临床分期及病理分级的增高而升高，且
MMPO活性明显高于 MMPz。MMPz及 MMPO在初发与复发及单发
与初发膀既移行细胞癌间的比较无显著差异 p＞0．05，肿瘤直径
＞3．ocm的膀既移行细胞癌＊MPz及＊＊民活性明显高于直径＜
3．ocm的膀脱移行细胞癌，MMP。及 MMPO活性，P＜0．05。
二、膀就移行细胞癌尿液中M＊巳、＊＊ 及尿细胞学敏感性
比较
40例膀既移行细胞癌中MMPz阳性32例，敏感性80％，38例
MMPg阳性，敏感性 95％，尿细胞学阳性 13例，敏感性 32．5％。
MMPg及 MMP。敏感性明显高于尿细胞学检查，p＜0·05。同时
MMPg敏感性明显高于 MMPZ，p＜0．05。MMPZ活性在 1级与 I十
皿级及 Tl与 T：叶之间比较 P＜of．05，在其它分期、分级间比较 P＞
0．05。MMPg在各分期、分级间比较 p＞0．05。MMPZ及 MMP。敏
感性在初发与复发、单发与多发以及肿瘤大小之间比较无明显差
异，p＞0．05。
·3·
讨 论
浸润、转移是肿瘤细胞、宿主细胞及细胞外基质（ECM）之间
一系列主动、复杂、多步骤相互作用的过程，肿瘤细胞与周围基质
细胞相互作用决定了恶性肿瘤细胞的生物学行为。肿瘤的浸润转
移首先必须发生基底膜pM）的降解，基底膜是主要由IV型胶原
组成的致密的网状结构。肿瘤的浸润、转移是一个主动过程山Ot－
ta把肿瘤的浸润、转移过程概括为三个步骤：①肿瘤细胞与细胞外
基质成分特异性粘附；②肿瘤细胞或基质细胞分泌蛋白水解酶，降
解细胞外基质（ECM）和基底膜，破坏BM屏障；③肿瘤细胞穿透
血管或淋巴管壁向远处转移或直接侵人到周围组织中。肿瘤细胞
在多种生长因子的作用下形成转移性?
Preface
Bladder cancer is the most common malignant tumor of urological system. Recurrence and metastasis are their main biological character-istics. Base - membrane, mainly composed of type IV collagen, is a nature barrier preventing the metastasis of tumor. Type IV collagenase MMP2 ( molecular num of gelatin enzyme A is 72 KD. ) and MMP9 (molecular num of gelatin enzyme B is 92KD. ) , relative to the blad-der cancer, can degrade type IV collagen specifically to promote the metastasis of tumor. Immunohistological method and hybridization in situ were usually used in previous studies about the relation between MMP2, MMP9 and bladder cancer, while the report, in which MMP2 and MMP9 in urine of bladder cancer patients were examined is few. Eymography method was used in our study to detect the expression of MMP2 and MMP9 in the urine of different grade and stage bladder cancer, and the results will be compared with the results of urine shedding cell method to investigate the relation between the level of MMP2, MMP9 in urine and bladder cancer and clarify the value of MMP2 and MMP9 in earlier diagnosis and the selection of operation pa-tern.
Materials and methods
1. General data
40 patients with bladder carcinoma hospitalized from September to November in 2001 were enrolled in this study. There were 30 men and 10 women whose age were from 36 to 77 years old and the mean age was 62 years old. After being confirmed by pathology, these sam-ples were all transitional carcinoma of bladder. According to the standard of WHO, there were 11 cases of grade I , 23 cases of grade II and 6 cases of grade III. According to the standard of UICC, there were 21 cases of stage T_(1) , 12 cases f T3 and 7 cases of T3.
2. Equipments and reagents
Equipments: electrophoresis apparatus of DYY - III type electrophoresis slot of DYY - III type QUEVE caloristat
Homoiothermy cleansing bath of LKB2219 type Overdrive and hypothermia centrifugal machine Reagents; acrylamide
methyleneacrylamide
gelatin
10% SDS -PAGE glue( including 1 rag/ml gelatin)
2.5% Triton X-100
Coomassie brilliant blue R250
3. Methods
(1) Samples collection
50 ml morning urine of patients with bladder carcinoma was col-lected and centrifugated for 10 minutes at 3000r/min under 4T!. The deposition was also collected and stored under - 201 waiting for use.
At the same time, routine desquamated cells examination was conducted.
( 2 ) zymography
(1)Samples were defrosted under room temperature and then lyase was added to make homogenate..
(2)Cetrifugating for 60 minutes at 15000r/min under 4CC.
(3)The upper clear fluid was collected and fixed protein quantity by phenol reagent method. 25#J sample was collected at the same pro-tein concentration and add to 10% SDS - PAGE glue ( polyacrylamide gel) which had l.0mg/ml gelatin. 5% concentrated glue was add to the upper layer as separating glue. Electrophoresis was conducted for 3 hours at 30 mA constant current.
(4)After electrophoresis, the gel was soaked in 2. 5% Triton X -100 for 3 hours.
(5)Then incubated at 50Mm Tris - Hcl, 200mmol/L NaCl, l0mmol/L CaCl2 fluid whose PH value was 7. 6 for 30 hours.
(6)After incubation, stained in 0. 5% coomassie brilliant blue R250, 30% methanol and 10% acetic acid for 4 hours.
(7)Discolorated by discoroloration fluid( methanol: acetic acid; water = 3 :1:6) for 4 hours.
(3)1 mage analysis
The results of Zymography electrophoresis were gray scanned by UVP gel image analysis system. The gray area represented the activity of enzyme and the enzyme activity of cells was quantitatively com-pared.
(4) Statistical analysis
The mean value between two groups were analyzed by t test and the rates between two groups were analyzed by x~?2? test.
Results
1. The comparison of the activity of MMP2 and MMP9 in the urine
of bladder carcinoma
The positive results of MMP2 and MMP9 were the penetrating band under the blue background. 32 cases expressed MMP2 in 40 ca-ses while 38 cases expressed MMP9. 2 cases neither expressed
膀胱移行细胞癌是泌尿系统最常见的恶性肿瘤，复发及转移为其显著的生物学特征，基底膜(base-ment membrane,BM)是肿瘤浸润转移的一道天然屏障，基底膜的主要成分是Ⅳ型胶原。Ⅳ型胶原酶MMP_2(明胶酶A分子量72KD)和MMP_9(明胶酶B分子量92KD)与膀胱移行细胞癌的关系最为密切，能够特异性地降解基底膜(BM)的主要成份Ⅳ型胶原，而促进肿瘤的浸润转移，以往研究MMP_2和MMP_9与膀胱移行细胞癌的关系多采用免疫组化和原位杂交的方法，膀胱移行细胞癌尿液MMP_2及MMP_9的检测报道较少，本实验采用明胶酶谱法(Zymograpky 法)检测不同分期、分级膀胱移行细胞癌患者尿液中MMP_2和MMP_9的表达，并与尿脱落细胞学检查进行对比，探讨膀胱移行细胞癌患者尿液中MMP_2及MMP_9与膀胱移行细胞癌分期分级的关系，以明确MMP_2及MMP_9是否具有早期诊断价值，以及对膀胱移行细胞癌的术式选择是否有价值，报告如下。
材料与方法
一、一般资料
2001.09～11月住院的膀胱移行细胞癌患者40例，其中男30例，女10例，年龄36～77岁，平均62岁，40例膀胱移行细胞癌患者均经术后病理证实，均为移行细胞癌，其中按WHO标准Ⅰ级11例，Ⅱ级23例，Ⅲ级6例，按UICC标准T_1 21例，T_2 12例，T_(3-4) 7例。
二、实验所需仪器及试剂
仪器：DYY-Ⅲ型电泳仪
DYY一皿28C型电泳槽
QUEVE恒温箱
LKB2219型恒温水浴箱
超速低温高心机
试剂：丙烯酸胺
甲叉双丙烯酸胺
明胶
10助 SDS－PA防胶（含 1．omg／Inl明胶）
2．5％Titon X－100
考马斯亮兰R250
三、方法
二．标本收集
留取膀就移行细胞癌患者晨尿50nil，4℃条件下300r／min离
心10分钟，收集沉淀，－20℃保存备用，同时常规行尿脱落细胞检
查。
2．明胶酶活性分析（Zymoonho）
①常温下标本解冻，加裂解液，匀浆。
欧℃条件下15000r／min，离心60分钟。
③收集上清，酚试剂法进行蛋白定量，在相同蛋白浓度条件下
取 25 gi样本上样于含 1．omg／rrd明胶的 10％SDS－PAGE胶（聚
丙烯酸胺凝胶X上层覆以 5％的浓缩胶作为分离胶，以 30毗恒
流电流电泳3小时。
④电泳后凝胶于 2．5％Triton X－100中浸泡 3 ／J’时。
⑤然后在 50Mm Tis—Hcf，200rnmVL NaCI，10nunol／L
CaC12＊H值 7．5溶液中孵育 30h。
⑤孵育结束后以0．5％考马斯亮兰R25030％甲醇上0％乙酸
染色4小时。
①脱色液（甲醇：乙酸：水一3：l：6）脱色4小时。
·2·
3．图像分析
利用UVP凝胶成像分析系统对ZymograPhy电泳结果进行灰
度扫描，以灰度面积代表酶活性，对各细胞的酶活性进行定量比
较。
四、统计学分析
两组间均数比较采用 t检验，两组间率的比较采用X‘检验。
结 果
一、膀脱移行细胞癌尿液 MMPZ及 MMPg活性比较
MMPz及MMPg阳性结果为蓝色背景下的透亮带。40例膀fi
移行细胞癌中 MMPz表达者 32例，MMPO表达者 38例，MMPz和
MMPO同时未表达者2例，同时表达者32例。MMPz及 MMPO的
活性随着膀航移行细胞癌临床分期及病理分级的增高而升高，且
MMPO活性明显高于 MMPz。MMPz及 MMPO在初发与复发及单发
与初发膀既移行细胞癌间的比较无显著差异 p＞0．05，肿瘤直径
＞3．ocm的膀既移行细胞癌＊MPz及＊＊民活性明显高于直径＜
3．ocm的膀脱移行细胞癌，MMP。及 MMPO活性，P＜0．05。
二、膀就移行细胞癌尿液中M＊巳、＊＊ 及尿细胞学敏感性
比较
40例膀既移行细胞癌中MMPz阳性32例，敏感性80％，38例
MMPg阳性，敏感性 95％，尿细胞学阳性 13例，敏感性 32．5％。
MMPg及 MMP。敏感性明显高于尿细胞学检查，p＜0·05。同时
MMPg敏感性明显高于 MMPZ，p＜0．05。MMPZ活性在 1级与 I十
皿级及 Tl与 T：叶之间比较 P＜of．05，在其它分期、分级间比较 P＞
0．05。MMPg在各分期、分级间比较 p＞0．05。MMPZ及 MMP。敏
感性在初发与复发、单发与多发以及肿瘤大小之间比较无明显差
异，p＞0．05。
·3·
讨 论
浸润、转移是肿瘤细胞、宿主细胞及细胞外基质（ECM）之间
一系列主动、复杂、多步骤相互作用的过程，肿瘤细胞与周围基质
细胞相互作用决定了恶性肿瘤细胞的生物学行为。肿瘤的浸润转
移首先必须发生基底膜pM）的降解，基底膜是主要由IV型胶原
组成的致密的网状结构。肿瘤的浸润、转移是一个主动过程山Ot－
ta把肿瘤的浸润、转移过程概括为三个步骤：①肿瘤细胞与细胞外
基质成分特异性粘附；②肿瘤细胞或基质细胞分泌蛋白水解酶，降
解细胞外基质（ECM）和基底膜，破坏BM屏障；③肿瘤细胞穿透
血管或淋巴管壁向远处转移或直接侵人到周围组织中。肿瘤细胞
在多种生长因子的作用下形成转移性?
Preface
Bladder cancer is the most common malignant tumor of urological system. Recurrence and metastasis are their main biological character-istics. Base - membrane, mainly composed of type IV collagen, is a nature barrier preventing the metastasis of tumor. Type IV collagenase MMP2 ( molecular num of gelatin enzyme A is 72 KD. ) and MMP9 (molecular num of gelatin enzyme B is 92KD. ) , relative to the blad-der cancer, can degrade type IV collagen specifically to promote the metastasis of tumor. Immunohistological method and hybridization in situ were usually used in previous studies about the relation between MMP2, MMP9 and bladder cancer, while the report, in which MMP2 and MMP9 in urine of bladder cancer patients were examined is few. Eymography method was used in our study to detect the expression of MMP2 and MMP9 in the urine of different grade and stage bladder cancer, and the results will be compared with the results of urine shedding cell method to investigate the relation between the level of MMP2, MMP9 in urine and bladder cancer and clarify the value of MMP2 and MMP9 in earlier diagnosis and the selection of operation pa-tern.
Materials and methods
1. General data
40 patients with bladder carcinoma hospitalized from September to November in 2001 were enrolled in this study. There were 30 men and 10 women whose age were from 36 to 77 years old and the mean age was 62 years old. After being confirmed by pathology, these sam-ples were all transitional carcinoma of bladder. According to the standard of WHO, there were 11 cases of grade I , 23 cases of grade II and 6 cases of grade III. According to the standard of UICC, there were 21 cases of stage T_(1) , 12 cases f T3 and 7 cases of T3.
2. Equipments and reagents
Equipments: electrophoresis apparatus of DYY - III type electrophoresis slot of DYY - III type QUEVE caloristat
Homoiothermy cleansing bath of LKB2219 type Overdrive and hypothermia centrifugal machine Reagents; acrylamide
methyleneacrylamide
gelatin
10% SDS -PAGE glue( including 1 rag/ml gelatin)
2.5% Triton X-100
Coomassie brilliant blue R250
3. Methods
(1) Samples collection
50 ml morning urine of patients with bladder carcinoma was col-lected and centrifugated for 10 minutes at 3000r/min under 4T!. The deposition was also collected and stored under - 201 waiting for use.
At the same time, routine desquamated cells examination was conducted.
( 2 ) zymography
(1)Samples were defrosted under room temperature and then lyase was added to make homogenate..
(2)Cetrifugating for 60 minutes at 15000r/min under 4CC.
(3)The upper clear fluid was collected and fixed protein quantity by phenol reagent method. 25#J sample was collected at the same pro-tein concentration and add to 10% SDS - PAGE glue ( polyacrylamide gel) which had l.0mg/ml gelatin. 5% concentrated glue was add to the upper layer as separating glue. Electrophoresis was conducted for 3 hours at 30 mA constant current.
(4)After electrophoresis, the gel was soaked in 2. 5% Triton X -100 for 3 hours.
(5)Then incubated at 50Mm Tris - Hcl, 200mmol/L NaCl, l0mmol/L CaCl2 fluid whose PH value was 7. 6 for 30 hours.
(6)After incubation, stained in 0. 5% coomassie brilliant blue R250, 30% methanol and 10% acetic acid for 4 hours.
(7)Discolorated by discoroloration fluid( methanol: acetic acid; water = 3 :1:6) for 4 hours.
(3)1 mage analysis
The results of Zymography electrophoresis were gray scanned by UVP gel image analysis system. The gray area represented the activity of enzyme and the enzyme activity of cells was quantitatively com-pared.
(4) Statistical analysis
The mean value between two groups were analyzed by t test and the rates between two groups were analyzed by x~?2? test.
Results
1. The comparison of the activity of MMP2 and MMP9 in the urine
of bladder carcinoma
The positive results of MMP2 and MMP9 were the penetrating band under the blue background. 32 cases expressed MMP2 in 40 ca-ses while 38 cases expressed MMP9. 2 cases neither expressed
引文
1． Zuckers, Rita M, Lysik, Mohommad H, Zanabi, and Utc Mou, et al. Mr9200 type IV collagenase Isincreased in plasma of atient swithcolon cancer and reast. Cancer Res, 1993 , 53:140-146．
2． Omstein DL, Macndb J, Cohn KH, et al. Evidence for tumor-host cooperation in reg-ulating matrix metalloproteinase 2 expres-sion human cocon cancer. Clin Exp Metastasis, 1999, 17:205-212．
3． Liotal LA.Tumor invasion and metastasis-Role of the extrace llualr matrix Rhoads memorial award lectare. Cancer Res, 1986, 46(1) :1-7．
4． Woodhouse EC, Chuaqui RF, Liotta LA. General mechanisms of metastasis.Cancer, 1997, 80( suppl): 1529-1537
5． Davies B, Waxman J, Wasan H, et al. Levels of matrix metallo-pro ternases inbladder cancer correlate with tamor grade and invasion.Cancer Res, 1993, 53:5365-5369．
6． Ozdemir E, Kakehi Y, Okuno H, et al. Role of matrix metallop roteinase 9 in the basement membrane destrauction of superficial urothelial carcinoma.J Urol, 1999, 161:1359-1363．
7． Bianco FJ. Gervasi DC, Tigvert R, et al. Matrix Metalloproteinase 9 expression in bladder washes from bladder cancer Pa-tients predicts patological stage and grade.Chin Cancer Res, 1998, 4:3011-3016．
8． Kanayama H, Yokota K, Korokawa Y, et al.Prognostic values of matrix metallopro-teinas 2 and tissue inhibitor of matrix met-alloproteinase 2 expression in bladder cancer. Cancer 1998, 82: 1359-1366．
9．宋波，金锡御，潘进洪，等．MMP_2及MMP_9在膀胱移行细胞癌中的表达及在侵袭转移中的作用．中华泌尿外科杂志，1999，20：400—402．
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