p21 mRNA在原发食管癌（鳞癌）中的表达和临床意义
摘要
前言
p21是目前已知的具有最广泛CDK抑制活性的细胞周期抑制蛋白，广泛抑制cyclin-复合物，负性调节CDK的功能预示其在抑制肿瘤方面的作用。p21与肿瘤发生、发展及其生物学行为有关，有可能成为估计肿瘤恶性行为及预后的新指标。p21表达的缺失可能意味着肿瘤的迅速生长，p21表达与肿瘤的分化程度，肿瘤浸润深度及有无淋巴结转移密切相关。食管癌是常见的恶性肿瘤之一，严重的威胁着人类的健康和生命，本实验旨在应用分子生物学的方法检测和分析原发食管癌中p21的表达情况，探讨其在食管癌中的临床意义。
实验材料
收集2001年1-10月的中国医科大学附属第一临床医院胸外科和辽宁省肿瘤医院胸外科共54例食管癌手术患者的癌组织和癌旁6cm以上的正常食管组织。手术切除后立即送至-80℃深低温冰箱中冻存。所有病例术前均未发现远处转移，未行放疗和化疗，术后均有病理证实(均为鳞癌)。
实验方法
1．总mRNA提取：参照TRIzol说明书分别提取食管癌组织和癌旁正常食管组织的总RNA。
2．逆转录合成cDNA第一链：取总RNA 2μl用于cDNA第一
链合成。
3．PCR扩增件 的引物，根据则基因结构，选择能扩增 CD－
NA的碱基序列。上游引物为：5’－TACTCCCCTGCCCTCAACAA．
GA－3’；下游引物为：5’－CGCTATTGAGCAGCGCTCAT－3’；
pZI的扩增条件为：95T ZInin；94t lImn；55T lmin；72t＊5
rn；28个循环；最后 72℃延伸 7ndn。
4．PCR产物检测：取10gi PCR产物进行 l．8％琼脂糖凝胶电
泳，进行EB染色。凝胶经UPV凝胶成像分析系统进行扫描分析。
5．统计分析：采用 X‘检验，P＜0．05有意义。
实验结果
1．p21 mRNA的表达与原发食管癌：在54例癌组织中，有16
例表达 PZI mRNAp9．6％人癌旁正常组织中，有 50例表达 P21
mRNAp2．6％人统计结果分析：pZI mRNA在两者之间的表达
有非常显著性的差异k＜o刀＊。
2．p21 mRNA的表达与淋巴结转移及’I’NM分期：在ZI例有
区域淋巴结转移病例中，PZI mRNA表达3例门4．2％人在33例
无淋巴结转移的病例中，P21 rnRNA表达 13例c9．4％人统计学
结果分析：pZI mRNA在有淋巴结转移组和无淋巴结转移组中的
表达有显著性差异瞩＜队05人在54例原发食管癌中，TN M分期
1期2二例，11期17例，见期16例，统计学结果分析：PZI rnRNA表
达在 1期与皿期之间差异有显著性＊’值为 4．00 P＜0．05入 11期
与皿期之间差异无显著性k’值为 1．41P＞0．05X而 1期与 11期
之间差异无显著性（X‘值为 0．73 P＞0．05人
·2·
讨 论
本实验研究中涉 蛋白在正常食管组及鳞癌组中，其阳性表
达分别为92．6％和29．6％，另外J ZI蛋白阳性表达与食管鳞癌
分期呈负相关及易于表达于无淋巴结转移的病灶中。以上结果提
示，p ZI基因失活以及其蛋白表达缺失在食管鳞癌发生机制起重
要作用，并且昨二蛋白表达可作为判断肿瘤分化程度的参考指标。
生 论
1一 在原发食管癌（鳞癌）组织中的表达与癌旁正常组织相
比明显减少。
2一 在有淋巴结转移组织表达低于无淋巴结转移组。
3．临床分期 1期者 P21蛋白强阳性率高于皿期者。这表明
pZI低表达与食管癌（鳞癌V分期、病程及淋巴结转移密切相关。
Prefaces
Many studies have shown that p21 was a cyclin-dependent ki-nase inhibitor ( GDI) , inhibit the cyclin - compound, and plays an important role in the growth, progression, and biology behavior of tumor, may be a new indicator to evaluate malignant level of carcino-ma. Absence of p21 may significant the carcinoma growth quickly, p21 expression have closely associated with the TNM stage, the be-havior of invasion and metastasis of carcinoma. Esophageal carcinoma is more and more threatening the health and life of human. In this study, we examined and analyzed the expression of p21 mRNA in pri-mary esophageal carcinoma by reverse trascription polymerase chain reaction ( RT - PCR) to define the role of p21 in this kind of cancer.
Materials and Methods
Esophageal carcinoma tissues and surrounding esophageal tissues from patients who received surgery at the First Affilated Hospital of China Medical University and the Liao Ning Tumor Hospital between 2001 January to October . The tissues were frozen at - 80℃. The pa-
tients had no detectable metastases in distant organs at the time of sur-gery. No patient had received chemotherapy or radiation therapy be-fore surgery.
1. Extraction of total RNA; Total RNA was extracted from tissue samples using a RNA extraction reagent, Trizol reagent(GIBCOBRL, Int). The concentration of total RNA was adjusted to l|xg/|xl.
2. Reverse Transcription ( RT) ;2μJ of total RNA were incubated in 12μl of reaction buffer.
3. Polymerase Chain Reaction ( PCR ) : PCR primers ( Sangon, China) for p21cDNA according to the p21 gene structure. The PCR reaceion was carried out in three steps as follows; 95℃ for 2min(l cycle) ; 94℃ for Imin; 55℃ for Imin; 72℃ for 1.5min(28 cycle) ; and 72℃ for7min(l cycle)
4. PCR fragments were analyzed by electrophoresis on 12% poly-acrylamide gels, and DNA was visualized by ethidium bromide stai-ning.
Results
1. RT -PCR analysis of p21mRNA in esophageal carcinoma and surrounding esophageal carcinoma tissues: in 54 esophageal carcinoma tissues, there were 16(29.6% ) cases expressed p21 mRNA, in sur-rounding esophageal carcinoma tissues, there were 50(92.6% ) cases expressed p21mRNA. Statistical, analysis shows significant difference between the expression of p21mRNA in carcinomas and surrounding e-sophageal carcinoma tissues ( P <0.01).
2. p21 expression and tumor progression; in 54 esophageal carci-noma tissues were divided in two groups; one with lymph node metas-
tasis; the other without lymph node metastasis. p21 mRNA was more expressed in cases without lymph node metastasis (13/33 39. 4% ) than those with lymph node metastasis (3/21 14.2% ). Statistical a-nalysis shows significant difference between the expression of p21mRNA in without lymph node metastasis and with lymph node me-tastasis ( P < 0. 05 ). Statistical analysis shows significant difference between the expression of p21mRNA in TNM stage I and HI, (P < 0.05) , no significant difference between I and II ( P >0.05) , and II and IH(P>0.05).
Disscussion
In this study, p21 high expression in normal surrounding esopha-geal carcinoma tissues than esophageal carcinoma tissues. P21 expres-sion have opposite relation with esophageal carcinoma TNM stage, and p21 low expression in the tissue that with lymph node metastasis, these result imply'; absence expression and lose function of p21 play an important role in esophageal carcinoma happen; the expression of p21 protein may be an reference indictor to determine differentiation level of carcinoma.
Conclusion
1. p21 was high expression in surrounding esophageal carcinoma tissues than esophageal carcinoma tissues.
2. p21 was high expression in without lymph node metastasis group than with lymph node metastasis group.
3. The expression of p21 is closely associated with the TNM
stage, the behavior of invasion and metastasis of primary esophageal carcinoma.
p21是目前已知的具有最广泛CDK抑制活性的细胞周期抑制蛋白，广泛抑制cyclin-复合物，负性调节CDK的功能预示其在抑制肿瘤方面的作用。p21与肿瘤发生、发展及其生物学行为有关，有可能成为估计肿瘤恶性行为及预后的新指标。p21表达的缺失可能意味着肿瘤的迅速生长，p21表达与肿瘤的分化程度，肿瘤浸润深度及有无淋巴结转移密切相关。食管癌是常见的恶性肿瘤之一，严重的威胁着人类的健康和生命，本实验旨在应用分子生物学的方法检测和分析原发食管癌中p21的表达情况，探讨其在食管癌中的临床意义。
实验材料
收集2001年1-10月的中国医科大学附属第一临床医院胸外科和辽宁省肿瘤医院胸外科共54例食管癌手术患者的癌组织和癌旁6cm以上的正常食管组织。手术切除后立即送至-80℃深低温冰箱中冻存。所有病例术前均未发现远处转移，未行放疗和化疗，术后均有病理证实(均为鳞癌)。
实验方法
1．总mRNA提取：参照TRIzol说明书分别提取食管癌组织和癌旁正常食管组织的总RNA。
2．逆转录合成cDNA第一链：取总RNA 2μl用于cDNA第一
链合成。
3．PCR扩增件 的引物，根据则基因结构，选择能扩增 CD－
NA的碱基序列。上游引物为：5’－TACTCCCCTGCCCTCAACAA．
GA－3’；下游引物为：5’－CGCTATTGAGCAGCGCTCAT－3’；
pZI的扩增条件为：95T ZInin；94t lImn；55T lmin；72t＊5
rn；28个循环；最后 72℃延伸 7ndn。
4．PCR产物检测：取10gi PCR产物进行 l．8％琼脂糖凝胶电
泳，进行EB染色。凝胶经UPV凝胶成像分析系统进行扫描分析。
5．统计分析：采用 X‘检验，P＜0．05有意义。
实验结果
1．p21 mRNA的表达与原发食管癌：在54例癌组织中，有16
例表达 PZI mRNAp9．6％人癌旁正常组织中，有 50例表达 P21
mRNAp2．6％人统计结果分析：pZI mRNA在两者之间的表达
有非常显著性的差异k＜o刀＊。
2．p21 mRNA的表达与淋巴结转移及’I’NM分期：在ZI例有
区域淋巴结转移病例中，PZI mRNA表达3例门4．2％人在33例
无淋巴结转移的病例中，P21 rnRNA表达 13例c9．4％人统计学
结果分析：pZI mRNA在有淋巴结转移组和无淋巴结转移组中的
表达有显著性差异瞩＜队05人在54例原发食管癌中，TN M分期
1期2二例，11期17例，见期16例，统计学结果分析：PZI rnRNA表
达在 1期与皿期之间差异有显著性＊’值为 4．00 P＜0．05入 11期
与皿期之间差异无显著性k’值为 1．41P＞0．05X而 1期与 11期
之间差异无显著性（X‘值为 0．73 P＞0．05人
·2·
讨 论
本实验研究中涉 蛋白在正常食管组及鳞癌组中，其阳性表
达分别为92．6％和29．6％，另外J ZI蛋白阳性表达与食管鳞癌
分期呈负相关及易于表达于无淋巴结转移的病灶中。以上结果提
示，p ZI基因失活以及其蛋白表达缺失在食管鳞癌发生机制起重
要作用，并且昨二蛋白表达可作为判断肿瘤分化程度的参考指标。
生 论
1一 在原发食管癌（鳞癌）组织中的表达与癌旁正常组织相
比明显减少。
2一 在有淋巴结转移组织表达低于无淋巴结转移组。
3．临床分期 1期者 P21蛋白强阳性率高于皿期者。这表明
pZI低表达与食管癌（鳞癌V分期、病程及淋巴结转移密切相关。
Prefaces
Many studies have shown that p21 was a cyclin-dependent ki-nase inhibitor ( GDI) , inhibit the cyclin - compound, and plays an important role in the growth, progression, and biology behavior of tumor, may be a new indicator to evaluate malignant level of carcino-ma. Absence of p21 may significant the carcinoma growth quickly, p21 expression have closely associated with the TNM stage, the be-havior of invasion and metastasis of carcinoma. Esophageal carcinoma is more and more threatening the health and life of human. In this study, we examined and analyzed the expression of p21 mRNA in pri-mary esophageal carcinoma by reverse trascription polymerase chain reaction ( RT - PCR) to define the role of p21 in this kind of cancer.
Materials and Methods
Esophageal carcinoma tissues and surrounding esophageal tissues from patients who received surgery at the First Affilated Hospital of China Medical University and the Liao Ning Tumor Hospital between 2001 January to October . The tissues were frozen at - 80℃. The pa-
tients had no detectable metastases in distant organs at the time of sur-gery. No patient had received chemotherapy or radiation therapy be-fore surgery.
1. Extraction of total RNA; Total RNA was extracted from tissue samples using a RNA extraction reagent, Trizol reagent(GIBCOBRL, Int). The concentration of total RNA was adjusted to l|xg/|xl.
2. Reverse Transcription ( RT) ;2μJ of total RNA were incubated in 12μl of reaction buffer.
3. Polymerase Chain Reaction ( PCR ) : PCR primers ( Sangon, China) for p21cDNA according to the p21 gene structure. The PCR reaceion was carried out in three steps as follows; 95℃ for 2min(l cycle) ; 94℃ for Imin; 55℃ for Imin; 72℃ for 1.5min(28 cycle) ; and 72℃ for7min(l cycle)
4. PCR fragments were analyzed by electrophoresis on 12% poly-acrylamide gels, and DNA was visualized by ethidium bromide stai-ning.
Results
1. RT -PCR analysis of p21mRNA in esophageal carcinoma and surrounding esophageal carcinoma tissues: in 54 esophageal carcinoma tissues, there were 16(29.6% ) cases expressed p21 mRNA, in sur-rounding esophageal carcinoma tissues, there were 50(92.6% ) cases expressed p21mRNA. Statistical, analysis shows significant difference between the expression of p21mRNA in carcinomas and surrounding e-sophageal carcinoma tissues ( P <0.01).
2. p21 expression and tumor progression; in 54 esophageal carci-noma tissues were divided in two groups; one with lymph node metas-
tasis; the other without lymph node metastasis. p21 mRNA was more expressed in cases without lymph node metastasis (13/33 39. 4% ) than those with lymph node metastasis (3/21 14.2% ). Statistical a-nalysis shows significant difference between the expression of p21mRNA in without lymph node metastasis and with lymph node me-tastasis ( P < 0. 05 ). Statistical analysis shows significant difference between the expression of p21mRNA in TNM stage I and HI, (P < 0.05) , no significant difference between I and II ( P >0.05) , and II and IH(P>0.05).
Disscussion
In this study, p21 high expression in normal surrounding esopha-geal carcinoma tissues than esophageal carcinoma tissues. P21 expres-sion have opposite relation with esophageal carcinoma TNM stage, and p21 low expression in the tissue that with lymph node metastasis, these result imply'; absence expression and lose function of p21 play an important role in esophageal carcinoma happen; the expression of p21 protein may be an reference indictor to determine differentiation level of carcinoma.
Conclusion
1. p21 was high expression in surrounding esophageal carcinoma tissues than esophageal carcinoma tissues.
2. p21 was high expression in without lymph node metastasis group than with lymph node metastasis group.
3. The expression of p21 is closely associated with the TNM
stage, the behavior of invasion and metastasis of primary esophageal carcinoma.
引文
1. Maniatis T, Molecular cloning[M]. New York: Ccold Spring Habor Lab, 1982.282
2. Kao GD, Mckerma G, Maity A, et al. Cancer Res, 1997; 25:2098 - 2105
3. Kiyokawa H, Kineman RD, Manova - Todorova KO, et al. Cell, 1997 ;85:721 - 732
4. Dynlacht BD. Nature, 1997 ;389:149 - 152
5. Farrow SV, White JHM, Martinou L, et al. Nature, 1995 ;374:731 -733
6. Grpss. The concept of viral etiology of cancer and allied disease with particular reference to cancer of the colon[J]. Eur J Cancer Clin Oncol, 1987,23:343.
7. Peter ME, Heufelder AE, Hengartner MO. Proc Nail Acad Sci USA, 1997 ;94:12736 - 12737
8. Yasui W, Akama Y, Kuniyasu H, et al. Expression of cyclin - dependent kinase inhibitor p21~(WAF1CIP1) in non- neoplastic mucosa and neoplasia of the stomach: relationship with p53 status and proliferative activity. J Pathol, 1996; 180:122 - 128
9. Yasui W, Akama Y, Yokozaki H, et al. Expression of p21 (waf1/cip1/sdi1), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma. Cancer, 1997; 79 (11): 2067 - 2072
10. Marchetti A, Doglioni C, Barbareschi M, et al. p21 RNA and protein expression in non- small cell lung carcinomas: evidence of p53 -independent expression and association with tumoral dif
ferentiatioin. Oncogene, 1996; 12:1319 - 1324
11. Palazzo JP, Mercer WE, Kovatich AJ, et al. Immunohistoehemical localization of p21~(WAF1CIP1) innormal, hyperplastie, and neoplastic uterine tissues. Hum Pathol, 1997;28(1) :60 -66
12．刘伟，张亚历．RT-PCR技术在胃肠癌外周血微转移检测中的应用．《中国癌症杂志》，2000，10(2)：175-177
13. Palazzo JP, Mercer WE, Kovatich AJ, et al. Immunohistoehemicai localization of p21~(WAF1CIP1) in normal, hyperplastic, and neo plastic uterine tissues. Hum Pathol, 1997 ;28(1) :60-66
14. Gomyo Y, Ikeda M, Osaki M, et al. Expression of p21 (waf1/cipl/sdil), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma. Cancer, 1997; 79 (11):2067 - 2072
15. Lukas J, Groshen S, Saffari B, et al. WAF1/CIP1 gene polynorphism and expression in carcinomas of the breast, ovary, and endometrium. Am J Pathol, 1997 ;150(1) :167 - 175
16. Slebos RJC, Baas IO, Clement M, et al. Clinical and pathological association with p53 tumour- suppressor gene mutations and expression of p21~(WAF1CIP1) in colorectal carcinoma. Br J Cancer, 1996 ;74:165 - 171
17. Lukas J, Groshen S, Saffari B, et al. WAF1/CIP1 gene polynorphism and expression in carcinomas of the breast, ovary, and endometrium. Am J Pathol, 1997; 150(1) "167 - 175
2. Kao GD, Mckerma G, Maity A, et al. Cancer Res, 1997; 25:2098 - 2105
3. Kiyokawa H, Kineman RD, Manova - Todorova KO, et al. Cell, 1997 ;85:721 - 732
4. Dynlacht BD. Nature, 1997 ;389:149 - 152
5. Farrow SV, White JHM, Martinou L, et al. Nature, 1995 ;374:731 -733
6. Grpss. The concept of viral etiology of cancer and allied disease with particular reference to cancer of the colon[J]. Eur J Cancer Clin Oncol, 1987,23:343.
7. Peter ME, Heufelder AE, Hengartner MO. Proc Nail Acad Sci USA, 1997 ;94:12736 - 12737
8. Yasui W, Akama Y, Kuniyasu H, et al. Expression of cyclin - dependent kinase inhibitor p21~(WAF1CIP1) in non- neoplastic mucosa and neoplasia of the stomach: relationship with p53 status and proliferative activity. J Pathol, 1996; 180:122 - 128
9. Yasui W, Akama Y, Yokozaki H, et al. Expression of p21 (waf1/cip1/sdi1), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma. Cancer, 1997; 79 (11): 2067 - 2072
10. Marchetti A, Doglioni C, Barbareschi M, et al. p21 RNA and protein expression in non- small cell lung carcinomas: evidence of p53 -independent expression and association with tumoral dif
ferentiatioin. Oncogene, 1996; 12:1319 - 1324
11. Palazzo JP, Mercer WE, Kovatich AJ, et al. Immunohistoehemical localization of p21~(WAF1CIP1) innormal, hyperplastie, and neoplastic uterine tissues. Hum Pathol, 1997;28(1) :60 -66
12．刘伟，张亚历．RT-PCR技术在胃肠癌外周血微转移检测中的应用．《中国癌症杂志》，2000，10(2)：175-177
13. Palazzo JP, Mercer WE, Kovatich AJ, et al. Immunohistoehemicai localization of p21~(WAF1CIP1) in normal, hyperplastic, and neo plastic uterine tissues. Hum Pathol, 1997 ;28(1) :60-66
14. Gomyo Y, Ikeda M, Osaki M, et al. Expression of p21 (waf1/cipl/sdil), but not p53 protein, is a factor in the survival of patients with advanced gastric carcinoma. Cancer, 1997; 79 (11):2067 - 2072
15. Lukas J, Groshen S, Saffari B, et al. WAF1/CIP1 gene polynorphism and expression in carcinomas of the breast, ovary, and endometrium. Am J Pathol, 1997 ;150(1) :167 - 175
16. Slebos RJC, Baas IO, Clement M, et al. Clinical and pathological association with p53 tumour- suppressor gene mutations and expression of p21~(WAF1CIP1) in colorectal carcinoma. Br J Cancer, 1996 ;74:165 - 171
17. Lukas J, Groshen S, Saffari B, et al. WAF1/CIP1 gene polynorphism and expression in carcinomas of the breast, ovary, and endometrium. Am J Pathol, 1997; 150(1) "167 - 175