血清多胺在肺癌诊断中的应用
作者：吴逸明 张振中 史香林 王新朝
单位：史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV 65055USA)；吴逸明 张振中 王新朝(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)
关键词：肿瘤；大鼠；多胺；血清；RP-HPLC
河南医科大学学报000305
摘要 目的：探讨血清多胺水平在肺癌诊断中的应用价值。 方法：建立肺癌大鼠模型, 运用RP-HPLC方法测定大鼠血清多胺水平, 采用SPSS软件包进行统计学处理。结果： 实验组出现肿瘤的16只大鼠血清精胺水平((5.70±1.77) μmol/L)显著高于正常对照组大鼠血清精胺水平((4.83±1.26) μmol/L)(P<0.05)。实验组未出现肿瘤的大鼠(14只)精胺水平((4.61±1.83) μmol/L)与正常对照组精胺水平相比差异无显著性(P>0.05)，实验组出现肿瘤的大鼠(16只)血清精胺水平显著高于实验组未出现肿瘤的大鼠(14只)血清精胺水平(P<0.05)。实验组出现肿瘤的大鼠(16只)血清精脒水平((8.33±2.64) μmol/L)显著高于正常对照组大鼠血清精脒水平((5.53±2.30) μmol/L)(P<0.05)，实验组未出现肿瘤的大鼠(14只)精脒水平((5.39± 2.30) μmol/L)与正常对照组大鼠血清精脒水平相比差异无显著性(P>0.05)，而实验组出现肿瘤的大鼠(16只)精脒水平显著高于实验组未出现肿瘤的大鼠血清精脒水平(P<0.05)。 精胺和精脒2项综合指标阳性率为68.8%。 结论：血清多胺水平是肺癌诊断的一个有用指标。
, http://www.100md.com
分类号 R446.11
The application of polyamines in serum to diagnosis of lung cancer
WU Yiming WANG Xinchao
(Department of Occupational Health and Health Toxicology, Henan Medical University，Zhengzhou 450052 P.R.China)
ZHANG Zhenzhong
(Department of Pharmacy, Henan Medical University ,Zhengzhou 450052 P.R.China)
, http://www.100md.com
SHI Xianglin
( Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
Abstract Aim: To study the diagnostic value of polyamines in serum for lung cancer. Methods:The levels of polyamines in serum of rat-bearing lung cancer were determined by RP-HPLC and the data of polyamines in serum were processed by SPSS software. Results:The serum spermine level was significantly higher in rat-bearing tumor((5.70±1.77) μmol/L) than that in control rat serum ((4.83±1.26) μmol/L) . There was no significant difference between the polyamine levels in serum of rat non-tumor of experiment group (( 4.61±1.83) μmol/L) and of control group (P>0.05). The spermine level in serum was obviously higher in rat-bearing tumor of experiment group than that in rats without tumor of experiment group (P<0.05). The spermidine level was significantly higher in tumor mouse serum ((8.33±2.64) μmol/L) than that in control mouse serum ((5.53±2.30) μmol/L)(P<0.05) and markedly higher in tumor mouse serum than that in non-tumor mouse serum of experiment group (( 5.39±2.30) μmol/L)(P<0.05), there was no significant difference between the polyamines levels in non-tumor mouse serum and control mouse serum (P>0.05).Conclusion:The level of polyamines in mouse serum is a valuable index for the diagnosis of lung cancer.
, http://www.100md.com
Key words lung neoplasms; rats; polyamines; serum; RP-HPLC
The naturally occurring polyamines such as putrescine, spermine, spermidine and their derivatives , are normal constituents of both human and animal cells and play an important role in cell proliferation and differentiation. Polyamines have multifunctions of physiology , enhance cell growth and affect nucleic acid synthesis, expression of oncogene and antioncogene. More polyamines products of synthesis are one of the most early changes in the biological bodies while carcinogenic substances act biological bodies. If polyamines synthesis were controlled, the tumor cell growth would stop. Probably polyamines is a derestrainer of oncogene, more polyamines products of synthesis can stimulate oncogene and if polyamines levels decreased, oncogene expression would be controlled. There is a relationship of adjustment and control between polyamines levels and oncogene expression and it is necessary for oncogene expression to maintain polyamines levels. Many studies have shown that polyamines levels were associated with tumor development and progression more or less[1，2，3].
, http://www.100md.com
1 Materials and method
1.1 Animal model Weanling Wistar rats at the age of 4 weeks and weighing 120～200 g were obtained from Laboratory Animal Center of Henan Province. Those rats were divided into two groups at random. Physiological saline suspensions of coal tar pitch (CTP) were poured into the bronchi of the experiment group (n = 30). Physiological saline suspensions of carbon powder were poured into bronchi of control rats (n = 20), both groups were poured successively every 10 days for 6 times and all rats were killed 13 months after being poured. Blood samples from rats were taken and centrifuged immediately, and supernatant was stored at -20 ℃ until analysis. All the rats were examined by pathology
, 百拇医药
1.2 Apparatus Waters HPLC system (including Waters 600 pump, Waters 486 detector whose wavelength can be changed, Waters 800 integration), high-speed centrifugal machine ( Shanghai Medical Centrifugal Factory), Millipore pure water system(American Millipore Company)
1.3 Reagents Spermidine hydrochloride (Sigma Company), Spermine hydrochloride (Sigma Company), Putrescine hydrochloride (Sigma Company), methanol (Chromatography pure) (Shandong Yuwang Industrial Company), triethylamine (Shanghai No. 3 Reagents Factory), phosphoric acid (A.R) (Tianjin Reagents Factory). All water used was purified by Millipore pure system.
, 百拇医药
1.4 Chromatography analysis Column, ODS-C18 column (20 mm × 4.6 mm I.D, 2 μm), column temperature, 35 ℃, mobile phase, solution A,V(water) : V(phosphoric acid)∶ V(triethylamine) = 360 ∶ 9 ∶ 6 ， solution B, methanol, gradient elution, flow rate, 0.8 ml/min, detection wavelength, 338 nm.
1.5 Statistics Data reported were mean ± standard derivation . Statistical comparisons were made by student′s t-test.
2 Results
, 百拇医药
The levels of polyamines in serum were listed in Tab1.The data of polyamines in serum were processed by SPSS software. The serum spermine level was significantly higher (P<0.05) in tumor (( 5.70±1.77) μmol/L) than that in control ( (4.83±1.26)μmol/L) . There was no significant difference between the serum polyamine levels in non-tumor of experiment group ( (4.61±1.83)μmol/L) and in control group (P>0.05). The serum spermine level was obviously higher (P<0.05) in tumor of experiment group than that in non-tumor of experiment group. The serum spermidine level was significantly higher in tumor ((8.33±2.64) μmol/L) than that in control (( 5.53± 2.30) μmol/L)(P<0.05) and markedly higher in tumor than that in non-tumor of experiment group((5.39±2.30) μmol/L)(P<0.05), there was no significant difference between the serum polyamines levels in non-tumor in and control (P>0.05).
, http://www.100md.com
Tab1 Polyamines results in rats serum μmol/L Control(n = 20)
Experiment(n = 30)
Non-tumor(n=14)
Tumor(n=16)
Spermine
4.83 ± 1.26
4.61 ±1.83
5.70 ±1.77*△
Spermidine
, 百拇医药 5.53 ± 2.30
5.39 ±2.30
8.33 ±2.64*△
* vs control, P<0.05 ; △ vs non-tumor, P<0.05
3 Discussion
Polyamines involved in cell differentiation and proliferation, are fatty family compounds of lower chain in bodies. Malignant tumor is a disease of cell abnormal differentiation and the result of out of cell control and dedifferentiation, and polyamines play an important role in adjustment and control of cell proliferation and differentiation. It has been shown that elevated polyamines levels could lead to maladjustment of cell proliferation and tumor development and progression are related closely to polyamines levels. Polyamines could control cell growth and affect protein expression through DNA and RNA synthesis in the cells. At any stage metabolism of nucleic acid is related to polyamines. In vitro experiments proved that adding spermidine to cell culture solution of mouse mammary gland , the products of tyrein synthesis increased significantly. At different phase of cell division, two peaks of polyamines levels were observed from the last G1 phase to the early S phase and from the last S period to the early G2 phase[4]. So the results demonstrated that polyamines levels were involved in cell division. Many studies point out that polyamines levels in body liquid and serum of malignant tumor patients were significantly higher than those in normal persons, suggesting and proposing that polyamines may be a tumor biomarker. The results of the present study also proved that polyamines used as diagnosis index of tumor have important value in the diagnosis of tumors. Polyamines levels play an important role in cell growth and affect activation or suppression of oncogene. External factors (physical or chemical factor) acting biological body resulted in elevated levels of polyamines to activate oncogene and inactivate tumor suppress antioncogene, so the cells grow malignantly. The detailed mechanism of polyamines related to cell carcianogensis will be to further study deeply.
, http://www.100md.com
基金项目：河南省自然科学基金资助项目 204022300
研究方向：肺癌的病因学、预防、早期诊断和综合治疗，作者简介：吴逸明男，55岁，教授，博士生导师，Reference
[1]Shah N, Thomas T, Shirahata A, et al. Activation of nuclear factor kappaB by polyamines in breast cancer cells. Biochemistry, 1999,38(45):14 763
[2]Manni A, Wright C, Hsu CJ, et al. Polyamines and autocrine control of tumor growth by prolactin in experimental breast cancer in culture. Endocrinology, 1986, 119: 2 033
, http://www.100md.com
[3]Zhao JW , Zhu GB. Polyamines significance in tumor research. Amino Acid and Biological Resource, 1995, 17(1):46
[4]Guan J, Fan MZ, Cao SL. Reversion of malignant phenotypes of human lung squamous carcinoma cells by ortithine decarboxylase. Chinese Journal of Oncology, 1996,18(2): 81
2000-01-05收稿, 百拇医药
单位：史香林(美国职业安全卫生研究所病理生理学研究室 摩根墩 WV 65055USA)；吴逸明 张振中 王新朝(河南医科大学劳动卫生学与卫生毒理学教研室 郑州 450052)
关键词：肿瘤；大鼠；多胺；血清；RP-HPLC
河南医科大学学报000305
摘要 目的：探讨血清多胺水平在肺癌诊断中的应用价值。 方法：建立肺癌大鼠模型, 运用RP-HPLC方法测定大鼠血清多胺水平, 采用SPSS软件包进行统计学处理。结果： 实验组出现肿瘤的16只大鼠血清精胺水平((5.70±1.77) μmol/L)显著高于正常对照组大鼠血清精胺水平((4.83±1.26) μmol/L)(P<0.05)。实验组未出现肿瘤的大鼠(14只)精胺水平((4.61±1.83) μmol/L)与正常对照组精胺水平相比差异无显著性(P>0.05)，实验组出现肿瘤的大鼠(16只)血清精胺水平显著高于实验组未出现肿瘤的大鼠(14只)血清精胺水平(P<0.05)。实验组出现肿瘤的大鼠(16只)血清精脒水平((8.33±2.64) μmol/L)显著高于正常对照组大鼠血清精脒水平((5.53±2.30) μmol/L)(P<0.05)，实验组未出现肿瘤的大鼠(14只)精脒水平((5.39± 2.30) μmol/L)与正常对照组大鼠血清精脒水平相比差异无显著性(P>0.05)，而实验组出现肿瘤的大鼠(16只)精脒水平显著高于实验组未出现肿瘤的大鼠血清精脒水平(P<0.05)。 精胺和精脒2项综合指标阳性率为68.8%。 结论：血清多胺水平是肺癌诊断的一个有用指标。
, http://www.100md.com
分类号 R446.11
The application of polyamines in serum to diagnosis of lung cancer
WU Yiming WANG Xinchao
(Department of Occupational Health and Health Toxicology, Henan Medical University，Zhengzhou 450052 P.R.China)
ZHANG Zhenzhong
(Department of Pharmacy, Henan Medical University ,Zhengzhou 450052 P.R.China)
, http://www.100md.com
SHI Xianglin
( Pathology and Physiology Research, NIOSH, Morgantown, WV 26505, USA)
Abstract Aim: To study the diagnostic value of polyamines in serum for lung cancer. Methods:The levels of polyamines in serum of rat-bearing lung cancer were determined by RP-HPLC and the data of polyamines in serum were processed by SPSS software. Results:The serum spermine level was significantly higher in rat-bearing tumor((5.70±1.77) μmol/L) than that in control rat serum ((4.83±1.26) μmol/L) . There was no significant difference between the polyamine levels in serum of rat non-tumor of experiment group (( 4.61±1.83) μmol/L) and of control group (P>0.05). The spermine level in serum was obviously higher in rat-bearing tumor of experiment group than that in rats without tumor of experiment group (P<0.05). The spermidine level was significantly higher in tumor mouse serum ((8.33±2.64) μmol/L) than that in control mouse serum ((5.53±2.30) μmol/L)(P<0.05) and markedly higher in tumor mouse serum than that in non-tumor mouse serum of experiment group (( 5.39±2.30) μmol/L)(P<0.05), there was no significant difference between the polyamines levels in non-tumor mouse serum and control mouse serum (P>0.05).Conclusion:The level of polyamines in mouse serum is a valuable index for the diagnosis of lung cancer.
, http://www.100md.com
Key words lung neoplasms; rats; polyamines; serum; RP-HPLC
The naturally occurring polyamines such as putrescine, spermine, spermidine and their derivatives , are normal constituents of both human and animal cells and play an important role in cell proliferation and differentiation. Polyamines have multifunctions of physiology , enhance cell growth and affect nucleic acid synthesis, expression of oncogene and antioncogene. More polyamines products of synthesis are one of the most early changes in the biological bodies while carcinogenic substances act biological bodies. If polyamines synthesis were controlled, the tumor cell growth would stop. Probably polyamines is a derestrainer of oncogene, more polyamines products of synthesis can stimulate oncogene and if polyamines levels decreased, oncogene expression would be controlled. There is a relationship of adjustment and control between polyamines levels and oncogene expression and it is necessary for oncogene expression to maintain polyamines levels. Many studies have shown that polyamines levels were associated with tumor development and progression more or less[1，2，3].
, http://www.100md.com
1 Materials and method
1.1 Animal model Weanling Wistar rats at the age of 4 weeks and weighing 120～200 g were obtained from Laboratory Animal Center of Henan Province. Those rats were divided into two groups at random. Physiological saline suspensions of coal tar pitch (CTP) were poured into the bronchi of the experiment group (n = 30). Physiological saline suspensions of carbon powder were poured into bronchi of control rats (n = 20), both groups were poured successively every 10 days for 6 times and all rats were killed 13 months after being poured. Blood samples from rats were taken and centrifuged immediately, and supernatant was stored at -20 ℃ until analysis. All the rats were examined by pathology
, 百拇医药
1.2 Apparatus Waters HPLC system (including Waters 600 pump, Waters 486 detector whose wavelength can be changed, Waters 800 integration), high-speed centrifugal machine ( Shanghai Medical Centrifugal Factory), Millipore pure water system(American Millipore Company)
1.3 Reagents Spermidine hydrochloride (Sigma Company), Spermine hydrochloride (Sigma Company), Putrescine hydrochloride (Sigma Company), methanol (Chromatography pure) (Shandong Yuwang Industrial Company), triethylamine (Shanghai No. 3 Reagents Factory), phosphoric acid (A.R) (Tianjin Reagents Factory). All water used was purified by Millipore pure system.
, 百拇医药
1.4 Chromatography analysis Column, ODS-C18 column (20 mm × 4.6 mm I.D, 2 μm), column temperature, 35 ℃, mobile phase, solution A,V(water) : V(phosphoric acid)∶ V(triethylamine) = 360 ∶ 9 ∶ 6 ， solution B, methanol, gradient elution, flow rate, 0.8 ml/min, detection wavelength, 338 nm.
1.5 Statistics Data reported were mean ± standard derivation . Statistical comparisons were made by student′s t-test.
2 Results
, 百拇医药
The levels of polyamines in serum were listed in Tab1.The data of polyamines in serum were processed by SPSS software. The serum spermine level was significantly higher (P<0.05) in tumor (( 5.70±1.77) μmol/L) than that in control ( (4.83±1.26)μmol/L) . There was no significant difference between the serum polyamine levels in non-tumor of experiment group ( (4.61±1.83)μmol/L) and in control group (P>0.05). The serum spermine level was obviously higher (P<0.05) in tumor of experiment group than that in non-tumor of experiment group. The serum spermidine level was significantly higher in tumor ((8.33±2.64) μmol/L) than that in control (( 5.53± 2.30) μmol/L)(P<0.05) and markedly higher in tumor than that in non-tumor of experiment group((5.39±2.30) μmol/L)(P<0.05), there was no significant difference between the serum polyamines levels in non-tumor in and control (P>0.05).
, http://www.100md.com
Tab1 Polyamines results in rats serum μmol/L Control(n = 20)
Experiment(n = 30)
Non-tumor(n=14)
Tumor(n=16)
Spermine
4.83 ± 1.26
4.61 ±1.83
5.70 ±1.77*△
Spermidine
, 百拇医药 5.53 ± 2.30
5.39 ±2.30
8.33 ±2.64*△
* vs control, P<0.05 ; △ vs non-tumor, P<0.05
3 Discussion
Polyamines involved in cell differentiation and proliferation, are fatty family compounds of lower chain in bodies. Malignant tumor is a disease of cell abnormal differentiation and the result of out of cell control and dedifferentiation, and polyamines play an important role in adjustment and control of cell proliferation and differentiation. It has been shown that elevated polyamines levels could lead to maladjustment of cell proliferation and tumor development and progression are related closely to polyamines levels. Polyamines could control cell growth and affect protein expression through DNA and RNA synthesis in the cells. At any stage metabolism of nucleic acid is related to polyamines. In vitro experiments proved that adding spermidine to cell culture solution of mouse mammary gland , the products of tyrein synthesis increased significantly. At different phase of cell division, two peaks of polyamines levels were observed from the last G1 phase to the early S phase and from the last S period to the early G2 phase[4]. So the results demonstrated that polyamines levels were involved in cell division. Many studies point out that polyamines levels in body liquid and serum of malignant tumor patients were significantly higher than those in normal persons, suggesting and proposing that polyamines may be a tumor biomarker. The results of the present study also proved that polyamines used as diagnosis index of tumor have important value in the diagnosis of tumors. Polyamines levels play an important role in cell growth and affect activation or suppression of oncogene. External factors (physical or chemical factor) acting biological body resulted in elevated levels of polyamines to activate oncogene and inactivate tumor suppress antioncogene, so the cells grow malignantly. The detailed mechanism of polyamines related to cell carcianogensis will be to further study deeply.
, http://www.100md.com
基金项目：河南省自然科学基金资助项目 204022300
研究方向：肺癌的病因学、预防、早期诊断和综合治疗，作者简介：吴逸明男，55岁，教授，博士生导师，Reference
[1]Shah N, Thomas T, Shirahata A, et al. Activation of nuclear factor kappaB by polyamines in breast cancer cells. Biochemistry, 1999,38(45):14 763
[2]Manni A, Wright C, Hsu CJ, et al. Polyamines and autocrine control of tumor growth by prolactin in experimental breast cancer in culture. Endocrinology, 1986, 119: 2 033
, http://www.100md.com
[3]Zhao JW , Zhu GB. Polyamines significance in tumor research. Amino Acid and Biological Resource, 1995, 17(1):46
[4]Guan J, Fan MZ, Cao SL. Reversion of malignant phenotypes of human lung squamous carcinoma cells by ortithine decarboxylase. Chinese Journal of Oncology, 1996,18(2): 81
2000-01-05收稿, 百拇医药