高侵袭转移Lovo细胞亚系的筛选与建立
摘要
大肠癌转移是一个复杂的过程，是肿瘤和宿主相互作用的共同结果，
其中肿瘤细胞在肿瘤侵袭的过程中起主导作用，研究瘤细胞生物学特性不
仅对研究肿瘤的发生及其发展具有重要意义，而且对了解它们的转移特性
具有重要作用。Lovo细胞是大肠癌未分化的高转移腺癌细胞，其转移期
间较长，4周的转移率约为40-50％。目前国内应用的Lovo细胞由于经
过多次的体外传代培养，细胞已出现退化现象，转移率有逐渐下降的趋势，
转移周期偏长。本实验采用体内连续传代方法，应用免疫缺陷动物裸鼠，
经脾反复接种瘤细胞，建立肝转移模型，从转移灶分离瘤细胞，经过多次
重复接种，使某些高转移亚系瘤细胞逐渐适应并呈优势生长和稳定下来，
同时去除不适应和不稳定亚系，获得了较稳定的具有较强转移能力的亚
系，该亚系具有在某器官形成高选择转移灶特性，经该方法筛选出的Lovo
细胞亚系可用于各种实验性大肠癌的实验和治疗研究。
本研究主要内容包括：
1、应用裸鼠体内连续传代方法，从脾接种建立肝转移模型，通过这
种实验性转移，选择并建立高侵袭转移的Lovo细胞亚系。
2、进行Lovo细胞亚系体外相关生物学特性的检测，通过调查瘤细胞
电泳泳动速率、运动能力、生长活力、细胞周期和DNA含量及体外侵袭
能力等参数，综合分析Lovo亚系细胞的侵袭能力。
3、进行Lovo细胞亚系裸鼠脾脏种植的体内观察与鉴定，探讨它的遗
传稳定性、对环境的适应性和转移潜能。
主要方法及结果：
l、应用体内连续传代方法，把L。V。细胞接种入裸鼠脾脏，选择肝转
移灶，进行瘤细胞分离培养，重复3次脾接种，筛选出高侵袭转移的L。VO
细胞亚系。
二、对高侵袭转移L。V。亚系细胞进行裸鼠脾种植实验，结果显示L。VO
亚系细胞有遗传很稳定、环境适应性强和转移潜能高等特点。
3、对高侵袭转移L。V。亚系细胞和母系L。V。细胞进行瘤细胞电泳泳
动速率检测，研究瘤细胞表面电荷性质和密度变化，结果显示LOVO亚系
细胞电泳泳动速率明显高于母系LOV。细胞。
4、应用MTT法测定高侵袭转移L。v。亚系细胞和母系L。v。细胞的
活力，研究瘤细胞的生长速度，结果显示L叫。亚系细胞和母系LOVO细
胞生长速度有显著差别。
5、应用琼脂糖滴法测定瘤细胞的运动能力，结果显示高侵袭转移LOVO
亚系细胞移出琼脂糖滴的细胞数和距离明显高于母系L。V。细胞。
6、应用单层NIH3T3细胞法测定瘤细胞的体外侵袭能力，结果显示
高侵袭转移LOvO亚系细胞侵袭能力明显高于母系L。v。细胞。
7、应用 FCM测定瘤细胞的细胞周期和 DNA含量，结果显示高侵袭
转移LOV。亚系细胞SP明显高于其母系L。V。细胞，说明DNA合成增加。
本研究结果提示：
L。V。亚系细胞是L。VO细胞一高侵袭转移细胞亚系。
Metastasis of human colorectal carcinoma(HCC),which is the
interaction result between tumor and its host, is a complicated
process. Tumor cell is the main part of tumor metastasis.Study on
biological feature of tumor cell is significant in the research of
tumor’s genesis and development.Lovo is HCC tumor cell and
undifferentiated adenocarcinoma cell.Although it’s a strong invasive
cell line,its metastasis period is rather long with a high metastasis
rate which is up to 40-50% in 4w’s metastasis.Lovo cell applied
nowadays begins to decline because of the repeated culture of go-
down-to future generation in vitro,metastasis rate tends to decrease
gradually with a longer metastasis period.This experiment is to
construct a model of liver metastasis by means of continous culture
of go-down-to future generation in vivo using nude mouse to
repeatedly inoculate tumor’s cell to its spleen and then separate
tumor cell from metastasis field to make some subline tumor cells
adapt gradually and show its dorminant growth and become stable
after several times inoculation,Simultaneously,we remove those
4
unstable subline to obstain relatively stable ones with a
comparatively strong metastasis ability.In addition,this subline
possesses a high function of selecting one organ to form metastasis
field,Lovo cell chosen by this way can both be cultured in vitro and
grow easily in proper organ in vivo.Therefore the lovo cell can be
used in various experiment and treatment study for HCC.
Main contents of this research include:
1. Apply a continous go-down-to the future generation method
of mouse, constructing a liver-metastasized nude mouse’s model
from spleen inoculation to select strong invasive lovo cell subline
through repeated experimental metastasis.
2. From its electrophoresis migration rate. its movement
ability its growth curve cell cycline. quantity of DNA and
metastasis in vitro to examine lovo cell and analyse its metastasis
ability.
Main methods and results:
1. Construction of strong invasive lovo cell subline by
continous go-down-to future generation in vivo.To found a liver
metastasis model by inoculating love cell in nude mouse spleen.
5
2. Make tumor cell’s eletrophoresis migration rate examination
for strong invasive lovo cell subline and its mother love cell and to
study charge properties and density of tumor cell’s surface.Result
shows the electrophoresis migration rate of lovo cell subline is
obviously higher than that of its mother lovo cell.
3. Use MTT method to examine growth curve of strong
invasive lovo cell subline and its mother lovo cell in order to study
cell’s growth speed.The result shows a great difference in growth
speed between lovo cell subline and its mother lovo cell.
4. Apply agrose drop method to examine tumor cell’s ability to
move.Result shows the cell’s amount and distance of emigrating
agrose drop is obviously higher than that of its mother lovo
cell.Apply NIH3T3 cell to test tumor cell’s metastasis ability in
vitro.The result shows strong invasive lovo cell subline’s ability of
invasing NIH3T3 cell is obviously higher than that of its mother
lovo cell.
5. Apply FCM to examine
其中肿瘤细胞在肿瘤侵袭的过程中起主导作用，研究瘤细胞生物学特性不
仅对研究肿瘤的发生及其发展具有重要意义，而且对了解它们的转移特性
具有重要作用。Lovo细胞是大肠癌未分化的高转移腺癌细胞，其转移期
间较长，4周的转移率约为40-50％。目前国内应用的Lovo细胞由于经
过多次的体外传代培养，细胞已出现退化现象，转移率有逐渐下降的趋势，
转移周期偏长。本实验采用体内连续传代方法，应用免疫缺陷动物裸鼠，
经脾反复接种瘤细胞，建立肝转移模型，从转移灶分离瘤细胞，经过多次
重复接种，使某些高转移亚系瘤细胞逐渐适应并呈优势生长和稳定下来，
同时去除不适应和不稳定亚系，获得了较稳定的具有较强转移能力的亚
系，该亚系具有在某器官形成高选择转移灶特性，经该方法筛选出的Lovo
细胞亚系可用于各种实验性大肠癌的实验和治疗研究。
本研究主要内容包括：
1、应用裸鼠体内连续传代方法，从脾接种建立肝转移模型，通过这
种实验性转移，选择并建立高侵袭转移的Lovo细胞亚系。
2、进行Lovo细胞亚系体外相关生物学特性的检测，通过调查瘤细胞
电泳泳动速率、运动能力、生长活力、细胞周期和DNA含量及体外侵袭
能力等参数，综合分析Lovo亚系细胞的侵袭能力。
3、进行Lovo细胞亚系裸鼠脾脏种植的体内观察与鉴定，探讨它的遗
传稳定性、对环境的适应性和转移潜能。
主要方法及结果：
l、应用体内连续传代方法，把L。V。细胞接种入裸鼠脾脏，选择肝转
移灶，进行瘤细胞分离培养，重复3次脾接种，筛选出高侵袭转移的L。VO
细胞亚系。
二、对高侵袭转移L。V。亚系细胞进行裸鼠脾种植实验，结果显示L。VO
亚系细胞有遗传很稳定、环境适应性强和转移潜能高等特点。
3、对高侵袭转移L。V。亚系细胞和母系L。V。细胞进行瘤细胞电泳泳
动速率检测，研究瘤细胞表面电荷性质和密度变化，结果显示LOVO亚系
细胞电泳泳动速率明显高于母系LOV。细胞。
4、应用MTT法测定高侵袭转移L。v。亚系细胞和母系L。v。细胞的
活力，研究瘤细胞的生长速度，结果显示L叫。亚系细胞和母系LOVO细
胞生长速度有显著差别。
5、应用琼脂糖滴法测定瘤细胞的运动能力，结果显示高侵袭转移LOVO
亚系细胞移出琼脂糖滴的细胞数和距离明显高于母系L。V。细胞。
6、应用单层NIH3T3细胞法测定瘤细胞的体外侵袭能力，结果显示
高侵袭转移LOvO亚系细胞侵袭能力明显高于母系L。v。细胞。
7、应用 FCM测定瘤细胞的细胞周期和 DNA含量，结果显示高侵袭
转移LOV。亚系细胞SP明显高于其母系L。V。细胞，说明DNA合成增加。
本研究结果提示：
L。V。亚系细胞是L。VO细胞一高侵袭转移细胞亚系。
Metastasis of human colorectal carcinoma(HCC),which is the
interaction result between tumor and its host, is a complicated
process. Tumor cell is the main part of tumor metastasis.Study on
biological feature of tumor cell is significant in the research of
tumor’s genesis and development.Lovo is HCC tumor cell and
undifferentiated adenocarcinoma cell.Although it’s a strong invasive
cell line,its metastasis period is rather long with a high metastasis
rate which is up to 40-50% in 4w’s metastasis.Lovo cell applied
nowadays begins to decline because of the repeated culture of go-
down-to future generation in vitro,metastasis rate tends to decrease
gradually with a longer metastasis period.This experiment is to
construct a model of liver metastasis by means of continous culture
of go-down-to future generation in vivo using nude mouse to
repeatedly inoculate tumor’s cell to its spleen and then separate
tumor cell from metastasis field to make some subline tumor cells
adapt gradually and show its dorminant growth and become stable
after several times inoculation,Simultaneously,we remove those
4
unstable subline to obstain relatively stable ones with a
comparatively strong metastasis ability.In addition,this subline
possesses a high function of selecting one organ to form metastasis
field,Lovo cell chosen by this way can both be cultured in vitro and
grow easily in proper organ in vivo.Therefore the lovo cell can be
used in various experiment and treatment study for HCC.
Main contents of this research include:
1. Apply a continous go-down-to the future generation method
of mouse, constructing a liver-metastasized nude mouse’s model
from spleen inoculation to select strong invasive lovo cell subline
through repeated experimental metastasis.
2. From its electrophoresis migration rate. its movement
ability its growth curve cell cycline. quantity of DNA and
metastasis in vitro to examine lovo cell and analyse its metastasis
ability.
Main methods and results:
1. Construction of strong invasive lovo cell subline by
continous go-down-to future generation in vivo.To found a liver
metastasis model by inoculating love cell in nude mouse spleen.
5
2. Make tumor cell’s eletrophoresis migration rate examination
for strong invasive lovo cell subline and its mother love cell and to
study charge properties and density of tumor cell’s surface.Result
shows the electrophoresis migration rate of lovo cell subline is
obviously higher than that of its mother lovo cell.
3. Use MTT method to examine growth curve of strong
invasive lovo cell subline and its mother lovo cell in order to study
cell’s growth speed.The result shows a great difference in growth
speed between lovo cell subline and its mother lovo cell.
4. Apply agrose drop method to examine tumor cell’s ability to
move.Result shows the cell’s amount and distance of emigrating
agrose drop is obviously higher than that of its mother lovo
cell.Apply NIH3T3 cell to test tumor cell’s metastasis ability in
vitro.The result shows strong invasive lovo cell subline’s ability of
invasing NIH3T3 cell is obviously higher than that of its mother
lovo cell.
5. Apply FCM to examine
引文
1．丁彦青等。结肠癌淋巴结微转移免疫组化检测的预后价值。中华误诊学杂志1999；3(3)：315
2. Kumar CC. Signaling by integrin receptors.Oncogene 1998;17(11 reviews):1365
3．高进。癌的侵袭与转移—基础与临床。北京医科大学中国协和医科大学联和出版社 1996年第1版
4. Price IE. Influence of organ environment on metastasis Cancer Bull 1987;39:162
5. Sobel ME. Metastasis suppressor gene.J Natl Cancer Inst 1990;82:247
6. Nicolson GL.Organ specificity of tumor metastasis:role of preferential adhesion, invasion and growth of maligant cells at specific seconddddary sites. Cancer Metastasis Rev 1988;7:143
7. Werner A et al. Inhibition of experimental liver metastasis by combined treatment with tamoxifen and interferon. Anticancer-Drugs 1996;7(3):307
8. Talmadge JE. The selective nature of metastasis. Cancer Metatasis Reviews 1983;2:25
9. Hanna N et al. Relationship between metastastic potential and resistance to natural killercell-mediated cytotoxing in three mucine systems.J Natl Cancer Inst 1981;66:1183
10. Liotta LA.Tumor invasion and metastasis:role of basement membrane.Am J Pathol 1984;117:339
11. Schiffmann E. Motility as aprincipal requirement for metastasis. Cancer Invest 1990;8(16):673
12. Mareel M et al. Invasion Experimental and Clinical Implications. Oxford, New York, Tokyo:Oxford university press 1984:252
13. Giavizzi R et al. Experimental nude mouse model of human colorectal cancer liver metastasis J Natl Cancer Inst 1986;77:1903
14．邱红明等。实验性人大肠癌裸鼠肝转移模型的建立。广东第六届病理年会 1995
15．方福德等。现代医学实验技巧全书。北京医科大学中国协和医科大学联和出版社 1995年第1版：469
16. Fidler IJ. Tumor heterogenity and biology of cancer invasion and metastasis. Cancer Res 1978;38:2651
17. Miner KM et al. Clonal drift of cell surface, melanogenic, and experimental metastatic properties of in vivo-selected, brain mening-colonizing murine B16 melanoma. Cancer Res 1982;42:4631
18．薛克勋等。人肺腺癌移植于裸鼠不同部位侵袭能力的观察。中国医学科学院学报 1986；8：128
19．李学农等。人直肠癌高侵润力亚系的体外选择及其生物学特性比较研究。中华肿瘤学杂志 1992；20(4)：301
20. Fidler IJ et al. Metastasis results from preexisting variant cells with a malignant tumor.Science 1977;197:893
21. Nagashima A et al. Establishment and characterization of high-and low-metastatic clones derived from a methycholanthrene-induced rat fibrosarcoma.. Cancer Res 1986;46:4420
22. Fidler IJ et al. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis.Cancer Metastasis Rev 1991;10:229
23. Radinsky R et al. Paracrine growth regulation of human colon carcinoma organ-specific metastasis.Cancer Metastasis Rev 1993;12:345
24. Rochefort H et al. Cathepsin D in breast cancer from molecular and cellular biology to clinical applications.Cancer Cells 1990;2(12):383
25. Honn KV et al. Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix.Cancer Metastasis Rev 1990;11:353
26. Dicolson GL et al. Preferential organ attachment and invasion in vitro by B16 melanoma cells selected for differing metastatic colonization and invasive properties.Invasion metastasis 1985;5:1144
27. Aznavoorian S et al. Molecular aspects of tumor cell invasion and metastasis. Cancer 1993;71(4):1368
28. Jessup JM et al. The biology of colorectal carcinoma. Curr Probl Cancer 1992;16:263.
29. Gohji K et al. Regulation of gelatinase production in metastatic renal cell carcinoma by organ-specific fibroblast.Jpn J Cancer Res 1994;85:152
30．潘李珍 等译。R．I．费雷什尼 编著 实用动物细胞培养技术 世界图书出版公司北京公司出版 1996第1版
31．徐国超等。人牙龈成纤维细胞电泳行为的研究。华西口腔医学杂志 1996；14(3)：183
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2. Kumar CC. Signaling by integrin receptors.Oncogene 1998;17(11 reviews):1365
3．高进。癌的侵袭与转移—基础与临床。北京医科大学中国协和医科大学联和出版社 1996年第1版
4. Price IE. Influence of organ environment on metastasis Cancer Bull 1987;39:162
5. Sobel ME. Metastasis suppressor gene.J Natl Cancer Inst 1990;82:247
6. Nicolson GL.Organ specificity of tumor metastasis:role of preferential adhesion, invasion and growth of maligant cells at specific seconddddary sites. Cancer Metastasis Rev 1988;7:143
7. Werner A et al. Inhibition of experimental liver metastasis by combined treatment with tamoxifen and interferon. Anticancer-Drugs 1996;7(3):307
8. Talmadge JE. The selective nature of metastasis. Cancer Metatasis Reviews 1983;2:25
9. Hanna N et al. Relationship between metastastic potential and resistance to natural killercell-mediated cytotoxing in three mucine systems.J Natl Cancer Inst 1981;66:1183
10. Liotta LA.Tumor invasion and metastasis:role of basement membrane.Am J Pathol 1984;117:339
11. Schiffmann E. Motility as aprincipal requirement for metastasis. Cancer Invest 1990;8(16):673
12. Mareel M et al. Invasion Experimental and Clinical Implications. Oxford, New York, Tokyo:Oxford university press 1984:252
13. Giavizzi R et al. Experimental nude mouse model of human colorectal cancer liver metastasis J Natl Cancer Inst 1986;77:1903
14．邱红明等。实验性人大肠癌裸鼠肝转移模型的建立。广东第六届病理年会 1995
15．方福德等。现代医学实验技巧全书。北京医科大学中国协和医科大学联和出版社 1995年第1版：469
16. Fidler IJ. Tumor heterogenity and biology of cancer invasion and metastasis. Cancer Res 1978;38:2651
17. Miner KM et al. Clonal drift of cell surface, melanogenic, and experimental metastatic properties of in vivo-selected, brain mening-colonizing murine B16 melanoma. Cancer Res 1982;42:4631
18．薛克勋等。人肺腺癌移植于裸鼠不同部位侵袭能力的观察。中国医学科学院学报 1986；8：128
19．李学农等。人直肠癌高侵润力亚系的体外选择及其生物学特性比较研究。中华肿瘤学杂志 1992；20(4)：301
20. Fidler IJ et al. Metastasis results from preexisting variant cells with a malignant tumor.Science 1977;197:893
21. Nagashima A et al. Establishment and characterization of high-and low-metastatic clones derived from a methycholanthrene-induced rat fibrosarcoma.. Cancer Res 1986;46:4420
22. Fidler IJ et al. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model for the biology and therapy of metastasis.Cancer Metastasis Rev 1991;10:229
23. Radinsky R et al. Paracrine growth regulation of human colon carcinoma organ-specific metastasis.Cancer Metastasis Rev 1993;12:345
24. Rochefort H et al. Cathepsin D in breast cancer from molecular and cellular biology to clinical applications.Cancer Cells 1990;2(12):383
25. Honn KV et al. Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix.Cancer Metastasis Rev 1990;11:353
26. Dicolson GL et al. Preferential organ attachment and invasion in vitro by B16 melanoma cells selected for differing metastatic colonization and invasive properties.Invasion metastasis 1985;5:1144
27. Aznavoorian S et al. Molecular aspects of tumor cell invasion and metastasis. Cancer 1993;71(4):1368
28. Jessup JM et al. The biology of colorectal carcinoma. Curr Probl Cancer 1992;16:263.
29. Gohji K et al. Regulation of gelatinase production in metastatic renal cell carcinoma by organ-specific fibroblast.Jpn J Cancer Res 1994;85:152
30．潘李珍 等译。R．I．费雷什尼 编著 实用动物细胞培养技术 世界图书出版公司北京公司出版 1996第1版
31．徐国超等。人牙龈成纤维细胞电泳行为的研究。华西口腔医学杂志 1996；14(3)：183
32. Sherbt GV. Cell electrophoresis:the biophysical characterisation of the cell surface.London: Academic Press 1978;36
33. Yongde Shi et al. Macrophage electrophoretic mobility test for brain tumors and malignancies Chn Med 1979:3(92):211
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