CN1202243C - Human liver cancer cell line - Google Patents
Human liver cancer cell line Download PDFInfo
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- CN1202243C CN1202243C CNB011268182A CN01126818A CN1202243C CN 1202243 C CN1202243 C CN 1202243C CN B011268182 A CNB011268182 A CN B011268182A CN 01126818 A CN01126818 A CN 01126818A CN 1202243 C CN1202243 C CN 1202243C
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- nude mice
- lung
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- screening
- shifts
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Abstract
The present invention belongs to the field of microbiological animal cell lines, and more specifically relates to a bel7402 with metastatic phenotype in pulmonary metastatic nidi. The bel7402 HCCLM3 which has special metastatic phenotype and can generate high pulmonary metastasis once a nude mouse is hypodermically inoculated is obtained by adopting a nude mouse lung to transfer nidus tumors and screening with a method of continuous pulmonary metastasis inside a nude mouse. The present invention provides suitable experimental materials for the in-depth research of the mechanism of hepatoma pulmonary metastasis and for the prevention and the cure of pulmonary metastasis.
Description
Technical field
The invention belongs to technical field of microbe cell line, be specifically related to from the lung metastasis, establish the Bel7402 of metastatic phenotype.
Background technology
Primary hepatocarcinoma is one of modal malignant tumour of China.Excision is therapy measure.Even but diameter is less than the small liver cancer of 5cm, recurrence rate was also up to 40-50% in postoperative 5 years.Liver cancer invasion and attack and transfer are the biggest obstacles of successfully treating.In hepatoma Metastasis, lungs are modal transfer organs, also are one of lethal major reasons of patient.Be pernicious biological behaviours such as the invasion and attack of understanding liver cancer cell in depth and transfer, must set up suitable research model.People's metastasis of cancer model of widespread usage is that people's cancer is inoculated in the immune deficiency animal at present, the human carcinoma cell line who mainly is meant the people's cancer nude mice metastasis model that forms and can takes place to shift in the immune deficiency animal body in the nude mouse body.Fudan University's liver cancer research had once been set up human hepatocellular carcinoma nude mouse model (tumour 1986,6:10-13), high-transfer human hepatocellular carcinoma nude mouse model (Int J Cancer 1996,66:239-243), (the Br J Cancer 1999 of high-transfer human hepatocellular carcinoma cells system, 81:814-21), the human liver cancer cell clone strain (Chinese liver and gall surgical department magazine is waited to deliver) of different metastatic potentials.These provide desirable experiment material for the research hepatoma Metastasis.Yet, make a general survey of people's liver cancer model both domestic and external, still there is not Bel7402 from the lung metastasis, can't compare research to the biological behaviour of primary tumor clone and lung metastasis clone, also just be difficult to seek the effective control strategy that shifts at lung.
Summary of the invention
The objective of the invention is from nude mice lung metastasis, to set up special phenotype Bel7402, be beneficial to further investigate the mechanism that the liver cancer lung shifts, and provide suitable experiment material for the control lung shifts.Another object of the present invention is the method that establishes special metastatic phenotype Bel7402.The present invention adopts step sizing method in the nude mouse, and high metastatic human hepatoma monoclonal cell strain MHCC97-H is inoculated in the nude mice liver, and then lung takes place shifts, and step sizing three times establishes the Bel7402 HCCLM3 of metastatic phenotype.Concrete grammar is as follows:
1. first round nude mice lung shifts screening, gets MHCC97-H cell 5 * 10
6It is subcutaneous that/0.2mL is inoculated in nude mice.Nude mouse is BALB/C-nu/nu, and is male, purchases in Shanghai Pharmaceutical Inst., Chinese Academy of Sciences age in 4-6 week, and the specific-pathogen free environment is raised down.Subcutaneous tumors grows to the about 1.0cm of diameter after 2 weeks, get tumor tissue fritter in-situ inoculating that subcutaneous tumors is cut into diameter 1mm in the nude mice liver, nude mice depletion after 42 days is taken out the multiple cropping of lung metastasis and is planted in nude mice subcutaneous, after 72 days, the lung metastasis is in the subcutaneous diameter 1.5cm tumour that increases into of nude mice.
2. second take turns nude mice lung transfer screening, get above-mentioned nude mice subcutaneous tumors by first round method multiple cropping nude mice liver, row second is taken turns screening, and after 28 days, the lung metastasis is in the subcutaneous diameter 1.5cm tumour that increases into of nude mice.
3. third round nude mice lung shifts screening, takes out second and takes turns the subcutaneous tumors that nude mice lung transfer screening obtains, and repeats said process, and after 25 days, the lung metastasis is in the subcutaneous diameter 1.5cm tumour that increases into of nude mice.
4. cell cultures is built and is, take out the subcutaneous amplification knurl of lung metastasis and carry out cell cultures, nutrient solution be high sugared DMEM substratum (GIBCOBRL, Grand Is land, NY, USA) add 10% (v/v) foetal calf serum (Hyclone, Utah, USA).Culture temperature is 37 ℃, and atmosphere surrounding is 5%CO
2/ 95% air, humidity are saturated humidity.The cell growth is stable, changes nutrient solution every other day, goes down to posterity once in per 5 days, and the recovery of conventional frozen back survives good.
The present invention shifts screening through above-mentioned continuous 3 nude mice lungs, has set up special metastatic phenotype Bel7402 HCCLM3 (Human hepatocellular carcinoma cell linelung metastasis, 3rd selection).
Clone of the present invention after testing, its general biological characteristics shows that this cell is the Polygons epithelioid cell, adherent growth, the forfeiture of contact growth-inhibiting, the hypo-triploid caryogram, karyomit(e) scope 37-60 bar, 44.5 hours cell colony doubling times, cloning efficiency 32.4 ± 3.2%, cell random motion speed 20 ± 2 μ m/h.Flow cytometry shows, the cell cycle each the time phase percentage be respectively apoptotic cell 9.18%, G1 phase 56.37%, S phase 23.09%, G2-M phase 8.88%.Immunocytochemical stain shows that MHCCLM3 cell AFP, albumin, Cytokeratin 8 are strong positive, the P16 positive, P53, nm23, HBsAg feminine gender.The fluorescent PCR method detects has HBV DNA to integrate in the cellular genome.
Cell of the present invention is tied to form knurl and the transitivity detection shows 5 * 10
6Cell/0.2ml dose inoculation nude mice is subcutaneous, tumor formation rate 100% (10/10), becoming knurl latent period is 11.0 ± 1.0 days, the subcutaneous tumors growth rapidly, inoculation back the 15th day, obvious vasoganglion promptly appears in tumor surface; Gross tumor volume reaches 3568.8 ± 450mm after 30 days
3The transfer of 100% lung takes place in nude mice after 5 weeks, and the median of lungs metastasis is 121 a/lung.
The tumor tissue fritter in-situ inoculating that subcutaneous tumors is cut into diameter 1mm is in the nude mice liver after 35 days, the popularity metastasis of cancer appears in nude mice: stomach wall invades 10/10, intraabdominal metastasis (hepatogastric ligament, nethike embrane, pancreas etc.) 8/10, shift 10/10 in the liver, diaphram shifts 7/10, lung shifts 10/10, and lungs metastasis median is 268 a/lung.
Table 1 is the one-tenth knurl and the metastatic test data of clone of the present invention.
The above results shows that clone of the present invention has been showed special metastatic phenotype, promptly be inoculated in subcutaneous 100% lung that promptly takes place of nude mice and shift, and metastasis is many, is to have Bel7402 special metastatic phenotype, that the transfer of height lung promptly takes place in subcutaneous vaccination.The mechanism that shifts for further investigation liver cancer lung and prevent and treat the liver cancer lung and shift suitable experiment material is provided.
Table 1
The test item result
Swollen dosage of inoculation 5 * 10
6Cell/0.2ml
Knurl
Several 10 of inoculation nude mice
Carefully
Born of the same parents
Tumor formation rate 100% (10/10)
Naked
Mouse become knurl latent period (my god) 11 ± 1
Skin
Inoculate subcutaneous tumors volume (mean+SD) 3568.125 ± 450mm after 30 days
3
Down
Connect
Inoculate the lungs rate of transform 100% (10/10) after 5 weeks
Kind
121/the lung of median of nude mice lungs metastasis
Rind gall tissue block dosage of inoculation 1 * 1 * 1mm
3/ nude mice
Down
Several 10 of inoculation nude mice
Knurl
Group
35 days observing times
Knit
Average-volume (mean+SD) 5080 ± 160mm of liver neoplasm after little 35 days
3
Piece
The transfer case of 35 days postabdomens
Connect
Stomach wall shifts 100% (10/10)
Kind
Intraabdominal metastasis 80% (8/10)
Naked
Shift 100% (10/10) in the liver
Mouse
Diaphram shifts 70% (7/10)
Liver
The lung rate of transform 100% (10/10) after dirty 35 days
268/the lung of median of lung metastasis
Description of drawings
The HCCLM3 cell of Fig. 1 for cultivating, Giemsa dyeing, 200 *.The Polygons epithelioid cell, adherent growth, the big and engrain of nucleus, kernel is obvious, visible pathologic mitosis picture.
Fig. 2 is a HCCLM3 cell 5 * 10
6Behind subcutaneous 35 days of cell/0.2ml dose inoculation nude mice, the formation gross tumor volume is 3188mm
3
Fig. 3 is behind the HCCLM3 cell subcutaneous tumors cancerous tissue inoculation liver 35 days, and the arrow indication is stomach wall and intraabdominal metastasis kitchen range.
Fig. 4 is behind the HCCLM3 cell subcutaneous tumors cancerous tissue inoculation liver 35 days, and each leaf of liver is seen a plurality of transfer tubercles.
Fig. 5 is HCCLM35 * 10
6Behind subcutaneous 35 days of cell/0.2ml dose inoculation nude mice, form big metastasis, surround Bronchiole in lung, HE dyeing, 200 *.
Claims (2)
1, a kind of Bel7402 is characterized in that deriving from nude mice lung metastasis, is inoculated in subcutaneous 100% lung that can take place of nude mouse and shifts, and described clone is to shift screening by continuous nude mice lung to set up, and concrete steps are:
(1) first round nude mice lung shifts screening, and the MHCC97-H cell inoculation is subcutaneous in nude mice, and after the one-tenth knurl, in-situ inoculating subcutaneous tumors fritter after the nude mice depletion, takes out the multiple cropping of lung metastasis in the subcutaneous knurl that increases into of nude mice in the nude mice liver,
(2) second take turns the nude mice lung shifts screening, gets the multiple cropping of above-mentioned nude mice subcutaneous tumors in the nude mice liver, and method is the same,
(3) third round nude mice lung shifts screening, gets second and takes turns the subcutaneous tumors that the nude mice lung shifts screening, repeats said process,
(4) cell cultures is built and is, gets the subcutaneous amplification knurl of third round lung metastasis and carries out primary cell culture, and nutrient solution is the high sugared DMEM substratum that contains 10% foetal calf serum, culture condition: 37 ℃, and 5%CO
2/ 95% air, saturated humidity is changed nutrient solution every other day, goes down to posterity once in per 5 days.
2, by the described Bel7402 of claim 1, it is characterized in that described clone also possesses Bel7402's morphology and biological characteristics, have HBV DNA to integrate in its cellular genome, can express AFP, albumin, Cytokeratin 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011268182A CN1202243C (en) | 2001-09-20 | 2001-09-20 | Human liver cancer cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011268182A CN1202243C (en) | 2001-09-20 | 2001-09-20 | Human liver cancer cell line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1338512A CN1338512A (en) | 2002-03-06 |
| CN1202243C true CN1202243C (en) | 2005-05-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011268182A Expired - Fee Related CN1202243C (en) | 2001-09-20 | 2001-09-20 | Human liver cancer cell line |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966170A (en) * | 2014-05-12 | 2014-08-06 | 浙江大学 | Highly metastatic hepatoma cell line with GFP (Green Fluorescent Protein)-labeled PNC and application of highly metastatic hepatoma cell line |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100595273C (en) * | 2006-11-29 | 2010-03-24 | 复旦大学附属肿瘤医院 | Pancreatic carcinoma cell lines with highly metastatic potential in the liver |
| CN102115730A (en) * | 2010-09-29 | 2011-07-06 | 复旦大学附属中山医院 | High metastatic potential hepatoma cell line capable of steady autophagy indication, and establishment method and application method thereof |
| CN103087989A (en) * | 2013-02-01 | 2013-05-08 | 湖南省肿瘤医院 | Human hepatoma cell line HLCZ01 and application thereof |
| CN103305467B (en) * | 2013-05-31 | 2016-06-15 | 湖南大学 | A kind of human liver cancer cell system HLCZ02 and application thereof |
| CN108467855B (en) * | 2017-02-23 | 2021-09-07 | 中国科学院上海营养与健康研究所 | Novel lung-specific metastatic hepatoma cell and preparation thereof |
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2001
- 2001-09-20 CN CNB011268182A patent/CN1202243C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966170A (en) * | 2014-05-12 | 2014-08-06 | 浙江大学 | Highly metastatic hepatoma cell line with GFP (Green Fluorescent Protein)-labeled PNC and application of highly metastatic hepatoma cell line |
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| CN1338512A (en) | 2002-03-06 |
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