CN1982441A - Human lung adenocarcinoma self-transferring cell strain and its construction - Google Patents
Human lung adenocarcinoma self-transferring cell strain and its construction Download PDFInfo
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Abstract
A spontaneous transferring cell strain SPC-A-1BM is taken as human-lung adenocarcinoma SPC-A-1 cell strain. It has high spontaneous transferring rate and missed rate, lung transferring rate reaches to 80%, nodus lymphoideus transferring rate reaches to 85% and bone transferring rate reaches to 25%. It has excellent DNA cell stability and can be used to make research for human-lung adenocarcinoma transferring mechanism.
Description
Technical field
The invention belongs to biotechnology, technical field of microbe cell line, be specifically related to a kind of human lung adenocarcinoma cell line and establishment method and purposes with spontaneous transfer high potential.
Background technology
Lung cancer is one of the most common, that grade malignancy is the highest tumour, occupies preceding 3 of global malignant tumour M ﹠ M.In the especially big or middle city of China, lung cancer is the first malignant tumour killer from the 1980s all the time.At present, the clinical treatment principle of lung cancer is the multidisciplinary synthesis treatment based on surgical operation.Unremitting effort through decades, at present existing quite a few patient can take diagnosis early, early treatment and positive complex therapy (comprising operation, radiotherapy, chemotherapy and biotherapy etc.) and obtain long-term survival, but most patients has belonged to late period when going to a doctor, tumour extensively shifts, wherein the bone metastasis rate is up to 52.3% and 49.6%[Moriwaki, GanTo Kagaku Ryoho, 1,987 14; 1680-87], especially adenocarcinoma of lung is unexpectedly up to 62.1%[Yang Rui product, Chinese Journal of Nuclear Medicine, 1,992 12; 24-26].This class patient's result of treatment is very poor, and the annual rate of depositing is no more than 20%.Lack effective diagnosis and treatment method and medicine for lung cancer metastasis at present.Therefore, molecule mechanism that the further investigation human lung adenocarcinoma shifts and the effective diagnosis and treatment means of positive searching have very important significance.Because above advanced lung cancer of IIIa phase belongs to the operation taboo, be difficult to obtain experiment material metastasis cancer cell is carried out systematic study, thereby the metastasis model of setting up the human body adenocarcinoma of lung is the unique selection of carrying out this type of research at present.
At present, existing mouse metastasis model is to be planted tumour cell and formed experiment metastasis model [Wakabayashi, Oncology, 2000 Jun that many internal organs shift by left ventricle, tail vein road; 59 (1): 75-80; Sasaki, Anticancer Res, 1998 May-Jun; 18 (3A): 1579-84; Iguchi, Cancer Res, 1996 Sep1; 56 (17): 4040-3; Miki, Oncol Res, 2,000 12 (5): 209-217.], after also being arranged, bronchial instillation or lung puncture implantation tumour cell form lung cancer original position metastasis model [March, Vet Pathol, the 200138:483-490 that internal organs shift; Hastings, Anticancer Res, 2000 20:3625-30; McLemore, Cancer Res, 1987 47:5132-40].
In recent years, spontaneous metastasis model more and more is subjected to tumor research mechanism and scholar's attention, liver cancer spontaneous metastasis model [Gao is arranged, World J Gastroenterol, 2004 10:3107-11], mammary cancer spontaneous metastasis model [Wong, Am J Pathol, 2002 161:749-753], sarcoma spontaneous metastasis model [Yamamoto, Clin ExpMetastasis, 2003 20:181-185], the lung cancer spontaneous metastasis model, Inufusa[Nippon Geka GakkaiZasshi.1988 Jul; 89 (7): 1075-82] only shifting hypotype cell generation lung at the subcutaneous plantation human lung adenocarcinoma of nude mice shifts and plants in blood and muscle and do not shift; Teraoka[Jpn J Cancer Res.1995 May; 86 (5): 419-23] it is subcutaneous human lung tissue to be planted in the chest of SCID mouse, and subcutaneous then plantation people's lung squamous cancer and adenocarcinoma cell relatively are planted in the SCID human lung tissue on one's body and the transfer case of mouse lung tissue; Kozaki[Cancer Res, 200060:2535-40] the nodus lymphoideus transferring rate hypotype cell H460-LNM35 of people's lung large cell carcinoma H460 cell and it is planted in subcutaneous comparison two groups of SCID mouse sentinel lymph node of SCID mouse and lung transfer case respectively, sentinel lymph node group obvious difference and lung transfer group is not seen notable difference as a result; Miyahara[Thorac Cardiovasc Surg.2005Apr; 53 (2): 118-21] build Lweis mouse lung carcinoma cell spontaneous metastasis model and study certain curative effect of medication.
Domestic and foreign literature there is no has the report that the high metastatic human lung adenocarcinoma cell line of lung, lymphoglandula (mediastinum and axillary lymph knot) and osseous tissue is attacked in spontaneous transfer simultaneously.
The spontaneous in vivo transfer of the tumour cell in various sources is that the fixed target organ is arranged as a rule, be these have metastatic tumour ubcellular group (subpopulation) control well under the experiment condition, can in FX, forming the metastases kitchen range according to the fixed method, and these fixed metastasis models just the tumour worker as the instrument of medicine for anti transfer of tumor screening and the means and the model of oncological pathology man conduct understanding metastases pathologic process.[Lu Dayong, Henan medical research 20009 (3): 2822-51].
Summary of the invention
The purpose of this invention is to provide a kind of spontaneous transfer cell strain of human lung adenocarcinoma with spontaneous transfer high potential.Further purpose of the present invention provides the medical application of this human lung adenocarcinoma cell line.
The present invention adopts human lung adenocarcinoma SPC-A-1 cell strain as source cell, method by inside and outside step sizing of immunodeficient mouse body and nucleic bone video picture detection, set up lung adenocarcinoma cell line (Human Lung Adenocarcinoma cell Subpopulation with Highly Bone Metastasis with high potential of bone metastasis, SPC-A-1BM, CGMCC No.1226).The present invention is on the basis of human lung adenocarcinoma bone transfer cell strain SPC-A-1BM, human lung adenocarcinoma bone transitional cell carried down through experiment mice lung plantation back formation offside lung, mediastinum and axillary lymph again move, put to death after 60 days in the mouse lung plantation, get the mediastinal lymph nodes vitro culture.Treat to be planted in again after cell grows the lung of mouse.Through obtaining to have human lung adenocarcinoma cell subgroup (the Human Lung Adenocarcinoma Subline with SpontaneousMetastases Characters of the spontaneous transfer high potential of lung, lymphoglandula and bone after 3 circulations, SPC-A-1SM), in preservation on December 26 in 2005, depositary institution: CGMCC, the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number: CGMCC No.1577, classification name: the spontaneous transfer cell strain SPC-A-1-SM of human lung adenocarcinoma.
Cell strain of the present invention has following biological characteristics:
1, have the high rate of transform of the spontaneous transfer of lung, lymphoglandula and osseous tissue and high missed rate thereof,
The present invention compares parental cell and spontaneous transitional cell, and the result shows that cell strain of the present invention is obviously high in lung, lymphoglandula and the osseous tissue rate of transform and missed rate thereof, and missed rate reaches 100%; Lung shifts 80%; Nodus lymphoideus transferring rate 85%; Bone transferase 12 5%.Table 1 is parental generation and spontaneous transitional cell lung, lymphoglandula and the osseous tissue rate of transform (shifting mouse/plantation mouse) and missed rate comparison (%) thereof.
Table 1.
| Lung shifts | Nodus lymphoideus transferring rate | Bone shifts | Missed rate | ||||
| The cell seeding position | Left ventricle | Subcutaneous | Left ventricle | Subcutaneous | Left ventricle | Subcutaneous | Subcutaneous |
| SPC-A-1 | 0/10(0) | 0/10(0) | 3/10(30) | 3/10(30) | 2/19(10.5) | 0/10(0) | 8/10(80) |
| SPC-A-1SM | 8/8(100) | 16/20(80) | 8/8(100) | 17/20(85) | 8/8(100) | 5/20(25) | 20/20(100) |
2, chromosome karyotype analysis shows: the SPC-A-1SM cell belongs to hypo-triploid, and the bigger variation of the karyomit(e) of each cell existence, and the unstable of this cell DNA is described.
The karyomit(e) strip analysis of 100 cells of meter of the present invention and G divide band-karyotyping, the result shows: there is bigger variation in the karyomit(e) that the SPC-A-1SM cell belongs to hypo-triploid and each cell, the unstable of this cell DNA is described, wherein the 2nd, 7,13,15,17,22 increase by one, 2 of No. 23 increases; 9th, reduce one No. 16; One of No. 7 galianconism inversion; One of No. 15 translocation chromosome; Sex chromosome is respectively XX and Y.Table 2 is cytometer numerical table (counting 100 cells).
Table 2.
| Karyomit(e) bar number/cell | ≤0 | 41-50 | 51-60 | 61-70 | ＞70 |
| Cell count | 5 | 5 | 40 | 40 | 10 |
3, the growth of cell strain of the present invention reduces the dependency of EGFR signal path, and this reduction is accompanied by the rise of inhibitor of apoptosis protein survivin (Survivin) and Bcl-2 and angiogenesis factor VEGF-B, VEGF-C and VEGF-D genetic expression.
The present invention adopts quantitative PCR technique respectively to epithelial growth factor receptor (EGFR/HER) family among parental cell SPC-A-1 and the spontaneous transitional cell SPC-A-1SM, test analysis has been carried out in the genetic expression of vascular endothelial growth factor (VEGF) family and 3 inhibitor of apoptosis protein, the result shows, compare with parental cell, in this spontaneous transitional cell except that the erbB4/HER4 expression of receptor increases other expression of receptor significantly descend, point out the dependency reduction of the growth of this type of cell to the EGFR signal path, this reduction is accompanied by inhibitor of apoptosis protein survivin (Survivin) and Bcl-2 and angiogenesis factor VEGF-B, the rise of VEGF-C and VEGF-D genetic expression.Table 3 is gene expression analysis of parental cell and spontaneous transitional cell.
Table 3.
| Target gene | The genetic expression value | Ratio (%) | |
| SPC-A-1 | SPC-A-1SM | ||
| EGFR/HER1 | 7247.1 | 2198.3 | 30％ |
| ErbB2/HER2 | 11294 | 8362.1 | 74％ |
| ErbB3/HER3 | 9129 | 1755.7 | 19％ |
| ErbB4/HER4 | 27 | 45.4 | 168％ |
| VEGF-A | 52941.1 | 15316.1 | 29％ |
| VEGF-B | 21458.8 | 39080.5 | 182％ |
| VEGF-C | 2988.2 | 24368 | 816％ |
| VEGF-D | 10.3 | 175.0 | 1699％ |
| Survivin | 39764.7 | 43103.4 | 108％ |
| Bcl-2 | 1305.9 | 4281.6 | 328％ |
| Bcl-xL | 202.4 | 36.5 | 18％ |
The present invention sets up the spontaneous transfer cell strain of human lung adenocarcinoma with spontaneous transfer high potential by following method.
Adopt laboratory animal immunodeficient mouse (NIH-BNX mice), (national inventing patent application number: 200410093062.8) on the basis of human lung adenocarcinoma bone transfer cell strain of having set up and animal model thereof, human lung adenocarcinoma bone transitional cell carried down through experiment mice lung plantation back formation offside lung, mediastinum and axillary lymph again move, put to death after 60 days in the mouse lung plantation, get the mediastinal lymph nodes vitro culture.Treat to be planted in again after cell grows the lung of mouse.Through obtaining to have the spontaneous metastatic human lung adenocarcinoma cell subgroup of lung, lymphoglandula and high potential of bone metastasis after 3 circulations.The described strain process of building comprises the steps:
1. forming indivedual internal organs behind the immunodeficient mouse blood road injection parental generation lung adenocarcinoma cell SPC-A-1 shifts;
2. the gamma camera row bone video picture of the pinhole collimator in the special aperture of designing voluntarily by single photon emission computerized tomography instrument (SPECT) or configuration behind the mouse tail vein injection radio isotope skeletal imaging agent detects mouse bone metastatic lesion;
3. excision pathology osseous tissue carries out the transitivity lung adenocarcinoma cell subgroup of vitro culture acquisition based on the bone transfer;
4. repeating with cocycle with these cancer cells is the human lung adenocarcinoma cell subgroup of main high potential to obtain the having bone transfer;
5. will be through 7 circulations, the bone rate of transform reaches transitional cell more than 80% and is planted in mouse lung and gets the mediastinal lymph nodes vitro culture after 60 days;
6. the transitional cell that mediastinal lymph nodes is turned out remakes the mouse lung plantation, finishes the tenth circulation then, and the transitional cell of turning out reaches 100% through the mouse subcutaneous injection missed rate; Lung shifts 80%; Nodus lymphoideus transferring rate 85%; Bone transferase 12 5%.
The present invention has set up spontaneous transfer cell strain and the animal model thereof with human lung adenocarcinoma.Spontaneous transfer cell strain and animal model thereof are than other experimental transfer cell strain and the more approaching human pathogenesis of animal model.The foundation of this model not only provides a desirable material for the mechanism of further investigation lung cancer metastasis, and going deep into along with research, further anti-metastasis drug screens and develops novel effectively treatment means, and vast lung cancer patient is benefited, and has significant scientific meaning and using value.
Description of drawings:
Fig. 1 is parental cell SPC-A-1.Fig. 2 is cell strain the 10th a generation transitional cell of the present invention.Fig. 3 is cell strain the 12nd a generation transitional cell of the present invention.Fig. 4 is that cell strain Chromosome G of the present invention is divided band.Fig. 5 is a cell strain chromosome karyotype analysis of the present invention.Fig. 6 is that 60 days backbones of the subcutaneous plantation of cell strain of the present invention shift.Fig. 7 is that cell strain axillary lymph of the present invention is carried down and moved.Fig. 8 is a cell strain mediastinal lymph node metastasis of the present invention.Fig. 9 cell strain of the present invention is that backbone shifts pathology, HE * 100.Figure 10 is that cell strain of the present invention oxter, intercostal LN shift.Figure 11 is that cell strain lung of the present invention extensively shifts.Figure 12 is that cell strain slide pincer attack method of the present invention shows that lung shifts.
Embodiment
Embodiment 1
1, preliminary experiment human lung adenocarcinoma cell 1 * 10
6, it is subcutaneous to inject immunodeficient mouse, treats that tumor is long to take out tumor during to the 0.5-1 centimetre, gets peripheral knurl body tissue and shreds, smashes slurry, cell cultures, and the required cell concentration that increases after cell grows is standby.
2, the immunodeficient mouse body weight is greater than 20 grams, no matter male and female.Buying back SPF level raises in cages a few days.
3, the anaesthesia experiment animal is injected dilution Thiopental Sodium (0.75mg/ only) and ketamine (1.5mg/ only) respectively at abdominal cavity and the leg muscle of mouse.Stand-by (Venous system injection cancer cells need not anaesthetized).
4, cancer cell injection injects 0.8 * 10 at the left ventricle of anesthetized mice first
6The SPC-A-1 human lung adenocarcinoma cell.
5, weigh every other day after two weeks of injection of weighing.Body weight is lower than 16 grams and will tightly observes and prevent death.
6, per two weeks of video picture are made isotope bone scanning respectively, take out after the discovery bone metastasis and make vitro culture.Immunodeficient mouse forms behind left ventricle injection human lung adenocarcinoma cell and shifts, behind the mouse tail vein injection radio isotope, configuration voluntarily the aperture diameter of design, consigned processing be that bone metastatic lesion (the radio isotope bone scanning detects the bone metastatic lesion than individual month of the Zao 3-6 of X line) is determined in the gamma camera of pinhole collimator of 1mm and the ECT plane bone video picture of two probe siemens.
7, bone transitional cell subculture in vitro separately is cultivated the doubtful bone metastasis of mouse that will take out, is cut into small pieces and puts the cell cultures flask culture, waits to grow further amplification behind the cancer cells, then the bone transitional cell is injected in again mouse blood road.Obtain bone transitivity lung carcinoma cell subgroup.
8, will be through 7 circulations, the bone rate of transform reaches bone transitional cell more than 80% and is planted in mouse lung and gets the mediastinal lymph nodes vitro culture after 60 days.
9, the transitional cell that mediastinal lymph nodes is turned out remakes the mouse lung plantation.After 3 circulations of plantation vitro culture in the lung, obtain spontaneous transitional cell, called after: SPC-A-1SM.This spontaneous transitional cell is through mouse subcutaneous injection 2 * 10
6/ only, missed rate reaches 100%; After the lotus knurl 60 days, lung shifts 80%; Nodus lymphoideus transferring rate 85%; Bone transferase 12 5%.
Cell strain of the present invention with for source cell SPC-A-1 compares, result demonstration: SPC-A-1SM its soak into, migrate, invasive ability is that source cell is incomparable.A, behind the spontaneous transitional cell of subcutaneous plantation, mouse tumor formation rate 100%; Behind the subcutaneous plantation source cell SPC-A-1, mouse tumor formation rate 80%.B, the spontaneous transitional cell of subcutaneous plantation are after 60 days, and the lung metastatic nodules is 7-49, and mean number is 11, and the lung rate of transform reaches 80%; Nodus lymphoideus transferring rate kitchen range number is 6-12,8 of average out to, and the nodus lymphoideus transferring rate rate reaches 85%; Bone metastasis number is 1-3, and average 0.3, the bone rate of transform reaches 25%.And behind the subcutaneous plantation source cell SPC-A-1, the lung rate of transform is zero, nodus lymphoideus transferring rate rate 30%, the bone rate of transform are zero.C, behind spontaneous transitional cell of subcutaneous plantation and the source cell, other internal organs are not all found obviously to shift.D, behind the spontaneous transitional cell 2,000,000 of subcutaneous plantation, mouse existence fate is 66-92 days.And behind the subcutaneous plantation source cell 2,000,000, under equal conditions, mouse existence fate is 130-180 days.
Embodiment 2
1, spontaneous transitional cell 2,000,000 a cells/BNX mouse, standby.
2, immunodeficient mouse body weight 18 gram, 6-7 age in week, male and female dual-purpose.Buying back SPF level raises in cages a few days.
3, it is subcutaneous that the spontaneous metastasis model making is injected at the mouse left and right sides with each 1,000,000/0.1 milliliter of spontaneous transitional cell.
4, measure tumor and measure the tumor size weekly after three weeks.To tightly observe after 45 days and prevent death.
5, the subcutaneous cancer cells of video picture is planted 60 heaven-made isotope bone scannings, mouse deep anaesthesia before the scanning, and the big lung tubercle of part, lymphoglandula and bone metastasis present the dense poly-phenomenon of radioactivity in scanning process, must detect by emphasis.
6, measure the tumor size, the whole body digital photographing before dissecting.Dissection is observed position lymphoglandula such as the confession of tumor blood, outpost, oxter and mediastinum and is distributed from belly; Lung's transfer condition can be converged lung tissue with slide and be watched lung to shift, and calculates lung tubercle number, lymph footing (outpost, oxter and mediastinum) in case of necessity.Get the dense poly-osseous tissue of radioactivity and soak formalin censorship pathology.Each main points is all digital takes a picture.
7, fill in database and form.
Experiment of the present invention uses the video picture of BNX mouse preceding because of the long necessary injecting anesthetic medicine of time of developing, and emptying is urinated as far as possible; The cell culture medium that is adopted is 10% foetal calf serum RPMI, 1640 substratum well known in the art, pH=7, CO
2Incubating foster case cultivation is 37 ℃, CO
2Concentration is 5%.
Claims (5)
1, the spontaneous transfer cell strain of a kind of human lung adenocarcinoma is characterized in that described cell divide name: the spontaneous transfer cell strain SPC-A-1-BM of human lung adenocarcinoma, deposit number: CGMCC No.1577.
2, by the spontaneous transfer cell strain of the described human lung adenocarcinoma of claim 1, it is characterized in that described cell has following biological characteristics:
1) have lung, lymphoglandula and osseous tissue high spontaneous transferring rate and high missed rate thereof, wherein missed rate reaches 100%, and lung shifts 80%, nodus lymphoideus transferring rate 85%, bone transferase 12 5%;
2) karyotype belongs to hypo-triploid, and chromosomal variation is big, the cell DNA instability;
3) growth of cell strain reduces the dependency of EGFR signal path, is accompanied by the rise of inhibitor of apoptosis protein survivin and Bcl-2 and angiogenesis factor VEGF-B, VEGF-C and VEGF-D genetic expression.
3, by the spontaneous transfer cell strain of the described human lung adenocarcinoma of claim 1, the source cell that it is characterized in that described cell strain is the SPC-A-1 cell.
4, by the establishment method of the spontaneous transfer cell strain of the described human lung adenocarcinoma of claim 1, it is characterized in that by following step:
1) forming internal organs behind the immunodeficient mouse blood road infusion source cell shifts;
2) the gamma camera row bone video picture by single photon emission computerized tomography instrument or configuration pinhole collimator detects mouse bone metastasis behind the mouse tail vein injection radio isotope skeletal imaging agent;
3) the pathology osseous tissue carries out the transitivity lung adenocarcinoma cell subgroup of vitro culture acquisition based on the bone transfer;
4) cancer cells of step 3) repeats above-mentioned circulation and obtains to have that bone shifts is the human lung adenocarcinoma cell subgroup of main high potential;
5) bone of step 4) transfer human lung adenocarcinoma cell is planted in mouse lung and gets the mediastinal lymph nodes vitro culture after 60 days, gets the nodus lymphoideus transferring rate cell;
6) the nodus lymphoideus transferring rate cell with step 5) remakes the mouse lung plantation, finishes the tenth circulation, gets the spontaneous transfer cell strain of human lung adenocarcinoma;
7) with the subcutaneous plantation of the spontaneous transfer cell strain mouse of the human lung adenocarcinoma of step 6).
5, by the establishment method of the spontaneous transfer cell strain of the described human lung adenocarcinoma of claim 4, the spontaneous transitional cell 2,000,000/mouse of the subcutaneous plantation human lung adenocarcinoma of the mouse of step 7) wherein, 60 days.
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