CN1317386C - Lung cancer strain with high potential power of bone transference and its generation - Google Patents
Lung cancer strain with high potential power of bone transference and its generation Download PDFInfo
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- CN1317386C CN1317386C CNB2004100930628A CN200410093062A CN1317386C CN 1317386 C CN1317386 C CN 1317386C CN B2004100930628 A CNB2004100930628 A CN B2004100930628A CN 200410093062 A CN200410093062 A CN 200410093062A CN 1317386 C CN1317386 C CN 1317386C
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Abstract
The present invention belongs to the technical field of microorganism animal cell lines. In the present invention, cell strains SPC-A-1 of human lung adenocarcinoma are used as source cells; SPC-A-1BM for the cell strains of the lung adenocarcinoma, which has high potency of bone metastasis, is established through a method of in vivo and in vitro continuous screening of mice with immune defects and the detection of radionuclide bone imaging. The cell strains have the following biological characteristics that cells have small volume because the cell volume is reduced by 30% as compared with the source cells; rapid cell growth is realized; the infiltration capability and the migration capability of the cells are three times as great as those of the source cells; high tumor forming rate of naked mouse transplanting and high short-term tumor detaining rate are obtained; the malignant degree is high because the metastasis of multiple positions is caused by blood channels; and the positive marking rate of surface antibodies of human Her-2 tumors is 90.9%. The present invention provides a technology platform and an effective molecule detection method for systematic research into the frequent metastasis of lung cancer and the exploration diagnosis and treatment of the bone metastasis of lung cancer of human bodies.
Description
Technical field
The invention belongs to biotechnology, technical field of microbe cell line, be specifically related to a kind of lung adenocarcinoma cell line and establishment method and purposes with high potential of bone metastasis.
Background technology
Lung cancer is one of the most common clinical, tumour that grade malignancy is the highest, 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 still treated based on the multidisciplinary synthesis of surgical operation.Unremitting effort through decades, 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 transferrer is up to 40%, and the adenocarcinoma of lung bone rate of transform is up to 51.6%.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 bone metastases of lung cancer at present.Therefore, molecule mechanism and the positive searching that further investigation human lung adenocarcinoma bone shifts effectively detects, the diagnosis and treatment means, has very important significance.Because above advanced lung cancer of IIIa phase belongs to the operation taboo, be difficult to obtain experiment material the metastatic carcinoma of bone cell is carried out systematic study, thereby the bone metastasis model of setting up the human body adenocarcinoma of lung is the unique selection of carrying out this type of research at present.
Report is arranged, adopt the method for tumour cell ventricle injection mammary cancer and melanoma cell to prepare nude mice bone metastasis model, obtain better effects [Wakabayashi, Oncology, 2000 Jun; 59 (1): 75-80; Sasaki, Anticancer Res, 1998 May-Jun; 18 (3A): 1579-84].Document [Iguchi, Cancer Res 1996Sep 1; 56 (17): 4040-3] reported that the injection of personnel selection Lung Squamous Carcinoma Cells left ventricle forms the bone metastasis model, the effect of research parathyroid hormonerelated protein in bone shifts.Document [Miki, Oncol Res 2000; 12 (5): 209-217.] adopt the method for intravenous injection human small cell lung carcinoma to prepare the transfer of SCID mouse bone and other many internal organs metastasis model, and detect the bone transfer case with the X line.Document [Kjonniksen, Cancer Res, 1992 Mar1 are also arranged; 52 (5): 1347-51; Arguello, Int J Cancer, 1991 Jun 19; 48 (4): 583-90] report carries out ventricle injection to make a shortcoming of bone metastasis model is to cause multiple transfers such as brain, lung, adrenal cortex, and causes the experimental mouse bone to shift not occur as yet and shift dead because of other internal organs.Document [Nicolson, ExpCell Res, 1993 Feb; 204 (2): 171-80] report for the solution tumour cell causes many internal organs branch problem, can carry out the cell that strong bone metastasis forms ability repeatedly bone and shift making, the cell mutation that the simple bone that makes new advances in the hope of separation shifts.
Domestic and foreign literature there is no and uses the video picture of radio isotope bone to shift the report of seeking the bone metastasis in the animal model at bone.
Summary of the invention
The purpose of this invention is to provide a kind of lung adenocarcinoma cell line and establishment method thereof with high potential of bone metastasis.Further purpose of the present invention provides the medical application of this lung cancer 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 a kind of lung adenocarcinoma cell line with high potential of bone metastasis (Human Lung Adenocarcinoma cell Subpopulation with Highly Bone Metastasis, SPC-A-1BM).In preservation on September 23 in 2004, depositary institution: CGMCC, No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number: CGMCC No.1226, classification name: human lung adenocarcinoma bone transfer cell strain SPC-A-1BM.This cell strain has following biological characteristics:
1, cell volume is little, diminishes gradually through in vivo cell after the in vitro several cycles, can inject 2,000,000 bone transitional cells at the blood pipeline in one step after the 6th generation.
Then volume is bigger for parental cell (the low differentiation of SPC-A-1 lung adenocarcinoma cell), even also bigger than the cell of maxicell lung cancer.If blood road injection cell concentration must be less than 1,000,000 (70-80 a ten thousand/nude mice) first, otherwise can cause the nude mice embolism and death.
2, the cell growth rapidly.
Bone transitional cell after parental cell and 3-4 the circulation is cultivated under same cell amount, the same terms simultaneously, observe every day and can find the growth of bone transitional cell rapidly, also have many suspension cells in the culturing bottle except attached cell, this is rare in parental cell.
3, short-term missed rate height.
From beginning subcutaneous injection 100-200 ten thousand cancer cells, tumor growth to 1 centimeter needs 1.5-2 month, and the 70-80% knurl that stagnates only; Circulation 3-4 is for back subcutaneous injection 1,000,000 bone transitional cells, and tumor growth to 1 centimeter only needs week age and 100% knurl that stagnates.
4, the bone rate of transform improved by generation.
Table 1 is the comparison of each missed rate for cell, injection cell concentration and the bone rate of transform thereof.
5, the time that nude mice forms the bone transfer reduced by generation.
Table 2 is that different injection systems cause that the fate that tumor bearing nude mice formation bone shifts compares.
6, the ratio height that metastasis site occurs.
Table 3 is that different injection systems cause that the tumor bearing nude mice tissue shifts the comparison (%) of ratio.
7, the grade malignancy height of cell strain.
The bone transitional cell is with people Her-2 tumor surface antibody labeling, and through cells were tested by flow cytometry, positive rate is 90.9%, exceeds 10 percentage points of parental cells.Show that this cell strain is human archeocyte and grade malignancy height, poor prognosis.
Table 1.
| Go down to posterity | The subcutaneous injection missed rate | The left ventricle injection | Tail vein injection | ||
| Cell concentration (ten thousand) | Bone rate of transform % | Cell concentration (ten thousand) | Bone rate of transform % | ||
| Parental generation | 70-80％ | 70-80 | 30-40 | / | / |
| The s-generation | 80-90％ | 100 | 60-70 | / | / |
| The | 100％ | 100 | 70-80 | 100 | 30 |
| The | 100％ | 100 | 80 | 125 | 50 |
| The | 100％ | 125 | 80-90 | 150 | 50-60 |
| The | 100％ | 150 | 90± | 200 | 60 |
Wherein: 1, subcutaneous missed rate is added up after meaning every mouse bare subcutaneous injection 1,000,000 cells.
2, cell concentration means injectable maximum cell amount.Illustrate that repeatedly screening back cell progressively diminishes.
Table 2.
| SPC-A-1 | The 1st generation bone M | The 2nd generation bone M | The 3rd generation bone M | The 4th generation bone M | The 5th generation bone M |
| The injection of chamber, a left side | 80-96 days | 40-60 days | 28-35 days | 24-30 days | 21-28 days |
| Tail vein injection | / | 50-70 days | 35-40 days | 30-40 days | 30-39 days |
Table 3.
| Backbone | Jawbone | Four limbs | Rib | Brain | Submaxilary | Suprarenal gland | Cardiac muscle | Lung | LN | |
| The injection of chamber, a left side | 22.4 | 25.9 | 5.2 | 5.2 | 1.7 | 12.1 | 8.6 | 0 | 12.1 | 6.9 |
| Tail vein injection | 16.4 | 20 | 5.5 | 0 | 3.6 | 7.3 | 7.3 | 1.8 | 30.9 | 7.3 |
The present invention sets up the lung adenocarcinoma cell line with high potential of bone metastasis by following method.
Adopt laboratory animal immunodeficient mouse (nude mice, SCID mice, NIH-III BNX mice), shift animal model by the human lung adenocarcinoma bone of having set up, lung adenocarcinoma cell is formed transfer after the injection of experiment mice blood road, after the bone metastatic lesion was determined in the spike of radio isotope skeletal imaging agent, the vitro culture pathological tissues obtained bone transitivity lung carcinoma cell subgroup, repeated with the final lung adenocarcinoma cell that obtains to have high potential of bone metastasis of cocycle.Comprise the steps:
1, behind the immunodeficient mouse blood road injection parental generation lung adenocarcinoma cell, injects cell concentration 70-80 a ten thousand/nude mice first, form indivedual internal organs and shift;
2, behind the mouse tail vein injection radio isotope skeletal imaging agent, detect mouse bone metastatic lesion by the single photon emission computerized tomography instrument (SPECT) or the gamma camera row bone video picture of disposing specific pinhole collimator;
3, vitro culture pathology osseous tissue obtains bone transitivity lung adenocarcinoma cell subgroup;
4, above-mentioned cancer cells repeats to obtain to have the lung adenocarcinoma cell subgroup of high potential of bone metastasis with cocycle;
5, above-mentioned bone transitional cell is carried out biological property analysis, determine that the lung adenocarcinoma cell with high potential of bone metastasis comprises that ultrastructure relatively; The cell colony doubling time; Chromosome karyotype analysis; Tumorigenesis rate, the rate of transform and the specificity at tail vein, subcutaneous injection position.
Aforesaid method of the present invention is through 8 circulations, and the cancer cells animal bone rate of transform reaches more than 80%.
Adopt high potential bone of the present invention to shift the bone transfer animal model of lung adenocarcinoma cell line by human lung adenocarcinoma, the result shows the characteristics that the bone transfer has fast, specificity is high.The present invention has overcome the defective of prior art " the bone video picture is highly sensitive, specificity is low ", makes the bone migration period from initial 3 months 21-28 up till now days; Metastasis site from initial be difficult to form shift (because of all frozen lung cancer cell lines because prolonged and repeated frozen going down to posterity) so that active the reduction, or detect with the X line and promptly to enable to detect the bone metastasis and can't duplicate (the X line is taken the photograph the cancer cells that tablet amounts can shift mouse tissue and killed) also, therefore can't obtain the bone transitional cell, can detect the bone metastasis up till now and can raise bone transitional cell of new generation external former being commissioned to train again.The present invention injects doses in lotus knurl immunodeficient mouse body radio isotope makes that can detect the bone metastasis can raise bone transitional cell of new generation external former being commissioned to train with this metastasis again, and then inject in the mouse body, in the immunodeficient mouse body He after the external repeated screening, make tumor cell viability heighten thus.The injection of blood road mouth, head, lung, suprarenal gland, backbone, bones of limbs and rib occur after 3,4 generations morely to be shifted, and forms typical adenocarcinoma of lung transfer mode; Missed rate is from beginning subcutaneous injection 100-200 ten thousand cancer cells, and knurl length to 1 centimeter needed 1.5-2 month and 70-80% knurl subcutaneous injection 1,000,000 bone transitional cells up till now that stagnate only, and tumor length to 1 centimeter only needs week age and 100% knurl that stagnates.
The present invention has set up human lung adenocarcinoma metastatic series model and the high potential bone transfer lung adenocarcinoma cell line that shifts animal model based on the bone of human lung adenocarcinoma.Can carry out early diagnosis to the transfer of human lung adenocarcinoma bone provides reference data, also can further set up the genes involved chip technology, and assessment is explored various about things and comprised treatment curative effects such as chemistry, biology, immunity, Chinese materia medica, radiopharmaceuticals.Shift and explore diagnosis and treatment human body bone metastases of lung cancer for systematic study lung cancer is multiple technology platform is provided.Simultaneously, for further analyzing the genes involved that relevant functional genome of bone metastases of lung cancer and bone shift, deepen the understanding to the bone metastases of lung cancer molecule mechanism, the early diagnosis, the assessment curative effect that shift for bone provide a kind of effective molecular detecting method.
Description of drawings
Fig. 1, Fig. 2 are that normal nude mice goes up the video picture of lower part of the body bone.Fig. 3 is the SPC-A-1 cell.Fig. 4 be the 4th generation the bone transitional cell.Fig. 5 is nude mice skull, lumbar vertebrae tumour.Fig. 6, lumbar vertebrae tumour pathological section.Fig. 7, nude mice jawbone, left humerus tumour.Fig. 8, nude mice left humerus tumour pathological section.Fig. 9, upper and lower jawbone shifts.Figure 10, pathology show that jawbone shifts.Figure 11, right head enlargement.Figure 12, lower jaw soft-tissue tumor.Figure 13, maxilla and lower-left jaw M.Figure 14, jawbone shifts pathology.Figure 15, the enlargement of bilateral maxilla.Figure 16, submaxilary knurl M.Figure 17, rib shifts.Figure 18, pathological section confirms.Figure 19, submaxilary suprarenal gland M.Figure 20, suprarenal gland M pathology.
Figure 21, the 22nd, through the cells were tested by flow cytometry result, wherein, regional A is the diffusing point of cell density content, records the percentage that counting accounts for grand total, records the counting in the zone, X-axis, Y-axis average to the bone transitional cell; Area B is a fluorescein time period intensity, records the percentage that the peak region inside counting accounts for grand total, records B zone inside counting, X-axis, Y-axis average; Figure 21 wherein, murine antibody mark bone transitional cell is organized in contrast, and Figure 22 is that Her-2 tumor surface antibody labeling positive rate is 90.9%.
Embodiment
1, preliminary experiment human lung adenocarcinoma cell (SPC-A-1) 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 and ketamine respectively at abdominal cavity and the leg muscle of mouse,
4, cancer cell injection injects 0.7 * 10 at the left ventricle of anesthetized mice first
6Human lung adenocarcinoma cell, the SPF level is raised in cages.
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, find the bone metastasis, choose the bone material that meets lung cancer far-end metastasis characteristic and make vitro culture and pathological section.
Immunodeficient mouse forms behind left ventricle injection human lung adenocarcinoma cell and shifts, behind the mouse tail vein injection radio isotope, the bone metastatic lesion is determined in the gamma camera and the ECT plane bone video pictures of two probe siemens that in the configuration aperture diameter are the pinhole collimator of 1mm, and the result confirms to adopt the radio isotope bone scanning to detect the bone metastatic lesion than individual month of the Zao 3-6 of X line.Anaesthesia dosage during above-mentioned gamma camera pin hole video picture is once anaesthetized to repose with every mouse and is advisable more than 2 hours.Venous system injection cancer cells can not anaesthetized.
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 industry group, repeat 8-10 with the final lung carcinoma cell that obtains to have high potential of bone metastasis of cocycle with this cancer cells.
Detect demonstration the 6th generation bone transitional cell and dwindle 30% than source cell, its infiltration, the ability of migrating are 3 times of source cell, nude mice is migrated to the ratio of outflow height, the subcutaneous transplantation tumor formation rate is 100%, can cause after the injection of menses road that bone, lung, suprarenal gland, submaxilary, brain and cardiac muscle shift, especially bone shifts discovery time early, and the metastasis number is more.Left ventricle injection bone transitional cell causes that it is that mean transferred kitchen range number is 4 when inoculating for 3 weeks that lung, bone, kidney or suprarenal gland, submaxilary and lymphoglandula etc. are organized transfer time.
1, the former bone transitional cell of being commissioned to train and raising after the cellular immunity deficiency mouse left ventricle injection cancer cells secondary circulation, 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, tail vein injection injects 〉=10 from the tail vein
6Behind the cancer cells, the SPF level is raised in cages.
4, the injection of weighing was weighed weekly after three weeks, and body weight is lower than 16 grams and will tightly observes and prevent death.
5, video picture is made isotope bone scanning after one month in per two weeks, takes out after the discovery bone metastasis and makes vitro culture.
6, bone transitional cell subculture in vitro separately is cultivated and is repeated above work up to obtaining new bone transitional cell subgroup.
The result shows, the 4th generation tail vein injection adenocarcinoma of lung bone transitional cell the video picture of 36 days nucleic bones of mouse find that upper right jaw, left humerus head, rib, backbone radioactivity anomaly are dense poly-; Dissect and find lung, suprarenal gland and lower jaw soft tissue abnormality; Pathology confirms metastasis of cancer.Tail vein injection bone transitional cell causes transfer times such as lung, bone, lymphoglandula and suprarenal gland for inoculating 4-5 during week, and mean transferred kitchen range number is 3.
Cell culture medium of the present invention is 10% calf serum RPMI1640 substratum, pH=7, and culture temperature is 37 ℃.
Claims (3)
1, a kind of lung adenocarcinoma cell line with high potential of bone metastasis, source cell is the SPC-A-1 cell, it is characterized in that described cell is human lung adenocarcinoma bone transfer cell strain SPC-A-1BM, deposit number: CGMCC No.1226, have following biological characteristics: cell volume is little, dwindle 30% than source cell, the cell growth rapidly, its infiltration, the ability of migrating are 3 times of source cell, nude mice is migrated to ratio of outflow and short-term missed rate height, the menses road causes multi-section bit transition kitchen range, the grade malignancy height, and people Her-2 tumor surface antibody labeling positive rate is 90.9%.
2, the lung adenocarcinoma cell line with high potential of bone metastasis according to claim 1 is characterized in that described cell strain menses road causes multi-section bit transition kitchen range to comprise bone, lung, suprarenal gland, submaxilary, brain and cardiac muscle, wherein shifts early with bone, and number is many; Left ventricle injection bone transitional cell causes that organizing transfer time is that mean transferred kitchen range number is 4 when inoculating for 3 weeks, and tail vein injection bone transitional cell causes that be to inoculate 4-5 during week transfer time, and mean transferred kitchen range number is 3.
3, the lung adenocarcinoma cell line with high potential of bone metastasis according to claim 1 is characterized in that described short-term missed rate is a week age tumor growth to 1 centimeter, and described lung adenocarcinoma cell line short-term missed rate is 100% knurl that stagnates.
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| CN101717753B (en) * | 2008-10-09 | 2012-07-04 | 上海市胸科医院 | Chinese lung adenocarcinoma cell strain having metastatic potential of multiple organs |
| CN101955911B (en) * | 2009-07-14 | 2012-05-23 | 上海市胸科医院 | Chinese lung adenocarcinoma cell line with high metastases potentiality of bone, lever and adrenal gland |
| CN101955912B (en) * | 2009-07-14 | 2012-02-22 | 上海市胸科医院 | Chinese lung adenocarcinoma cell strain with bone metastasis characteristic |
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| CN102433307B (en) * | 2011-12-31 | 2014-07-16 | 广州呼吸疾病研究所 | Cell strain from human lung adenocarcinoma and preparation method thereof |
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