CN101993854B - Colorectal carcinoma cell line CJF from hepatic metastasis and construction method thereof - Google Patents
Colorectal carcinoma cell line CJF from hepatic metastasis and construction method thereof Download PDFInfo
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Abstract
The invention provides a colorectal carcinoma cell line CJF from hepatic metastasis and a construction method thereof. The obtaining method of the colorectal carcinoma cell line comprises the following steps: obtaining tissues of colorectal carcinoma hepatic metastasis; after digesting the tissues into single cells by using collagenase/hyaluronidase, separating and inoculating CD133+ cells under NOD (Non-Obese Diabetic)/SCID rat skin by MACS (magnetic-activated cell separation); nodulating, and taking out tumor tissues; and culturing in vitro to obtain the colorectal carcinoma cell line CJF which can stably passage and is rich in colorectal carcinoma stem cells. In the invention, CJF cells are obtained from hepatic metastasis of colonic carcinoma patients; compared with other colorectal carcinoma cell lines, the colorectal carcinoma cell line CJF has incomparable advantages for the study of the colonic carcinoma hepatic metastases; cell subsets of the CJF cells for stably expressing the CD133+ cells is about 10 percent; and the action and the research of the colorectal carcinoma cell line in a colorectal carcinoma hepatic metastases process and the tumor stem cells in a metastases mechanism have wide application prospects.
Description
(1) technical field
The present invention relates to a kind of colorectal carcinoma cell line CJF and construction process thereof that derives from the hepatic metastases kitchen range.
(2) background technology
Large bowel cancer comprises the colorectal carcinoma and the rectum cancer, is one of common cancer, occupies the 3rd of malignant tumour at developed country's sickness rate, and mortality ratio occupies second.China's colorectal cancer incidence rate occupies the 3rd and in rising trend, and mortality ratio occupies the 5th.Invasion and attack and transfer are to influence PATIENTS WITH LARGE BOWEL treatment failure and lethal major cause, and wherein modal metastasis site is a liver, so the hepatic metastases mechanism of large bowel cancer is the important topic of current tumor research.In recent years think that tumor stem cell has one type of undifferentiated cell colony of self and multidirectional differentiation potential, research shows that most cells is in G
0/ G
1Phase, express multiple drug resistant gene, so insensitive to multiple chemotherapeutics, traditional tumor chemotherapeutic drug can only be eliminated most non-tumor stem cell and can not kill tumor stem cell, these remaining cells are tumor recurrence, transfer and treatment failure cause.It is thus clear that eliminating tumor stem cell is the key of prevention of recurrence and transfer.This just needs us to further investigate in the tumor development process, the biological mechanism of the self of modulate tumor stem cell, differentiation and tumour cell metastasis.
According to some specific mark of cell surface, identify the stem cell or the stem cell-like cell of some solid tumors successively in recent years.O ' Brien utilizes CD133+ as the tumor stem cell molecule marker in large bowel cancer; The tumour cell of successfully isolating CD133+ has very strong one-tenth knurl ability; 100～1000 tumour cells just can be in the subcutaneous one-tenth knurl of immunodeficient mouse, and the CD133-of equal amts then can not become knurl.Think that at present tumour cell CD133+ is one of specific marker of large bowel cancer stem cell.
But since finding the large bowel cancer stem cell so far; Also do not set up the research platform of a good tumor stem cell; The report that does not all have the foundation of the human large intestine cancer clone that is rich in the large bowel cancer stem cell has both at home and abroad greatly hindered the large bowel cancer stem cell in the recurrence of large bowel cancer and the research of transfer related mechanism.
(3) summary of the invention
The purpose of this invention is to provide a kind of human large intestine cancer clone that is rich in the large bowel cancer stem cell that derives from the hepatic metastases kitchen range; Set up good experiment porch for the research of large bowel cancer stem cell, help the mechanism, transfer and relapse to large bowel cancer, the treatment that has reached the large bowel cancer stem cell to carry out deep research.
The technical scheme that the present invention adopts is:
A kind of colorectal carcinoma cell line CJF that derives from the hepatic metastases kitchen range; Obtain by following method: the tissue of getting the colorectal cancer with liver metastases kitchen range; With collagenase/Unidasa be digested to unicellular after; It is subcutaneous in the NOD/SCID mouse that MACS sub-elects the CD133+ cell inoculation, takes out tumor tissues after the one-tenth knurl, obtains to stablize the colorectal carcinoma cell line CJF that is rich in the large bowel cancer stem cell that goes down to posterity through vitro culture.
Concrete, colorectal carcinoma cell line CJF is human colon carcinoma hepatic metastases cell CJF, is preserved in Chinese typical culture collection center; Address: China, Wuhan, Wuhan University; 430072, deposit number: CCTCC No:C 200956, preservation date: on 01 26th, 2010.
The present invention utilizes human large intestine cancer hepatic metastases pathological tissues; Subcutaneous through sub-electing the CD133+ cell inoculation to the NOD/SCID mouse; Draw materials from the NOD/SCID mouse again; Adopt vitro culture to combine to inoculate growth pattern in the animal body and hocket, set up the clone CJF that is rich in the large bowel cancer stem cell that derives from the colorectal cancer with liver metastases kitchen range, particularly the stem cell correlative study of large bowel cancer and the transfer of tumour provide suitable experiment material for the research of large bowel cancer.
The invention still further relates to the method that makes up said colorectal carcinoma cell line CJF; Said method comprises: the tissue of getting the colorectal cancer with liver metastases kitchen range; With collagenase/Unidasa be digested to unicellular after; It is subcutaneous in the NOD/SCID mouse that MACS sub-elects the CD133+ cell inoculation, takes out tumor tissues after the one-tenth knurl, obtains to stablize the colorectal carcinoma cell line CJF that is rich in the large bowel cancer stem cell that goes down to posterity through vitro culture.
Concrete, said method is following:
(1) gets the tissue of colorectal cancer with liver metastases kitchen range, be digested to unicellular with collagenase/Unidasa;
(2) cell that obtains of step (1) digestion is hatched 30min with the CD133 magnetic bead; Then through MACS sorting post; With PBS the CD133+ cell that is adsorbed is washed to collection tube from the sorting post, centrifugal supernatant discarded, get the deposition resuspended to the RPMI1640 of serum-free; The NOD/SCID mouse that was seeded to for 4～5 ages in week is subcutaneous, and postvaccinal NOD/SCID mouse is cultivated in standard SPF environment;
When (3) NOD/SCID mouse growth of xenografted to diameter was the 2cm left and right sides, cervical vertebra was put to death mouse from disconnected method, isolates tumor tissues under the aseptic condition, processes single cell suspension behind the collagenase digesting;
(4) get step (3) single cell suspension and carry out the amplification in vitro cultivation: seed cells into the RPMI1640 substratum earlier, cultivate after 3～5 days, after the substratum suction is gone; Add fresh RPMI1640 substratum and continue to cultivate, about 70% the time at the bottom of being cultured to cell and covering with bottle, inhale and remove substratum; Add tryptic digestive juice, place 37 ℃ of digestion, under inverted microscope, observe; Treat cell rounding, stop digestion when taking off wall, centrifugal, get deposition and add fresh RPMI1640 substratum continuation enlarged culturing; 80%～90% o'clock had digestive transfer culture at the bottom of every RPMI1640 substratum at a distance from replacing 1/2 in 3～4 days, cell cover with bottle; Vitro culture is after 20 generations, and the cell growth is stable, promptly obtains the stable colorectal carcinoma cell line CJF that is rich in the large bowel cancer stem cell that goes down to posterity.
The invention discloses a kind of clone that is rich in the large bowel cancer stem cell and construction process that derives from the hepatic metastases kitchen range; Its beneficial effect is mainly reflected in: 1, the CJF cell is the clone of drawing materials and setting up from colorectal carcinoma people hepatic metastases kitchen range; Have the special phenotype of colorectal cancer cells hepatic metastases, suitable material is provided colorectal cancer with liver metastases mechanism; 2, the cell subsets of CJF cytotostatic expression CD133+ is about 10%, and this strain clone is with a wide range of applications in colorectal cancer with liver metastases process and the effect research of tumor stem cell in metastasis; 3, be commissioned to train the period of supporting former; Since one of tumour cell experience from the body to the change process of external microenvironment, tumour cell is not easy survival, it is very difficult down tumour cell being gone down to posterity at general condition of in vitro culture; The present invention has used the method for magnetic bead sorting to isolate the CD133+ cell; Inoculation NOD/SCID mouse is subcutaneous, can effectively improve tumor formation rate, thereby can make possibly increasing greatly of its being immortalized property; After external stable 20 generations of going down to posterity, general culture condition just can make cell survival and go down to posterity.
(4) description of drawings
Fig. 1: sub-elect the subcutaneous back of former generation CD133+ cell inoculation NOD/SCID mouse and form transplanted tumor.
Fig. 2: the 20th generation CJF cell dissociation becomes single cell suspension, and after 14 days, the suspension clone ball forms in growth under RPMI1640 (II) culture condition.
Fig. 3: the suspension clone ball is containing the differentiation of 10% foetal calf serum RPMI1640 (I) culture medium condition.
Fig. 4: the chromosome karyotype analysis of CJF cell, most of cell are 48 karyomit(e)s, and the part cell is tetraploid.
Fig. 5: the growth curve chart of CJF cell, under the RPMI1640 that contains 10% foetal calf serum (I) condition, the cell colony doubling time is 48 hours.
Fig. 6: the cell subsets streaming figure of the 20th generation CJF cell CD133+, the CD133+ cellular component is 10% in this clone, and curve A is the homotype contrast, and curve B is CD133+ dyeing.
Fig. 7: the 20th generation CJF cell inoculation forms figure in 0.5% soft agar soft-agar cloning.
Fig. 8: the CD133+ cell becomes knurl ability map with the CD133-cell, black arrow becomes knurl for the CD133+ cell, and white arrow is that the CD133-cell does not become knurl.
(5) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
One, material
1. the compound method of cell culture fluid
1.1 nutrient solution RPMI1640 (I) forms as follows: 10% (v/v) foetal calf serum (Hangzhou SIJIQING company), 4ug/ml Regular Insulin, 100U/ml penicillium mould, 100ug/ml Streptomycin sulphate, solvent are RPMI1640 (1: 1) substratum (U.S. GIBCO company);
Nutrient solution RPMI1640 (II) forms as follows: 20ng/ml B27 (U.S. SIGMA company); 20ng/mlEGF (U.S. SIGMA company); 10ng/ml bEGF (U.S. SIGMA company), 4ug/ml Regular Insulin, 100u/ml penicillium mould; 100ug/ml Streptomycin sulphate, solvent are RPMI1640 (1: 1) substratum (U.S. GIBCO company).
1.2 Digestive system: the RPMI1640 substratum that contains 0.1% (w/v) collagenase and 0.1% (w/v) Unidasa.
2. other cell culture materials
The cell sieve; Trysinization liquid: 0.25% (w/v) pancreatin+0.02% (w/v) EDTA solution; Cells frozen storing liquid (FBS: DMSO=9: 1); The PBS damping fluid;
3. sample is originated
2nd Affiliated Hospital Zhejiang University School of Medicine surgical oncology patient Chen (admission number 553705) 43 years old, obtains the patient and agrees, signature Informed Consent Form, the colorectal cancer with liver metastases sample that excises in the art before the art.Pathological diagnoses is differentiation gland cancer companion large stretch of downright bad (pathological number 2008-20226) in the liver left-external side leaf transitivity.
4. laboratory animal
The NOD/SCID mouse, mouse age was 4～5 weeks, was provided by Chinese Academy of Sciences's Shanghai Si Laike Experimental Animal Center.
Two, method
From human colon carcinoma hepatic metastases kitchen range, draw materials respectively; Adopt digestion method to obtain cancer cells; Carry out culturing in vivo through magnetic bead sorting method sorting CD133+ cell inoculation NOD/SCID mouse; Steps such as tumour cell subculture in vitro separately cultivation are set up a strain can be in the external stable clone that goes down to posterity, and concrete grammar is following:
1. hepatic metastases kitchen range cancer cells obtains
Fresh colorectal cancer with liver metastases tissue is removed necrotic tissue with eye scissors, be cut into about 1mm after washing 4 times with containing 5 times of concentration two anti-(adding 500u/ml penicillium mould and 500ug/ml Streptomycin sulphate) PBS
3Size is with containing 0.1% collagenase and 0.1% Unidasa (Sigma Chemical Co., St.Louis; MO) RPMI1640 substratum digested 2 hours down at 37 ℃, with the cell sieve filtration in 40um aperture, removed not digestion tissue; In filtrating, add PBS liquid 3ml, the centrifugal 5min of 1000rpm, supernatant discarded; Be resuspended in the RPMI1640 substratum with the rinsing of PBS liquid, treat further processing.
2. sorting CD133+ cell inoculation NOD/SCID mouse carries out culturing in vivo
The primary cell that obtains of digestion hatches with the CD133 magnetic bead that (Miltenyi-Biotec GmbH, Germany) 30min make cell suspension through placing the MACS sorting post on the sorter then; Again the sorting post is withdrawn from sorter, the CD133+ cell that is adsorbed is washed to collection tube from the sorting post, the centrifugal 5min of 1000rpm with PBS; Supernatant discarded; Resuspended to the RPMI1640 substratum of 200ul, the NOD/SCID mouse that was seeded to for 4～5 ages in week is subcutaneous, and the NOD/SCID mouse is cultivated in standard SPF environment (with reference to " laboratory animal environment and facility " GB14925-2001); Observe the tumor growth situation weekly, measure the diameter of tumor size.
3. tumour cell subculture in vitro separately
When the about 2cm of NOD/SCID mouse growth of xenografted to diameter was big or small, cervical vertebra was put to death mouse from disconnected method, isolates tumor tissues under the aseptic condition, processes single cell suspension behind the collagenase digesting, and the tumour cell amplification in vitro is cultivated.
Concrete grammar is following: seed cells into culturing bottle earlier, cultivate after 3～5 days, some eugonic " cell islands " appear in the part apoptosis of tumor cells, after the substratum suction is gone, add fresh culture and continue to cultivate.About 70% the time at the bottom of being cultured to cell and covering with bottle, inhale and remove substratum, add the 2ml tryptic digestive juice; Place 37 ℃ of digestion; Under inverted microscope, observe, treat cell rounding, take off wall, add the RPMI1640 (I) that contains serum and stop digestion; The centrifugal 5min of 1000rpm adds fresh RPMI1640 (I) substratum and continues enlarged culturing.
According to the state and the nutrient solution change in color of cell, 80%～90% at the bottom of every nutrient solution (I) substratum at a distance from replacing 1/2 in 3～4 days, cell cover with bottle i.e. timely had digestive transfer culture, the ratio 1: 2 of going down to posterity, and inoculating cell density is 5 * 10
5/ bottle.Frozen vitro culture is after 20 generations, and the cell growth is stable.
Three, experimental result
One) it is many half a year in external continuous passage to derive from the colorectal cancer cells of hepatic metastases kitchen range, has reached generation more than 60 at present, called after CJF cell (CCTCC No:C 200956).
Specific as follows:
1. obtaining of former generation colorectal cancer cells
The colorectal cancer with liver metastases tissue obtains single cell suspension through after digesting.
2. amplification in the inoculation NOD/SCID mouse body after the colorectal cancer cells CD133+ sorting
The cell suspension that obtains sub-elects the CD133+ cell with the MACS method, and it is subcutaneous all to be seeded to the NOD/SCID mouse, the tumour of about 2 liang of week back formation diameter 0.5cm, the 6 week back about 2.0cm of diameter (Fig. 1).
3. the tumour cell subculture in vitro separately is cultivated
Through the tumour cell of amplification in vitro,, be 25cm a bottom surface through after the trysinization
2Culturing bottle in cultivate, preceding 15 days, treat that cell grows to 80%, goes down to posterity by 1: 2.After this cell growth begins to quicken, and whenever goes down to posterity once at a distance from 3 days, and the ratio that goes down to posterity is 1: 3～4, and nutrient solution is RPMI1640 (I), at present subculture in vitro separately cultivate about 60 surplus generation.
Two). characteristics of cell biology
1. morphology
Cell inoculation is after the RPMI1640 that contains 10% foetal calf serum (I) cultivates 2 days, and inverted microscope is observed big many cells down and is fusiformis, and endochylema is abundant, and nuclear is big, circle, and kernel is a plurality of and obvious.After being digested to single cell suspension, in RPMI1640 (II), cultivated 7 days down, the unicellular clump cells that grows up to the about 0.1～0.3mm of dense spherical diameter, tumour cell are spherical suspension growth, identical with document description (Fig. 2).The cell ball is adherent growth after the RPMI1640 that contains 10% foetal calf serum (I) cultivates 7 days, is divided into spindle cell (Fig. 3) again.
2. chromosome karyotype analysis
The mitotic figure cell of selecting karyomit(e) finely disseminated 100 mid-terms carries out karyotyping.Statistics shows, 48 of the most of one-tenth of cell chromosome, and wherein No. 3, No. 5 karyomit(e) is triploid, and No. 2 karyomit(e) has distortion, and few part cell chromosome is tetraploid (Fig. 4).
3. cytokinetics
The drafting of cell growth curve
CD133+ cell and the CD133-cell growth curve (Fig. 5) in the RPMI1640 that contains 10% foetal calf serum (I) substratum.Utilizing half altitude valve to calculate the cell colony doubling time is 48 hours.
4. tumor stem cell marker detection
It is 10% (curve A is the homotype contrast, and curve B is represented CJF cell CD133+ mark) that the 20th generation CJF cell uses flow cytometer to detect the CD133+ ratio.
5. frozen with the recovery
Cell recovery after frozen, the survival rate of cell is about 70%, and the viable cell cultivation conditions is good.
6. cloning efficiency
The 20th generation CJF cell inoculation contains 0.5% soft agar in lower floor; The upper strata contains 24 orifice plates of 0.2% soft agar, and 1000 cells in every hole were cultivated 21 days in 37 ℃ of incubators; Inverted microscope is observed the counting clone and is formed number, is 21 ± 4% (Fig. 7) through the cloning efficiency that calculates tumour cell.
7. allosome becomes knurl
Inoculation equal amts CD133+ (black arrow) and CD133-(white arrow) cell are subcutaneous to the NOD/SCID mouse, observe tumour and form.2000 CD133+ have tumor growth, and tumor growth (Fig. 8) is not seen at the CD133-place.10000 also visible tumor growths of CD133-, but more late than the CD133+ cell formation tumour of equal amts, and also volume is little.
Claims (1)
1. a colorectal carcinoma cell line CJF who derives from the hepatic metastases kitchen range is characterized in that said colorectal carcinoma cell line CJF is human colon carcinoma hepatic metastases cell CJF, is preserved in Chinese typical culture collection center; Address: China; Wuhan, Wuhan University, 430072; Deposit number: CCTCC No:C 200956, preservation date: on 01 26th, 2010.
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| CN103387963B (en) * | 2013-08-12 | 2015-05-20 | 厦门大学附属中山医院 | Ming classification human expansive type gastric cancer cell line and application thereof |
| CN108070561A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of the primary colon cancer cell of people |
| CN108685947A (en) * | 2017-04-11 | 2018-10-23 | 上海尚泰生物技术有限公司 | Colorectal cancer hepatic metastases primary tumor and transfer stove match model |
| CN108130311B (en) * | 2017-12-19 | 2020-12-08 | 柴怡 | Human primary colon cancer liver metastasis cell line HCS1220 |
| CN110760477A (en) * | 2019-10-10 | 2020-02-07 | 杭州广科安德生物科技有限公司 | Establishment method of mouse liver high-metastasis intestinal cancer cell strain, cell strain and application |
| CN117070462B (en) * | 2023-08-17 | 2024-02-23 | 镜像绮点(上海)细胞技术有限公司 | Isolation and purification of primary cells |
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