CN104164406A - Mesenchymal stem cell in-vivo mutant tumor cell lung transitional cell strain B6 and application thereof - Google Patents
Mesenchymal stem cell in-vivo mutant tumor cell lung transitional cell strain B6 and application thereof Download PDFInfo
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Abstract
The invention discloses a mesenchymal stem cell in-vivo mutant tumor cell lung transitional cell strain B6, and a construction method and application thereof. The construction method comprises the following steps: injecting a mouse bone marrow mesenchymal stem cell in-vivo mutant tumor cell strain K3 into a nude mouse shin marrow cavity to cause lung transition, carrying out separate primary culture on the lung transition tumor body cells to obtain the in-vitro cells, carrying out monoclonal screening, in-vitro amplification and tumor transition, and repeating screening twice to obtain the in-vivo mutant tumor cell strain K3 lung transitional cell strain B6. The identification on the successful construction of the in-vivo mutant tumor cell strain K3 lung transitional cell strain B6 by a series of biological verification methods proves that the lung transitional cell strain B6 has higher invasion, transition, proliferation and clone formation capacities and tumor stem cell characteristics as compared with the in-vivo mutant tumor cell strain K3. The construction of the K3 in-vivo mutant tumor cell strain lung transitional cell strain B6 provides research bases for researching tumor stem cell biological characteristics and searching new ways for treating tumors, and has important scientific research value.
Description
Technical field
The present invention relates to interstital stem cell vivo mutations tumour cell lung transfer cell strain B6 and application thereof, more specifically, the present invention relates to a kind of interstital stem cell vivo mutations tumor cell line K3 lung transfer cell strain and application thereof of rat bone marrow-derived, belong to the vitro culture field of cell.
Background technology
Before half a century, just there is scholar to propose cancer stem-cell hypothesis (cancer stem cells, CSCs), think that tumour has heterogeneity, no matter be all to exist the few cell subsets of content in leukemia cell or solid tumor cell, these cells have induced tumor and generate, maintain self, be considered to the arch-criminal of tumour origin and tumour metastasis and recurrence.This theory thinks that CSCs originates from the sudden change of normal stem cell or progenitor cell, and they serving as the role of stem cell, and in tumour, other cells do not have into knurl ability in tumour cell, can not long-term surviving.Tumor stem cell is found the earliest in hemopathy, at present, more and more studies and finds that various solid tumor tissues exist tumor stem cell.Along with deepening continuously of tumor research, CSCs will become comparatively ideal oncotherapy target cell.
The separation of tumor stem cell is mainly according to the difference of the various membranins of tumor cell surface, adhesion molecule and acceptor, by immunological magnetic bead sorting method or airflow classification method separation of C SCs.The authentication method of CSCs generally comprises experiment two aspects in experiment in vitro and body.The many cytokines such as EGF, LIF that add in serum free medium of experiment in vitro are cultivated CSCs, by colony, form the mensuration of experiment, dryness related gene expression and induce its biological characteristicses of evaluation such as differentiation, the difference between comparison of tumor stem cell and non-tumor stem cell.In body, experiment has strong tumorigenicity in immunodeficient mouse body according to CSCs, and only a small amount of cell gets final product tumorigenesis, and the transplanted tumor kitchen range feature identical with primary tumo(u)r morphological feature.At present, by the inside and outside experiment of body, determine that tumour cell has self-renewal capacity, Multidirectional Differentiation ability and strong tumorigenesis ability and can be accredited as tumor stem cell.
Metastases is by tumour, at original site, to break through stratum basale directly to organize towards periphery diffusion or in remote organization's survival, form the process of new tumour by blood road or lymphatic metastasis oncocyte.The tumour cell in various sources shifts in vivo has organ specificity as a rule, and these have metastatic tumour ubcellular group can in specific region, form neoplasm metastasis under certain experiment condition.Metastases is the product of a multi-step cytobiology process, and this process is called as invasion and attack-transfer cascade effect, comprises that tumor cell migration is in remote organs, and the adaptive process to new environment.
Studies have found that, although 90% metastases cell can depart from primary tumor tissue, and the final organ at a distance that arrives, but only less than 2% cell, can form micrometastasis kitchen range, and finally can produce the metastases cell of obvious metastasis clinically, be only 0.02%.This has absolutely proved that metastases is an extremely inefficient behavior, and what can in the microenvironment of transfer place, maintain oneself's growth a large amount of propagation is exactly probably tumor stem cell.
The built vertical interstital stem cell vivo mutations tumor cell line K3(deposit number in this laboratory: CGMCC NO.9158, number of patent application: 201410236304.8), adopt rat mesenchymal stem cells tail vein repeatedly in injecting body, to become knurl, the cell in vitro that knurl somatocyte separation and Culture is obtained, then screening obtains interstital stem cell vivo mutations tumor cell line through mono-clonal.In conjunction with the current research about tumor stem cell and metastases relation; the cell that interstital stem cell vivo mutations tumour cell filters out after interior generation shifts is repeatedly proposed; have more height metastatic potential; the tumor stem cell characteristics such as tumorigenesis ability; therefore the patent protection of we this application transfer cell strain B6 of K3 lung (the hepatic metastases cell strain that F4 is K3, for the application example of B6 makes additional remarks).
Summary of the invention
Based on this this patent, a kind of new interstital stem cell vivo mutations tumour cell lung transfer cell strain B6 and application thereof have been set up.
Interstital stem cell vivo mutations tumour cell lung transfer cell strain of the present invention, called after B6, deposit number CGMCC NO.9157.
The construction process of interstital stem cell vivo mutations tumour cell lung transfer cell strain of the present invention, comprises the following steps:
(1) cultivation of male rat marrow interstital stem cell vivo mutations tumor cell line K3: (contain at DMEM nutrient solution
10% FBS) in culturing bottle, 37 ℃, 5% CO2, saturated humidity is cultivated.Go down to posterity every three days once;
(2) foundation of interstital stem cell vivo mutations tumour cell metastatic tumor model: phase K3 cell 0.25% tryptic digestion of taking the logarithm, platform is expected blue dyeing counting, the above person of viable cell 90% is for experiment.The resuspended one-tenth 1 * 10 of serum-free medium
7individual cell/mL, gets 0.1mL cell suspension inoculation in female BALB/C nude mice ligament tibia end medullary space.After 23-30 days, there is emaciation in nude mice, is moribund state, dissects to survey and see: medullary space injection group is shown in Pulmonary metastasis focuses.Former culture after the enzymic digestion of metastasis taking-up type Ⅳ collagen, causes metastatic tumor again after amplification in vitro, repeat 2 and take turns screening, obtains the lung transfer cell strain B6 in K3 source.
Step 2) described medullary space injection, its method is as follows: by nude mice with 0.05g/mL ketamine by after a 0.1 mL/ intraperitoneal anesthesia, dorsal position is fixed on animal plate, sterilization bilateral hind leg is stand-by.Cut skin, push muscle tissue aside and expose right side hind leg tibial tubercle, with Kirschner wire, rotate gently to there being the sense that falls through to stop.With 1mL syringe, drawing standby cell suspension again enters medullary space along puncturing hole and slowly injects 0.1mL.Left hind injects the resuspended diluent of 0.1mL cell (serum-free DMEM) with method, after injection finishes, seals puncturing hole immediately, skin suture with bone wax.After operation, unclamp nude mice, observe to its second wind and put back to rearging cage raising.
The Identification of Biological Characteristics method of interstital stem cell vivo mutations tumour cell lung transfer cell strain of the present invention, comprises several lower several aspects:
(1) paraffin section is made and HE dyeing: when the dying shape of emaciation appears in nude mice, with the neck method of breaking, put to death nude mice, dissect and detect transfer case.Getting primary tumor tissue and metastatic tumor tissue puts into and is added with antibiotic PBS Central Plains culture.Rest part is put into fixedly 48h of 4% paraformaldehyde, and paraffin embedding is cut into the thick section of 5 μ m, microscopic examination after HE dyeing.
(2) immunohistochemical staining analysis: the expression of interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 tumor stem cell genes involved ABCG2;
(3) cell monoclonalization experiment obtains monoclonal interstital stem cell vivo mutations tumour cell pulmonary metastases cell strain B6;
(4) source of lung transitional cell is identified in male sex determination (SRY) gene amplification;
(5) experiment of Transwell migration and invasion detects the invasion and attack transfer ability of interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6;
(6) total RNA extraction, reverse transcription and pcr amplification detect CXCR4 and the MMP2 expression conditions of interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6;
(7) total protein extraction, Western-blot analyze: the total protein that extracts interstital stem cell vivo mutations tumour cell K3, K3 lung transfer cell strain (K3-B6) and K3 hepatic metastases cell strain (K3-F4), Western-blot analyzes ABCG2 (GENE ID:312382), CD133 (GENE ID:60357), CD166(GENE ID:79559), Bmi-1(GENE ID:307151) expression, GAPDH (GENE ID:24383) gene of take is internal reference.
(8) interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 transfer case in the experiment of nude mice tumorigenesis and small animal living body imaging nude mouse;
(9) whether soft-agar cloning formation observation interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 has tumor stem cell characteristic clone Formation and characteristics;
(10) nude mice by subcutaneous tumorigenesis experimental observation interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 growth characteristics, for the transitional cell research of interstital stem cell sudden change tumour cell provides foundation.
In the Identification of Biological Characteristics method of cell strain of the present invention, lung shifts the tissue paraffin section de HE visible tumour cell that dyes, and immunohistochemical methods shows that ABCG2 protein expression increases; By limiting dilution, obtained the K3-B6 in mono-clonal source, pcr amplification sry gene (GENE ID:25221) shows that the sry gene of K3-B6 is positive, points out its source male sex cell; Compare with K3 cell, the experiment of Transwell migration and invasion shows that the migration and invasion ability of K3-B6 is higher, qRT-PCR analyzes and shows that CXCR4 (GENE ID:60628) and MMP2 (GENE ID:81686) genetic expression strengthen, and Western-blot analyzes and shows that CXCR4 (GENE ID:60628) protein expression of K3-B6 increases; The lung transfer ability of the visible K3-B6 of small animal living body imaging is higher, and it is stronger that soft-agar cloning forms ability; Tumor stem cell genes involved is as ABCG2 (GENE ID:312382), CD133 (GENE ID:60357), CD166 (GENE ID:79559), the protein expressions such as Bmi-1 (GENE ID:307151) increase, and the experiment of nude mice by subcutaneous tumorigenesis also shows that the subcutaneous tumorigenesis ability of K3-B6 strengthens.The tumour source of above-mentioned experimental result prompting vivo mutations tumor cell line K3 lung transfer cell strain B6, the expression of stem cell genes involved and invasion and attack, migration, propagation, tumorigenesis ability are compared K3 and are all significantly improved.
Accompanying drawing explanation
Fig. 1 is that result figure is seen in the transfer of medullary space injection lung substantially.
Fig. 2 is the metastasis HE of lung coloration result figure after medullary space injection.
Fig. 3 is tumor tissue immunohistochemistry (ABCG2) staining analysis result.
Fig. 4 is the cultivation figure of liver pulmonary metastases histocyte mono-clonal result and each strain cell.
Fig. 5 is sry gene amplification electrophorogram, in figure, and M:DNA marker LC: blank ♀: female rats DNA ♂: male rat DNA.
Fig. 6 is Transwell experiment K3-F4, K3-B6 cells in vitro migration and invasion potentiality result, and in figure, A: the cell quantity statistical graph of lower chamber.* representative has significant difference (P < 0.05, compares with K3 groups of cells, n=3); B: the cellular form figure of lower chamber, above Transwell migration experiment, below Transwell Matrigel (* 200).
Fig. 7 is K3, K3-F4, CXCR4 in K3-B6 cell, the expression of MMP2, in figure, A: fluorescence quantitative RT-RCR detects K3, K3-F4, (* representative has significant difference (P < 0.05 compares with K3 groups of cells, n=3)) in the expression of CXCR4 mRNA in K3-B6 cell; B: fluorescence quantitative PCR detection K3, K3-F4, in K3-B6 cell, the expression * of MMP2 mRNA representative has significant difference (P < 0.05 compares with K3 groups of cells, n=3)); C:Western-blot detects K3, K3-F4, the protein expression of CXCR4 in K3-B6 cell.
Fig. 8 is K3-F4, and in K3-B6 cell nude mouse, transfer ability detects, in figure, and A: red fluorescent protein labeled cell, stably express (take K3 cell as representative, * 100) after G418 screening; B: animal imaging system is observed K3, K3-F4, K3-B6 cell lung transfer case.
Fig. 9 is that soft-agar cloning forms experiment, in figure, and A:K3-F4, K3-B6 cell and the comparison of K3 cell clonal formation rate: each bar shaped post represents the mean value (± SD) of three independent experiments, * P < 0.005; B: clone ball form (* 40) under microscope.
Figure 10 is that Western blot analyzes ABCG2, CD133, and CD166, BMI gene is at K3, K3-F4, protein expression level in K3-B6 cell.
Figure 11 is K3, K3-F4, and tumorigenesis experimental result in K3-B6 cell paste, in figure, A:K3, K3-F4, subcutaneous tumors growth curve due to K3-B6 cell; B:K3, K3-F4, subcutaneous tumors tissue due to K3-B6 cell, shows that in the body of K3-B6, tumorigenesis ability is the strongest.
concrete embodiment
embodiment 1: the concrete application of the present invention one implementation step is described below:
(1) cultivation of male rat marrow interstital stem cell vivo mutations tumor cell line K3: in the culturing bottle of DMEM nutrient solution (containing 10% FBS), 37 ℃, 5% CO2, saturated humidity is cultivated.Go down to posterity every three days once;
(2) foundation of interstital stem cell vivo mutations tumour cell metastatic tumor model: phase K3 cell 0.25% tryptic digestion of taking the logarithm, platform is expected blue dyeing counting, the above person of viable cell 90% is for experiment.The resuspended one-tenth 1 * 10 of serum-free medium
7individual cell/ml, gets 0.1ml cell suspension inoculation in female BALB/C nude mice ligament tibia end medullary space.After 23-30 days, there is emaciation in nude mice, is moribund state, dissects to survey and see: medullary space injection group is shown in Pulmonary metastasis focuses.Former culture after the enzymic digestion of metastasis taking-up type Ⅳ collagen, causes metastatic tumor again after amplification in vitro, repeat 2 and take turns screening, obtains the lung transfer cell strain B6 in K3 source.
Cause pulmonary metastases scheme as follows: after using 0.05g/mL ketamine by a 0.1 mL/ intraperitoneal anesthesia nude mice, dorsal position is fixed on animal plate, and sterilization bilateral hind leg is stand-by.Cut skin, push muscle tissue aside and expose right side hind leg tibial tubercle, with Kirschner wire, rotate gently to there being the sense that falls through to stop.With 1mL syringe, drawing standby cell suspension again enters medullary space along puncturing hole and slowly injects 0.1mL.Left hind injects the resuspended diluent of 0.1mL cell (serum-free DMEM) with method, after injection finishes, seals puncturing hole immediately, skin suture with bone wax.After operation, unclamp nude mice, observe to its second wind and put back to rearging cage raising.
embodiment 2:the concrete application of the present invention two implementation steps are described below:
(1) cultivation of male rat marrow interstital stem cell vivo mutations tumour cell K3 and pulmonary metastases cell B6 thereof: in the culturing bottle of DMEM nutrient solution (containing 10% FBS), 37 ℃, 5% CO2, saturated humidity is cultivated.Go down to posterity every three days once;
(2) foundation of interstital stem cell vivo mutations tumour cell metastatic tumor model: phase K3 cell 0.25% tryptic digestion of taking the logarithm, platform is expected blue dyeing counting, the above person of viable cell 90% is for experiment.With the resuspended one-tenth 1 * 10 of serum-free medium
7individual cell/ml, gets 0.1ml cell suspension inoculation in female BALB/C nude mice spleen.After 30 days, there is emaciation in nude mice, is moribund state, dissects to survey and see: spleen group is shown in hepatic metastases kitchen range, and other positions have no transfer.Former culture after the enzymic digestion of metastasis taking-up type Ⅳ collagen, after amplification in vitro, cause again metastatic tumor, repeat 2 and take turns screening, the experiment of Liver m etastases cell monoclonalization is obtained to monoclonal interstital stem cell vivo mutations tumour cell diagnosis of hepatic metastases cell strain F4;
The interstital stem cell vivo mutations tumour cell diagnosis of hepatic metastases cell strain F4 and the pulmonary metastases cell strain B6 that obtain are carried out to investigation and comparison, for illustrating the transfer of tumour cell, provide experimental cell material.
(1) paraffin section is made and HE dyeing:
When there is the dying shape of emaciation in nude mice, with the neck method of breaking, put to death nude mice, dissect and detect transfer case, the results are shown in Figure 1, visible local obviously tumor tissue.Getting primary tumor tissue and metastatic tumor tissue puts into and is added with the standby former culture of antibiotic PBS.Rest part is put into fixedly 48h of 4% paraformaldehyde, and paraffin embedding is cut into the thick section of 5 μ m, and microscopic examination after HE dyeing, the results are shown in Figure 2, and right figure is the partial enlarged drawing of left figure, visible obviously oncocyte.
(2) immunohistochemical staining is analyzed the expression of interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 and diagnosis of hepatic metastases cell F4 tumor stem cell genes involved ABCG2:
Paraffin section de-waxing enters water, adds 3%H
2o
25min, sealing endogenous peroxydase, TBS washing; Water-bath heating 0.01M Sodium Citrate buffered soln (PH6.0), to 95 ℃, is put into tissue heating 10min and is repaired antigen.5% bovine serum albumin (BSA) sealing 20min, discards BSA.Add 37 ℃ of primary antibodie ABCG2 (mouse Chinese People's Anti-Japanese Military and Political College mouse, 1:50) and hatch 1h, abandon primary antibodie, TBS washing; Add anti-(goat anti-mouse) 37 ℃ of bioid two and hatch 20min, discard TBS washing; Add 37 ℃ of SABC and hatch 20min, washing; DAB colour developing, Hematorylin is redyed.Neutral gum sealing, Microscopic observation after horizontal airing.The ABCG2 expression that the results are shown in Figure visible metastatic tumor in 3, figure is higher, particularly Pulmonary artery.
(3) cell monoclonalization experiment obtains monoclonal interstital stem cell vivo mutations tumour cell pulmonary metastases cell strain B6 and hepatic metastases cell strain F4: through 5-6 the oncocyte purifying that goes down to posterity, the lung transitional cell of the former culture that the phase of taking the logarithm grows, with making individual cells suspension after 0.25% trysinization, the cell suspension that takes a morsel, with Trypan Blue living cell counting.Cell suspension is moved to Consecution multiple dilution in graduated centrifuge tube, be diluted to 1000 cell/mL, after mixing, add 96 orifice plates, make every porocyte suspension be transferred to 0-3 cell, then add 0.1ml containing the DMEM of 10%FBS, put into 37 ℃, in 5% CO2 incubator, under saturated humidity environment, cultivate.Microscopic observation record is containing the hole of a cell, after 6 d, under inverted microscope, observe and have single cell clone growth, supplemental medium continues to cultivate, and after at the bottom of covering with hole, digestion moves to 24 orifice plates, 6 orifice plates, finally move in culturing bottle and cultivate, obtain monoclonal cell strain B6; In the preparation of metastatic tumor model, after some medullary space injection, find hepatic metastases, same method obtains monoclonal hepatic metastases cell strain F4(F4 for making additional remarks for the application example of B6).The results are shown in Figure 4.
(4) source of hepatic metastases cell F4 and lung transitional cell B6 is identified in male sex determination (SRY) gene amplification: with the negative contrast of female rats genomic dna, the positive contrast of male rat genomic dna, pcr amplification lung transfer cell strain (K3-B6), the sry gene of hepatic metastases cell strain (K3-F4) (GENE ID:25221), the results are shown in Figure 5, show K3, K3-F4, the SRY of K3-B6 is positive, points out its source male sex cell.
(5) experiment of Transwell migration and invasion detects the invasion and attack transfer ability of interstital stem cell vivo mutations tumour cell diagnosis of hepatic metastases cell F4 and pulmonary metastases cell B6:
Transwell Matrigel: matrigel (Matrigel) is put 4 ℃ of thawings of spending the night, on ice, serum-free DMEM dilution (1:3) for Matrigel, the Matrigel having diluted adds Boyden Charmber cell upper strata gently, avoid Bubble formation, place 1h for 37 ℃, cell is resuspended in serum-free DMEM (1 * 10
5/ 200 μ L), each cell upper strata adds the above-mentioned cell suspension of 200 μ L, and lower floor's cell adds 500 μ L containing the DMEM of 10%FBS, establishes three multiple holes for every group.37 ℃, under 5%CO2 standard humidity, cultivate 6h.Cotton swab is softly wiped the cell of upper strata cell away, and PBS washes, and 4% paraformaldehyde is 30min fixedly, and PBS washes; Violet staining 15min, PBS washes.Microscopy under inverted microscope, takes pictures in 6 visuals field.
Transwell moves experiment: method is with transwell Matrigel, and just cell does not spread Matrigel.
The results are shown in Figure 6, result shows that the external migration and invasion potentiality of K3-B6 obviously strengthen compared with K3.
(6) CXCR4 and the MMP2 expression conditions in total RNA extraction, reverse transcription and qRT-PCR augmentation detection interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 and diagnosis of hepatic metastases cell F4:
Extract total RNA of interstital stem cell vivo mutations tumour cell, hepatic metastases cell strain (K3-F4), lung transfer cell strain (K3-B6), qRT-PCR detects CXCR4 (GENE ID:60628) and MMP2 (GENE ID:81686) expression conditions, take β-actin(GENE ID:81822) gene is internal reference.The results are shown in Figure 7, show that CXCR4 and the MMP2 genetic expression of K3-B6 is increased.
Table 1. amplification SRY, CXCR4, MMP2, the primer sequence of β-actin genomic dna
(7) total protein extraction, Western-blot analyze:
Extract the total protein of interstital stem cell vivo mutations tumour cell K3, K3 lung transfer cell strain (K3-B6) and K3 hepatic metastases cell strain (K3-F4), Western-blot analyzes ABCG2 (GENE ID:312382), CD133 (GENE ID:60357), CD166(GENE ID:79559), Bmi-1(GENE ID:307151) expression, GAPDH (GENE ID:24383) gene of take is internal reference.See Figure 10, show that the stem cell related gene expression of K3-B6 strengthens.
(8) interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 and diagnosis of hepatic metastases cell F4 transfer case in the experiment of nude mice tumorigenesis and small animal living body imaging nude mouse:
Take the logarithm K3, K3-F4, K3-B6 cell 0.25% tryptic digestion of phase red fluorescent protein (RFP) mark, platform is expected blue dyeing counting, the above person of viable cell 90% is for experiment.The resuspended one-tenth of serum-free medium 1x10
6cell/mL.Get 0.1mL cell suspension through tail intravenous inoculation in BALB/c-nu nude mouse, with method blank group injection PBS, 4 every group, metastatic potential in the body of two kinds of cells relatively.After 4 weeks, nude mice is pressed after a 0.1 mL/ intraperitoneal anesthesia with 0.05g/mL ketamine, adopt small animal living body imaging system (U.S. CRi Maestro company) to observe transfer case in nude mouse.The results are shown in Figure A in 8, figure is red fluorescent protein labeled cell, stably express (take K3 cell as representative, * 100) after G418 screening; B is that animal imaging system is observed K3, K3-F4, and K3-B6 cell lung transfer case, shows that K3-B6 lung transfer ability is the strongest.
(9) whether soft-agar cloning formation observation interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 and diagnosis of hepatic metastases cell F4 have tumor stem cell characteristic clone Formation and characteristics:
3% agar-agar soln, standby with physiological saline configuration autoclaving.Blank DMEM dilution agar concentration is in 0.6%, 6 orifice plate, respectively to add 1mL, solidifies stand-by under placement room temperature.Collect the cell of logarithmic growth, after PBS washing, trysinization counting.That gets 37 ℃ of insulations contains 1 * 10
4individual cell suspension 1.8mL, add 50 ℃ of 3% agar-agar soln 0.2mL to mix rapidly, be made into 0.3% nutrient agar, in 6 orifice plates, respectively add 1mL immediately, in culture plate, cultivate, after 14 days, take out methyl alcohol and fixedly after compressing tablet, use Giemsa stain dyeing, the clone that under microscope, counting cells number is greater than 50.Colony forming efficiency=average colony number/inoculating cell number * 100%.The results are shown in Figure 9, show that the stem cell related gene expression of K3-B6 strengthens;
(10) nude mice by subcutaneous tumorigenesis experimental observation interstital stem cell vivo mutations tumour cell pulmonary metastases cell B6 and diagnosis of hepatic metastases cell F4 growth characteristics, for the transitional cell research of interstital stem cell sudden change tumour cell provides foundation:
Get K3, K3-F4, K3-B6 cell dissociation counting, cell concn is adjusted into 5 * 10
6/ mL, 0.1mL injects subcutaneous right side, BALB/c-nu nude mice back.Under SPF condition, raise, when tumour is tangible by the time, use vernier callipers periodic measurement gross tumor volume.The calculation formula of gross tumor volume is 1/2 * a * b
2, wherein a represents long diameter, b represents short diameter.The results are shown in Figure 11, show that the interior tumorigenesis ability of body of K3-B6 is the strongest.
The above example has only been expressed several implementation method of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction of the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (4)
1. an interstital stem cell vivo mutations tumour cell lung transfer cell strain, called after B6, deposit number CGMCC NO.9157.
2. a kind of interstital stem cell vivo mutations tumour cell lung transfer cell strain as claimed in claim 1, is characterized in that, described cell strain derives from the interstital stem cell vivo mutations tumor cell line K3 of rat marrow.
3. a kind of interstital stem cell vivo mutations tumour cell lung transfer cell strain as claimed in claim 1, is characterized in that, the construction process of described cell strain, comprises the following steps:
(1) cultivation of male rat marrow interstital stem cell vivo mutations tumor cell line K3: at DMEM nutrient solution, contain
In the culturing bottle of 1 0%FBS, saturated humidity is cultivated; Go down to posterity every three days once;
(2) foundation of interstital stem cell vivo mutations tumour cell metastatic tumor model: the phase K3 cell tryptase protease digestion of taking the logarithm, platform is expected blue dyeing counting, the above person of viable cell 90% is for experiment; Serum-free medium is resuspended, and obtained cell suspension is inoculated in female BALB/C nude mice ligament tibia end medullary space; After 23-30 days, there is emaciation in nude mice, is moribund state, dissects to survey and see: medullary space injection group is shown in Pulmonary metastasis focuses; Former culture after the enzymic digestion of metastasis taking-up type Ⅳ collagen, causes metastatic tumor again after amplification in vitro, repeat 2 and take turns screening, obtains the lung transfer cell strain B6 in K3 source.
4. the application of interstital stem cell vivo mutations tumour cell lung transfer cell strain B6 in metastases and the correlative study at tumor stem cell described in claim 1.
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| CN114686434A (en) * | 2022-03-14 | 2022-07-01 | 北京航空航天大学 | Model for evaluating tumor cell invasion in vitro and application thereof |
| CN114686434B (en) * | 2022-03-14 | 2024-05-28 | 北京航空航天大学 | Model for in vitro evaluation of tumor cell invasion and application thereof |
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