CN108467854A - New bone transspecific liver cancer cells and its preparation - Google Patents
New bone transspecific liver cancer cells and its preparation Download PDFInfo
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- CN108467854A CN108467854A CN201710099943.8A CN201710099943A CN108467854A CN 108467854 A CN108467854 A CN 108467854A CN 201710099943 A CN201710099943 A CN 201710099943A CN 108467854 A CN108467854 A CN 108467854A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- A—HUMAN NECESSITIES
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Abstract
The present invention relates to a kind of bone transspecific liver cancer cells 3B BM, and specifically, the cell 3B BM are that the preserving number of China typical culture collection center is CCTCC NO：The HEP 3B organ specificity transfer cell lines BM of C2016174.
Description
Technical field
The invention belongs to biology and oncology, in particular it relates to which a kind of bone transspecific liver cancer is thin
Born of the same parents.
Background technology
Worldwide, hepatocellular carcinoma is to lead to the second largest factor of tumour associated death, and the high metastatic of liver cancer is led
Hepatocarcinoma patient is caused to keep the prognosis after drug therapy very poor, clinical common hepatoma Metastasis turns for Intrahepatic metastasis, Lung metastases and bone
It moves.
The transfer of liver cancer is related to multiple processes, including tumour cell falling off in hepatocyte in situ, invades surrounding tissue, enters
Hematological system, survival in hematological system and goes out blood vessel and then is cloned in remote organization's orga- nogenesis, and each process relates to
And the interaction between the variation and tumour and microenvironment of tumour cell itself, specific mechanism of action are also not fully clear
Chu.
Tumour cell, which ties up to, has critical role in the basic research of tumour, cultured tumor cells in vitro be it is more difficult,
Especially establish can long term growth, passage, and the human tumor cell line with certain feature, it will usually it is unstable to meet with passage feature
Calmly, situations such as Tumor formation is poor, genetic background is inconsistent or Specific marker differential expression is big.For liver cancer cells,
Condition of in vitro culture is generally very harsh, and usually not have in vitro culture steady for the cell (especially metastatic cells) obtained at random
It is qualitative.
Therefore, there is an urgent need in the art to develop one kind to be suitble to modern liver cancer cells research and can stablize build for animal model
Vertical liver cancer cell lines, especially common metastatic hepatic carcinoma cell line.
Invention content
The present invention provides a kind of liver cancer cells of bone transspecific, special with extremely strong one-tenth knurl ability and Bone tumour
The opposite sex, and passage and hereditary feature are stablized.
First aspect present invention provides a kind of bone transspecific liver cancer cells 3B-BM, during the cell 3B-BM is
The preserving number of state's Type Tissue Collection is CCTCC NO：The HEP-3B organ specificity transfer cell lines of C2016174
BM。
Second aspect of the present invention, the filial generation for additionally providing bone transspecific liver cancer cells described in first aspect present invention are thin
Born of the same parents, the daughter cell can result in the liver cancer of nude mice and/or humanoid skeletonization transspecific.
In another preferred example, substantially in another preferred example, the daughter cell protects the daughter cell substantially
Stay (>=95%, >=96%, >=97%, >=98%, >=99%) or whole bone transspecific liver cancer cells for remaining parental generation
The structure and characteristic of 3B-BM.
In another preferred example, the daughter cell is 3B-BM cells by within 15 generations, preferably, within 10 generations,
Within 5 generations, the daughter cell more preferably, within 3 generations passed on.
In another preferred example, the daughter cell retains or all remains the bone transspecific liver of parental generation substantially
The characteristic of cancer cell 3B-BM.
In another preferred example, cell (or daughter cell of second aspect of the present invention) as described in the first aspect of the invention,
The bone transspecific liver cancer cells (or daughter cell) have following one or more characteristics：
(a) cell described in has specific miRNA express spectras, and the miRNA express spectras include following characteristics
(b) Bone tumour rate >=70% of the cell；And/or
(c) Organ relative weight rate ＜ 30% other than the bone of the cell.
Third aspect present invention, provide bone transspecific liver cancer cells described in first aspect present invention (or the present invention
Daughter cell described in second aspect) purposes, be used to prepare the bone transspecific liver cancer model of non-human mammal or be used for
The candidate compound of screening treatment bone transspecific liver cancer.
It is characterized in that, the mammal is selected from rat, mouse, rabbit, sheep, dog, monkey.
In another preferred example, the mammal is immune deficiency experimental animal.
In another preferred example, the animal is nude mice, preferably T cell defect nude mice.
Fourth aspect present invention provides a kind of method for establishing bone transspecific liver cancer cells animal model, including
Step：
(i) by 5 × 105-1×106Bone tumour liver cancer cells described in a first aspect present invention (or its daughter cell) are inoculated with
In nude mice；
(ii) nude mice in culture (i) 21-42 days, take out mouse hind leg, shred bone both ends joint, 1ml injection needles
Serum-free DMEM is drawn, is eluted from bone one end, repeatedly, is cultivated in obtained cell mixing DMEM culture mediums, wait for cell
Selected by flow cytometry apoptosis GFP positive cells are carried out after adherent, to obtain Bone tumour liver cancer cells animal model.
In another preferred example, the mammal is immunodeficient mouse, and such as nude mice is cultivated 21-42 days.
In another preferred example, which increases very fast, and 10cm dish 1 pass 4 can reach the density of 80% effect every other day,
The cell carries GFP and m-cherry selection markers simultaneously.
In another preferred example, the inoculation is inoculated in lower portion：Tail vein, abdominal cavity, tail vein, subcutaneous part
Position, liver or combinations thereof.
Fifth aspect present invention provides a kind of method of the candidate compound of screening treatment bone transspecific liver cancer,
Including step：
(a) by 5 × 105—1×106Bone transspecific liver cancer cells described in first aspect present invention (or its filial generation is thin
Born of the same parents) it is inoculated in mammal；
(b) mammal of incubation step (a) 21-42 days obtain bone transspecific liver cancer animal model；With
(c) compound will be tested and is applied to the bone transspecific liver cancer animal model of step (b), and tested with not application
The bone transspecific liver cancer animal model of the step of compound (b) is compared, wherein leading to bone transspecific after application
The test compound that liver cancer symptom improves or cures is exactly to treat the candidate compound of bone transspecific liver cancer；
Or the method includes：
(a1) in test group, add in the cultivating system of the bone transspecific liver cancer cells described in first aspect present invention
Add test compound, and observes the quantity and/or growing state of bone transspecific liver cancer cells；In control group, in bone spy
Test compound is not added in the cultivating system of opposite sex transfer liver cancer cells, and observes the quantity of bone transspecific liver cancer cells
And/or growing state；
Wherein, if the quantity of bone transspecific liver cancer cells or the speed of growth are less than control group in test group, with regard to table
The bright test compound, which is the growth to bone transspecific liver cancer cells or proliferation, has the treatment bone specificity of inhibiting effect to turn
Move the candidate compound of liver cancer.
Sixth aspect present invention provides a kind of method of the potential liver cancer Lung metastases related gene of screening, including step：
By the gene expressed in bone transspecific liver cancer cells (or its daughter cell) described in first aspect present invention with
The genetic comparison expressed in normal liver cell is filtered out and is being united in bone transspecific liver cancer cells described in first aspect present invention
Meter learns the gene of upper up-regulated expression or downward, which is potential bone transspecific liver cancer related gene.
In another preferred example, the method further includes：
Further cell experiment and/or animal are carried out to the potential bone transspecific liver cancer related gene obtained
Experiment, to select the gene that liver cancer Lung metastases are risen with definite effect.
Seventh aspect present invention provides a kind of method of in vitro culture bone transspecific liver cancer cell lines, including step
Suddenly：In suitable culture medium, the bone transspecific liver cancer cells described in first aspect present invention or its daughter cell are cultivated.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows characteristic of the first and second the screened generation liver cancer cell lines still with the transfer of random multiple organ.
Fig. 2A shows that after screening, 3B-BM cell lines of the present invention have extremely strong specific Bone tumour ability；And scheme
When 2B is that 3B-BM cell lines of the present invention passed on for 15 generation, still there is extremely strong specific Bone tumour ability.
Fig. 3 shows that the proliferative capacity of cell line of the present invention has significant enhancing compared with its parent cell.
Specific implementation mode
The present inventor has carried out a large amount of sieve after extensive and in-depth study, to the cell line of tens of metastatic hepatic carcinomas
Choosing and culture, finally establish a kind of liver cancer cells of new bone transspecific, and Tumor formation is good, and Bone tumour rate is high,
And passage characteristic is highly stable, can be used for the treatment of Animal Model and intractable liver cancer (or drug resistance liver cancer).It is heavier
Want, the present invention also provides a comparison of the characteristic of each transspecific cell, find out liver cancer enter after blood shift it is key
The factor, so illustrate hepatoma Metastasis this process mechanism.On this basis, the present invention is completed.
Term
As used herein, term " liver cancer cells of bone transspecific ", " HEP-3B organ specificity transfer cell lines
BM " " liver cancer cells of people's bone transspecific ", " liver cancer cells of the present invention ", " cell of the present invention ", " 3B-BM " is interchangeable makes
With, refer both to the liver cancer cell lines 3B-BM of bone transspecific of the present invention, on October 18th, 2016 be preserved in Chinese Typical Representative training
Object collection (CCTCC) (Wuhan University, Wuhan, China) is supported, deposit number is CCTCC NO：C2016174.
Cell line selection method and its feature
For liver cancer cells, condition of culture is typically more harsh, for example, being studied through the present inventor, finds this hair
The condition of in vitro culture generally use 10%FBS+DMEM+1% of the hep3B cells of bright use is dual anti-, cell density 70-
Or so 80% 2 days generation times.When using the liver cancer cells under the condition of culture, it should be noted digestion condition, utilize 0.25%
Pancreatin, whole temperature is maintained at 37 DEG C or so, and should carry out metastatic cells immediately in cell mixing to 70% degree
Inoculation.
However, even if cell culture operations are carried out in accordance with careful and harsh condition of culture, the inventors discovered that hep-3B
The transition probability of cell is extremely low, and it is multiple to shift site.The present inventor is by 4 × 7 (4 generations screened, per 7 repetitions of generation) batches
Metastatic cells culture is repeatedly screened by double labelling (GFP and luciferase), can not only be in live body horizontal location tumour cell
Position, and tumour cell can be precisely separating under ex vivo situation, to obtain hep-3B bones/Lung metastases cell of the present invention
System finds that hep-3B bones/Lung metastases cell line of the present invention has the subculture in vitro separately characteristic of exceptional stability, is passing on 15-20 instead of
Afterwards, which still there is strong bone/lung to orient transfer characteristic.
One kind preferably screening technique includes step：The Bone tumour that liver cancer-specific is screened by the continuous mode of two steps is thin
Born of the same parents are that the luciferase substrate for enzymatic activity carried first with cell shines in the transfer of live body horizontal location certain organs
Stove takes out transfer stove out of Mice Body, reaffirms it is the liver cancer cell lines marked under fluorescence microscope；Shred thigh bone two
End draws serum-free DMEM with 1ml syringes and rinses thigh bone repeatedly, obtains single cell suspension, and culture is adherent up to cell, growth
The tumour cell with GFP is screened to 70% or so density, then by flow cytometer, repeatedly tens of batches, until obtaining
The liver cancer cell lines of transfer must be stablized.
Screening operation is as follows：Thigh bone both ends are shredded, serum-free DMEM is drawn with 1ml syringes and rinses thigh bone repeatedly, obtain
Erythrocyte cracked liquid is added in single cell suspension, removes red blood cell, and pbs washes 3 times, cultivated in dmem culture mediums 2-3 days it is close to cell
Degree reaches 80% or so, and mild vitellophag is control with common mice organs cell, and the threshold value of setting GFP sortings sub-elects
GFP positive cells, as primary screening finally obtain the liver cancer cell lines for stablizing transfer as a result, orientation repeats screening.
The liver cancer cells of bone transspecific of the present invention have one or more of feature：
(a) cell described in has specific miRNA express spectras, and the miRNA express spectras include following characteristics：
(b) Bone tumour rate >=70% of the cell, preferably 75%；And/or
(d) Organ relative weight rate ＜ 30% other than the bone of the cell.
Those skilled in the art can be according to the conventional method of this field cell secondary culture, to 3B-BM cells of the present invention
System is passed on to obtain the daughter cell of 3B-BM.Certainly, in order to keep the homogeneity of 3B-BM hereditary capacities, it is preferred to use
3B-BM passes daughter cell of the cell line as 3B-BM (more preferably within 10 generations, within 5 generations, within 3 generations) within 15 generations,
When it is more than generation to be passaged to 15, then need to determine the homology of holding daughter cell by the way that identification is sequenced.In general, this field
The daughter cell with 95% or more homology of parental cell system can be selected, and the daughter cell must keep or protect substantially
Hold the biological nature of parental cell.
Experiments verify that the liver cancer cells of bone transspecific of the present invention passage 15-20 instead of after, still retain 70% and
More than, such as 75%, 80%, 85%, 90%, 95%, 98% or 100% Bone tumour rate.
Using
The bone transspecific liver cancer cell lines of the present invention can be used for preparing bone transspecific liver cancer animal model, may be used also
Bone transspecific liver cancer drug candidate is treated for screening.
A method of preferably establishing bone transspecific liver cancer animal model, including step：
(i) by 5 × 105-1×106Bone transspecific liver cancer cells described in a first aspect present invention (or its filial generation is thin
Born of the same parents) it is inoculated in non-human mammal；
(ii) mammal in culture (i) 21-42 days, to obtain bone transspecific liver cancer cells animal model.
Wherein, the inoculation is inoculated in lower portion：Liver, abdominal cavity, tail vein, subcutaneous location or combinations thereof；Institute
The mammal stated is immune deficiency experimental animal.
A kind of method of the candidate compound of preferred screening treatment bone transspecific liver cancer, including step：
(a) by 5 × 105—1×106Bone transspecific liver cancer cells described in first aspect present invention (or its filial generation is thin
Born of the same parents) it is inoculated in mammal；
(b) mammal of incubation step (a), such as nude mice obtain bone transspecific liver cancer animal model；With
(c) compound will be tested and is applied to the bone transspecific liver cancer animal model of step (b), and tested with not application
The bone transspecific liver cancer animal model of the step of compound (b) is compared, wherein leading to bone transspecific after application
The test compound that liver cancer symptom improves or cures is exactly to treat the candidate compound of bone transspecific liver cancer；
Or the method includes：
(a1) in test group, add in the cultivating system of the bone transspecific liver cancer cells described in first aspect present invention
Add test compound, and observes the quantity and/or growing state of bone transspecific liver cancer cells；In control group, in bone spy
Test compound is not added in the cultivating system of opposite sex transfer liver cancer cells, and observes the quantity of bone transspecific liver cancer cells
And/or growing state；
Wherein, if the quantity of bone transspecific liver cancer cells or the speed of growth are less than control group in test group, with regard to table
The bright test compound, which is the growth to bone transspecific liver cancer cells or proliferation, has the treatment bone specificity of inhibiting effect to turn
Move the candidate compound of liver cancer.
Preferably, the mammal is immunodeficient mouse (nude mice), and incubation time is 35 days.
Make in addition, bone transspecific liver cancer cell lines of the present invention can also shift liver cancer cell lines pairing with liver specificity
With the drug screening of gene and treatment different parts metastatic hepatic carcinoma for screening potential metastatic hepatic carcinoma.
A kind of method of the preferred potential bone transspecific liver cancer related gene of screening, including step：
By the gene expressed in the bone transspecific liver cancer cells or its daughter cell and table in conventional liver cancer cells
Statistically up-regulated expression or the gene of downward, the gene are selected in bone transspecific liver cancer cells in the genetic comparison reached
For potential bone transspecific liver cancer related gene, in addition, this method further includes turning to the potential bone specificity obtained
Move liver cancer related gene and carry out further cell experiment and/or zoopery, with select for bone transspecific liver cancer its
The gene of definite effect.
According to the measurement of mRNA, miRNA spectrum to bone transspecific liver cancer of the present invention and data analysis, following institute
Show：
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
The screening and identification of 1 bone transspecific liver cancer cell lines of embodiment
GFP is marked in the cell first, to carry out the separation of Bone tumour cell, secondly, the engineered stable table of the cell
Up to luciferase enzymes, mouse living imaging detection cell position and quantitative analysis can be carried out, is existed simultaneously because changing cell
M-cherry is marked while building luciferase enzymes, the selection markers as luciferase positive cells.The cell
Transfer ability is remarkably reinforced compared with former hep-3B cells.Specifically, the hep-3B cell tail veins of luciferase is marked to note
Nude mice is penetrated, the 20-40 days liver lesions for occurring random transferring in Mice Body namely primary hep-3B cells do not have transfer
Organ specificity.The mouse that target organ transfer is selected in the mouse of random transferring, shreds thigh bone both ends, with 1ml syringes
It draws serum-free DMEM and rinses thigh bone repeatedly, obtain single cell suspension, culture is adherent up to cell, and it is close to grow to 70% or so
Degree, tumour cell of the flow cytometry sorting with GFP, as a result shows there was only the swollen of only a few in obtained single cell suspension
Oncocyte；Thereafter, culture is enlarged to the GFP cells sub-elected, and continues with the m-cherry that the cell carries and carries out
Secondary sorting, it is ensured that is obtained is tumour cell rather than mouse stromal cell.This obtained cell is that first generation Bone tumour is thin
Born of the same parents are that, using the cell, what is obtained in the same way is the second continuous cell line.First and second generation Bone tumour cell line (totally 14
Batch) do not have stable Bone tumour ability, Bone tumour rate is respectively 30% and 40% or so, feeds back in Mice Body and finds still
So there is the phenomenon that random transferring, shifted outside bone it is more, as shown in fig. 1.
By bioluminescence imaging technology and morphological observation, repeatedly above step, it is stronger to filter out Bone tumour ability again
Cell, feed back obtained third and fourth generation Bone tumour cell line (totally 14 batch) of mouse, gradually show stronger transfer energy
Power, but most cells strain proliferative capacity after passage gradually weakens.As a result one plant is obtained in forth generation Bone tumour, and there is pole
The tendentiousness of strong Bone tumour, Bone tumour rate about 75% or more, the cell strain pass on the bone turn of (cell line i.e. of the present invention) after 2 generations
It moves result and sees Fig. 2A, and the outer transfer ability of bone reduces.This plant of cell is still as characterized above after stable pass on 15 times, that is, stablizes
Bone tumour characteristic.
The foundation and identification of 2 bone transspecific liver cancer cells mouse model of embodiment
2.1 by 5 × 105-1×106A bone transspecific liver cancer cells (or its daughter cell) are inoculated in nude mice, obtain
Bone transspecific liver cancer cells mouse.It takes out Mouse Bone within 21-42 days, shreds thigh bone both ends, serum-free is drawn with 1ml syringes
DMEM rinses thigh bone repeatedly, obtains single cell suspension, and culture is adherent up to cell, grows to 70% or so density, carries out streaming
Cell instrument sorts GFP positive cells, to obtain bone transspecific liver cancer cells.
The morphology of obtained transfer specific cell is identified, as a result proves its morphological feature and original liver cancer
Cell no significant difference is confirmed as liver cancer cells, but the competence for added value of the cell is significantly enhanced, such as Fig. 3.
2.2 passage activity identifications
Identified, preferably, in passage 15-20 generations, remain able to stable specific Bone tumour to cell passage activity, such as Fig. 2 B.
The each of 3 bone transspecific liver cancer cells of embodiment is identified for gene expression profile
Culture hep-3B cells and Bone tumour specific cell pass on 2 rear portion cells and are sent to preservation, parallel biography
A part of cell carries out hereditary feature identification in generation, includes the difference detection of gene sequencing, miRNA spectrum and mRNA spectrums, carefully
Born of the same parents are adherent, and pancreatin digests when reaching 50-70% in culture dish, and cell, trizol method lytic cells is collected by centrifugation, and RNA is extracted,
Reverse transcription simultaneously carries out mRNA expression chips (Bai Hao Bioisystech Co., Ltd) detection and gene sequencing.As a result, it has been found that preservation is thin
Born of the same parents and each filial generation (2-15 generations) cytogenetics feature are identical.
4 kinds of embodiment plants tumor formation rate identification
The tumor formation rate of Bone tumour specific cell of the present invention is determined according to a conventional method, as a result, it has been found that, tail is quiet
Arteries and veins tumor formation rate is 100%, and wherein Bone tumour specificity is 75% or more.
Cell line preservation
The HEP-3B organ specificity transfer cell line BM of the present invention, Chinese Typical Representative culture is deposited in 2016.10.18
Collection (CCTCC) (Wuhan University, Wuhan, China), preserving number is CCTCC NO：C2016174.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1. a kind of bone transspecific liver cancer cells 3B-BM, which is characterized in that the cell 3B-BM is Chinese Typical Representative culture
The preserving number of collection is CCTCC NO：The HEP-3B organ specificity transfer cell lines BM of C2016174.
2. cell described in claim 1, the bone transspecific liver cancer cells (or daughter cell) have with next or more
A characteristic：
(a) cell described in has specific miRNA express spectras, and the miRNA express spectras include following characteristics
(b) Bone tumour rate >=70% of the cell；And/or
(c) Organ relative weight rate ＜ 30% other than the bone of the cell.
3. the purposes of bone transspecific liver cancer cells described in claim 1, which is characterized in that be used to prepare non-human mammal
Bone transspecific liver cancer model or for screen treatment bone transspecific liver cancer candidate compound.
4. purposes as claimed in claim 3, which is characterized in that the mammal be selected from rat, mouse, rabbit, sheep, dog,
Monkey.
5. purposes as claimed in claim 3, which is characterized in that the mammal is immune deficiency experimental animal.
6. a kind of method of the candidate compound of screening treatment bone transspecific liver cancer, which is characterized in that including step：
(a1) in test group, test is added in the cultivating system of bone transspecific liver cancer cells described in claim 1
Compound, and observe the quantity and/or growing state of bone transspecific liver cancer cells；In control group, turn in bone specificity
Move liver cancer cells cultivating system in do not add test compound, and observe bone transspecific liver cancer cells quantity and/or
Growing state；
Wherein, if the quantity of bone transspecific liver cancer cells or the speed of growth are less than control group in test group, this is indicated that
Test compound is the treatment bone transspecific liver that the growth to bone transspecific liver cancer cells or proliferation have inhibiting effect
The candidate compound of cancer.
7. a kind of method of the potential liver cancer Lung metastases related gene of screening, which is characterized in that including step：
The gene ratio that will be expressed in the gene and normal liver cell expressed in bone transspecific liver cancer cells described in claim 1
Compared with, statistically up-regulated expression or the gene of downward are filtered out in bone transspecific liver cancer cells described in claim 1, it should
Gene is potential bone transspecific liver cancer related gene.
8. the method for claim 7, which is characterized in that the method further includes：
Further cell experiment is carried out to the potential bone transspecific liver cancer related gene obtained and/or animal is real
It tests, to select the gene that liver cancer Lung metastases are risen with definite effect.
9. a kind of method of in vitro culture bone transspecific liver cancer cell lines, which is characterized in that including step：In suitable training
It supports in base, cultivates bone transspecific liver cancer cells described in claim 1.
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