CN103881974B - A kind of low aggressive transitional cell bladder carcinoma cell line of people - Google Patents
A kind of low aggressive transitional cell bladder carcinoma cell line of people Download PDFInfo
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Abstract
The invention provides a kind of low aggressive transitional cell bladder carcinoma cell line of people, is a kind of low aggressive bladder cancer cell lines CH 1 of new people specifically, China typical culture collection center (CCTCC is deposited in August in 2013 within 26th, China, Wuhan), preserving number is CGMCC No.C2013128.Cell line cell form of the present invention and passage feature are stable, Tumor formation is good, and the cell line occurs without DISTANT METASTASES IN, foundation available for animal model, and it can be used in combination with multiple organ metastatic bladder cancer cell lines, for screening the medicine for heterogeneity carcinoma of urinary bladder, and it is related to shifting to gene for screening.
Description
Technical field
The invention belongs to biology and oncology, in particular it relates to a kind of new low aggressive carcinoma of urinary bladder
The foundation and its application of cell.
Background technology
The occurrence and development of the various diseases of the mankind are sufficiently complex, the pathogenesis of the various diseases of discussion to be goed deep into
And its curative effect mechanism can not be tested with patient.But Animal Protection Law can be followed, by animal to various diseases
Studied with biological phenomena, and then push away and unravel silk the secret that the mankind explore human life, explore the rule of human diseases occurrence and development
Rule, to control the generation of human diseases, development, cure disease and mitigate the ill pain of the mankind, improve life quality, extend the mankind
Life-span.
For a long time it has been found that using people in itself as experimental subjects come promote the development of medical science be it is difficult, clinically
The experience accumulated is not only over time and space there is limitation, and many experiments are also in ethics and methodology
There is various limitations.
The animal model (Animal model of human diseases) of human diseases is biomedical sciences research
It is middle to establish zoopery object and material with human diseases simulation sex expression.The use of animal model is modern biomedical
Particularly important an experimental method and means in research, help to be more convenient, more effectively recognize human diseases generation,
The rule of development and research prophylactico-therapeutic measures.
Tumour cell, which is tied up in the basic research of tumour, has critical role, cultured tumor cells in vitro be it is more difficult,
Especially establish can long term growth, passage, again have certain feature human tumor cell line.
Carcinoma of urinary bladder is one of most common urological cancer, directly threatens the existence of patient.In world wide, wing in 2008
Guang cancer new cases 386300, there are 150200 patients to die from carcinoma of urinary bladder.Carcinoma of urinary bladder can be largely classified into two types：Muscle layer is soaked
Profit and non-Myometrial involvement.The bladder cancer patients that 70%-80% newly makes a definite diagnosis are non-Myometrial involvement cancer, even if giving associated treatment in time
Also it is Myometrial involvement cancer to have 50%-70% recurrence rate and 10%-30% patient progress.And local leaching easily occurs for the latter
Profit or DISTANT METASTASES IN, although row radial cystectomy art and lymph node dissection, patient is also often difficult to cure, and is to cause patient dead
The main reason for dying.Not exclusively carcinoma of urinary bladder, for whole human malignancies, still lack effectively prevention and treatment at present
The method of lymph node or distant metastasis of human cancer, it is also indefinite to the mechanism of the organ metastasis of malignant tumour.
For the research field of carcinoma of urinary bladder, although existing bladder cancer cell lines at present, because its settling time is more early,
Its passage situation is unstable and Tumor formation is poor, and the characteristic and genetic background of cell line are also inconsistent, cause carcinoma of urinary bladder special
Property marker expression is uneven does not express even, and existing cell line is classified indefinite, therefore mesh to cancer cell invasion degree
Preceding conventional bladder cancer cell lines can not meet the requirement of modern scientific research.
Therefore, there is an urgent need to develop to meet that modern carcinoma of urinary bladder variation situation, genetic background are clear, can stablize and pass for this area
Generation and Tumor formation is good, and possesses the bladder cancer cell lines model of certain people of bladder tumor feature, to be applicable modern medicine to animal
Model establishes demand.
The content of the invention
There are low aggressive bladder cancer cell lines the invention provides a kind of.
First aspect present invention, there is provided a kind of human bladder cancer cell CH-1, the cell are China typical culture collections
The preserving number at center is CCTCC NO：The C2013128 low aggressive transitional cell bladder carcinoma cell line of people.
Second aspect of the present invention, there is provided the daughter cell of transitional cell bladder carcinoma cell line described in a kind of first aspect present invention, it is described
Daughter cell can result in nude mice and form low aggressive carcinoma of urinary bladder.
In another preference, described daughter cell retain substantially (>=95%, >=96%, >=97%, >=98%, >=
99%) the low aggressive transitional cell bladder carcinoma cell line CH1 of people of parental generation structure and characteristic or is all remained.
In another preference, described daughter cell is that CH-1 cell lines were passed through within 10 generations, more preferably, within 5 generations
The daughter cell of passage.
In another preference, described daughter cell retains or all remained the low aggressive bladder of people of parental generation substantially
Cancer cell CH-1 characteristic.
In another preference, the transitional cell bladder carcinoma cell line cell or daughter cell have following one or more characteristics：
(a) compared with existing cell line (such as T24), described cell has specific cancer related gene (including former cancer
Gene and tumor suppressor gene) express spectra；For example, suppressor gene p53 and tumor suppressor gene P27 are significantly high expression；
(b) rate of transform of the cell is less than 60%；
(d) plantation tumor formation rate in situ is more than 75%.
Third aspect present invention, there is provided filial generation described in first aspect present invention transitional cell bladder carcinoma cell line or second aspect of the present invention
The purposes of cell, for preparing the bladder cancer models of non-human mammal or the candidate compound of carcinoma of urinary bladder being treated for screening,
Wherein, described carcinoma of urinary bladder is low aggressive carcinoma of urinary bladder.
In another preference, described mammal is selected from rat, mouse, rabbit, sheep, dog, monkey.
In another preference, described mammal is immune deficiency experimental animal.
In another preference, described animal is nude mice (T cell defect), NON/SCID mouse (T cell, B cell and
NK cells combine defect).
Fourth aspect present invention, a kind of method for establishing low aggressive bladder carcinoma animal model, including step：
(i) by 1 × 104-1×109Transitional cell bladder carcinoma cell line described in individual first aspect present invention or its daughter cell are inoculated in inhuman
Mammal；
(ii) mammal in culture (i) 7-100 days, so as to obtain bladder carcinoma animal model；
It is preferred that described mammal is immunodeficient mouse, cultivated days are 35 days.
In another preference, described inoculation is inoculated in lower portion：Bladder, abdominal cavity, tail vein, subcutaneous location or
It is combined.
In another preference, described mammal is immune deficiency experimental animal.
Fifth aspect present invention, there is provided a kind of method for screening the candidate compound for treating low aggressive carcinoma of urinary bladder, bag
Include step：
(a) by 1 × 104—1×109Human bladder cancer cell or its daughter cell described in first aspect present invention are inoculated in
Mammal；
(b) mammal of incubation step (a) 7-100 days, bladder carcinoma animal model is obtained；With
(c) test compound is applied to the bladder carcinoma animal model of step (b), and with not applying the step of test compound
Suddenly the bladder carcinoma animal model of (b) is compared, wherein causing the test compound that carcinoma of urinary bladder symptom improves or cured after applying
Exactly treat the candidate compound of carcinoma of urinary bladder；
Or methods described includes：
(a1) in test group, add and survey in the cultivating system of the human bladder cancer cell CH-1 described in first aspect present invention
Compound is tried, and observes transitional cell bladder carcinoma cell line CH-1 quantity and/or growing state；In control group, in human bladder cancer cell CH-
Test compound is not added in 1 cultivating system, and observes transitional cell bladder carcinoma cell line CH-1 quantity and/or growing state；
Wherein, if transitional cell bladder carcinoma cell line CH-1 quantity or the speed of growth are less than control group in test group, the survey is indicated that
Examination compound is growth to transitional cell bladder carcinoma cell line or propagation have inhibitory action treatment carcinoma of urinary bladder candidate compound.
The aspect of the present invention six, there is provided the purposes of transitional cell bladder carcinoma cell line described in first aspect present invention, for screening carcinoma of urinary bladder
Aggressive related gene；It is preferred that described transitional cell bladder carcinoma cell line CH-1 is used to build bladder carcinoma in situ animal model.
Seventh aspect present invention, there is provided a kind of method for screening potential carcinoma of urinary bladder invasion related gene, including step：
By the gene expressed in transitional cell bladder carcinoma cell line described in first aspect present invention or its daughter cell and high aggressive bladder
The genetic comparison expressed in cancer cell or normal bladder cell, filters out in transitional cell bladder carcinoma cell line described in first aspect present invention and is uniting
Meter learns the gene of upper up-regulated expression or downward, and the gene is potential carcinoma of urinary bladder invasion related gene,.
In another preference, described method also includes：
Further cell experiment is carried out to the potential carcinoma of urinary bladder invasion related gene obtained and/or animal is real
Test, to select the gene that definite effect is played for carcinoma of urinary bladder invasion.
In another preference, described high aggressive transitional cell bladder carcinoma cell line is selected from：Wherein, in China typical culture collection
The preserving number of the heart is CCTCC NO：C2013129 high aggressive human bladder cancer cell CH-2 or existing bladder cancer cell lines T24.
Eighth aspect present invention, there is provided a kind of method of the low aggressive transitional cell bladder carcinoma cell line of in vitro culture, including step：
In suitable culture medium, the human bladder cancer cell 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, so as to form new or preferable technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 shows CH-1 cell lines form under different amplification.Upper figure shows that CH-1 cells are in not advise as seen from the figure
Then polygon or fusiformis, meet epithelial cell phenotype.
Wherein, Figure 1A is CH-1 cell lines (20 generation), 40 ×；Figure 1B is CH-1 cell lines (20 generation), 100 ×.
Fig. 2 shows the increment curve of each cell line, wherein, (about 1000/ empty) is every 24 small after 24 hours after plating cells
When with CCK-8 reagents detect cell-proliferation activity (24-120 hours) prompting CH-1 cell lines be in logarithm about in 48h-72h
Growth.
Fig. 3 shows the foundation (embodiment 2) of animal model, it is seen that cell line success lotus knurl, do not occur lymph node or
The transfer of person's other organs.
SABC detection shows nude mice primary tumor and patient's bladder cancerous tissue CK in Fig. 4AE1Expression, wherein, figure
4A shows the high expression keratin CK of patient's bladder cancerous tissue cancer epithelial cellAE1, and interstitial tissue has no expression；Fig. 4 B are shown
The high expression CK of nude mice primary tumorAE1Albumen.
Embodiment
The present inventor is cultivated and sieved to tens of T1 phase carcinoma of urinary bladder primary tumor cells by in-depth study extensively
Choosing, a kind of new low aggressive bladder cancer cell lines CH-1 is finally established, its Tumor formation is good, passes on stability of characteristics, can be used for
The foundation of animal model, and can be used in combination with multiple organ metastatic bladder cancer cell lines, it is directed to heterogeneity wing for screening
The medicine of Guang cancer, and can be used for screening is related to transfer to give gene.In addition, the present inventor has also screened T2-T4 phases carcinoma of urinary bladder original
Stove and transfer stove cell are sent out, and establishes a kind of metastatic bladder cancer cell lines CH-2 of multiple organ, on this basis, is completed
The present invention.
Specifically, tens of T1 phase carcinoma of urinary bladder primary tumor cells are inoculated in immunodeficient mouse by the present inventor respectively, are passed through
After culture growth, tumor tissues are taken to carry out subculture in vitro separately culture after carrying out nude mice continuous passage, and examined by related biological
After survey, screening obtains a kind of passage stabilization, low aggressive bladder cancer cell lines.Experiment of Zoology shows that the cell line is inoculated with
Cause nude mice to produce tumour after nude mice, but do not produce DISTANT METASTASES IN, and its Pathomorphology and former patient tumors cytomorphology
It is consistent.
Term
As used herein, term " low aggressive transitional cell bladder carcinoma cell line ", " the low aggressive transitional cell bladder carcinoma cell line of people ", " wing of the present invention
Guang cancer cell ", " cell of the present invention ", " cell CH-1 " of the present invention is used interchangeably, and it is thin to refer both to the low aggressive carcinoma of urinary bladder of the present invention
Born of the same parents system CH-1, it was preserved in China typical culture collection center (CCTCC) (Wuhan, China), preservation on the 26th in August in 2013
Numbering is CCTCC NO：C2013128.
As used herein, term " high aggressive transitional cell bladder carcinoma cell line ", " transitional cell bladder carcinoma cell line CH-2 " is used interchangeably, and refers both to produce
The high aggressive bladder cancer cell lines CH-2 of raw multiple organ transfer, it was preserved in Chinese Typical Representative culture on 26th in August in 2013
Collection (CCTCC) (Wuhan, China), deposit number are CCTCC NO：C2013129.
Cell line feature
Low aggressive Pan of the present invention irrigates cell compared with existing cell line, has one or more of feature：
(a) compared with existing cell line (such as T24), described cell has specific cancer related gene (including former cancer
Gene and tumor suppressor gene) express spectra；For example, suppressor gene p53 and tumor suppressor gene P27 are significantly high expression；
(b) rate of transform of the cell is less than 60%；
(d) plantation tumor formation rate in situ is more than 75%.
In addition, those skilled in the art can be according to the conventional method of this area passage culture, to CH-1 cell lines
Passed on so as to obtain CH-1 daughter cell.Certainly, in order to keep the homogeneity of CH-1 hereditary capacities, it is preferred to use CH-1
Daughter cell of the cell line in (within more preferably for 5 generations) as CH-1 is passed within 10 generations, when it is more than generation to be passaged to 10, is then needed
It can determine to keep the homology of daughter cell by sequencing identification.Generally, this area, which can be selected, has parental cell system
The daughter cell of more than 95% homology, and described daughter cell must keep or keep the biology of parental cell special substantially
Property.
Using
The low aggressive bladder cancer cell lines of the present invention can be used for preparing low aggressive bladder carcinoma animal model, it may also be used for
Low aggressive carcinoma of urinary bladder drug candidate is treated in screening.
A kind of method for preferably establishing low aggressive bladder carcinoma animal model, including step：
(i) by 1 × 104-1×109Transitional cell bladder carcinoma cell line described in individual first aspect present invention or its daughter cell are inoculated in lactation
Animal；
(ii) mammal in culture (i) 7-100 days, so as to obtain bladder carcinoma animal model.
Wherein, described inoculation is inoculated in lower portion：Bladder, abdominal cavity, tail vein, subcutaneous location or its combination；Institute
The mammal stated is immune deficiency experimental animal.
A kind of method that the candidate compound of low aggressive carcinoma of urinary bladder is treated in preferable screening, including step：
(a) by 1 × 104—1×109The above-mentioned human bladder cancer cell of the individual present invention or its daughter cell are inoculated in lactation and moved
Thing；
(b) mammal of incubation step (a) 7-100 days, bladder carcinoma animal model is obtained；With
(c) test compound is applied to the bladder carcinoma animal model of step (b), and with not applying the step of test compound
Suddenly the bladder carcinoma animal model of (b) is compared, wherein causing the test compound that carcinoma of urinary bladder symptom improves or cured after applying
Exactly treat the candidate compound of carcinoma of urinary bladder；
Or methods described includes：
(a) in test group, test compound is added in human bladder cancer cell CH-1 of the present invention cultivating system,
And observe transitional cell bladder carcinoma cell line CH-1 quantity and/or growing state；In control group, in human bladder cancer cell CH-1 culture body
Test compound is not added in system, and observes transitional cell bladder carcinoma cell line CH-1 quantity and/or growing state；
Wherein, if transitional cell bladder carcinoma cell line CH-1 quantity or the speed of growth are less than control group in test group, the survey is indicated that
Examination compound is growth to transitional cell bladder carcinoma cell line or propagation have inhibitory action treatment carcinoma of urinary bladder candidate compound.
Preferably, described mammal is immunodeficient mouse (nude mice), and its incubation time is 35 days.
Used in addition, the low aggressive bladder cancer cell lines of the present invention can also match with multiple organ transfer bladder cancer cell line,
For screening the drug screening of potential bladder oncogene and the different invasion and attack degree carcinomas of urinary bladder for the treatment of.
A kind of method of the potential low aggressive bladder cancer-associated gene of preferable screening, including step：
The gene that will be expressed in the gene expressed in the transitional cell bladder carcinoma cell line or its daughter cell and conventional transitional cell bladder carcinoma cell line
Compare, select in low aggressive transitional cell bladder carcinoma cell line statistically up-regulated expression or the gene of downward, the gene is potential low
Aggressive bladder cancer-associated gene, in addition, this method also includes the potential low aggressive bladder cancer-associated gene to being obtained
Further cell experiment and/or zoopery are carried out, to select the gene for low aggressive its definite effect of carcinoma of urinary bladder.
Universal method
All carcinoma of urinary bladder primary tumors are taken from the patient for suffering from carcinoma of urinary bladder and surgery excision being carried out to the court, and carcinoma of urinary bladder is through inspection
The T1 phases are confirmed as after looking into, all patients or its family members sign the informed consent form that Postoperative Specimen is used for scientific research.
Primitive cell culture
Culture to carcinoma of urinary bladder primary cell can take this area conventional meanses, and a kind of preferable cultural method is as follows：
Patient's cancerous tissue about 0.5 × 0.5cm is taken, 15ml centrifuge tubes is put into and adds 37 after the 2-3ml of 0.5% clostridiopetidase A IV concussions
DEG C digestion 15min；Fully blown and beaten repeatedly to after without obvious tissue block with 1ml glass pipettes after digestion and be put into centrifuge 700g centrifugations
5min；Supernatant is abandoned, adds RPMI-1640 nutrient solutions 10ml and dual anti-(penicillin and streptomysin) 100 μ containing 10% hyclone
Piping and druming is uniformly put into two 25 ㎝ after l2In blake bottle foster case is incubated in U.S.'s Thermo carbon dioxide that 37 DEG C of CO2 concentration are 5%
Interior culture, liquid is changed after 24 hours and continues to cultivate.Change within 2-3 days liquid once, and it is thin with phase contrast microscope observation epithelial cell and interstitial
Intracellular growth state.Epithelial cell in two blake bottles is in irregular polygon and fusiformis, and interstitial cell is elongated or rope
Shape, the two equal adherent growth.Being observed after a couple of days, under phase contrast microscope can find that a large amount of cell clones occurs in cell culture bottom of bottle.
Interstitial cell is removed using selection digestion method.0.25% pancreatin dilutes 10 times with PBS, adds the micro- Microscopic observation interstitial of blake bottle
Cell from it is streak be changed into ball when gently blow and beat, it is interstitial cell now to blow and beat the cell to get off, and then routinely cell disappears
Change passage method by remaining cell by 25 ㎝2Blake bottle is transferred to 75 ㎝2Continue to cultivate.
Animal model
The animal model of the present invention can use the mammal of various immune deficiencies, it is preferable that be nude mice.It is a kind of preferable
Animal model preparation method of the present invention is as follows：When bladder cancer cell lines reached for 20 generation centrifugation RPMI-1640 is digested with pancreatin
Nutrient solution is made into every 100 μ l and contains 1-5 × 106Individual cell the μ l of suspension 300 it is standby.It is female to choose growth conditions good 4-6 weeks
Property nude mice be used for establish animal model.After nude mice is anaesthetized, belly and perineum sterilization, it is catheter to have 24G venous detaining needles
Insert and inject the μ l of pancreatin 100 in nude mice bladder after empty bladder, ligature nude mice urethra with Line 1 and exit conduit simultaneously.Pancreatin disappears
Change extract out after bladder 30min pancreatin and with 100 μ l PBS washed bladders once, inject the ready μ l of cell suspension 100, equally
Ligature urethra.Ligature is removed after two hours.Mice with tumor growth conditions are observed, are put to death within three weeks.Post mortem at mice with tumor, under
Carefully analyzing each internal organs of observation by system upwards has without exception or metastases.Once it was found that transfer stove, transfer stove one after taking pictures
It is divided into three, is fixed respectively with formaldehyde, liquid nitrogen freezes and collagenase digesting.After fixing 24 hours with 4% formaldehyde, swollen together with former patient
Tumor tissue is made by hospital pathology department dehydration embedding together is cut into 5 μm every after wax stone with paraffin slicing machine, for HE dyeing and
SABC detects.
SABC detects
After transfer stove fixes 24 hours after removing with 4% formaldehyde, together with former patient's tumor tissues by hospital pathology department
Dehydration embedding is made is cut into 5 μm every after wax stone with paraffin slicing machine (German Leica), is examined for HE dyeing and SABC
Survey.For immune group detection antibody have E-cadherin, Snail, Slung, β-catenin, Twist (being purchased from CST companies),
P53, Ki-67 (are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge).Secondary antibody is purchased from Long Island company.
Main advantages of the present invention are：
1. the bladder cancer cell lines CH-1 of present invention character is stable, it can stablize and repeatedly pass on.
2. the present invention bladder cancer cell lines CH-1 Tumor formation it is good, hereditary capacity is clear and definite, be carcinoma of urinary bladder basis and face
The ideal cell line (efficiency assay) of bed application study early stage.
3. the bladder cancer cell lines CH-1 of the present invention does not produce DISTANT METASTASES IN, available for the low transfer bladder cancer for preparing stabilization
Model.
4. the bladder cancer cell lines CH-1 of the present invention can be matched with multiple organ transfer bladder cancer cell line and used, for screening
Carcinoma of urinary bladder metastasis related gene and screening are directed to the medicine of different aggressive carcinomas of urinary bladder.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally 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 condition proposed by manufacturer.Unless otherwise indicated, it is no
Then percentage and number are percentage by weight and parts by weight.
The foundation of embodiment 1CH-1 cell lines
(a) source：
By stages：T1
Main suit：Blood urine
Cystoscopy：Trigone of urinary bladder 1 × 1cm occupy-places
Pathology：High-level bladder transitional cell carcinoma
Operation：Carcinoma of urinary bladder electrocision
(b) primitive cell culture：
0.5 × 0.5cm of cancerous tissue is taken to carry out primitive cell culture, method is referring to universal method.
About 5 hour cell can adherent growths after fresh bladder cancer tissue centrifugation bed board after collagenase digesting.5 days left sides
The right side may occur in which a large amount of cell clones, and now visible interstitial cell and epithelial cell mix growth.After being passed on 3 times with difference digestion method
Interstitial cell can be removed substantially.
(c) Animal Model：
Cell is taken to be inoculated in immunodeficient mouse, CH-1 cell lines plant 3 Female nude mices altogether in vivo for the first time, and 3 naked
The successful lotus knurl of mouse, does not occur the transfer of lymph node or other organs.
The low aggressive bladder cancer cell lines CH-1 of embodiment 2 biological characteristics
Morphological observation：The cell line is in fusiformis or polygon (Fig. 1), and cellular morphology is stable.
Proliferation activity：Using the proliferation activity of CCK-8 reagents detection CH-1 cell lines, it is 48- to find its multiplication section
72 hours (Fig. 2).
Passage activity：The cell line reached for the 86th generation, and cellular morphology and the speed of growth are constant, was recovered after freezing good
It is good.
The marker expression of correlative protein expression and low aggressive carcinoma of urinary bladder：
A. the ImmunohistochemistryMethods Methods anti-human keratin antibody CK of mouse is passed throughAE1Detect source and the kind of mouse primary tumor.As a result
It has been shown that, the high expression CK of patient's urinary tract urothelium cancerous tissueAE1Albumen, and interstitial tissue has no expression (Fig. 4 A).Nude mice primary tumor
Same high expression CKAE1Albumen (Fig. 4 B).Prompting (1) CH-1 cell lines derive from urothelium.
B. suppressor gene p53 and P27 are also have detected on protein level, the high expression compared with T24 cell lines.
The low aggressive bladder cancer cell lines CH-1 of embodiment 3 rate of transform and plantation tumor formation rate research
Cell is taken to be inoculated in immunodeficient mouse, CH-1 cell lines plant 40 Female nude mices altogether in vivo for the first time, 35
Nude mice success lotus knurl, wherein the transfer of lymph node or other organs occur in 19 mouse.The above-mentioned experiment of repetition 3 times, is averaged
Lotus knurl success rate and the rate of transform, the lotus knurl success rate for as a result finding to change cell line is about 75-80%, and the rate of transform is below
60%.
The low aggressive bladder cancer cell lines CH-1 daughter cell preparation and application of embodiment 4
4.1 pairs of CH-1 cells carry out passing on 5-10 generations, obtain the daughter cell of the CH-1 cell lines, and to different algebraically
Cell line be sequenced, as a result find that, when being passaged to for 10 generation, all daughter cells and CH-1 homology are all higher than
95%.
4.2 use the technique study 5-10 of embodiment 2 for the biological characteristics of daughter cell, as a result display and CH-1 parental generations
Cell is consistent.
Cell line preservation
The low aggressive bladder cancer cell lines CH-1 of people of the present invention, Chinese Typical Representative culture is deposited on 26th in August in 2013
Thing collection (CCTCC) (Wuhan, China), preserving number is CCTCC NO：C2013128.
The high aggressive bladder cancer cell lines CH-2 of the people of the present invention, Chinese Typical Representative culture is deposited on 26th in August in 2013
Thing collection (CCTCC) (Wuhan, China), preserving number is CCTCC NO：C2013129.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (7)
1. a kind of human bladder cancer cell, it is characterised in that the cell is that the preserving number of China typical culture collection center is
CCTCC NO：The C2013128 low aggressive transitional cell bladder carcinoma cell line CH-1 of people.
2. transitional cell bladder carcinoma cell line as claimed in claim 1, it is characterised in that the transitional cell bladder carcinoma cell line has following one or more
Characteristic：
(a) compared with existing cell line, the suppressor gene p53 and tumor suppressor gene P27 of described cell are significantly high expression；
(b) rate of transform of the cell is less than 60%；
(c) plantation tumor formation rate in situ is more than 75%.
3. the purposes of transitional cell bladder carcinoma cell line described in claim 1, it is characterised in that for screening the candidate compound for the treatment of carcinoma of urinary bladder
Thing, wherein, described carcinoma of urinary bladder is low aggressive carcinoma of urinary bladder.
4. the purposes of transitional cell bladder carcinoma cell line described in claim 1, it is characterised in that for screening carcinoma of urinary bladder invasion related gene.
A kind of 5. method for screening potential carcinoma of urinary bladder invasion related gene, it is characterised in that including step：
By in the gene expressed in transitional cell bladder carcinoma cell line described in claim 1 transitional cell bladder carcinoma cell line aggressive with height or normal bladder cell
The genetic comparison of expression, statistically up-regulated expression or the gene of downward are filtered out in transitional cell bladder carcinoma cell line described in claim 1,
The gene is potential carcinoma of urinary bladder invasion related gene.
6. method as claimed in claim 5, it is characterised in that described method also includes：
Further cell experiment is carried out to the potential carcinoma of urinary bladder invasion related gene obtained, to select for carcinoma of urinary bladder
Invasion plays the gene of definite effect.
7. the method for the low aggressive transitional cell bladder carcinoma cell line of a kind of in vitro culture, it is characterised in that including step：In suitable culture medium
In, cultivate the human bladder cancer cell described in claim 1.
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| Expression of Axl in lung adenocarcinoma and correlation with tumor progression;Yi-Shing Shieh et al.;《Neoplasia》;20051231;第7卷(第12期);图1及图1图注 * |
| Tumor progression and expression of matrix metalloproteinase-2(MMP-2) mRNA by human urinary bladder cancer cells.;Hamasaki T et al.;《Urol Res》;19981231;第26卷(第6期);第371-376页 * |
| 膀胱癌三种细胞株体外侵袭力和黏附性的测定;王元天等;《中华实验外科杂志》;20050430;第22卷(第4期);第506页 * |
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