CN105062973B - One plant carries HPV feminine gender penis squamous cell carcinomas system that TP53 is mutated and application thereof - Google Patents
One plant carries HPV feminine gender penis squamous cell carcinomas system that TP53 is mutated and application thereof Download PDFInfo
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Abstract
The present invention " one plant carries HPV feminine gender penis squamous cell carcinomas system that TP53 is mutated and application thereof " discloses one plant of people's penis squamous cell carcinoma system.This plant carries the HPV feminine gender penis squamous cell carcinomas system of TP53 mutation, is preserved in China typical culture collection center, deposit number CCTCC NO on June 17th, 2015：C201585.Cell line Penl1 stabilization in vitro passage through animal into knurl sample being differentiated squamous-cell carcinomas in penis after knurl, and has done complete detection from cytomorphology, hereditary feature, protein expression, biological behaviour and chemosensitivity etc. are many-sided up to 14 months to cell.This plant of penis squamous cell carcinoma system can represent the tumoral character of HPV feminine gender penis squamous carcinomas, have wide prospect in the inside and outside basis of the screening anti-tumor medicines such as clinical diagnosis, chemotherapy and the targeted drug of further investigation penis carcinogenesis, development, metastasis and carcinoma of penis and clinical Study on Transformation.
Description
Technical field
The invention belongs to technical field of microbe cell line, the more particularly to one plant HPV feminine gender penis for carrying TP53 mutation
Squamous cell carcinoma system and application thereof.
Background technology
Carcinoma of penis is a kind of rare male malignancy, late period poor prognosis.Carcinoma of penis is in Africa, Asia and South America portion
Point country, incidence is up to 4/,100,000, up to the 10% of male malignancy, and in West Europe and the U.S., only accounts for malignant tumour
0.4%-0.6%.China's carcinoma of penis accounts for the 0.07% of male malignancy, close with West Europe and the U.S..Carcinoma of penis it is dangerous because
Element includes low health level, foreskin phimosis, non-row circumcision, smoking and high-risk human papillomavirus (hrHPV) infection.
Clear and definite HPV is the hazards of morbidity first in version Europe uropoiesis association (EAU) updated Guidelines in 2014, with other X factors
Collective effect causes tumour, but its pathogenesis is unclear.
The metastatic rule of carcinoma of penis is progressively to be transferred to pelvic lymph node by inguinal region lymph node, and DISTANT METASTASES IN is few
See.Lymphatic metastasis is to determine the key factor of carcinoma of penis prognosis and treatment, and there are the patient of lymphatic metastasis, particularly pelvic cavity
The patient of lymphatic metastasis, 5 years survival rates that complex treatment is treated in surgeon's knot combination at present are less than 10%.The reactivity of chemotherapy has
Limit, but still be the primary treatment regimen of end-stage patients, the report of domestic and international anti-epidermal growth factor receptor (EGFR) targeted therapy
It is still few, it is mostly the case after chemotherapy failure, its curative effect needs the research of a large amount of cases of multicenter.Because the incidence of carcinoma of penis is low and
Case is disperseed, and joint multiple center clinical study is difficult to carry out, and lacks its morbidity and the mechanism of transfer and the in vitro study for the treatment of,
Clinical treatment is not substantially in progress.
Permanent cell line is as the essential research tool of tumor research, in lung cancer, breast cancer, prostate cancer and uterine neck
It is widely used in the research of the kinds of tumor such as cancer, biological behaviour and the exploitation such as mechanism, tumour growth apoptosis in tumour
Play an important role in antitumor drug.The penis cancerous cell line of whole world report is few at present, there is no Chinese population source cloudy
Stem cancerous cell line, and the regional disparity of primary tumor cell characteristics and carcinoma of penis is lost after being passed on repeatedly in vitro because of cell line,
It is few in the deep basic research of cellular level.Therefore, case is shifted for the late period of China's carcinoma of penis particularly poor prognosis, established
The new antineoplaston mode of further investigation and exploitation of the carcinoma of penis cell model of population of China for carcinoma of penis has important
Scientific meaning and social effect.
The foundation of tumor cell line model is mainly directly cultivated or through animal subcutaneous vaccination by clinical fresh tumor tissue
In vitro culture after enriching and purifying tumour cell after fresh tumor tissue or cell.It is thin that the former often mixes non-tumour during establishing
Born of the same parents, such as interstitial cell, success rate is relatively low, and the latter needs Dependent Animals into knurl, and process complicated difficult is to promote the use of, both abroad
The two methods of report combine that to establish the success rate of cell line be about 10%.Carcinoma of penis Establishment of Cell Line of the present invention passes through direct body
Outer culture obtains, and verifies tumour close to clinical biological characteristics, cell tumour by establishing animal model into knurl in vitro
The cell outer membrane protein EGFR and kidney podocytic process epicyte protein (PDPN) of specific TP53 mutation and cell expression can conducts
The target spot of tumor monitoring, diagnosis and targeted therapy, is research penis carcinogenesis molecular mechanism, biological behaviour and antitumor drug
The gedanken experiment material of screening.
The content of the invention
The purpose of the present invention is lack cell characteristics after Long Term Passages for the people's carcinoma of penis reported at present to change and can not be anti-
Reflect population of China feature, there is provided people's carcinoma of penis that a kind of new Chinese population, long-term in vitro subculture and property are stablized is thin
Born of the same parents system.The cell line is through comprehensive cell line identity authentication and determines that HPV is negative, while finds that TP53 is mutated.
People's penis cancerous cell line of the present invention, it preservation cell and backed up its STR information in China on June 17th, 2015
Type Tissue Collection CCTCC, address：Wuhan City, Hubei Province Wuhan University in the school, postcode：430072, culture title
For people penis squamous cell carcinoma system Penl1, deposit number is CCTCC NO：C201585.Cell line detection has been survived.
The clinical samples in the cell line source of the present invention are 41 years old Chinese male carcinoma of penis metastatic lymph node sample, warp
One plant of new people's penis cancerous cell line for stablizing passage is successfully obtained after external directly culture and screening, is named as Penl1.
The present inventor's penis cancerous cell line Penl1 has following biological characteristics：
1. cell line is in adherent growth in DMEM in high glucose contains 10% embryo cow's serum culture medium, contactless suppression, growth side
Formula is the common agglomerating growth of squamous carcinoma；
2. cellular morphology is typical Epithelial form, what is connected under transmission electron microscope between visible iuntercellular epithelium is super
Micro-structure, cell intermediate filament structural proteins are epithelium keratin；
3. cell line detects through PCR and finds that two are dashed forward without mycoplasma and HPV infection, the full extron of sequencing TP53 genes
Become, P72R and R283P, cell line fingerprint the Bu Yu world cell database overlap；
4. the epithelial surface marks such as cell expression pan-CK, CK5 and desmoplakin, do not express vimentin interstitial marks
Note, no 16/18 E6 and p16 cores specific positives of HPV dyeing, but have p53 core positive stainings, while express memebrane protein i.e. epidermis
Growth factor receptors (EGFR) and kidney podocytic process epicyte protein (PDPN), flow cytometry show up to 98.5% cell EGFR
Positive but only 93% cell CK5 is positive；
5. cell growth curve shows that the doubling time about 27 is small, in vitro culture can detect squama in the medium on the 5th day
Cancer associated antigen and its level raised with incubation time, adherent inoculation efficiency only 27.12%, with Human Laryngeal Cancer Cell Hep-2 phases
And clone colony weaker than dilution clonality is small；
6. compared with cervix adenocarcinoma cell line HeLa, the lateral transfer ability of cell is weak compared with strong but invasiveness, cell in vitro
It is that 2.5106 inoculation severe combined immune mouse (SCID) can be into knurl, one-tenth knurl ability is weaker than Hep-2 cell lines, histotomy prompting
Transplantable tumor is all middle differentiated squamous-cell carcinomas pathomorphism with primary tumo(u)r, both protein expression profiles of immunohistochemistry detection are identical；
7. cell line carries out Antibiotics resistance test discovery, sensitive to cis-platinum as Hep-2, while finds a kind of clinic
Chemotherapeutic epirubicin less can also produce cell overt toxicity effect.
Further aim of the present invention is that people's penis cancerous cell line Penl1 can provide in vitro study penis carcinogenesis, development
The new clinical diagnosis means of transfer of molecules mechanism, exploitation, establish Ex vivo animal model and find new antineoplaston mode
Purposes.
The present invention people's penis cancerous cell line Penl1 can long-term in vitro subculture, property stablize, cell present squamous on
Cell cancer cell lines growth agglomerating growth characteristic, through Electronic Speculum, cytochemical staining, PCR and gene sequencing, cell fingerprint and
Protein expression, verify the identity of tumour cell and compared with former tumoral character into knurl experiment, characteristics of cell biology in vitro, determines
Cell line can reflect the characteristic of human body source penis squamous carcinoma, and Primary Study cell line is to chemotherapeutic drugs Cisplatin and epirubicin
Sensitiveness.The people penis cancerous cell line Penl1 of the present invention provides the penis squama for representing clinically HPV feminine genders for penis squamous carcinoma
The cell research instrument of cancer.The cell line and animal model based on Establishment of Cell Line can be used for research penis carcinogenesis, development,
The basic research of metastasis, diagnoses the preclinical study of the auxiliary examination methods of carcinoma of penis and Lymph Node Metastasis, and exploration discovery is new
The purposes such as therapy target developing anti-tumor medicaments.
Brief description of the drawings
(200 ×) morphology under the light microscopic of Fig. 1 the present inventor penis squamous cell carcinoma Penl1；
The immunocytochemical stain morphology of Fig. 2 the present inventor penis squamous cell carcinoma Penl1；
The Ultrastructure Morphology of Fig. 3 the present inventor penis squamous cell carcinoma Penl1；
The transplantable tumor specimen morphology of Fig. 4 the present inventor penis squamous cell carcinoma Penl1；
The DNA content of Fig. 5 the present inventor penis squamous cell carcinoma Penl1；
The PCR detections of the mycoplasma and HPV of Fig. 6 the present inventor penis squamous cell carcinoma Penl1；
The full extron sequencing mutation result of TP53 genes of Fig. 7 the present inventor penis squamous cell carcinoma Penl1；
The protein expression western blot results of Fig. 8 the present inventor penis squamous cell carcinoma Penl1；
The flow cytometry results of Fig. 9 the present inventor penis squamous cell carcinoma Penl1；
The immunohistochemistry morphology of Figure 10 the present inventor penis squamous cell carcinoma Penl1；
The growth curve and SCC-RA of Figure 11 the present inventor penis squamous cell carcinoma Penl1 is horizontal；
The kind plate efficiency and dilution cloning efficiency of Figure 12 the present inventor penis squamous cell carcinoma Penl1；
The migration of Figure 13 the present inventor penis squamous cell carcinoma Penl1 and Matrigel；
The Antibiotics resistance test of Figure 14 the present inventor penis squamous cell carcinoma Penl1.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment the present invention will be further described and analysis (tested in following embodiments
Method is conventional method unless otherwise specified).
People's penis squamous cell carcinoma of the present invention, China typical culture collection center is preserved on June 17th, 2015
(CCTCC) (address：Wuhan City, Hubei Province Wuhan University in the school, postcode：430072), culture title behaviour penis squamous cell carcinoma
It is Penl1, deposit number is CCTCC NO：C201585.
The foundation of 1. cell line of embodiment
1.1 original cuiture
Sample comes from Zhongshan Univ. Cancer Cure Center, is obtained from the groin transfer leaching of 41 years old male penis squamous cell carcinoma patients
Fawn on, after lymph node sample is in vitro in art, is separated into 5mm or so tissue fritters, immerses trained equipped with serum-free DMEM in high glucose immediately
Support the EP pipes of base (addition 200U/ml penicillin, 200 μ g/ml streptomysins).In the cell special bio safety cabinet in laboratory,
Tumor specimen is rinsed 3 times with phosphate buffer (PBS), and Sterile ophthalmic cuts the fritter that tumor specimen is cut into 1-3mm diameters,
It is placed in 25cm to even distribution2Tissue culture flasks in, add about 1-3ml DMEM in high glucose culture medium (10% import tire ox blood of addition
(FBS), 200U/ml penicillin, 200 μ g/ml streptomysins clearly, use as growth medium), ensure that fritter moistening is not floatd at the same time
Arise from above culture medium, be carefully turned over blake bottle, be put into 37 degree of water-baths and 5%CO2Incubator in.4 it is small when after, upset culture
Bottle simultaneously adds culture medium to 5ml.24 it is small when after, change liquid and remove lymphocyte of not adherent tissue block and buoyant growth etc..Before
1-6 generations, the method by quickly using 0.25% pancreatin-ethylenediamine tetra-acetic acid (EDTA) to digest, removal are easily digested de- wall
Fibroblast, while it is adherent using multiple blake bottles to sequentially add same cell suspension (the completely postdigestive cell of blake bottle)
Method, remove easily adherent fibroblast in cell suspension and tumour cell be further purified.It is adherent to be separated into fiber process
In, the fibroblast isolated continues subculture in vitro separately culture, CAF is named as, as follow-up fibroblast and normal gene
The control of group.During seven generations, the neoplastic epithelial cells of screening form multiple clones, and it is more to choose the consistent growing number of cellular morphology
Clone continues to pass on as stable cell lines Penl1.
1.2 cells are passed on and frozen
Use 25cm2During blake bottle culture cell, when Penl1 cells coverage rate reaches 70-90%, progress cell passage：Go
Except old culture medium, add 5ml PBS and rinse twice, wash away culture medium, add 1ml or so 0.25% pancreas enzyme -EDTA digestive ferment, put
Enter in incubator and be incubated, about 5-10 minutes, the cell rounding it was observed that cell cytoplasm bounces back, space between cells became larger, and gently rocks training
Support bottle, after most cells take off wall, add the fresh growth medium of 3ml or so, blow and beat into cell suspension, collect cell from
The heart, is passed in 1: 4 to 1: 8 ratios, planted in original culture bottle or new blake bottle.The cell frozen, uses addition 5-10% dimethyl
The growth medium of sulfoxide (DMSO) and 50%FBS are as frozen stock solution, and progressively cool down freeze-stored cell.Survival ratio after cell recovery
About 30-60%, it is different because of the holding time.The 21-25 used in detection process is submitted for cell to CCTCC preservations.
1.3 cell culture features and form
Penl1 cell injuring models stablize passage up to 13 months in the present invention, reach for 88 generations.Culture cell is placed in inversion
Under microscope, cell such as Fig. 1 after taking pictures, Penl1 cells form under phase contrast microscope is more homogeneous in the present invention, and growth is without connecing
Touch and suppress, cell is in flat paving stone sample Epithelial morphology, agglomerating growth, consistent with squamous cell carcinoma system cell growth mode.Pass
During generation, needed 5-10 minutes using 0.25% pancreas enzyme -EDTA digestion time, be longer than existing tumor cell line, Long Term Passages
During the cell keep basically identical Epithelial morphology, growth pattern and need the digestion time of long enough (5-10 minutes) to disappear
The characteristics of changing de- wall.
Immunocytochemical stain and the ultrastructural morphology observation of embodiment 2.Penl1 cell lines
2.1 cytochemical staining
When cell was passaged to for 8 generation, it will be grown on 24mm coverslips that Penl1 cell attachments are grown in six orifice plates, cell
After completely adherent rear and growth conditions are good, six orifice plates are removed into incubator, remove nutrient solution, rinsed coverslip with PBS, add
4% paraformaldehyde fixes cell 10 minutes, uses 0.3%Triton X-100 penetration cells, 10% sheep blood of following cell
When room temperature closing 1 is small clearly, when 37 degree of incubation pan-CK and vimentin primary antibodies 1 are small, it are incubated polymerization HRP and mark anti-mouse IgG bis-
Anti- 37 degree are incubated 30 minutes, and DAB develops the color 5 minutes or so, and haematoxylin develops the color 3 minutes, dehydration and mounting.Above cytochemical staining
Such as carried out without explanation using common experimentation.Penl1 cellular morphologies such as Fig. 2 present invention are consistent with phase contrast microscope,
Epithelial cell intermediate filament protein pan-CK dyeing is positive in endochylema and is distributed along cytoskeleton, fibroblast intermediate filament protein
Vimentin is negative, and the CAF as fibroblast control is just opposite.
2.2 ultrastructural morphology
(the 25th generation cells of Penl1, hereafter detect the cell used such as without explanation, are 21- the good cell of growth conditions
25 generation Penl1 cells) fixed through 2.5% glutaraldehyde, then through 1% osmic acid (OsO4) fixed, the dehydration of alcohol of gradient concentration, resin
Embedding, routinely repaiies block, ultra-thin section simultaneously dyes, and uses Hitachi H-7000FA transmission electron microscope observations.Such as Fig. 3 institutes
Show, it is seen that the tension force silk (A) in cell cytosol connects (B) with the desmosome of cell membrane surface.
2.3 transplantable tumor morphology
For determine people's penis squamous cell carcinoma Penl1 one-tenth knurl ability, 2.5 × 106Penl1 cells are resuspended with 100 μ l PBS,
The subcutaneous vaccination of gained cell suspension to 4 weeks sizes severe combined immunodeficient mouse (SCID mice, purchased from Beijing dimension tonneau China
Animal Science and Technology Ltd.) right side inguinal region is subcutaneous, and the visible swollen thing of naked eyes, collects sample, with 10% good fortune after 6 weeks after 2 weeks
Your Malin fixes, routine paraffin wax embedding and Hematoxylin-eosin (HE) dyeing, micro- Microscopic observation.As shown in figure 4, cell inoculation
The big core of tumour cell, deep dye core and nuclear atypia, a small amount of keratin pearl, with primary tumor are shown in transplantable tumor size about 5mm afterwards, HE dyeing
Morphology is consistent with pathological diagnosis, is diagnosed as differentiated squamous-cell carcinomas in penis.
The science of heredity feature of embodiment 3.Penl1 cell lines
3.1 short tandem repeat (STR) cellular identification
The DNA of tissue specimen and cell uses the AllPrepDNA/RNA/Protein Mini set reagents of Qigen companies
Box extracts.The DNA for the metastatic lymph node knot of originating patient, Normal Lymph Nodes and the Penl1 cell lines of making a collection of specimens, it is public using basic point
The amplification that Goldeneye 20ASTR suit carries out 19 STR bit points and 1 Sex Determination site totally 20 sites is taken charge of, PCR expands
Increase production thing and carry out ABI3730xl electrophoresis, using 4.0 software analysis of GeneMapper, cell line identification will be included and commonly use 9 positions
The STR spectrums of point and three maxicell databases in the world (American Type Culture Collecti (ATCC), German living resources collection
(DSMZ) and Japan Cancer resources bank (JRCB)) STR databases be compared.As shown in table 1, metastatic lymph node and normal leaching
The STR spectrums fawned on are consistent, and the STR of cell line Penl1 spectrums are shown in that an electrophoresis peak is lost in D5S818 sites, prompt the something lost of Penl1
The full-length genome of material and source patient is passed there are the identical STR bit point of major part, but there is also difference.In addition, cell line STR
Compose, the STR information with any cell of STR databases in the world does not repeat, and determines the uniqueness of cell.
Table 1 shifts, the STR of Normal Lymph Nodes and cell line spectrums
3.2DNA content
Harvest 106HeLa the and Penl1 cells in exponential phase of growth, while normal human lymphocytes are as standard pair
According to winning cell cycle detection kit using shellfish and detect DNA content to specifications.Proliferating antigen Ki67 (PI) calculation formula is
PI=(G2M+S)/(G0G1+S+G2M).As shown in figure 5, compared with human lymphocyte and HeLa cells, the DNA of Penl1 cells
Content is polyploid, and the cell of higher proportion is in vegetative state when Penl1 proliferation indexes are higher than HeLa, i.e. exponential phase of growth.
3.3 cell mycoplasmas and HPV detections
Detection of mycoplasma uses the PCR detection of mycoplasma kit of parts of Huaan biology, to specifications, carries out just
The often PCR amplification rear electrophoresis detection of lymph node (LN), metastatic lymph node (LM), Penl1 cells and CAF cell DNAs.HPV is detected
Before, amplification reference gene GAPDH is used as Quality Control, the amplification of progress HPV viruse DNA after Quality Control qualification, is used respectively in Chao Shi PCR
The common HPV versatilities primer of MY09/MY11 and GP5+/GP6+ these two pairs, conventional electrophoretic gel imaging.As shown in fig. 6, branch is former
Electrophoresis result (A) display is surveyed in physical examination, and LN, LM, Penl1 cell and CAF cell DNAs do not amplify mycoplasma DNA, HPV detection
Electrophoresis result (B) show LN, LM, Penl1 cell and CAF cell DNAs it is amplifiable go out GAPDH products, but can not amplify
The DNA of HPV, that is, illustrate Penl1 cells without mycoplasma and HPV infection.
3.4 TP53 gene sequencing
LN, LM, Penl1 cell and CAF cell DNAs use each extron primer designed according to TP53 genes, product electricity
Swimming confirms to expand successfully Hou Song Invitrogen Corp. sequencing.As shown in fig. 7, the sequencing result of cell line and normal TP53 genes
(http：//asia.ensembl.org/) it is compared, and with being found after the TP53 sequencing results of LN, LM and CAF,
There are two point mutation by Penl1：P72R (CCC to CGC) and R283P (CGC to CCC).Wherein P72R is existed in normally
In lymph node and fibroblast, and in the database (http of TP53：//p53.iarc.fr/) confirm that this sports monokaryon
Nucleotide polymorphism.R283P mutation are existed only in metastatic lymph node and Penl1, may be not present in Normal Lymph Nodes and into fiber
Cell, and in metastatic lymph node, because containing some fibroblast, the sequencing peak of the point mutation is overlapping with normal base,
It can determine that the mutation for sporting tumour-specific.
The protein expression feature of embodiment 4.Penl1 cell lines
4.1 western blot are detected
The full cell pyrolysis liquid of Penl1 and CAF cells is collected, routinely westernblot experimental methods electrophoretic separation egg
In vain, electric transferring film, is incubated overnight primary antibody, 37 degree of incubation secondary antibodies, ECL methods development detection protein content.Primary antibody include GAPDH (1:
2500, Santa Cruz), pan-CK (1: 200, Santa Cruz), EGFR (1: 2500, Santa Cruz), p16 (1: 200,
Santa Cruz), PDPN (1: 200, Santa Cruz) and vimentin (1: 200, Santa Cruz), secondary antibody uses sheep
Anti- mouse or goat-anti rabbit (1: 2000, Santa Cruz), ECL methods development albumen use green skies BeyoECL Plus to cover
Dress.The results are shown in Figure 8, and (positives and negative control of the lung cancer cell line HCC827 and H460 as EGFR, infect the uterine neck of HPV
The positive control that cancer HeLa cell lines are expressed as p16), (A) Penl1 cells expressed epithelial cell intermediate filament protein pan-CK,
Memebrane protein EGFR is expressed at the same time, (B) Penl1 cells also express epithelium PROTEIN C K5, memebrane protein the PDPN egg related to HPV of squamous carcinoma
White p16, but fibroblast cellular intermediate filament protein vimentin is not expressed.
4.2 flow cytometry
After Penl1 cell dissociations in good condition, PBS is resuspended, and breakthrough experiment kit suit (BD is fixed according to cell
Cat.No.554715) illustrate to use.When detecting intracellular protein CK5, first fixed, penetration cell, is containing CK5 primary antibodies (10 μ
l/106Cells, Santa Cruz) and the cell washing lotion of isotype control Ab in respectively 4 degree incubation 45 minutes, used after rinsing
Contain IgG1-PE (10 μ l/106Cells, Miltenyi Biotec) 4 degree of cell washing lotion be incubated 30 minutes after rinse upper machine.
When detecting memebrane protein EGFR, directly containing anti-EGFR-FITC (10 μ l/106Cells, Santa Cruz) and Isotype control
Rabbit igg-FITC (10 μ l/106Cells, Miltenyi Biotec) PBS in 4 degree be incubated 30 minutes, upper machine after rinsing.Cell
Cell is analyzed using Gallios Flow cytometries, later data is analyzed using kaluza.As shown in figure 9, in Penl1 cells, table
Up to the cell proportion of EGFR up to 98.5%, and CK5 positive expressions cell proportion is up to 93.0%, the table of latter two albumen of statistical analysis
Up to there is significant difference (p=0.0095).
4.3 immunohistochemistry
LM, LN and Penl1 cell transplantation knurl sample routinely carry out formalin and fix, and prepare paraffin mass, and section is exempted from
Epidemic disease group.According to routine immunization group method, dewax after roasting piece, using 10mM EDTA antigen retrievals, 1%Triton-X100 is worn
Thoroughly, 3%H2O2 is blocked, 5%BSA closings, and 4 degree are incubated overnight primary antibody, and when 37 degree of incubation secondary antibodies 1 are small, DAB develops the color, and haematoxylin is multiple
Core is contaminated, it is conventional to be dehydrated resin mounting.The primary antibody used as above western blot and the primary antibody of cell streaming, in addition primary antibody also wrap
Include desmoplakin (1: 100, Santa Cruz), HPV 16/18E6 (1: 100, Santa Cruz), p15/p16 (1: 100,
Santa Cruz) and p53 rabbit monoclonal antibodies (1: 160, Cell Signaling Technology), secondary antibody used
Goat-anti rabbit/mouse (Wuhan doctor's moral) of oxide enzyme lotus root connection, DAB colour reagents box (Shanghai Gene Tech).LM as shown in Figure 10
With epithelium GAP-associated protein GAP pan-CK, CK5 and desmoplakin positive expression in epithelioma component in transplantable tumor sample, and interstitial
Label vimentin is negative；HPV GAP-associated protein GAP E6 radiolucent tables reach, and p16 diffuses the karyon overexpression suppression at the same time of endochylema weak expression
Oncogene p53 albumen, meets the expression characteristic of non-HPV albumen；In addition, Penl1 cells also express memebrane protein EGFR and PDPN.With
Control LN only have mesenchymal vimentin expression, other immune groups can label it is negative.
The biological behaviour feature of embodiment 5.Penl1 cell lines
5.1 cell growth curves and squamous carcinoma related antigen (SCC-RA) detection
Penl1 cells are pressed 6 × 106Cell/cm2Kind is in 21 25cm2In blake bottle, totally 7 times 3 repetitions when interval 24 is small
Ground test, digest and cell counted on the cell and lencocyte count plate of blake bottle, at the same collect renewal 24 it is small when after cell training
Yang Jisong clinical examinations section office carry out SCC-RA detections.As a result as shown in figure 11, cell after the incubation period of 1-2 days exponentially
Increase, the tends to plateau for 6-7 days, and at the same time, the SCC-RA in culture medium started to detect at the 5th day, with culture
Shi Wen rises in seven days reach 1.7ng/ml (the abnormal SCC-RA critical values of clinical detection are 1.5ng/ml).
5.2 cell kind plate efficiency and dilution cloning efficiency
Penl1 cells are inoculated with (3 repetitions) by the experiment of kind plate by 50,100,250,500,1000 cells per 6cm culture dishes,
10000 cell inoculations ensure cell inoculation as positive control at the same time.After 1 week after visible cell clone, contaminated using crystal violet
Color, and the clone for counting and being more than 16 cells that takes pictures.Kind plate efficiency as shown in figure 12 (A), is cultivated in 50-1000 cells per 6cm
Under the inoculum density of ware, the cell number and colony counts of cell kind plate are in a linear relationship, and kind plate efficiency is 27.12%.
Dilute cloning experimentation in, using Human Laryngeal Cancer Cell Hep-2 as compare, in six orifice plates inoculation 100 cells/
Hole, 6 repetitions are tested, and culture medium is growth medium, and the method dyeing of plate experiment is as above planted after 7 days, counts clone, is taken pictures simultaneously
Data analysis.As a result the dilution cloning efficiency of (B＆C) Penl1 cells is less than Hep-2, the colony counts formed as shown in figure 12
With size again smaller than HeLa cells.
5.3 migrations and Matrigel
Cell invasion experiment is carried out using Transwell methods.By 2 × 104Penl1 and HeLa cells be resuspended in 200 μ
In the serum free medium of l, the small interior in upper strata of the tranwell (healthy and free from worry) in 24 holes is inoculated in, the cell face of upper strata cell has been used
Matrigel (BD companies) processing, the FBS DMEM in high glucose culture mediums of lower room addition 600ul10%, 24 it is small when after remove upper chamber
Culture medium, the remaining cell face for not penetrating cell is wiped with cotton swab, violet staining, is taken pictures under phase contrast microscope and is counted low power
The cell number of cell is passed through in the visual field.(A＆C) as shown in figure 13, in same time, less Penl1 cells pass through matrigel
The film of processed upper chamber, the invasive ability of Penl1 cells are weaker than HeLa cells.
The method that the horizontal migration of cell uses scratch experiment.About 5-10 × 10 in inoculation in six orifice plates4Penl1 and
HeLa cells, when cell nearly 90-100% coverage rates, are drawn with the yellow pipette tips of 200 μ l in the bottom of six orifice plate culture cells
Trace, rinsing remove the cell in cut, with serum-free DMEM in high glucose culture 24 it is small when, before and after observing cut under phase contrast microscope
Distance change between cut, simultaneously analysis of accounts of taking pictures.As shown in figure 13 (B＆D) visible Penl1 cuts reduce apart from bigger, explanation
The distance of Penl1 cell migrations is more than HeLa cells, i.e. the lateral transfer ability of Penl1 cells is better than HeLa cells.
Application of the embodiment 6.Penl1 cell lines in carcinoma of penis cis-platinum and epirubicin chemotherapy research
Penl1, Hep-2 and MCF-7 cell are pressed into 2-3 × 103The density of cell per well is inoculated in 96 orifice plates, and next day is more
Change into containing gradient cis-platinum (0,0.15625,0.3125,0.625,1.25,2.5,5.0,10.0,20.0 μ g/ml) and the soft ratio of table
The fresh 10%FBS DMEM in high glucose culture medium of star (12.5,25,50,100,200,400,800,1600nM), drug powder nothing
Bacterium PBS dilutes.48 it is small when and 72 it is small when after use CCK-8 kit detection cell vigor, add CCK-8 to specifications, incubate
Educate 2.5 it is small when obtain the correspondence absorbance in each hole.Inhibiting rate (IR) calculation formula is IR=100%- survival rates (SR), survival rate
Calculation formula is SR=100% × (instrument connection absorbance-blank well absorbance)/(control wells absorbance-blank well absorbance).
As a result such as Figure 14 is shown, relative to the Hep-2 cells (A＆B) to cis-platinum sensitivity and the MCF-7 cells (C＆ to epirubicin sensitivity
D), 48 it is small when and 72 it is small when Penl1 cell survival rates be less than two control cells, i.e. sensitiveness of the Penl1 cells to two kinds of medicines
It is high.When Penl1 cells 72 are small to 50% inhibiting rate (IC50) of cis-platinum for 0.11 μ g/ml, 72 when small to the IC50 of epirubicin
For 44.46nM, the IC50 respectively less than to the control cell lines of medicaments insensitive.
Claims (3)
1. one plant of people's penis squamous cell carcinoma system, it is characterised in that it is deposited in Chinese Typical Representative culture guarantor on June 17th, 2015
Tibetan center (CCTCC), address：Wuhan City, Hubei Province Wuhan University in the school, postcode：430072, culture title behaviour penis squama
Cancerous cell line Penl1, deposit number are CCTCC NO：C201585.
2. one plant of people penis squamous cell carcinoma system as claimed in claim 1, its cell characteristic includes surpassing in Epithelial morphology and epithelium
Micro-structure, animal can be into knurl and positive for middle differentiated squamous-cell carcinomas, epithelium intermediate filament protein keratin on mouse in being tested into knurl；More times
Body DNA content, without mycoplasma and HPV infection, TP53 extrons 4 and 8 have mutation；Express EGFR, pan-CK, CK5, PDPN, p16
Albumen but do not express vimentin；Doubling time about 27h, lateral transfer ability is strong compared with HeLa cells but invasiveness is weak, right
Cis-platinum and epirubicin are sensitive；Cell determines that cell fingerprint is not believed with 3 maxicell databases in the world through short tandem repeat
Cease overlapping；This plant of penis squamous cell carcinoma system represents the tumoral character of HPV feminine gender penis squamous carcinomas.
3. one plant of people penis squamous cell carcinoma system as claimed in claim 1 or 2, it is characterised in that people's penis squamous cell carcinoma system
Penl1 represents HPV feminine gender penis squamous carcinoma features, in further investigation penis carcinogenesis, development, metastasis and carcinoma of penis
Have in the inside and outside basis of the screening anti-tumor medicines such as clinical diagnosis, chemotherapeutics and targeted drug and clinical Study on Transformation wide
Wealthy prospect.
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| 阴茎癌中p53表达与HPV16、18DNA的检测;拉莱·苏祖克等;《临床与实验病理学杂志》;20020630;第18卷(第3期);第304-306页 * |
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