CN105343897B - Rhesus liver cancer model, rhesus liver cancer cell strain and application thereof - Google Patents
Rhesus liver cancer model, rhesus liver cancer cell strain and application thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing a rhesus monkey liver cancer model, which is to apply diethyl nitrosamine to primates, wherein the application dosage of the diethyl nitrosamine is as follows: 40 mg/Kg/time. The invention also provides a rhesus monkey hepatoma cell strain and application thereof. The animal model can be used for evaluating new technologies such as liver cancer prevention factors, anti-liver cancer new drugs, liver cancer radiotherapy, liver cancer surgical treatment, imaging diagnosis and the like which cannot be completed by rodent animal models.
Description
Technical field
The present invention relates to a kind of rhesus macaque liver cancer models, rhesus macaque hepatoma cell strain and application thereof.
Background technology
Liver cancer, i.e. hepatocellular carcinoma Hepatocellular carcinoma, HCC are the common malignant tumours of liver, every year
New example of diagnosing a disease is up to 700,000, and global liver cancer new cases are 78.2 ten thousand within only 2012, and death is 74.6 ten thousand, at present because it is treated
Measure is extremely limited, and liver cancer seriously threatens human health and life.Inland of China onset of liver cancer has occupied alimentary canal evil at present
Property tumor mortality rate first of, number of the infected account for the whole world 55%, death toll be the whole world 45%.How the diagnosis and treatment of liver cancer are promoted
Quality of life that is horizontal, improving patient is the root problem for being badly in need of solving in liver cancer study on prevention.Liver cancer animal model, especially
It can more preferably predict that the liver cancer animal model of clinical efficacy is diagnosing cancer of liver, treats the research and development of New Measure, new technology and anti-cancer drugs
It is successfully crucial.So far, all it is to use grinding tooth in relation to the in vivo studies of tumour basic research and anti-cancer agent therapeutic evaluation research
Animal model, including the portable allogenic animal model of immune competent mice and immune deficiency or genetic engineering mice human body swell
Tumor portable heterogenous animal model.It is paid although rodent tumor models are tumour basic research and anti-cancer agent therapeutic evaluation
Tremendous contribution is gone out, in Related Research Domain extensive use, but still there are many defects, such as：The tumor animal time-to-live is short,
It is observed when not being suitable for long；It is poor that toy is resistant to surgical operation, radiotherapy etc., is not suitable for continuous dynamic observation；Institute's lotus tumour
It organizes small, is not suitable for biopsy dynamic observation etc..Smith etc. summarizes macaque, other primates, dog and rat drug metabolism
With the correlation of people, find have 71% compound through research and related human preferable in rhesus macaque, the experimental result of dog has
19% and rat to only have 14% approximate with the mankind.As it can be seen that using rodent model study of disease mechanism and research and development drug, institute
It obtains result and Human clinical's curative effect correlation is very low, most of new drugs is caused to be eliminated in clinical test.Rodent is solving
It cuts open, pathologic, physiologic and drug metabolism reaction etc. to differ greatly with the mankind, test result and clinical correlation or curative effect
There is very big gap in predictability, therefore establish new, the more efficiently big animal liver cancer animal model gesture of non-human primates and exist
It must go.
Rhesus macaque is in anatomy, pathologic, physiologic, immune and drug metabolism reaction etc. and similar, the gene of mankind's height
Sequence has high similarity with human diseases ortholog and drug target.Chemical carcinogens, such as：N-
Nitrosodiethylamine (DENA), 2-amino-3-methylimidazoquinoline (IQ), aflatoxin B1,
Sterimatocysti, HBV etc. can induce rhesus macaque liver cancer, wherein it is stronger to rhesus macaque carcinogenicity with DENA, relatively stablize, HBV+
The rhesus macaque hepatocellular carcinoma that DENA induces, pathology find similar to human liver cancer.Result of study prompts, and DENA etc. can be used for building
Vertical chemical induction rhesus macaque liver tumour model.But previously research is primarily upon the carcinogenesis of chemical carcinogens, does not suggest that logical
Cross Induced By Chemical Carcinogen rhesus macaque liver hepatocellular carcinoma, establish rhesus macaque liver cancer model, portable monkey hepatocellular carcinoma cell lines,
Internal xenogenesis dystopy and dystopy in situ or of the same race and Transplantable Hepatocellular Carcinoma model in situ, to be used for preclinical diagnosing cancer of liver, control
Treat the research and development of New Measure, new technology and anti-cancer drugs.
It there is no the rhesus macaque liver cancer model that repeatability is established and portable rhesus macaque hepatocellular carcinoma thin in the world at present
Born of the same parents' strain.
Invention content
The technical solution of the present invention is to provide diethylnitrosamines to prepare the animal for screening treatment liver-cancer medicine
Purposes in model.There is provided the foundation of rhesus macaque liver cancer model for another technical solution of the present invention.
The present invention provides a kind of methods preparing liver cancer animal model, it is that diethylnitrosamine is applied to primate
Animal, the method for administration and dosage of diethylnitrosamine are：40mg/Kg/ times.
Preferably, the primate is rhesus macaque.
Preferably, the method for administration of the diethylnitrosamine and dosage are：Intraperitoneal injection, each 40mg/Kg,
Once a week, it co-administers 20 times.
The present invention also provides the liver cancer animal models that this method is prepared.
The present invention also provides a kind of rhesus macaque hepatoma cell strain, the preservations of its China typical culture collection center preservation
Number：CCTCC NO：The rhesus macaque hepatoma cell strain M12353 of C2015142.
Rhesus macaque hepatoma cell strain M12353 of the present invention was deposited in China typical culture collection on the 9th in September in 2015
The heart, is referred to as CCTCC, and address is：The Chinese Wuhan Wuhan Universitys, preserving number are：CCTCC NO：C2015142.
Wherein, aforementioned cells strain is to isolate and purify preparation by aforementioned liver cancer model.
The present invention also provides purposes of the aforementioned rhesus macaque hepatoma cell strain in preparing liver cancer animal model.
Preferably, the liver cancer animal model is rhesus macaque liver cancer model.
The present invention also provides aforementioned liver cancer animal model, aforementioned rhesus macaque hepatoma cell strains in the medicine for screening treatment liver cancer
The research of object and/or prevention of hcc factor, anti-liver cancer and anti-new drug, Radiotherapy For Carcinoma of The Liver, Surgery For Hepatocellular Carcinoma operative treatment, imaging diagnosis
In purposes.
The present invention also provides a kind of methods of the drug of screening treatment liver cancer, include the following steps：
Candidate is applied to aforementioned liver cancer animal model, aforementioned rhesus macaque hepatoma cell strain by a；
B, with aforementioned liver cancer animal model, the drug of the potential treatment liver cancer of aforementioned rhesus macaque hepatoma cell strain evaluation.
The rhesus macaque liver cancer model that the method for the present invention is successfully established can be used for the impossible liver of rodent models
The experimental studies such as cancerous precaution factor, anti-liver cancer and anti-new drug, Radiotherapy For Carcinoma of The Liver, Surgery For Hepatocellular Carcinoma operative treatment, imaging diagnosis.
On the other hand, the present invention is also successfully established the rhesus macaque hepatoma cell strain M12353 that can be passed on steadily in the long term,
It can pass on steadily in the long term in vitro, at present >80, Chi Xushijian >16 months, immunodeficient animals (nude mouse) xenogenesis moved
Planting has Tumor formation, every inoculation 1-2 × 106Cell, tumor formation rate 100%, incubation period are 10-14 days；It can transplant in perseverance
Tumor formation in the monkey body of river prepares rhesus macaque liver cancer model of the same race, for prevention of hcc factor, anti-liver cancer and anti-new drug, Radiotherapy For Carcinoma of The Liver,
The experimental studies such as Surgery For Hepatocellular Carcinoma operative treatment, imaging diagnosis, application prospect are good.
Below by way of specific implementation mode, the present invention is described in further detail, but is not intended to limit the present invention, ability
Field technique personnel can make various changes and modifications according to the present invention, without departing from the spirit of the present invention, should all belong to this
Invention scope of the appended claims.
Description of the drawings
The multiple liver tumours of Fig. 1 monkeys M12393
Fig. 2 rhesus macaque liver cancer CT photos
The white aFP testing results 3A-2-H1-2 of Fig. 3 rhesus serums is internal reference standard；A3-7-E3-7 is different dilutions
Lotus liver cancer monkey serum；F3-7-H3-7 is normal monkey serum.
Fig. 4 M12393 Histopathology forms
Fig. 5 M12385 Histopathology forms
Fig. 6 rhesus macaque M12353 liver cancer general forms
Fig. 7 M12353 Histopathology forms
Fig. 8 M12353 liver cancer tissue nude mouses inoculate transplanting tumor formation
Fig. 9 M12353 liver cancer tissue Transplanted Into Nude Mice tumor formation histopathology forms
Figure 10 M12353 liver cancer tissue block in vitro culture forms
Figure 11 M12353 Morphology of Hepatocellular Carcinoma
Figure 12 M12353 liver cancer cells Transplanted Into Nude Mice tumor formation histopathology forms
Figure 13 monkey liver cancer cells M12353 growth curves
Growth inhibition effect of Figure 14 chemotherapeutics to monkey liver cancer cells M12353 and human liver cancer cell
Figure 15 chemotherapeutics 5 FU 5 fluorouracil (5-Fu) inhibits to make to monkey liver cancer cells M12353 and hepatoma cell growth
Morphologic observation
Specific implementation mode
The preparation of 1 animal model of the present invention of embodiment
1, materials and methods：
1.1 experiment material
Be born in 1 week healthy rhesus macaque, male, and weight 0.5-0.8kg, has purchased from this bold and unconstrained biotechnology of Chengdu Green by 6
Limit company.
1.2 main agents
Diethylnitrosamine：Purchased from Sigma companies.
Physiological saline is purchased from Xinan Pharmaceutical Co., Ltd.).
The preparation of 1.3 main solutions
The preparation of diethylnitrosamine solution：
DENA stostes (Sigma) 25ml is taken, adds injection physiological saline 75ml, is configured to 20%DENA aqueous solutions, is protected from light
It saves backup.
1.4 key instrument
Dispoable medical sterilizing syringe：Purchased from Sichuan Shuanglu Medical Apparatus & Instruments Co., Ltd..
1.5 modeling method
With into 20%DENA aqueous solution intraperitoneal injections, 40mg/kg, once a week, co-injection 20 times；Claim body every 2 weeks
Weight observes animal ordinary circumstance.
2 experimental results
2.1 DENA induce rhesus macaque hepatocellular carcinoma CT examination result
1 rhesus macaque death of experimental group, other 5 survivals.
1 monkey is in experiment the 432nd day (14 months) because of haemorrhagic shock liver failure, entembole death (become celestial discovery), solution
It cuts open and finds the liver multiple lump of diameter 0.5-4.5CM sizes (Fig. 1), it is similar to human liver cancer shape.
CT examination is carried out to remaining 5 rhesus macaque, 4 discovery Space occupation in liver venereal disease stoves, 1 uncertain.Its representativeness is shone
Piece such as figure (Fig. 2).
2.2 lotus hepatocellular carcinoma rhesus serum human liver cancer Research of predicting markers testing results
The experimental group rhesus macaque of 5 survivals of acquisition and the serum of 6 Normal Rhesus, following (table has been carried out with Elisa methods
1) liver cancer correlating markings analyte detection.
1 monkey liver cancer model serum mark analyte detection of table
As a result：
(1) testing result is shown in (table 2-5, Fig. 3)
2 alpha-fetoprotein of table (aFP)
3 glutathione-S-transferase of table (GSTP1)
4 heterogeneous nuclear ribonucleoprotein K of table (HNRNPK)
Table 5 takes off-γ-carboxyls factor (DCP)
The macaque model that it can be seen from table 2- tables 5 and Fig. 3 prepared by the method for the present invention, the marker of liver cancer：First
Fetoprotein, glutathione-S-transferase, heterogeneous nuclear ribonucleoprotein K, de--γ=equal great expression of carboxyl factor.
2.3 rhesus macaque hepatic cell carcinomatosis Physical examination results
It chooses
Two survival rhesus macaquies are taken, the liver cancer tissue on liver is taken, is fixed with 10% neutral formalin, conventional stone Na embedding,
It is sliced HE dyeing, micro- sem observation.
As a result as shown in Figure 4 and Figure 5：All tumor tissues are the hepatocellular carcinoma of different differentiation degrees, with human liver cell
Cancer form is similar.
There is Space occupation in liver venereal disease stove, the mark of height expression liver cancer in rhesus macaque liver cancer animal model prepared by the method for the present invention
Will object, meanwhile, there is canceration in liver, and cancer cell is similar to human hepatocellular carcinoma form, illustrates that rhesus macaque liver cancer model models successfully.
2 rhesus macaque hepatoma cell strain M12353 of embodiment is established
One, experimental method
1, rhesus macaque Tissues of Hepatocellular Carcinoma nude mouse xenograft
The lotus liver cancer rhesus macaque that survive in Example 1, number is M12353, kills lotus liver at carbon dioxide inhalation anesthesia
Cancer rhesus macaque M12353, takes liver tumour tissue to be respectively used to：(1) solid with 10% neutral formalin, it is examined for histopathology
It looks into；(2) serum-free medium is set, nude mouse xenograft is used for；(3) serum-free medium is set, cell culture is used for.
2, the foundation of cell strain
It takes M12353 liver cancer tissues or Transplanted Into Nude Mice at tumor tissue, removes hemorrhagic necrosis tissue, be trimmed to 0.1-0.5mm
Tissue block is placed in culture dish by tissue block at a certain distance, and HM culture solutions (Hepatocyte is added after standing 30 minutes
Medium, Sciencell, 5201), liquid or use were changed every 1-2 days according to cell growth status
(Gibco, A11105-01) had digestive transfer culture, liquid nitrogen cryopreservation.
Two, experimental result
1, rhesus macaque Tissues of Hepatocellular Carcinoma nude mouse xenograft
(1) observation of rhesus macaque M12353 liver cancer general form is as shown in Figure 6.
(2) observation of rhesus macaque M12353 Histopathologies is as shown in Figure 7.
(3) rhesus macaque M12353 liver cancer nude mouse subcutaneous transplantation tumor formation
It takes the M12353 liver cancer tissues without downright bad bleeding are sterile to be fabricated to 0.5-1.0mm tissue blocks, or adds equivalent serum-free
Culture solution is fabricated to tissue suspension, then adds equivalent Matrigel (BD), inoculates and transplants to nude mouse.
As a result：
The direct nude mouse of M12353 liver cancer tissues, which inoculates transplanting, has Tumor formation (Fig. 8).At present in nude mice
10 generations of interior continuous biography, transplanting tumor formation rate are 90-100%.
M12353 liver cancer tissue Transplanted Into Nude Mice tumor formation pathological studies (Fig. 9), hepatocellular carcinoma form and former Henghe
Monkey M12353 liver cancer or human hepatocellular carcinoma are similar.
2, the foundation of cell strain
(1) M12353 liver cancer tissues block in vitro culture was to 72 hours, it is seen that tissue block peripheral cell is grown (Figure 10).
(2) the M12353 hepatoma cell strains obtained extend with incubation time, and passage number increases cell activity enhancing (figure
11), pleomorphism paving stone sample is grown, and cellular morphology is uniform, is pure cell line.
The M12353 hepatoma cell strains that the present invention obtains are named as rhesus macaque hepatoma cell strain M12353, in September, 2015
It was deposited in China typical culture collection center in 9th, is referred to as CCTCC, address is：The Chinese Wuhan Wuhan Universitys,
Preserving number is：CCTCC NO：C2015142.
The present invention is from the liver cancer tissue of rhesus macaque liver cancer animal model prepared by embodiment 1, isolated rhesus macaque
Hepatoma cell strain M12353.
The Tumor formation and growth characteristics of 3 rhesus macaque hepatoma cell strain M12353 of the present invention of embodiment detects
1, in vitro culture rhesus macaque hepatoma cell strain M12353 nude mouses subcutaneous transplantation tumor formation
It is CCTCC NO to harvest in vitro culture to 50 generations preserving number of the present invention：The rhesus macaque hepatoma cell strain of C2015142
M12353, with serum-free medium and matrigel (1:1) resuspension prepares cell suspension, is inoculated with and is moved with cell suspension nude mice by subcutaneous
It plants, 1.5-5 × 106A inoculation animal, each 2-4 point, is repeated 5 times.
The results show that each point can be with tumor formation every time, and tumor formation rate 100%, tumor formation histopathology form such as Figure 12
It is shown, it is identical as the pathological characters of liver cancer tissue.
Experimental result illustrates that preserving number of the present invention is CCTCC NO：The rhesus macaque hepatoma cell strain M12353 tools of C2015142
There is Tumor formation, it is hepatoma cell strain also to illustrate that the present invention obtains really.
2, in vitro culture M12353 rhesus macaquies liver cancer cell growth curve determination
2 × 10 are pressed after the monkey liver cancer cells M12353 digestion of logarithmic growth phase3A/hole is inoculated in 96 orifice plates, is placed
96 orifice plates took 8 holes that 10 μ l MTT are added per hole in being cultivated in incubator every 24 hours, continued to cultivate 4h, added 100 μ l
10%SDS/0.01N HCl in incubator overnight measure OD values with microplate reader (λ=570nm), draw cell growth curve.
The results show that preserving number of the present invention is CCTCC NO：The rhesus macaque hepatoma cell strain M12353 growths of C2015142
It is in good condition, 30 ± h of doubling time (Figure 13).
4 rhesus macaque hepatoma cell strain M12353 of the present invention of embodiment is tested as the pharmaceutical intervention of cell model
1, experimental method
The preserving number of the present invention of logarithmic growth phase is CCTCC NO：The rhesus macaque hepatoma cell strain M12353 of C2015142
And human liver cancer cells Hep G2, HepG2, Hep3B, 2 × 10 are pressed after digestion3A/hole is inoculated in 96 orifice plates, per 100 μ l of hole
Culture for 24 hours, is handled with the 5 FU 5 fluorouracil of various dose (5-Fu), adriamycin (ADM), oxaliplatin (L-OHP) respectively respectively,
3 multiple holes of each dosage group place 96 orifice plates after cultivating 72h in incubator, 10 μ l MTT are added per hole, continue to cultivate 4h,
100 μ l 10%SDS/0.01N HCL are added, are stayed overnight in incubator, OD values are measured with microplate reader (λ=570nm), take it flat
Mean value.Cell survival rate, cell survival rate=processing group OD value ÷ control group OD value × 100% are calculated, experiment is repeated 3 times.
2, experimental result：
The common chemotherapy of hepatocellular carcinoma drug fluorouracil (5-Fu) of clinic, adriamycin (ADM), oxaliplatin (L-OHP) can press down
System preserving number of the present invention is CCTCC NO：The growth of the rhesus macaque hepatoma cell strain M12353 of C2015142；Preserving number of the present invention
For CCTCC NO：The rhesus macaque hepatoma cell strain M12353 of C2015142 chemotherapy of hepatocellular carcinoma drug fluorouracil (5-s common to clinic
Fu), adriamycin (ADM), the sensibility of oxaliplatin (L-OHP) are similar to human liver cancer cells Hep G2, HepG2, Hep3B
(Figure 14,15).
The experiment results show that preserving number of the present invention is CCTCC NO：The rhesus macaque hepatoma cell strain M12353 of C2015142
The drug that can be used for screening treatment liver cancer, also further demonstrating present invention acquisition rhesus macaque hepatoma cell strain M12353 is exactly
Hepatoma cell strain.
To sum up, the liver cancer animal model that prepared by the present invention models successfully, and can be used for rodent models can not complete
Biotechnology new drug evaluation and cellular replacement therapy technology evaluation.Meanwhile be also successfully established can be long-term by the present invention
The rhesus macaque hepatoma cell strain M12353 for stablizing passage, can pass on steadily in the long term in vitro, at present >80, continue
Shi Jian >16 months, immunodeficient animals (nude mouse) heterograft had Tumor formation, every inoculation 1-2 × 106A cell, tumor formation
Rate is 100%, and incubation period is 10-14 days, can transplant in tumor formation in rhesus monkeys, prepare rhesus macaque liver cancer model of the same race, also
It is real to can be used for prevention of hcc factor, anti-liver cancer and anti-new drug, Radiotherapy For Carcinoma of The Liver, Surgery For Hepatocellular Carcinoma operative treatment, imaging diagnosis etc.
Research is tested, application prospect is good.
Claims (6)
1. a kind of rhesus macaque hepatoma cell strain, it is characterised in that：The preserving number of its China typical culture collection center preservation：
CCTCC NO：The rhesus macaque hepatoma cell strain M12353 of C2015142.
2. cell strain according to claim 1, it is characterised in that：It is to isolate and purify preparation by liver cancer model；
Wherein, the preparation method of the liver cancer model is as follows：Diethylnitrosamine is applied to primate, diethyl nitrous
The administration dosage of amine is：40mg/Kg/ times to get；
Further, the primate is rhesus macaque；
The method of administration and dosage of the diethylnitrosamine be：Intraperitoneal injection, each 40mg/Kg are applied altogether once a week
With 20 times.
3. purposes of the rhesus macaque hepatoma cell strain as claimed in claim 1 or 2 in preparing liver cancer animal model.
4. purposes according to claim 3, it is characterised in that：The liver cancer animal model is rhesus macaque liver cancer model.
5. rhesus macaque hepatoma cell strain as claimed in claim 1 or 2 screening treatment liver cancer drug and/or prevention of hcc because
Element, anti-liver cancer and anti-new drug, Radiotherapy For Carcinoma of The Liver, Surgery For Hepatocellular Carcinoma operative treatment, imaging diagnosis research in purposes.
6. a kind of method of the drug of screening treatment liver cancer, includes the following steps：
A, candidate is applied to rhesus macaque hepatoma cell strain as claimed in claim 1 or 2；
B, the drug of potential treatment liver cancer is evaluated with rhesus macaque hepatoma cell strain as claimed in claim 1 or 2.
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