CN102524180A - Method for establishing microcystin MC-LR promoted diethyl nitrosamine DEN induced rat liver cancer model - Google Patents
Method for establishing microcystin MC-LR promoted diethyl nitrosamine DEN induced rat liver cancer model Download PDFInfo
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Abstract
The invention discloses a method for inducing a rat liver cancer model by injecting diethyl nitrosamine DEN in coordination with microcystin MC-LR into a rat body through intraperitoneal injection. Indexes such as liver weight to body weight ratio, liver appearance, liver histopathological slicing and GST-Pi (Glutathione S-transferase) protein expression level clearly show liver cell injury phase, liver cell hyperplasia-hepatocirrhosis phase and liver cell carcinomatous change process in a rat liver cancer occurring process, and find that the liver cancer model induced by the DEN in coordination with the microcystin MC-LR has a more obvious effect at the same time than that induced by DEN independently; the tumor formation time is shorter; and the model is an ideal model for researching occurrence of the liver cancer of a human body. The model has the characteristic of clearly showing each stage for occurrence of the liver cancer and contributing to research of a liver cancer occurrence process mechanism, detection of precancerous lesion and development of treatment medicaments.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to the short diethylnitrosamine DEN of a kind of microcapsule phycotoxin MC-LR and bring out the method that the rat liver cancer model is set up.
Background technology
Liver cancer is one of China's common malignancy, and the title of " cancer king " is arranged.Therefore need to select a kind of suitable liver cancer animal model to simulate the research of the pathological process help liver cancer of human hepatocellular.Bringing out property liver cancer model is meant the liver cancer model of using chemistry, physics or biological carcinogenic factor to act on animal and forming.Wherein, diethylnitrosamine (DEN) is that animal liver cancer is a kind of inducing agent of extensive use, and liver cancer process and human body cancer evolution process that it brings out are closely similar.DEN is by the definite carcinogen of international tumor research central authority at present.Microcystin MC is a kind of hepatotoxin, and the target organ of bio-toxicity effect is a liver.Can from blood, transfer to liver, mainly show as and make the congested enlargement of liver, can cause hepatorrhagia and necrosis when serious.
The method for building up of rat liver cancer model is divided into two kinds of transplantation type and primary; The transplantation type liver cancer model normally is expelled to mouse peritoneal with HCC; Separate into cell suspension after extracting ascites, open mouse peritoneal again and cell suspension is expelled to mouse liver and set up model.This model becomes the knurl time shorter, but can't observe the process that liver cancer takes place.Primary liver cancer model is to utilize chemical carcinogen to bring out rat liver cancer slowly to take place, though this model time is longer, can observe the Main Stage of liver cancer genesis and development, helps the detection of the early stage preneoplastic lesion of liver cancer.The model that much brings out the primary rat liver cancer with DEN is arranged at present both at home and abroad, generally adopt the 0.25%DEN aqueous solution to irritate stomach or the method for freely drinking water is set up the rat liver cancer model.The problem that this model mainly exists is that poisoning dosage is inaccurate, and the time is longer, contains diethylnitrosamine (DEN, 80 * 10 like humans such as Li Xiaoyan
-6) drink water and set up the rat liver cancer model, its model 1-8 week is the hepatocellular injury phase, and 9-16 week is hyperplasia-hardening period, and 17-25 week is the canceration phase.And Zhao Jinming etc. start agent; The algae toxin is a promoter; Make up rat tumor and promote model, observe the hepatic pathology form and change, use SABC method, in situ hybridization method to detect GST-Pi albumen and GST-Pi mRNA expression in the liver cell respectively.The result shows that the experimental group animal's liver produces the nodular hyperplasia kitchen range, and the independent effect of algae toxin can not induce GST-Pi albumen and GST-Pi mRNA to express, but can promote DEN to induce the GST-Pi mRNA and the protein expression of generation.It is active that prompting algae toxin has short cancer; But used algae toxin is the primary extract from bloom blue algae; Do not have the data that algae toxin purity and classification are identified; And the algae toxin has kind more than 60, the water body algae appearance that different time, place are collected in the content and the classification of algae toxin be discrepant, these have increased the uncertainty of experimental result.In addition, used algae toxin dose is 20mg/kg.bw (high dose), 5mg/kg.bw (low dosage) (being equivalent to dried frustule quality), does not carry out the series concentration experiment.
Act on to algae toxin selectivity liver, the activity of main CKIs phosphatase PP1, PP2, have tangible dose-effect relationship: the algae toxin of low dosage has the promoting growth of cell effect, and high dose can cell death inducing, cell growth inhibiting.Therefore, confirm that algae toxin type and activity scope seem particularly important for the foundation of short cancer model.
Summary of the invention
Technical problem to be solved by this invention is through chronic toxicological experiment; Under low concentration, set up the short diethylnitrosamine (DEN) of algae phycotoxin MC-LR and bring out the rat liver cancer model; In this process, can observe the Main Stage that liver cancer forms through morphology, histopathology and protein expression level; Comprise the hepatocellular injury phase, liver cell proliferation-cirrhosis phase and canceration of hepatic cell phase.
Technical scheme of the present invention is following:
A kind of microcapsule phycotoxin MC-LR is urged the method for building up that diethylnitrosamine (DEN) brings out the rat liver cancer model, and step is:
1. the foundation of rat liver cancer model
A, choose 15 of the SD male rats that body weight is 150 ± 5g, give the adaptation time in a week, give sufficient food between feeding period, and carry out each item disinfectant measure;
B, to use pure water that DEN is made into concentration be 0.25% solution;
C, with absolute methanol with the pure article powder dissolution of algae toxin, be mixed with the algae toxin soiutions of 1mg/ml;
The algae toxin soiutions of D, the DEN solution with 0.25% and 1mg/ml is expelled in the rat abdominal cavity, and making the DEN final concentration is 10mg/kg, and algae toxin final concentration is 10 μ g/kg;
E, said process carried out once coprocessing 4 months in per five days;
F, model are set up the appetite of observing rat in the process all the time, drinking-water, fur, situation such as spirit;
2. morphologic detection
Rat is put to death with the cervical vertebra dislocation method in the end in every month, opens the abdominal cavity, use the PBS buffer solution for cleaning after intactly taking out liver, and the record body weight, liver is heavy and calculate liver weighs body weight and when observe the color and the quality of liver, and takes pictures;
3. histopathology
Get fixedly 24h of part lobe of the liver warp 10% neutral formalin solution, FFPE, section is after HE dyes the variation of microscopically observation of cell;
4.GST-Pi protein expression content detection
Frozen hepatic tissue in-80 ℃ of refrigerators is got 0.5g in 5ml PBS buffer solution and after adding the enzyme inhibitor PMSF of 100mM, carry out ultrasonic degradation, the ultrasonic time is 2S, stops 6S, altogether 14min.12000rpm/min is centrifugal 10 minutes under the back 4 ℃ of conditions of ultrasonic end, gets supernatant, detects the expression contents of GST-Pi with Western Blot method.
Be with the prolongation in algae phycotoxin MC-LR processing time by the heavy weight ratio of the rats'liver of the visible model group of accompanying drawing 1 and obviously increase progressively trend; Wherein, (DEN+MC-LR) processed group increases comparatively fast than the heavy weight ratio of DEN processed group liver, explains that algae toxin 10 μ g/kg MC-LR have the effect of tangible promotion liver cell proliferation.
When luring cancer to 1 month, liver is not seen obviously unusual, but does not have color by the visible model group of Fig. 2; When luring cancer to 2 month, the liver quality changes software strategy into software-hardware strategy, and the surface is coarse gradually; During to 3 months, the liver quality is harder, the cirrhosis phenomenon occurs, and demonstrates that quantity does not wait, the circular focus of canescence not of uniform size; During to 4 months, liver surface is coarse, a plurality of canescence cancerous nodes not of uniform size, and visible hemorrhage and downright bad, when dissected ascites phenomenon is serious.
HE PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM result:
Roughly be divided into for three phases by the visible rat liver pathological change of Fig. 3: (1) is the hepatocellular injury phase when luring cancer to 1 month, and cell is arranged relatively more disorderly, and endochylema is loose, and being dispersed in property of inflammatory cell is distributed in the leaflet.(2) luring cancer is liver cell proliferation-hardening period in the time of 2 months to 3 months, wherein 2 the end of month liver cell begin the sex change oedema, it is big that volume obviously becomes, to invade profit serious for visible inflammatory cell in the leaflet; The little bile duct proliferation phenomenon of hepatic tissue cirrhosis is serious during 3 the end of month, is held by fibr tissue to form the pseudolobuli structure and the cancer cell hyperplasia phenomenon occurs.(3) be the canceration of hepatic cell phase to 4 months, cell nucleus obviously increases, and the karyon ratio increases, and nuclear division increases mutually, still visible multinuclear and megacaryocyte.
By Fig. 4 different times GST-Pi protein expression content detection as a result figure can see: normal structure is expressed GST-Pi hardly; DEN associating algae phycotoxin MC-LR (10 μ g/kg) processed group GST-Pi in the time of 1 month begins to express; But expression seldom, is about 1.5 times of normal group; This with morphology and pathology on detect for hepatocellular injury phase result consistent.In the time of 2 months, GST-Pi expresses enhancing, and expression is about 3 times of normal group; This is consistent with the result that morphology and pathology detected to the liver cell proliferation phase.GST-Pi expresses obviously and strengthens in the time of 3 months, and expression is about 4 times of normal group; This detects with morphology and pathology is that cirrhosis phase result is consistent.GST-Pi expression and compared with normal are the highest in the time of 4 months, and this detects with morphology and pathology is that canceration of hepatic cell phase result is consistent.
Beneficial effect of the present invention
1. successfully set up the rat liver cancer model that the short diethylnitrosamine (DEN) of algae phycotoxin MC-LR brings out;
2. a series of processes of rat liver canceration are clearly observed in the section of rat liver outward appearance and pathology; Be hepatocellular injury phase (1 month), liver cell proliferation-hardening period (2-3 month) and the tissue level of canceration of hepatic cell phase (4 months) and the change procedure of cellular level.
3. GST-Pi Protein Detection result shows, in each period that the liver cancer in the model group takes place, the expression of GST-Pi prolongs in time to be and obviously increases progressively trend.
This achievement is to utilize microcapsule phycotoxin MC-LR as a kind of carcinogenic promoting agent, truly brings out primary liver cancer model fast.This technology not only can be used for studying the mechanism of primary liver cancer genesis and development, also is applicable to the exploitation of various index detections, diagnosing cancer of liver, treatment and medicine in the neoplastic process simultaneously.Therefore, no matter be for human health, still aspect economical, societal benefits, all have good popularizing application prospect.
Description of drawings
Fig. 1 is that the model group rat is at the heavy body weight ratio of different time sections liver variation diagram;
Fig. 2 is the apparent figure of model group different time sections rat liver;
The histologic section figure of Fig. 3 model group liver cell different times;
Fig. 4 model group liver organization different times GST-Pi protein expression content figure.
Embodiment
1. selecting male body weight for use is 15 of SD rats about 150 ± 5g, and gives the adaptation time in 1 week, during rat feeding, regularly with 0.01% stupid bundle bromine ammonium salt solution wiping soaking disinfection mouse cage, and in time changes bedding and padding.Raising temperature is controlled at about 25 ℃, and gives sufficient food.
2. the DEN solution dilution being become concentration is 0.25% DEN solution.
3. with the pure article powder of dissolve with methanol algae toxin, shake 5min under the lucifuge condition, be mixed with the algae toxin mother liquor of 1mg/ml.
4. 0.25% DEN solution and algae toxin soiutions are expelled in the rat abdominal cavity, making the DEN final concentration is 10mg/kg, and algae toxin final concentration is 10 μ g/kg.
5. said process carried out once coprocessing 4 months in per 5 days.
6. model is set up the appetite of observing rat in the process all the time, drinking-water, fur, situation such as spirit.
7. dissect rat, get liver, a series of morphologic observations such as weigh in, liver is heavy and take pictures.
8. get fixedly 24h of part lobe of the liver warp 10% neutral formalin, FFPE, section is after HE dyes the variation of microscopically observation of cell.
9. frozen hepatic tissue in-80 ℃ of refrigerators is got 0.5g and in 5ml PBS buffer solution and after adding the enzyme inhibitor PMSF of 100mM, carry out ultrasonic degradation, the ultrasonic time is 2S, stops 6S, altogether 14min.12000rpm/min is centrifugal 10 minutes under the back 4 ℃ of conditions of ultrasonic end, gets supernatant and carries out electrophoresis, utilizes the expression contents of the antibody test GST-Pi of GST-Pi.
Claims (5)
1. the short diethylnitrosamine DEN of microcapsule phycotoxin MC-LR brings out the method that the rat liver cancer model is set up, and it is characterized in that, may further comprise the steps:
(1) DEN associating microcapsule phycotoxin MC-LR is set up the rat liver cancer model;
(2) morphologic detection;
(3) pathology detect;
(4) GST-Pi protein expression content detection.
2. the short diethylnitrosamine (DEN) of a kind of microcapsule phycotoxin MC-LR according to claim 1 brings out the method for building up of rat liver cancer model, and it is characterized in that: the step that said step (1) is more concrete is:
A, to choose body weight be 15 of 150 ± 5g SD male rats; Give the adaptation time in a week, during rat feeding,, in time change bedding and padding regularly with 0.01% stupid bundle bromine ammonium salt solution wiping soaking disinfection mouse cage; Raising temperature is controlled at about 25 ℃, and gives sufficient food and water source;
The DEN solution of B, preparation 0.25% and the algae phycotoxin MC-LR methanol solution of 1mg/ml;
C, model group are the algae phycotoxin MC-LR methanol solution of lumbar injection 0.25%DEN solution and 1mg/ml, and making the DEN final concentration is 10mg/kg, and algae toxin final concentration is 10 μ g/kg;
D, above experiment were administered once coprocessing 4 months in per five days;
E, at different times, note to observe rat appetite, drinking-water, fur, situation such as spirit.
3. the short diethylnitrosamine (DEN) of a kind of microcapsule phycotoxin MC-LR according to claim 1 brings out the method for building up of rat liver cancer model; It is characterized in that: the step that said step (2) is more concrete is: rat is put to death with the cervical vertebra dislocation method in the end in every month, opens the abdominal cavity, uses the PBS buffer solution for cleaning after intactly taking out liver; The record body weight; Liver weight and calculating liver weigh the ratio with body weight, observe the color and the quality of liver simultaneously, and take pictures.
4. the short diethylnitrosamine (DEN) of a kind of microcapsule phycotoxin MC-LR according to claim 1 brings out the method for building up of rat liver cancer model; It is characterized in that: the concrete operation method that the pathology of said step (3) detect is: get the part lobe of the liver through with the fixing 24h of 10% neutral formalin solution; FFPE; Cut into slices after HE dyes, with the variation of om observation cell.
5. the short diethylnitrosamine (DEN) of a kind of microcapsule phycotoxin MC-LR according to claim 1 brings out the method for building up of rat liver cancer model; It is characterized in that: the concrete operations of the described protein content determination of step (4) are: after-80 ℃ of refrigerator hepatic tissues take out, carry out ultrasonic degradation with frozen; 12000rpm/min is after centrifugal 10 minutes under 4 ℃ of conditions; Get supernatant and carry out protein immunoblotting, detect the expression contents of GST-Pi.
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Cited By (6)
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| CN104515758A (en) * | 2014-12-15 | 2015-04-15 | 北京大学 | Cancer promotion activity quantitative determination method and screening method of cancer promoter |
| CN104904660A (en) * | 2015-05-08 | 2015-09-16 | 贵阳中医学院 | Method for establishing mouse model with hepatic preneoplastic lesion caused by double factors of alcohol and HBV |
| CN107372302A (en) * | 2017-07-07 | 2017-11-24 | 遂宁市中心医院 | Screening has an impact the animal model of large biological molecule to cellular biology of tumor in vivo |
| WO2018099148A1 (en) * | 2016-11-30 | 2018-06-07 | 南京大学 | Use of microcystins in preparation of drugs for preventing or treating organ and tissue fibrosis diseases |
| CN111248149A (en) * | 2018-12-03 | 2020-06-09 | 凯斯艾生物科技（苏州）有限公司 | Method for constructing mouse model of diabetes mellitus accompanied with severe non-alcoholic steatohepatitis |
| CN112674028A (en) * | 2020-12-30 | 2021-04-20 | 汉姆德(宁波)智能医疗科技有限公司 | Method for establishing cancer model of animal induced by cancer inducer |
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Cited By (9)
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| CN104515758A (en) * | 2014-12-15 | 2015-04-15 | 北京大学 | Cancer promotion activity quantitative determination method and screening method of cancer promoter |
| CN104515758B (en) * | 2014-12-15 | 2017-12-15 | 北京大学 | Promote the quantitative detecting method of cancer activity and the screening technique of carcinogenic promoting agent |
| CN104904660A (en) * | 2015-05-08 | 2015-09-16 | 贵阳中医学院 | Method for establishing mouse model with hepatic preneoplastic lesion caused by double factors of alcohol and HBV |
| WO2018099148A1 (en) * | 2016-11-30 | 2018-06-07 | 南京大学 | Use of microcystins in preparation of drugs for preventing or treating organ and tissue fibrosis diseases |
| CN107372302A (en) * | 2017-07-07 | 2017-11-24 | 遂宁市中心医院 | Screening has an impact the animal model of large biological molecule to cellular biology of tumor in vivo |
| CN111248149A (en) * | 2018-12-03 | 2020-06-09 | 凯斯艾生物科技（苏州）有限公司 | Method for constructing mouse model of diabetes mellitus accompanied with severe non-alcoholic steatohepatitis |
| CN111248149B (en) * | 2018-12-03 | 2022-01-28 | 凯斯艾生物科技（苏州）有限公司 | Method for constructing mouse model of diabetes mellitus accompanied with severe non-alcoholic steatohepatitis |
| CN112674028A (en) * | 2020-12-30 | 2021-04-20 | 汉姆德(宁波)智能医疗科技有限公司 | Method for establishing cancer model of animal induced by cancer inducer |
| CN112674028B (en) * | 2020-12-30 | 2022-06-28 | 汉姆德(宁波)智能医疗科技有限公司 | Method for establishing cancer model of animal induced by cancer inducer |
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Application publication date: 20120704 |