CN101810151A - Method and dedicated inducer for building nudemice malignant ascites model associated with human ovarian cancer - Google Patents
Method and dedicated inducer for building nudemice malignant ascites model associated with human ovarian cancer Download PDFInfo
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Abstract
The invention discloses a method and a dedicated inducer for building a nudemice malignant ascites model associated with human ovarian cancer. The inducer for building the malignant ascites model ssociated with mouse ovarian cancer consists of Ho8910 cells and a 1640 culture medium, wherein the volume of each inducer is 0.2ml and the number of the Ho8910 cells of each inducer is 2x106. One whole inducer is used for the nudemice aged from 3-5 years old at a time. Since the incidence of the mouse model is much like the middle or late period symptoms of human ovarian cancer, the model successfully simulates the development process of the middle or late period human ovarian cancer. The model building provides a powerful tool for systematic study on the biological behavior of ovarian cancer and research on cell treatment, novel drug screening and effect observation and the like.
Description
Technical field
The present invention relates to a kind of method and special-purpose inducing agent thereof of the HOC of structure nude mice ascites tumor model.
Background technology
Oophoroma is the female sex organ common malignancy, and the incidence of disease is only second to cervical carcinoma and carcinoma of uterine body, but its lethality accounts for the gynecological tumor first place.The trend that oophoroma incidence of disease appearance is in recent years increased gradually has a strong impact on women's health.Oophoroma course of disease concealment lacks effective method of early diagnosis, most of patients near late period during discovery.Though conventional operation, chemotherapy and radiation can relief of symptoms, are difficult to improve patient's survival rate.Molecular biology and tumor immunology research provide theoretical foundation and technical method for the cell biological treatment of oophoroma.But cell therapy is used for must carrying out safety and efficiency evaluation before clinical, and the oophoroma animal model is particularly important, particularly can reflect the people mouse follow board of evolution of oophoroma pathology and Clinical symptoms.Carried out since bare mouse different species is implanted into malignant tumour and succeeds from Rygaard in 1969 etc., nude mice has become the instrument that people study the malignant tumour biological property.In recent years, existing research reported nude mice oophoroma subcutaneous transplantation knurl model (Zhang Changying, written coloured silk. the foundation [J] of HOC nude mice subcutaneous transplantation knurl model. Wuhan University Of Technology's journal (natural science edition) .2001,24 (1): 99; Gao Guolan, Zou Chunfang, etc. the foundation of people's epithelial ovarian cancer nude mice subcutaneous transplantation knurl model and the evaluation of biological character. practical cancer magazine, 2004,19 (5): 454-457).But subcutaneous transplantation knurl model can not well react the pathology progress of oophoroma, has been subjected to very big restriction so further use.People such as Zhang Shu make suspension with high metastatic human ovarian cancer cell (Ho-8910PM), and to inject nude mice subcutaneous, obtain the subcutaneous transplantation knurl, get the tumor tissue piece then and carry out the ovary orthotopic transplantation, observe graft appearance transfer through 77 days and (open very Lin Qide, Di Wen, the foundation of oophoroma orthotopic transplantation-transfer animal model, China's journal of obstetrics and gynecology, 2004,39 (12): 835-837).But set up this model and have following problem: the nude mice ovary is less, no matter be inoculation also piece of tissue transplant all very difficult and the cycle is longer, and to plant in " original position " be in the ovarian epithelium, technical difficulty is bigger, has limited this application of model scope.Other has the research report, the ascites that extracts clinical ovarian cancer patients is directly injected nude mice abdominal cavity (Chen Aiping, the king and, Peng Zhilan etc., the foundation and the morphological observation of HOC's transplanted tumor in nude mice and ascites tumor model, tumour, 2001,21 (2): 82-84), belong to former generation transplanting, become knurl long latent period, graft obtains and has certain difficulty.In sum, structure had both met the oophoroma Clinical symptoms, and being convenient to test the HOC nude mice ascites tumor model of observing and analyzing has again become the problem of needing solution badly.
Summary of the invention
An object of the present invention is to provide a kind of inducing agent that is used to make up HOC nude mice ascites tumor model.
The inducing agent that is used to make up HOC nude mice ascites tumor model provided by the present invention is made up of Ho8910 cell and 1640 medium, and the volume of every inducing agent is 0.2ml, and the number of Ho8910 cell is 2 * 10 in every inducing agent
6Individual, the mouse in the corresponding 3-5 of every inducing agent age in week is once finished using.
In the above-mentioned inducing agent, per 1 liter of described 1640 medium are prepared as follows: with 10.4 gram 1640 dehydrated mediums and 2.0 gram NaHCO
3Soluble in water, regulating the pH value is 7.2, and water is settled to 1 liter, obtains 1 liter of 1640 medium.
In the above-mentioned inducing agent, described mouse is the BALB/C nude mice.
Another object of the present invention provides a kind of method of the HOC of structure nude mice ascites tumor model.
The method of structure HOC nude mice ascites tumor model provided by the present invention, comprise the steps: above-mentioned arbitrary described inducing agent lumbar injection in the 3-5 mouse in age in week, obtain inoculating the mouse of Ho8910 cell, the mouse of described inoculation Ho8910 cell was raised 25-45 days, mouse to described inoculation Ho8910 cell possesses following (1) and (2) proterties, obtains HOC nude mice ascites tumor model:
(1) [(the mouse abdominal circumference of inoculation Ho8910 cell-do not inoculate the mouse abdominal circumference of Ho8910 cell)/do not inoculate the mouse abdominal circumference of Ho8910 cell]＞10%; (2) the mouse belly of inoculation Ho-8910 cell touches the knurl body.
In the said method, the condition of described raising is: SPF level environment, 22 ℃ of temperature, humidity 55% and 12 hours daytime/circadian rhythms.
In the said method, described mouse is the BALB/C nude mice.
The present invention has made up model by the ovarian cancer cell of lumbar injection to the nude mice specific quantity.Experiment showed, 25-45 days 100% one-tenth knurls behind the nude inoculation; Belly is tangible to tumor nodule, and abdominal circumference increases, and ascites volume is 2-6ml, average out to 3.5ml, the visible tumour cell of smear; Inoculation back The average survival time live time is 60.4 days.Dissect into the knurl mouse, the visible solid tumor tissue in abdominal cavity, and have tumor tissues to be attached to organs such as intestines, liver, spleen; Pathologic finding sees that there is tumor cell invasion in internal organs such as liver, spleen, the low differentiation slurry of tumor tissues behaviour ovary sexual gland cancer.The constructed HOC nude mice ascites tumor model of the present invention has tumor formation rate height, one-tenth knurl weak point in latent period, the model proterties is remarkable and stable, longer feature of mean survival time.And the incidence utmost point of mouse model of the present invention is similar to human oophoroma middle and advanced stage symptom, illustrate model success of the present invention simulation in the middle and advanced stage evolution of human ovarian carcinoma.Being established as systematic Study oophoroma biological behaviour, carrying out cell therapy and screening researchs such as new drug and observation of curative effect of this model provides strong tool.
Description of drawings
Fig. 1. nude mice picture after the one-tenth knurl.
Fig. 2. pathology testing results (200 *) such as ascites, one-tenth knurl mouse intraperitoneal tumor tissues, liver, spleen.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Proliferation of Human Ovarian Cell (Ho8910 cell) is available from Chinese Academy of Sciences's cell bank, and catalog number is TCHU24; 1640 dehydrated mediums are available from GIBCO company; Hyclone is available from Shanghai Excell Biology Product Co., Ltd.; Trypsase dry powder was available from Amresco company in 1: 250; Micropipettor is available from French Gilson company, and low speed centrifuge is available from Sigma company, and the 50ml centrifuge tube is available from Coring company, and 78-1 type magnetic force heating stirrer is pacified general electronic engineering Co., Ltd, CO available from Jiangsu
2Incubator is available from ThermoForma company.Nude mice (BALB/C background, age in 3-5 week, female) is available from zootype research institute of Nanjing University (credit number: SCXK (Soviet Union) 2005-0002).
The composition of embodiment 1, inducing agent and preparation
One, forms
Every inducing agent is made up of Ho8910 cell and 1640 medium; The volume of every inducing agent is 0.2ml, and the number of Ho8910 cell is 2 * 10 in every inducing agent
6Individual; The corresponding 3-5 of every inducing agent age in week tried mouse, a shot finishes.
Two, preparation
(1) deactivation of hyclone: the hyclone that will not have bacterium, mycoplasma and viral pollution places 56 ℃ of water bath with thermostatic control 30min, and the deactivation complement is put in 4 ℃ of refrigerators afterwards and preserves, and is standby.
The preparation of (2) 1640 perfect mediums: get 1 bag of 1640 dehydrated medium (10.4 gram), join in the beaker that the 950ml tri-distilled water is housed, magnetic stirrer is dissolved it fully, adds 2.0 gram NaHCO
3, continuing to be stirred to dissolving, the HCl adjusting pH value 7.2 with 1M uses tri-distilled water to be settled to 1000mL, the aseptic membrane filtration degerming of 0.22um, 4 ℃ of preservations.Add the hyclone of 10% volume before using, be 1640 perfect mediums.
That is: each rises 1640 medium and prepares as follows: with 10.4 gram 1640 dehydrated mediums and 2.0 gram NaHCO
3Soluble in water, regulating the pH value is 7.2, and water is settled to 1 liter, obtains 1 liter of 1640 medium;
Add hyclone in 1640 medium, the volume ratio that makes 1640 medium and hyclone is 9: 1, then obtains 1640 perfect mediums.
The preparation of (3) 0.25% pancreatin: the trypsase dry powder of getting 2.5 grams is dissolved in 1 * PBS solution of 1000ml, stirs it is dissolved fully, and the aseptic membrane filtration degerming of 0.22um, 4 ℃ of preservations, standby.
(4) Ho8910 cell culture and preparation: with the Ho8910 cell inoculation in 1640 perfect mediums, 37 ℃, 5%CO
2Cultivate, when treating that cell is in exponential phase, use 0.25% trypsinization, collecting cell, use 1640 medium washed cells twice (800rpm, 10min, 25 ℃), the trypan blue decoration method is calculated cell motility rate, cell motility rate 〉=90% as a result;
(5) behind the viable count, use 1640 medium re-suspended cells, make that cell concentration is 1.0 * 10 in the cell suspension
7Cells/ml, every inducing agent is this cell suspension of 0.2ml, cell concentration is 1.0 * 10 in every inducing agent
7Cells/ml, number of cells is 2 * 10 in every inducing agent
6Individual, standby.
The foundation and the evaluation of embodiment 2, mouse oophoroma ascites tumor model
Used mouse is nude mice (a BALB/C background, female age in 3-5 week, SPF level) in the present embodiment, and the body weight of every mouse is 14-18g.
All experiments all meet " China Science ﹠ Technology University's management of laboratory animal regulations ".
One, construction method
(1) inoculation: get 20 of nude mices (BALB/C background), female age in 3-5 week, SPF level, 22 ℃ of raising temperatures, 55%, 12 hour daytime/circadian rhythm of humidity.Be divided into 2 groups at random by body weight, 10 every group.The once above-mentioned inducing agent of experimental group lumbar injection, injected dose are 1 inducing agent/mouse (i.e. 200 μ l cell suspension/mouse); 1640 medium (being 200ul 1640 medium/mouse) of a same dose of every mouse peritoneal injection of control group.
(2) feed the inoculation back: experimental group and control group inoculation back nude mice all give normal diet, continue to cultivate under the following conditions: 22 ℃ of SPF level environment, raising temperatures, 55%, 12 hour daytime/circadian rhythm of humidity.Observe the mouse state of mind, active situation and survival condition, measure mouse body weight and abdominal circumference and change, whether touch in the mouse peritoneal tumor nodule, judge whether mouse becomes knurl and one-tenth knurl time.
(3) judge whether inoculation back mouse becomes knurl: increase obviously (greater than 10%) and belly and touch the knurl body and promptly be judged as into knurl when abdominal circumference appears in inoculation back nude mice, become the knurl mouse also to have movable minimizing, the symptom of becoming thin.
Abdominal circumference increases tangible authentication method: mouse abdominal circumference＞10% before (mouse abdominal circumference before inoculation back mouse abdominal circumference-inoculation)/inoculation, think that promptly inoculation back mouse abdominal circumference increases obviously;
(4) pathology of mouse detect after the one-tenth knurl
1, the detection method of ascites: get into the knurl mouse, pluck the eyeball bloodletting, disconnected afterwards marrow is put to death, use eye scissors to open mouse peritoneal, use asepsis injector to draw intraperitoneal liquid, liquid is transferred in the aseptic centrifuge tube, accurately measure its volume, be ascites volume, with ascites 3000rpm, 10min is centrifugal, supernatant discarded, carry out HE dyeing behind the precipitation smear, detect whether there is tumour cell.
2, the detection method of abdominopelvic cavity internal organs (liver): after the ascites of above-mentioned mouse sopped up, separate liver, perusal, the visible tumor nodule of liver surface, use ophthalmology clip part hepatic tissue to be put in 10% formalin solution, carry out pathology and detect the tumor cell invasion situation.
3, the detection method of abdominopelvic cavity internal organs (spleen): after the ascites of above-mentioned mouse sopped up, separating spleen, perusal, the visible tumor nodule in spleen surface, use ophthalmology clip part spleen tissue to be put in 10% formalin solution, carry out pathology and detect the tumor cell invasion situation.
4, the detection method of abdominopelvic cavity tumor nodule: after the mouse after the above-mentioned dissection separates liver and spleen, the perusal abdominal cavity, as seen a plurality of tumor planting kitchen ranges, be tumor nodule (cancerous tissue), whether get tumor nodule and be put in 10% formalin solution, carrying out the pathology detection is that tumour cell constitutes.
Two, result
1. become the knurl time: experimental group, inoculation back 25-45 days, 100% nude mice becomes knurl.Control group does not all become knurl.As shown in Figure 1, left side figure is a control group mice abdominal cavity situation, and right figure is an experimental mice abdominal cavity situation.
2. life span: nude mice becomes 100% death in 25-40 days after the knurl, and the inoculation back mean survival time is 60.4 days.Control group mice does not all occur dead.
3. life quality:
Experimental group:
The 0-14 days mouse Non Apparent Abnormalities in inoculation back, whole mouse become knurl in 25-45 days, show as abdominal distention, and belly touches the knurl body.
Mouse becomes after the knurl 7 days, dissects mouse, and there is ascites in visible intraperitoneal, and ascites volume is 2-6ml, average out to 3.5ml, visible tumour cell (Fig. 2 ascites) in the ascites; Detect abdominopelvic cavity internal organs (liver), the visible tumor nodule in surface, there is tumor cell invasion in pathology detection result shown in Fig. 2 liver; Detect abdominopelvic cavity internal organs (spleen), the visible tumor nodule in surface, there is tumor cell invasion in pathology detection result in the spleen shown in Fig. 2 spleen; The metastases kitchen range that has dispersivity in the abdominal cavity, pathological section show that it is inner for the tumour gathering, shown in Fig. 2 cancerous tissue.
Control group: the 0-14 days mouse in inoculation back are all normal, and are also all normal in 25-45 days, all normal in the follow-up time, do not have into knurl person.Select to dissect the period identical and the pathology experiment with experimental group.The result does not all have ascites to produce, and does not all have tumour to occur in liver, the spleen.
Above-mentioned sample is through HE dyeing (h and E dyeing), and the result shows the low differentiation of the tumour cell behaviour serosity adenocarcinoma ovaries cell of infiltration, and is consistent with the Ho8910 cell pathology somatotype in being injected into the mouse body.
Above-mentioned pathology detection result shows that the incidence utmost point of mouse model of the present invention is similar to human oophoroma middle and advanced stage symptom, illustrate model success of the present invention simulation in the middle and advanced stage evolution of human ovarian carcinoma, and proterties is stablized.
Claims (6)
1. an inducing agent that is used to make up HOC nude mice ascites tumor model is made up of Ho8910 cell and 1640 medium, and the volume of every inducing agent is 0.2ml, and the number of Ho8910 cell is 2 * 10 in every inducing agent
6Individual, the mouse in the corresponding 3-5 of every inducing agent age in week is once finished using.
2. inducing agent according to claim 1 is characterized in that: per 1 liter of described 1640 medium are prepared as follows: with 10.4 gram 1640 dehydrated mediums and 2.0 gram NaHCO
3Soluble in water, regulating the pH value is 7.2, and water is settled to 1 liter, obtains 1 liter of 1640 medium.
3. inducing agent according to claim 1 and 2 is characterized in that: described mouse is the BALB/C nude mice.
4. method that makes up HOC nude mice ascites tumor model, comprise the steps: claim 1 or 2 described inducing agent lumbar injections in the 3-5 mouse in age in week, obtain inoculating the mouse of Ho8910 cell, the mouse of described inoculation Ho8910 cell was raised 25-45 days, mouse to described inoculation Ho8910 cell possesses following (1) and (2) proterties, obtains HOC nude mice ascites tumor model:
(1) [(the mouse abdominal circumference of inoculation Ho8910 cell-do not inoculate the mouse abdominal circumference of Ho8910 cell)/do not inoculate the mouse abdominal circumference of Ho8910 cell]＞10%; (2) the mouse belly of inoculation Ho-8910 cell touches the knurl body.
5. method according to claim 4 is characterized in that: the condition of described raising is: SPF level environment, 22 ℃ of temperature, humidity 55% and 12 hours daytime/circadian rhythms.
6. according to claim 4 or 5 described methods, it is characterized in that: described mouse is the BALB/C nude mice.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013923A (en) * | 2012-12-29 | 2013-04-03 | 四川大学华西第二医院 | Method for creating animal model of human ovarian cancer |
| CN106754719A (en) * | 2016-11-17 | 2017-05-31 | 上海市胸科医院 | A kind of method of inoculation liquid composition and xenograft tumor animal model of being originated using its structure malignant pleural effusion |
| CN110663637A (en) * | 2019-09-16 | 2020-01-10 | 承德医学院 | Construction method of human choriocarcinoma nude mouse orthotopic transplantation tumor model |
-
2010
- 2010-03-26 CN CN 201010134976 patent/CN101810151A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 《山东医药》 20100212 马娟等 "裸鼠荷人卵巢癌腹水瘤模型的构建" 第27页 1-6 第50卷, 第6期 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013923A (en) * | 2012-12-29 | 2013-04-03 | 四川大学华西第二医院 | Method for creating animal model of human ovarian cancer |
| CN106754719A (en) * | 2016-11-17 | 2017-05-31 | 上海市胸科医院 | A kind of method of inoculation liquid composition and xenograft tumor animal model of being originated using its structure malignant pleural effusion |
| CN106754719B (en) * | 2016-11-17 | 2020-04-07 | 上海市胸科医院 | Inoculation liquid composition and method for constructing malignant pleural effusion source xenograft tumor animal model by using same |
| CN110663637A (en) * | 2019-09-16 | 2020-01-10 | 承德医学院 | Construction method of human choriocarcinoma nude mouse orthotopic transplantation tumor model |
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Application publication date: 20100825 |