CN104783921A - Building method for nude mouse skin winkle model for observing tumor vessel microcirculation - Google Patents
Building method for nude mouse skin winkle model for observing tumor vessel microcirculation Download PDFInfo
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- CN104783921A CN104783921A CN201510065553.XA CN201510065553A CN104783921A CN 104783921 A CN104783921 A CN 104783921A CN 201510065553 A CN201510065553 A CN 201510065553A CN 104783921 A CN104783921 A CN 104783921A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
Abstract
The invention discloses a building method for a nude mouse skin winkle model for observing tumor vessel microcirculation. The building method is characterized in that the method is carried out according to the following steps, CBRH-7919 hepatoma cells of a log phase are taken, digested through 0.25% of trypsin, and concentrated in a centrifugal mode to obtain CBRH-7919 hepatoma cell suspension, the certain amount of the CBRH-7919 hepatoma cell suspension is injected into the epidermis of a nude mouse, and it is ensured that the number of the CBRH-7919 hepatoma cells entering the epidermis of the nude mouse ranges from 5*105 per ml to 4*106 per ml; 2-3 days after the injection is finished, the nude mouse with the tumor body formed is taken, and the skin winkle model is built on the face with the tumor body growing.
Description
Technical field
The present invention relates to a kind of method for building up of nude mice skin fold model, particularly a kind of method for building up observing tumor vessel microcirculatory nude mice skin fold model.
Background technology
Malignant tumor is one of principal disease of harm humans health, and within 2008, number of the infected 1,279 ten thousand is estimated in the whole world.Wherein, in developed country, malignant tumor causes dead first cause, and in developing country, be also in second.The socio-economic development of cancer to various countries causes huge disease and financial burden.Therefore, if capture cancer, Therapeutic cancer, becomes the focus of current tumor research.
Angiogenesis around tumor has played important function in the generation, transfer of tumor.Tumor growth has clear and definite blood vessel dependency, and tumor obtains nutritional labeling by new vessels from host, carries tumor cell again through blood vessel to host, strengthens the metastasis ability of tumor stove.The growth of tumor can be divided into without blood vessel phase and blood vessel phase.Volume is more than the tumor of 2mm3 and intravasation phase, and need new vessels to maintain nutrition supply and excretion metabolism product does not have new vessels to grow into if it continues generation, maintenance resting state is just even degenerated by tumor.Therefore, enough vascularization are to provide sufficient nutrition and oxygen for the essential condition being tumor growth.
At present, animal model is the important research instrument of research tumor-blood-vessel growth.But, build situ tumor model research angiogenesis and there is certain limitation.First, part deep tumor, as cancer of pancreas, the tumor models such as hepatocarcinoma, structure difficulty is high, specimen sampling is difficult, and not easily observes; Secondly, cannot carry out Study on real-time performance to tumor, thus cause tumor vessel mechanism, generation, evolution can not be carried out inquiring in detail, fully.
In nineteen twenty-four, Sandison mentions applying animal observation window model to study biopsy cell first time under live body micro-observation technology, after this, be variously intended to investigate the method that animal (mice, hamster, rat, rabbit etc.) microcirculatory research starts to introduce form observation.Endrich in 1980 etc. propose Mus skin of back gauffer model first, and this discovery is widely used in microcirculatory research field in the past in 30 years, and is used to do a large amount of experimentatioies.This model has outstanding advantage in following research field: 1) microcirculation physiology; 2) inflammation is with downright bad; 3) ischemia-reperfusion; 4) angiogenesis; 5) transplant.Therefore, a crease in the skin model has advantageous advantage to research tumor microcirculation.
Previously use a crease in the skin scale-model investigation tumor microcirculation, mainly with tumor cell inoculation within form, and then simulation tumor microenvironment.But this can not throw a flood of light on microcirculatory formation around tumor tumor body, progression.Therefore, we study invention a kind of observation tumor vessel microcirculatory nude mice skin fold model.
Summary of the invention
The present invention is the above-mentioned deficiency in order to solve existing for prior art, proposes a kind of observation tumor vessel microcirculatory nude mice skin fold model, and this model can more press close to simulate microcirculation generation, the evolution of tumor growth, is convenient to immediate observation simultaneously.
Technical solution of the present invention is: a kind of method for building up observing tumor vessel microcirculatory nude mice skin fold model, is characterized in that: described method is carried out according to following steps:
To take the logarithm the CBRH-7919 hepatoma carcinoma cell of phase, after being used the trypsinization of 0.25%, centrifugal concentrating obtains CBRH-7919 hepatoma carcinoma cell suspension, then, under a certain amount of CBRH-7919 hepatoma carcinoma cell suspension being expelled to nude mice epidermis, ensure that the quantitative range entering the subepidermal CBRH-7919 hepatoma carcinoma cell of nude mice is 5 × 10
5to 4 × 10
6individual/ml.
Injected rear 2-3 days and got the nude mice forming tumor tumor body, set up a crease in the skin model in the one side of tumor tumor bulk-growth, concrete grammar is as follows:
Anesthesia: pentobarbital sodium physiological saline solution is mixed with the sterile solution of 1%, press the capable intraperitoneal injection of 45mg/kg according to body weight and anaesthetizes, if anaesthetized the shallow sterile solution adding 0.1ml in art in abdominal cavity,
Preserved skin: the microvillus at nude mice back is thoroughly removed totally by application depilatory,
Preoperative Method: nude mice is positioned on operating board, nude mice back wrinkled skin has been pinched along its spinal column, get the median line position that its head rhicnosis portion of central authorities and afterbody hit exactly between extended line and do a labelling, 2 points are got respectively in median line both sides, distance between this 2 and median line mid point is 2cm, skinfold is passed from these 2 respectively with 2 chorda serica chirurgicalis sterilises, and utilize this 2 chorda serica chirurgicalis sterilis pull-up experimental mouse skins, patient's sight line is perpendicular to experimental mouse median line, under the irradiation of offside transmitted light source, see Major Vessels trend and position making marks clearly
Cell external member is installed: first cell frame is put in the skin upper limb place, center, back that nude mice leans against nearly patient side, tied fixing with chorda serica chirurgicalis sterilis, after under the irradiation of transmittance light source, with marking pen according to the scope of circular viewfinder area of side of installing cell frame, mark the viewfinder area scope that offside is corresponding
For hanging the silk thread of nude mice skin during dismounting Preoperative Method, experiment nude mice is then allowed to lie on one's side towards deviating from patient side, under experiment nude mice is positioned over gimbals microscope, under being padded on wrinkled skin with sterile gauze, make viewfinder area be in level, and regulate microscope to focus and marked region
Apply aseptic microsurgery equipment and remove the skin of label side, subcutaneous tissue and bilateral retractor under microscopic visualization, excision extension is the prototype area of diameter 15mm,
After having excised the skin of field of vision, glass frame is positioned over viewfinder area central authorities, second cell frame is fixed to corresponding position, separated to avoid oppressing supply vessels by anti-extrusion pad between two panels external member, two panels cell frame is fixed afterwards with nut and suture, in aforesaid operations process, window area warm saline keeps moistening
Cover the dismountable aseptic circular lid slide of one deck in glass frame in two panels cell frame and fix, and drip enough warm salines in placement coverslip forward direction glass frame.
By CBRH-7919 hepatoma carcinoma cell with the trypsinization of 0.25% after, get a certain amount of cell suspension to count under light microscopic, then by centrifugal for tumor cell, concentrated, resuspended, and to adjust CBRH-7919 hepatoma carcinoma cell number in CBRH-7919 hepatoma carcinoma cell suspension be 5 × 106/and ml to 2 × 107/ml, under the CBRH-7919 hepatoma carcinoma cell suspension of 0.1ml-0.2ml is expelled to nude mice epidermis.
Compared with the existing technology, tool has the following advantages in the present invention:
1. this experimental model is drawn materials convenient, and method for building up is simple, and success rate is high;
2. this experimental model can more real simulation tumor growth environment, occurs, evolution more presses close to tumor vessel;
3. this experimental model can to the capable immediate observation of tumor peripheral vessels, and observational technique is simple, convenient.
Accompanying drawing explanation
Fig. 1 is the process schematic one utilizing this method Modling model.
Fig. 2 is the process schematic two utilizing this method Modling model.
Fig. 3 is the process schematic three utilizing this method Modling model.
Fig. 4 is the side schematic diagram of the model utilizing this method to set up.
Fig. 5 is the opposite side schematic diagram of the model utilizing this method to set up.
Fig. 6 is blood vessel microcirculation schematic diagram under the light microscopic of the model utilizing this method to set up.
Fig. 7 is blood vessel microcirculation schematic diagram under the fluorescence of the model utilizing this method to set up.
Detailed description of the invention
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described.As shown in Figures 1 to 5: a kind of method for building up observing tumor vessel microcirculatory nude mice skin fold model, carry out according to following steps:
To take the logarithm the CBRH-7919 hepatoma carcinoma cell of phase, after being used the trypsinization of 0.25%, the Digestive system getting 20ul carries out the counting of hepatoma carcinoma cell under light microscopic, then by Digestive system centrifugal concentrating, and in Digestive system, add solvent according to count results, make the hepatoma carcinoma cell concentration of Digestive system be adjustment 5 × 10
6individual/ml to 2 × 10
7the cell suspension of individual/ml, then the pallium cell injection of 0.1ml-0.2ml is got with the syringe of 1ml under nude mice epidermis, the enclosed mass that diameter is about 2mm is formed at nude mice by subcutaneous, injection site should be chosen at around skin of back trunk as far as possible, care should be used to avoids syringe needle to touch blood vessel simultaneously, prevent angiorrhexis, cause hemorrhage.
Injected rear 2-3 days and got the nude mice forming tumor tumor body, set up a crease in the skin model in the one side of tumor tumor bulk-growth, concrete grammar is as follows:
Anesthesia: pentobarbital sodium physiological saline solution is mixed with the sterile solution of 1%, namely join and namely use, the capable intraperitoneal injection anesthesia of 45mg/kg is pressed according to body weight, select right lower abdomen inserting needle in the outer part, pumpback before injection, prevent medicine from directly injecting abdominal viscera or blood vessel, shallowly added 0.1ml(10mg if anaesthetize in art) sterile solution in abdominal cavity
Preserved skin: the microvillus at nude mice back is thoroughly removed totally by application depilatory, note not directly rejecting microvillus here, directly shave except fine hair can bring out mechanical irritation and skin micro-damage, this can not ensure the quality of model,
Preoperative Method: nude mice is positioned on cosy operating board, nude mice back wrinkled skin has been pinched along its spinal column, get its head rhicnosis portion of central authorities and afterbody and hit exactly median line between extended line, 2 points are got respectively in median line both sides, distance between this 2 and median line mid point is 2cm, skinfold is passed from these 2 respectively with 2 chorda serica chirurgicalis sterilises, and utilize this 2 chorda serica chirurgicalis sterilis pull-up experimental mouse skins, patient's sight line is perpendicular to experimental mouse median line, under the irradiation of offside transmitted light source, see Major Vessels trend and position making marks clearly, here make marks is to avoid being damaged to any Major Vessels and microvessel structure when fixedly mounting back cell external member afterwards.
Cell external member is installed: first cell frame is put in the skin upper limb place, center, back that nude mice leans against nearly patient side, tied fixing with chorda serica chirurgicalis sterilis, after under the irradiation of transmittance light source, with marking pen according to the scope of circular viewfinder area of side of installing cell frame, mark the viewfinder area scope that offside is corresponding, it should be noted that nude mice dorsal tumors and peripheral vessels thereof should be positioned at form as far as possible
For hanging the silk thread of nude mice skin during dismounting Preoperative Method, experiment nude mice is then allowed to lie on one's side towards deviating from patient side, under experiment nude mice is positioned over gimbals microscope, under being padded on wrinkled skin with sterile gauze, make viewfinder area be in level, and regulate microscope to focus and marked region
Apply aseptic microsurgery equipment and remove the skin of label side, subcutaneous tissue and bilateral retractor under microscopic visualization, excision extension is the circular area of a diameter 15mm, should be noted, in operation must accurate operation, avoid the offside field of vision blood vessel injury in institute's excision extension, hemorrhage, thus have impact on the quality of model.And limit excision extension and also can avoid when installation second cell frame, cause two panels cell frame tension to be expressed to both sides skin histology, and then compressing supply vessels finally cause the situation of tissue ischemia model failure to occur; Do so simultaneously and can also limit skin edge granulation tissue and be implanted to graft area.
After having excised the skin of field of vision, glass frame is positioned over viewfinder area central authorities, second cell frame is fixed to corresponding position, separated to avoid oppressing supply vessels by anti-extrusion pad between two panels external member, two panels cell frame is fixed afterwards with nut and suture, in aforesaid operations process, window area warm saline keeps moistening
Cover the dismountable aseptic circular lid slide of one deck in glass frame in two panels cell frame and fix, in order to directly to observe microvessel growth situation under microscope, and placing enough warm salines in coverslip forward direction glass frame, produce to prevent bubble in coverslip.
Claims (2)
1. observe a method for building up for tumor vessel microcirculatory nude mice skin fold model, it is characterized in that: described method is carried out according to following steps:
To take the logarithm the CBRH-7919 hepatoma carcinoma cell of phase, after being used the trypsinization of 0.25%, centrifugal concentrating obtains CBRH-7919 hepatoma carcinoma cell suspension, then, under a certain amount of CBRH-7919 hepatoma carcinoma cell suspension being expelled to nude mice epidermis, ensure that the quantitative range entering the subepidermal CBRH-7919 hepatoma carcinoma cell of nude mice is 5 × 10
5to 4 × 10
6individual/ml;
Injected rear 2-3 days and got the nude mice forming tumor tumor body, set up a crease in the skin model in the one side of tumor tumor bulk-growth, concrete grammar is as follows:
Anesthesia: pentobarbital sodium physiological saline solution is mixed with the sterile solution of 1%, press the capable intraperitoneal injection of 45mg/kg according to body weight and anaesthetizes, if anaesthetized the shallow sterile solution adding 0.1ml in art in abdominal cavity,
Preserved skin: the microvillus at nude mice back is thoroughly removed totally by application depilatory,
Preoperative Method: nude mice is positioned on operating board, nude mice back wrinkled skin has been pinched along its spinal column, get the median line position that its head rhicnosis portion of central authorities and afterbody hit exactly between extended line and do a labelling, 2 points are got respectively in median line both sides, distance between this 2 and median line mid point is 2cm, skinfold is passed from these 2 respectively with 2 chorda serica chirurgicalis sterilises, and utilize this 2 chorda serica chirurgicalis sterilis pull-up experimental mouse skins, patient's sight line is perpendicular to experimental mouse median line, under the irradiation of offside transmitted light source, see Major Vessels trend and position making marks clearly
Cell external member is installed: first cell frame is put in the skin upper limb place, center, back that nude mice leans against nearly patient side, tied fixing with chorda serica chirurgicalis sterilis, after under the irradiation of transmittance light source, with marking pen according to the scope of circular viewfinder area of side of installing cell frame, mark the viewfinder area scope that offside is corresponding
For hanging the silk thread of nude mice skin during dismounting Preoperative Method, experiment nude mice is then allowed to lie on one's side towards deviating from patient side, under experiment nude mice is positioned over gimbals microscope, under being padded on wrinkled skin with sterile gauze, make viewfinder area be in level, and regulate microscope to focus and marked region
Apply aseptic microsurgery equipment and remove the skin of label side, subcutaneous tissue and bilateral retractor under microscopic visualization, excision extension is the circular area of diameter 15mm,
After having excised the skin of field of vision, glass frame is positioned over viewfinder area central authorities, second cell frame is fixed to corresponding position, separated to avoid oppressing supply vessels by anti-extrusion pad between two panels external member, two panels cell frame is fixed afterwards with nut and suture, in aforesaid operations process, window area warm saline keeps moistening
Cover the dismountable aseptic circular lid slide of one deck in glass frame in two panels cell frame and fix, and drip enough warm salines in placement coverslip forward direction glass frame.
2. the method for building up of observation tumor vessel according to claim 1 microcirculatory nude mice skin fold model, it is characterized in that: by CBRH-7919 hepatoma carcinoma cell with the trypsinization of 0.25% after, get a certain amount of cell suspension to count under light microscopic, then by centrifugal for tumor cell, concentrated, resuspended, and to adjust CBRH-7919 hepatoma carcinoma cell number in CBRH-7919 hepatoma carcinoma cell suspension be 5 × 106/and ml to 2 × 107/ml, under the CBRH-7919 hepatoma carcinoma cell suspension of 0.1ml-0.2ml is expelled to nude mice epidermis.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106618729A (en) * | 2017-01-03 | 2017-05-10 | 中国科学院化学研究所 | Device for real-time dynamic observation of tumor microenvironment change in radio frequency treatment |
| CN106821532A (en) * | 2017-02-28 | 2017-06-13 | 上海市第六人民医院 | A kind of mouse ridge skin wing form frame |
| CN108645683A (en) * | 2018-05-16 | 2018-10-12 | 绍兴文理学院 | A kind of palmitin fixer and its application |
| CN108913661A (en) * | 2018-06-22 | 2018-11-30 | 安徽 | A kind of preparation and extracting method of human research's candidate stem cell |
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2015
- 2015-02-09 CN CN201510065553.XA patent/CN104783921A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106618729A (en) * | 2017-01-03 | 2017-05-10 | 中国科学院化学研究所 | Device for real-time dynamic observation of tumor microenvironment change in radio frequency treatment |
| CN106618729B (en) * | 2017-01-03 | 2019-06-25 | 北京福纳康生物技术有限公司 | A kind of device for tumor microenvironment variation in the RF therapy of dynamic observation in real time |
| CN106821532A (en) * | 2017-02-28 | 2017-06-13 | 上海市第六人民医院 | A kind of mouse ridge skin wing form frame |
| CN108645683A (en) * | 2018-05-16 | 2018-10-12 | 绍兴文理学院 | A kind of palmitin fixer and its application |
| CN108913661A (en) * | 2018-06-22 | 2018-11-30 | 安徽 | A kind of preparation and extracting method of human research's candidate stem cell |
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Application publication date: 20150722 |
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