CN110622922A - Method for establishing mouse animal model with ascites due to pancreatic cancer - Google Patents
Method for establishing mouse animal model with ascites due to pancreatic cancer Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
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- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for establishing a mouse animal model with ascites due to pancreatic cancer, which comprises the steps of carrying out intraperitoneal injection on a C57BL/6 mouse by using pancreatic cancer Panc02 cells, extracting ascites after the ascites occurs, directly carrying out intraperitoneal injection on a new generation of mouse, transmitting the ascites to a 10 th generation of mouse, simultaneously extracting pancreatic cancer cells in the ascites of the 1 st generation mouse, the ascites of the 5 th generation mouse and the ascites of the 10 th generation mouse, carrying out purification culture, carrying out in vitro experiments and in vivo experiments on the pancreatic cancer cells after the purification culture, and groping the optimal cell amount for modeling and the number of extracted cell generations. Compared with the existing nude mouse pancreatic cancer ascites model, the invention initiates the pancreatic cancer ascites model of the C57BL/6 mouse with autonomous immunity, the modeling method has the characteristics of simple and convenient operation, short modeling time and high success rate, the method can quickly and efficiently establish the mouse pancreatic cancer ascites model, and a proper carrier tool is provided for the elucidation of pathogenesis, diagnosis and treatment of pancreatic cancer ascites.
Description
Technical Field
The invention belongs to a method for establishing an animal model, and particularly relates to a method for establishing a mouse animal model with ascites due to pancreatic cancer.
Background
The mortality rate of pancreatic cancer can rise to the second of all malignancies by the year 2030, as predicted by experts. Pancreatic cancer is easy to be transferred far away in the early stage of the disease, the pancreatic cancer is transferred to peritoneum and can be accompanied with malignant ascites and complicated symptoms such as intestinal obstruction, the quality of life of a patient is seriously affected by the generation of pancreatic cancer ascites, and the survival time of the patient is shortened.
At present, most of the mouse animal models with ascites due to pancreatic cancer are nude mouse ascites due to pancreatic cancer which lacks T cell immunity, the nude mouse ascites due to pancreatic cancer model is used for exploring the level of immune factors in the ascites due to pancreatic cancer and researching the immunotherapy of the ascites due to pancreatic cancer has limitations, and the model with ascites due to pancreatic cancer of a C57BL/6 mouse with autonomous immunity is rarely reported. Most of the existing methods for establishing the mouse animal model with the pancreatic cancer ascites are in-situ tumor transplantation methods, and the methods are complex in operation, high in difficulty and low in modeling success rate.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for establishing a pancreatic cancer ascites mouse animal model, which is simple and convenient to operate, short in modeling period and high in success rate.
In order to solve the technical problems, the invention adopts the following technical scheme:
the method for establishing the mouse animal model with ascites due to pancreatic cancer comprises the steps of carrying out C57BL/6 mouse intraperitoneal injection by using pancreatic cancer Panc02 cells, extracting ascites after ascites occurs, directly carrying out injection on a new batch of mice, simultaneously extracting pancreatic cancer cells in ascites of g1, g5 and g10 mice, carrying out purification culture on the pancreatic cancer cells after the purification culture, carrying out in-vitro experiments and in-vivo experiments on the pancreatic cancer cells, and groping the optimal cell amount for modeling and the number of extracted cell generations.
The method for establishing the mouse animal model with ascites due to pancreatic cancer comprises the steps of carrying out intraperitoneal injection on a C57BL/6 mouse by using pancreatic cancer Panc02 cells G0, extracting pancreatic cancer cells in ascites after ascites occurs, carrying out purification culture on the pancreatic cancer cells extracted from ascites, naming the mouse injected with the pancreatic cancer cells in the first batch as G1, naming the pancreatic cancer cells obtained by purification culture from G1 as G1, and naming the mouse injected with the ascites due to the pancreatic cancer cells in the second batch as G2.
The injection cell amount of G0 of pancreatic cancer Panc02 cells is 5X 106The injection amount is 0.2ml per mouse; the injection cell amount of the pancreatic cancer cell G1 is 1 × 106The injection amount is 0.2ml per tube.
The method for establishing the mouse animal model with ascites due to pancreatic cancer comprises the steps of injecting C57BL/6 mice into abdominal cavities by using pancreatic cancer Panc02 cells G0, extracting pancreatic cancer cells in abdominal water after ascites occurs, purifying and culturing the pancreatic cancer cells, naming the mice injected with pancreatic cancer cells in the first batch as G1, naming the pancreatic cancer cells obtained by purifying and culturing the mice injected with pancreatic cancer cells in G1 as G1, naming the mice injected with pancreatic cancer cells in the second batch as G2, carrying out passage till G10, and naming the pancreatic cancer cells obtained by purifying and culturing the mice in G5 and G10 as G5 and G10 respectively. Aiming at the limitation of the existing mouse animal model with ascites due to pancreatic cancer, the inventor designs and establishes a method for establishing the mouse animal model with ascites due to pancreatic cancer, pancreatic cancer Panc02 cells are used for intraperitoneal injection of C57BL/6 mice, pancreatic cancer cells in ascites are extracted after ascites occurs and are purified and cultured, the mice injected with pancreatic cancer cells in the first batch are named as G1, the pancreatic cancer cells obtained by purification and culture of G1 are named as G1, and the mice injected with ascites due to pancreatic cancer cells in the second batch are named as G2. According to the circulation, G1, G5 and G10 mice were obtained, respectively, and pancreatic cancer cells in ascites of G1, G5 and G10 mice were isolated, purified and cultured in vitro, and named as G1, G5 and G10, respectively. Comparing the malignancy degree of the pancreatic cancer cells extracted from the ascites through in vitro experiments and in vivo experiments, investigating and comparing the proportion of the ascites, the ascites amount and the survival time of the mouse, and finally screening out the optimal cell G1 and the cell amount of 1 multiplied by 10 for establishing the animal model of the mouse with the ascites due to pancreatic cancer6One/only. Compared with the existing nude mouse pancreatic cancer ascites model, the invention initiates the pancreatic cancer ascites model of the C57BL/6 mouse with autonomous immunity, the modeling method has the characteristics of simple and convenient operation, short modeling time and high success rate, the method can quickly and efficiently establish the mouse pancreatic cancer ascites model, and a proper carrier tool is provided for the elucidation of pathogenesis, diagnosis and treatment of pancreatic cancer ascites.
Drawings
FIG. 1 is an experimental route chart for the establishment of a pancreatic cancer ascites mouse animal model of the present invention.
FIG. 2 is a graph comparing the ascites volume and survival time of g1, g5, and g10 mice, wherein: a is the mouse ascites volume result and B is the mouse survival time.
FIG. 3 is a graph of HE staining (600X) of G0, G1, G5 and G10 cells in which: (A) HE staining of G0 cells, (B) HE staining of G1 cells, (C) HE staining of G5 cells, and (D) HE staining of G10 cells.
FIG. 4 is a graph of ultrastructural comparison (10000X) of G0, G1, G5 and G10 cells, in which: (A) g0 cell, (B) G1 cell, (C) G5 cell, and (D) G10 cell.
FIG. 5 is a graph comparing the migration and invasion capacity of G0, G1, G5 and G10 cells, in which: a scratch experiment micrograph, b Transwell cell migration experiment micrograph, C Transwell cell invasion experiment micrograph, A scratch experiment histogram, BTranswell cell migration experiment histogram and C Transwell cell invasion experiment histogram.
FIG. 6 is a graph of mouse survival analysis of P.i. injected with varying cell amounts of G0, G1, G5, and G10 cells, in which: (A) mouse survival assay with G0 cells injected, (B) mouse survival assay with G1 cells injected, (C) mouse survival assay with G5 cells injected, and (D) mouse survival assay with G10 cells injected.
FIG. 7 is an abdominal anatomy of a normal mouse and a pancreatic cancer ascites mouse modeled using the present invention.
Detailed Description
Pancreatic cancer ascites mouse animal model research
1. Resuscitation and culture of Panc02 cells
Preheating a water bath to 37 ℃, quickly taking out Panc02 cells from a liquid nitrogen tank, checking whether a freezing tube is well sealed, quickly placing the freezing tube in the water bath at 37 ℃ after screwing down a cover of the freezing tube, continuously shaking the freezing tube to enable the freezing tube to be uniformly heated and completely melt within 1 minute, observing the cell liquid dissolving condition, quickly taking out the freezing tube when observing that the cells in the freezing tube are completely dissolved, wiping the outside of the freezing tube with 75% alcohol, and moving the freezing tube to an ultraclean cell workbench. Will be provided withThe cell suspension in the freezing tube is transferred into a 15ml centrifuge tube by a pipette gun, the PBS buffer solution is added to 10ml, and the cell suspension is gently mixed by the pipette gun. The centrifuge tube was placed in a centrifuge after the centrifuge tube cap was screwed down, and after trimming, the centrifuge tube was centrifuged at 1000 rpm/min for 5 minutes, and the supernatant was discarded to retain the cell pellet. Repeating the operation for 2 times to ensure that the cell frozen stock solution is completely cleaned, adding DMEM culture medium into the cell precipitate, mixing the cells, transferring the brain-washing suspension into a culture bottle, placing at 37 deg.C, and adding 5% CO by volume fraction2Cultured in an incubator. Observing the form and the growth state of cells under an inverted microscope every day, sucking old culture solution in a culture bottle when the cells grow to reach 80-90% of the area of the culture bottle, then sucking sterile PBS buffer solution by using a disposable sterile sucker for cleaning the cells for 2 times, finally adding 0.25% of trypsin containing EDTA into the culture bottle for digesting about 0.5ml of cells, slowly and lightly shaking the culture bottle to uniformly fill the bottom of the bottle with the trypsin, observing the shape of the cells under the inverted microscope simultaneously, observing the shape of the cells when the cells are reduced and become round and bright, shaking the bottle body to see that silt-like cell suspension flows down at the bottom of the bottle, indicating that the cells are completely digested, quickly adding a DMEM complete culture medium to stop digestion, and lightly blowing off adherent cells. Sucking the cell suspension into a 10mL high-pressure sterilized centrifugal tube by using a disposable sterile pipette, centrifuging for 5 minutes at the revolution of 1000 rpm/minute, sucking supernatant after centrifugation is finished, retaining cell sediment at the bottom of the tube, adding about 3mL of DMEM culture medium into the centrifugal tube, slightly blowing and beating the resuspended cell sediment according to the ratio of 1: and 3, subpackaging the mixture into new culture bottles again in proportion, adding DMEM complete culture medium into each new culture bottle to reach about 5mL, slightly shaking the bottle body to enable the cell suspension to be uniformly spread on the bottom surface of the culture bottle as much as possible, observing whether cell sediment is blown to be uniformly resuspended under an inverted microscope, and after the observation that the cells are made into single cell suspension, putting the culture bottles into an incubator to continue culturing. When the cells grow to the logarithmic phase, digesting, centrifuging and re-suspending the cells to prepare the cells into a single cell suspension.
2. Cell counting
Wiping the cell counting plate and the cover glass with clean filter paper soaked by 75% alcohol, placing the cover glass on the cell counting plate after the cell counting plate and the cover glass are naturally dried, and moving the cover glass to be aligned with the edge of a counting area. Digesting and centrifuging Panc02 cells in logarithmic phase, adding PBS buffer solution to dilute the cell precipitate to a proper multiple, gently mixing the cell precipitate with a pipette, sucking the cell suspension with a 10-microliter pipette gun, and slowly adding the cell suspension to be detected along the middle point of the upper edge of the cover glass to prevent the cell suspension from overflowing the counting plate and avoid generating bubbles. Standing for 30 seconds, observing under a microscope when the cell suspension is uniformly paved on a cover glass, and recording the total number of the cells in four large squares of the counting plate as n. Note that when the cells were right on the grid line of the counting plate, only the cells on the left and upper lines were counted, and the cells on the right and lower lines were not counted, to avoid repeated counting. After counting, the cell concentration is calculated according to the following formula:
cell concentration (number of cells/ml) — (total number of cells n/4) × dilution factor × 104；
Total number of cells in cell suspension equals cell suspension concentration × volume of cell suspension (ml).
3. Cell viability assay
Wiping the cell counting plate and the cover glass with clean filter paper soaked by 75% alcohol, naturally airing the counting plate and the cover glass, placing the cover glass on the cell counting plate, aligning the cover glass with the edge of a counting area, and preparing 0.4% trypan blue solution. Prepared at a concentration of 106The cell suspension was mixed with 0.4% of Taiwan phenol blue solution at a ratio of 9:1 to give a final concentration of Taiwan phenol blue of 0.04%. And slowly blowing the cell suspension, sucking 10 mu l of the cell suspension by using a 10 mu l pipette, and slowly adding the cell suspension to be detected along the middle point of the upper edge of the cover glass to prevent the cell suspension from overflowing the counting plate and avoid generating bubbles. The number of live and dead cells (dead cells stained blue; live cells did not stain) was counted under a light microscope within three minutes and after counting according to the following formula:
the viable cell rate (%) - (total cell number-stained cell number)/total cell number × 100%.
In order to ensure the successful establishment of the mouse pancreatic cancer ascites model, the viable cell rate of the Panc02 cells is more than or equal to 95 percent.
4. Establishing mouse pancreatic cancer ascites model
10 SPF male C57BL/6 mice, 10 weeks old and 24-26g in body mass were taken and adapted to feed for 3 days before modeling. Sterilizing mouse abdominal skin with 75% ethanol, sucking prepared Panc02 cell suspension with 1ml sterile injector, taking mouse left lower abdomen, obliquely feeding needle, injecting Panc02 cell suspension 0.2ml (equivalent to injection tumor cell number of 5 × 10) into mouse abdominal cavity after breakthrough feeling6One/only). The whole inoculation process is clean, and the inoculation process is completed within 1 hour. After completion of the inoculation, the mice were observed for activity and given sufficient water and feed. The mice injected with Panc02 cells in the first batch are marked as first generation mice with ascites due to pancreatic cancer (g1), ascites is extracted from the mice producing ascites from g1, 10 mice with C57BL/6 cells are injected intraperitoneally in the same way, each mouse is injected with 0.1ml of ascites, the newly injected mice are marked as second generation mice with ascites due to pancreatic cancer (g2), and the same way is carried out until the mice are passaged among mice to tenth generation mice with ascites due to pancreatic cancer (g 10). Observing the changes of the eating, activity and mental state of the mouse every day, observing the ascites generation condition, continuously observing for 60 days, and judging that the mouse has no ascites after 60 days. When a model mouse is in a cachexia state, disinfecting eyes of the mouse by using an alcohol cotton swab, cutting beards of the mouse to prevent influencing the collection of eyeball blood of the mouse, pinching the neck skin of the mouse by using a left hand to enable the eyeball of the mouse to protrude as much as possible, picking the eyeball of the mouse by using a bent forceps held by a right hand, collecting peripheral blood of the mouse by using an EP (EP) tube wetted by heparin sodium, soaking the mouse for 5 minutes by using 75% alcohol, and collecting ascites of the mouse. The specific method comprises the following steps: the abdominal cavity of the mouse is fully exposed by scissors, the ascites in the abdominal cavity of the mouse is completely absorbed by the syringe, and the syringe is used for completely absorbing the ascites in the abdominal cavity of the mouse and is arranged in a corresponding sterile centrifuge tube. The proportion of ascites appearing in each mouse generation, the amount of ascites and the survival time of the mice were recorded.
5. Morphological Observation of cells
(1) And (3) carrying out HE staining on cells:
centrifuging Panc02 cell suspension and ascites of mice g1, g5 and g10 at the rotating speed of 1000 rmp/min at the temperature of 4 ℃ for 10 minutes, discarding supernatant, keeping a small amount of supernatant in a tube, blowing and uniformly mixing cell precipitates by using a pipette, sucking a small amount of cell precipitates on a clean glass slide, covering another clean glass slide on the cell precipitates, evacuating air between the two glass slides, rapidly pulling the glass slides oppositely to ensure that the cell precipitates are uniformly distributed on the glass slides to prepare a cell smear, fixing the cell smear by using 95% alcohol for 8 hours, washing the cell smear by using tap water for 2 times, 3 minutes and 3 minutes, staining hematoxylin for 2 times, washing by using water for 2 times, 3 minutes and 3 minutes, soaking in 1% hydrochloric acid alcohol for 3 seconds, washing by using water for 2 times, returning blue in water for 2 minutes, observing cell nucleus blue staining under a microscope, staining by eosin for 3-5 seconds, washing by using water for 2 times, 3 minutes/time, finally soaking and dehydrating for 2 seconds by 95 percent alcohol, naturally drying the glass slide, and finally sealing the glass slide by neutral resin. HE staining results were independently observed and diagnosed by 2 highly experienced pathologists and agreed.
(2) Observation by a cell transmission electron microscope:
centrifuging Panc02 cell suspension and g1, g5 and g10 mouse ascites at 1000 rmp/min at 4 ℃ for 10 minutes, discarding the supernatant, (1) fixing the cell pellet: slowly adding 3% glutaraldehyde buffer solution to avoid dispersing the cell precipitate, fixing the cell precipitate at 4 deg.C for more than 2 hr, washing with PBS buffer solution for 3 times, 10 min/time, fixing with 1% osmium tetroxide buffer solution for 1-3 hr, and washing with PBS buffer solution for 3 times, 10 min/time. (2) Dehydration of the cell pellet: slowly adding 30% ethanol into the cell sediment for dehydration for 10-15 minutes, and performing 50% ethanol dehydration for 10-15 minutes, 70% ethanol dehydration for 10-15 minutes, 80% ethanol dehydration for 10-15 minutes, 90% ethanol dehydration for 10-15 minutes, and 100% ethanol dehydration for 10-15 minutes by the same method, wherein 100% ethanol needs to be repeated for 2-3 times to ensure complete dehydration. The ethanol in the sample was displaced 3 times with acetone for 5-10 minutes each. (3) Infiltration of cell pellet: firstly, mixing acetone and epoxy resin according to the weight ratio of acetone: mixing the epoxy resin at a ratio of 3:1, stirring thoroughly, infiltrating at room temperature for 1-3 hours, and mixing acetone and epoxy resin at a ratio of acetone: mixing epoxy resin at a ratio of 1:1, stirring thoroughly, infiltrating at room temperature for 1-3 hours, and mixing acetone and epoxy resin at a ratio of acetone: the epoxy resin was mixed at a ratio of 1:3 and stirred thoroughly, allowed to permeate at room temperature for 12 hours, and finally the pure epoxy resin was allowed to permeate overnight. (4) Embedding and polymerization of cell pellet: adding a catalyst into the epoxy resin according to the proportion of 1.5-2%, uniformly mixing, injecting into an embedding mold, placing the sample in the embedding mold according to the required direction, and then placing in a drying oven for polymerization. The polymerization was started for 12 hours at 35 ℃ and then for another 12 hours at 45 ℃ and finally for 24 hours at 60 ℃. And then, trimming the agar block, slicing the agar block by a slicer, and scanning and observing the ultrastructure of the pancreatic cancer cells on a transmission electron microscope on a machine after dyeing.
6. Extraction and purification of pancreatic cancer cells in ascites
Extracting ascites of model mice g1, g5 and g10 under strict aseptic operation, quickly adding 20 times volume of PBS buffer solution to dilute and rinse the ascites (the ascites: the PBS buffer solution is 1:20), centrifuging at the rotating speed of 1000 rmp/min for 10 minutes at 4 ℃, slowly sucking supernatant, repeatedly cleaning cell precipitates for 3 times by using the PBS buffer solution according to the method, washing red blood cells in the ascites as much as possible, suspending and uniformly mixing the cell precipitates by using DMEM (DMEM) containing 10% fetal calf serum completely, transferring the cell precipitates into a culture bottle, slightly shaking the bottle body to ensure that cell suspension is uniformly paved on the bottom of the bottle, putting the bottle body into an incubator to continue culture, after the cells are extracted to the wall of the cells after 8 hours after the cells are extracted to the wall of the culture bottle, replacing the cells with liquid, cleaning off the non-adherent suspended red blood cells, replacing the cell culture liquid for 1 time every 1-2 days, and when the cells grow to 80-90% of the culture area of the bottle, cell passaging was performed at a rate of 1-to-2. Purification of the cells was performed according to literature reported method procedures. Adding diluted low-concentration 0.125% trypsin digestive juice into a culture bottle to digest cells, slightly shaking the culture bottle to uniformly fill the bottom of the bottle with trypsin, slightly shaking the culture bottle under an inverted microscope to observe while slightly shaking the culture bottle, sucking out the digestive juice when most of matrix cells are rounded and fall off, digesting the cells still attached to the bottom of the culture bottle with 0.25% trypsin, observing under the inverted microscope, slightly shaking the culture bottle by hand, finishing digestion when silt-like precipitates at the bottom of the culture bottle slowly flow down, adding 10% fetal calf serum-containing DMEM complete culture medium to stop digestion, sucking out cell suspension into a centrifuge tube by a disposable sterile straw, centrifuging at 1000 rpm/min for 5 min, removing cell supernatant, adding DMEM complete culture medium to uniformly mix new cell precipitates again, subpackaging the cell suspension into a new culture bottle, and continuing culture in a culture box, usually, the ascites-extracted cells are substantially free of stromal cells after 4-6 generations of purification according to the above-described method. The purified and cultured cells are sent to Shanghai wing and applied to biotechnology limited company for Short Tandem Repeat (STR) cell genetic quality identification and detection.
7. Scratch test for detecting migration capacity of pancreatic cancer cells
Resuscitating pancreatic cancer cells G0, G1, G5 and G10, replacing the culture solution once every other day, subculturing the cells at a ratio of 1:2 when the cells grow to about 80-90% of the area of a culture bottle, extracting the cells from G0, G1, G5 and G10 ascites in the logarithmic growth phase, digesting the cells with pancreatin containing 0.25% EDTA, centrifuging at a revolution of 1000 rpm/min for 5 min, discarding the supernatant, resuspending and uniformly mixing the cell precipitate, and counting the cells, wherein the cell counting method is as described above and is performed according to a ratio of 5 × 105Inoculating the cell amount of each cell into a 6-hole plate, continuously culturing in an incubator, observing the growth condition of the cells under a microscope next day, after the cells are paved on the hole plate, rinsing the cells for 2 times by PBS buffer solution, then scribing a cross scratch on the longest diameter of the hole plate by a 10-microliter pipette tip, cleaning the cells by PBS buffer solution for 3 times, cleaning the floating and falling cells, slowly adding serum-free DMEM culture medium along the hole wall by the pipette tip for continuous culture, taking pictures of the same position at 0 hour and 24 hours respectively, and comparing and observing the migration capacity of the pancreatic cancer cells.
8. Transwell chamber experiment for detecting migration and invasion capacity of pancreatic cancer cells
(1) The migration experiment comprises the following specific steps:
selecting cells which grow to 80-90% of the bottom area of a culture bottle and have good growth state, digesting the cells by trypsin according to the method for digesting the cells, and digesting the digested cells inCentrifuging at 1000 rpm/min for 5 min, discarding supernatant, mixing with serum-free DMEM medium, mixing cell precipitate, and counting cells by the method described above, wherein the cell concentration is adjusted to 2.5 × 105One per ml.
Secondly, taking a sterile 24-hole plate, respectively putting Transwell cells (8 mu m) into the 24-hole plate holes according to experimental groups, respectively taking 200 mu l of G0, G1, G5 and G10 cell suspension with adjusted cell density, inoculating the cell suspension into the upper chamber of the Transwell cells, adding 600 mu l of DMEM complete culture medium containing 10% fetal calf serum into the lower chamber of the cells, then putting the cells into the holes of the 24-hole plate, putting the cells into the liquid level in the hole plate in an inclined manner in the process of placing the cells, avoiding bubbles generated between the bottom of the cells and the liquid level in the holes and influencing the migration of the cells to the lower chamber of the cells, sealing the periphery of the culture plate by using a sealing film after the Transwell cells are determined to be placed, and placing the culture plate at 37 ℃ and 5% CO2The incubator saturated with humidity was continued for 24 hours.
③ after the cell culture is enough time, taking out the 24-hole plate from the cell culture box, clamping the chamber by using a bent forceps, pouring out the redundant culture medium in the Transwell chamber, rinsing the chamber for 2 times by using PBS buffer solution, lightly wiping the residual cells of the indoor membrane on the chamber by using a cotton swab, putting the chamber into the 24-hole plate containing 95 percent alcohol, standing and fixing for 20 minutes, taking out the chamber, pouring out the alcohol, and reversing and drying.
Preparing 0.5% crystal violet solution: taking out crystal violet powder from a crystal violet powder box, placing 0.2g of the crystal violet powder on weighing paper, accurately weighing by using a precision electronic balance, placing the crystal violet powder in a 50ml centrifuge tube, adding 40ml of double distilled water into the tube, shaking the tube body left and right to fully dissolve the crystal violet powder, and storing the crystal violet powder at room temperature in a dark place.
Fifthly, taking a new centrifuge tube to absorb 2ml of 0.5% crystal violet solution, adding 8ml of PBS buffer solution to dilute the solution by 5 times, diluting the solution to 0.1% crystal violet solution for use, taking a new 24-pore plate, adding 600 mu l of 0.1% crystal violet solution into the pore plate, respectively placing the small chambers into the pores containing the crystal violet solution, taking out the small chambers after staining for 20-30 minutes, rinsing the small chambers for 2 times by the PBS buffer solution, and washing off the redundant crystal violet solution.
Sixthly, the chamber is naturally dried and then placed under an inverted microscope for observation and photographing, and cells penetrating through the polycarbonate membrane of the chamber are seen to be purple. 5 fields were counted randomly under each chamber mirror, and the number of cells in each field was counted and averaged. The experiment was repeated 3 times.
(2) The cell invasion assay procedure was as follows:
taking out matrigel matrix glue from a refrigerator at the temperature of-20 ℃, placing the matrigel matrix glue in an environment at the temperature of 4 ℃ overnight to be dissolved into liquid, pre-cooling disposable pipette tips used in all experimental processes at the temperature of 4 ℃ for standby, diluting the pre-cooled matrigel matrix glue with serum-free DMEM medium in a sterile cell workbench according to a proportion (DMEM: matrigel is 7:1), operating the whole dilution process on ice, sucking 100 mu l of diluted matrigel matrix glue by using a pipette gun, flatly paving the matrigel matrix glue on the bottom surface of an upper chamber of a small chamber to enable the matrigel to completely cover the bottom of the small chamber, placing the small chamber into a cell culture box, standing for 4 hours, sucking residual liquid in the small chamber after the matrigel matrix glue is solidified, adding 50ul of serum-free culture medium into each hole for hydration, and placing the small chamber in a culture box at the temperature of 37 ℃ for 30 minutes.
Selecting cells with the density of 80-90% and good growth state, digesting the cells by trypsin according to the method, centrifuging the digested cells for 5 minutes at the revolution of 1000 rmp/min, discarding cell supernatant, mixing the cells again by serum-free culture medium, counting by a cell counting plate, and adjusting the cell concentration to be 5 multiplied by 105One per ml.
Thirdly, sucking out serum-free culture medium for hydration in the small chamber, respectively inoculating 200 mul of G0, G1, G5 and G10 cell suspension with adjusted cell density into the upper chamber of the Transwell small chamber paved with matrix glue, adding 600 mul of DMEM (serum-free medium) containing 10% fetal calf serum into the lower chamber, then putting the small chamber into the hole of a 24-hole plate, putting the small chamber into the liquid level of the hole plate in an inclined way in the process of placing the small chamber, avoiding air bubbles from being generated between the bottom of the small chamber and the liquid level in the hole and influencing the migration of cells into the lower chamber of the small chamber, after the Transwell small chamber is well placed, sealing the periphery of the culture plate by using a sealing film, putting the culture plate into the hole at 37 ℃ and 5% CO2Was cultured in an incubator for 24 hours.
Fourthly, after the culture time is enough, the 24-hole plate is taken out, the chamber is clamped by a bending forceps, the residual culture medium in the chamber is poured out, the chamber is rinsed for 2 times by PBS buffer solution, cells and matrigel on the upper indoor membrane surface are slightly wiped off by a cotton swab, the chamber is placed into the 24-hole plate filled with 95 percent alcohol for fixing for 20 minutes, and then the chamber is taken out, the alcohol is poured out, and the chamber is turned over and naturally dried.
Fifthly, taking a new 24-pore plate, adding 600 mu l of 0.1% crystal violet solution into the pore plate, respectively putting the small chambers into the pore plate, taking out the small chambers after dyeing for 20-30 minutes, rinsing the small chambers for 2 times by PBS buffer solution, and washing off the redundant crystal violet solution.
Sixthly, after the chamber is naturally dried, placing the chamber under a microscope to observe and take a picture, and the cells passing through the polycarbonate membrane of the chamber are purple under the microscope. 5 fields were counted randomly under each chamber mirror, and the number of cells in each field was counted and averaged. The experiment was repeated 3 times.
9. In vivo experiments comparing the characteristics of G0, G1, G5, G10 cells
Taking G0, G1, G5 and G10 cells in logarithmic growth phase, adjusting cell number, and performing intraperitoneal injection of new C57BL/6 mice (5 × 10) according to different cell number gradients5、1×106、2×106、5×106One cell per mouse), injecting 5 mice per cell quantity gradient, continuously observing for 60 days, judging that the mice have no ascites if the mice do not have ascites after 60 days, and killing the mice by adopting a cervical dislocation method when the mice have a cachexia state. And (4) counting the ascites proportion and survival time of the mice with each cell mass gradient.
10. Analysis of results
As shown in fig. 2, ascites was extracted from each generation of ascites mice for mouse-to-mouse passage, and the proportion of ascites appearing in the mice and the amount of ascites gradually increased, the survival time gradually decreased, and became stable as the 5 th generation of mice. Wherein the ascites volume of g5 and g10 mice is greater than the ascites volume of g1 mice (. about.P < 0.05); ascites was observed in 30% g1 mice and 100% g5 and g10 mice for 60 days. The survival time of the g1 mouse was significantly longer than that of the g5 and g10 mice.
As shown in fig. 4, the morphological results of ascites-extracted cells also showed that the increase of microvilli on the cell surfaces of G1, G5 and G10 cells was lengthened compared to G0 cells, while the organelles such as mitochondria and endoplasmic reticulum were not significantly changed. The results of transmission electron microscope observation of pancreatic cancer cells in the research show that after ascites is passed through abdominal cavity passage of mice, the microvilli on the surface of pancreatic cancer cells in the ascites is increased and lengthened, which indicates that the migration and invasion capacities of the pancreatic cancer cells purified by the ascites are enhanced after the ascites is passed through the abdominal cavity passage of the mice. Among them, little and short microvilli were observed on the surface of the G0 cells; g1 cells have short microvilli on their surface, but the number of microvilli is increased compared with G0 cells, but organelles such as mitochondria and endoplasmic reticulum have no significant changes compared with G0 cells; compared with G0 and G1 cells, the G5 cells have long and dense micro villi on the surface, and the mitochondria and the endoplasmic reticulum have no obvious change; compared with G0 and G1 cells, G10 cells had long and disorganized micro-villi on the surface, and no obvious changes were observed in the mitochondria and endoplasmic reticulum.
As shown in FIG. 5, the results of the scratch test and the Transwell chamber test also show that the migration and invasion capacity of pancreatic cancer cells is enhanced after the mice are passed through the abdominal cavity (FIG. 5). Wherein, the scratch test and the Transwell chamber migration test result show that the migration capacity of G1, G5 and G10 cells is enhanced compared with that of G0 cells (P < 0.05); the results of Transwell chamber invasion experiments showed that G1, G5 and G10 cells had enhanced invasion capacity compared to G0 cells (P < 0.05).
As shown in FIG. 6, the results of in vivo experiments showed that G0 cells were injected at 5X 105,1×106And 2X 106Cell Mass groups No mice produced ascites, 5X 106Only 20% of the mice in the cell mass group produced ascites (fig. 6A). G1 cells 5X 105Cell Mass group 60% mice produced ascites, whereas 1X 106,2×106And 5X 106Cell mass group all mice produced ascites (fig. 6B). Ascites was produced in mice of all cell populations of G5 and G10, and mice injected with G5 and G10 cells survived significantly shorter than mice injected with G0 and G1 cells.
12. Conclusion
The pancreas of the invention is obtained by the research, comparison and analysisIn the establishment of the adenocarcinoma ascites mouse animal model, the first generation ascites extraction cell (G1) is optimally injected in the abdominal cavity, and the optimal cell amount is 1 multiplied by 106One/only.
Claims (4)
1. A method for establishing a pancreatic cancer ascites mouse animal model is characterized by comprising the following steps: pancreatic cancer Panc02 cells were used for intraperitoneal injection in C57BL/6 mice, and ascites containing pancreatic cancer cells were extracted after the appearance of ascites for intraperitoneal injection in C57BL/6 mice.
2. The method for establishing a mouse animal model with ascites due to pancreatic cancer according to claim 1, wherein: the pancreatic cancer Panc02 cell G0 is used for intraperitoneal injection of C57BL/6 mice, the pancreatic cancer cells in ascites are extracted after ascites occurs and are subjected to purification culture, the mice injected with the pancreatic cancer cells in the first batch are named as G1, the pancreatic cancer cells obtained by purification culture of G1 are named as G1, the mice injected with the ascites of the pancreatic cancer cells in the second batch are named as G2, and the pancreatic cancer cells obtained by purification culture of G5 and G10 are respectively named as G5 and G10 after the passage is carried out until the passage reaches G10.
3. The method for establishing a mouse animal model with ascites due to pancreatic cancer according to claim 2, wherein: the injection cell amount of the pancreatic cancer Panc02 cell G0 is 5 multiplied by 106The injection amount is 0.2ml per mouse; the injection cell amount of the pancreatic cancer cell G1 is 1 × 106The injection amount is 0.2ml per tube.
4. The method for establishing a mouse animal model with ascites due to pancreatic cancer according to claim 2, wherein: the pancreatic cancer Panc02 cell G0 is used for intraperitoneal injection of C57BL/6 mice, the pancreatic cancer cells in ascites are extracted after ascites occurs and are subjected to purification culture, the mice injected with the pancreatic cancer cells in the first batch are named as G1, the pancreatic cancer cells obtained by purification culture of G1 are named as G1, the mice injected with the ascites of the pancreatic cancer cells in the second batch are named as G2, and the pancreatic cancer cells obtained by purification culture of G5 and G10 are respectively named as G5 and G10 after the passage is carried out until the passage reaches G10.
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