Rat liver macrophage cultural methodTechnical field
The present invention relates to technical field of cell culture, and in particular to a kind of rat liver macrophage cultural method.
Background technique
Macrophage is one of three big antigen presenting cells, is to participate in non-specific and specific immunity important cells.ItMajor function be to carry out phagocytosis to cell debris and pathogen in the form of fixing cell or free cell (to swallowAnd digestion), and lymph corpuscle or other immunocytes are activated, enable it to react pathogen.Due to macrophage have compared withStrong phagocytic function and antigen presentation function, so having biggish researching value in immunology.
Hepatic macrophages, also known as Kupffer cell (Kupffer cells, KC), survive in liver sinus shape pipe, itLiver can be protected by the reaction to pathogen and metastatic cells, while can also endure the damage of itself and foreign antibodies,In, foreign antibodies can enter blood by portal vein and arteria hepatica.Recent research indicate that hepatic macrophages fibrosis,It plays an important role in the research of hepatitis, liver obesity disease and liver transfer operation.Hepatic macrophages are a kind of research normal physiological and diseaseThe preferable model that macrophage acts under the conditions of reason.
Summary of the invention
The present invention provides a kind of rat liver macrophage cultural method, specific technical solution is as follows:
Rat liver macrophage cultivation, comprising the following steps:
(1) 100mg VI Collagenase is dissolved in the dedicated basal medium of 100ml Kupffer cell and is made 0.1%Digestive juice, 40 DEG C of water-baths are interior to preheat 10min；
(2) 300mg heparin sodium is dissolved in be made in 100ml Kupffer cell special culture media 0.3% cleaning solution liquid, 4010min is preheated in DEG C water-bath；
(3) Healthy female SD rat 1, weight 200g or so is taken, isoflurane Anesthesia machine anesthetized rat, disinfectant tamed iodine wipesRat body skin；
(4) rat is fixed in dorsal position, and cross type cut opens abdominal cavity, is pushed stomach, intestines to rats with left, is exposed liver；
(5) remaining needle is inserted into vena portae hepatica and ligatures note retaining needle flexible tube with operation suture thread, quickly dissection cavity of resorption from belowVein, is perfused 80ml cleaning solution liquid from remaining needle at once in 3S after dissection inferior caval vein, and perfusion flow velocity is 12ml/min；
(6) inferior caval vein notch is blocked to be perfused sufficiently with sterile cotton balls during being perfused, and does not have 30s to replace a poor quality cotton during perfusionBall；
(7) 50ml digestive juice is poured into after the completion of cleaning again, flow velocity 10ml/min, every 30s replaces a cotton balls during perfusion；
(8) it cuts liver to be placed in culture dish, eye scissors cut liver to 1.5mm3Size is transferred in 30ml digestive juice, 37DEG C 190rmp/ml shakes digestion 18min；
(9) 70um cell sieve filters the cell suspension digested, and 100g/min is centrifuged 3min；
(10) take supernatant in 50 new centrifuge tubes, 400g/min is centrifuged 8min, abandons supernatant, and cell is resuspended in 10ml cleaning solution；
(11) new 50ml centrifuge tube one is taken, the percoll separating liquid of 10ml 65% is added, then 10ml 35% is added toward upper layerPercoll separating liquid, 10ml cell suspension is finally added to top layer；
(12) 950g/ml is centrifuged 20min, and 35%, 65%percoll separating liquid intersection tunica albuginea layer is carefully drawn with pasteur pipet,Cell is resuspended with 10ml cleaning solution, 400g/min is centrifuged 8min and collects cell, abandons supernatant；
(13) cell is resuspended with the dedicated complete medium of 2ml Kupffer, counts, adjustment concentration to 105A cell/ml, inoculationIn the T25 bottle that 5ml cell suspension was coated with overnight to 100ug/ml poly-D-lysine (ten thousand unit of 15-30), 37 DEG C, 5%CO2TrainingSupport culture in case；
(14) culture medium is discarded after being inoculated with 2.5h, dedicated complete medium gently cleans attached cell 3 times, it is complete to add 5mlContinue to cultivate after full culture medium.
Rat liver macrophage is cultivated by extracting, establishes hepatic macrophages action model, it is thin for liver macrophageBorn of the same parents play an important role in fibrosis, hepatitis, liver obesity disease and the research of liver transfer operation.
Specific embodiment
Embodiment 1:
Rat liver macrophage cultivation, comprising the following steps:
(1) 100mg VI Collagenase is dissolved in the dedicated basal medium of 100ml Kupffer cell and is made 0.1%Digestive juice, 40 DEG C of water-baths are interior to preheat 10min；
(2) 300mg heparin sodium is dissolved in be made in 100ml Kupffer cell special culture media 0.3% cleaning solution liquid, 4010min is preheated in DEG C water-bath；
(3) Healthy female SD rat 1, weight 200g or so are taken.Isoflurane Anesthesia machine anesthetized rat, disinfectant tamed iodine wipingRat body skin；
(4) rat is fixed in dorsal position, and cross type cut opens abdominal cavity, is pushed stomach, intestines to rats with left, is exposed liver；
(5) remaining needle is inserted into vena portae hepatica and ligatures note retaining needle flexible tube with operation suture thread, quickly dissection cavity of resorption from belowVein, is perfused 80ml cleaning solution liquid from remaining needle at once in 3S after dissection inferior caval vein, and perfusion flow velocity is 12ml/min；
(6) inferior caval vein notch is blocked to be perfused sufficiently with sterile cotton balls during being perfused, and does not have 30s to replace a poor quality cotton during perfusionBall；
(7) 50ml digestive juice is poured into after the completion of cleaning again, flow velocity 10ml/min, every 30s replaces a cotton balls during perfusion；
(8) it cuts liver to be placed in culture dish, eye scissors cut liver to 1.5mm3Size is transferred in 30ml digestive juice, 37DEG C 190rmp/ml shakes digestion 18min；
(9) 70um cell sieve filters the cell suspension digested, and 100g/min is centrifuged 3min；
(10) take supernatant in 50 new centrifuge tubes, 400g/min is centrifuged 8min, abandons supernatant, and cell is resuspended in 10ml cleaning solution；
(11) new 50ml centrifuge tube one is taken, the percoll separating liquid of 10ml 65% is added, then 10ml 35% is added toward upper layerPercoll separating liquid, 10ml cell suspension is finally added to top layer；
(12) 950g/ml is centrifuged 20min, and 35%, 65%percoll separating liquid intersection tunica albuginea layer is carefully drawn with pasteur pipet,Cell is resuspended with 10ml cleaning solution, 400g/min is centrifuged 8min and collects cell, abandons supernatant；
(13) cell is resuspended with the dedicated complete medium of 2ml Kupffer, counts, adjustment concentration to 105A cell/ml, inoculationIn the T25 bottle that 5ml cell suspension was coated with overnight to 100ug/ml poly-D-lysine (ten thousand unit of 15-30), 37 DEG C, 5%CO2TrainingSupport culture in case；
(14) culture medium is discarded after being inoculated with 2.5h, dedicated complete medium gently cleans attached cell 3 times, it is complete to add 5mlContinue to cultivate after full culture medium.