CN101988048A - Preparation method of liver cells of laying hens - Google Patents
Preparation method of liver cells of laying hens Download PDFInfo
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- CN101988048A CN101988048A CN 201010528886 CN201010528886A CN101988048A CN 101988048 A CN101988048 A CN 101988048A CN 201010528886 CN201010528886 CN 201010528886 CN 201010528886 A CN201010528886 A CN 201010528886A CN 101988048 A CN101988048 A CN 101988048A
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Abstract
The invention relates to a preparation method of liver cells of laying hens, belonging to the technical field of medical biology. The method comprises the following three processes: ligating the blood vessels of the laying hens; carrying out perfusion in the livers; and washing the liver cells. The invention has the advantages that the quantity and motility rate of the separated liver cells are effectively improved; the method is suitable for laying hens or laying ducks or other laying domestic fowls; and the prepared liver cells can be widely used for the research of the liver cells of the poultry, the research of the lipid metabolism of the poultry and the like.
Description
Technical field
The invention belongs to medical biotechnology field, relate to the hepatocellular technology of preparing of laying hen.
Background technology
Liver is the important organ of bird, has functions such as synthetic, metabolism, detoxifcation.Because liver is the main place of poultry lipid metabolism, it is significant to the research lipid metabolism therefore to cultivate the poultry liver cell.Present hepatocellular cultural method mainly concentrates on the Mammals, but because anatomical structure and the Mammals difference of bird are big, therefore needs to set up the liver cell preparation method who is fit to bird.
At present isolated perfusion method and the interior perfusion methods of body of adopting of the liver cell preparation of bird more.Adopt the liver cell motility rate of isolated perfusion method preparation low, can stir liver when peeling off hepatic vein, liver is caused damage and pollution easily, reduced the success ratio of liver cell preparation.Therefore the isolated perfusion method also can't replace perfusion method in the body.Perfusion method concentrates on poultry and the hepatocellular preparation of young fowl at present in the body.Egg fowl is because of having flourishing vena oviductus clump, and vena oviductus communicates with hepatic vein, can cause perfusate can't enter liver fully by perfusion method in original body, liver cell preparation failure.Therefore the objective of the invention is to set up the interior perfusion method of body of laying hen liver, further seek high-quality laying hen liver cell isolation technique, provide more rational laying hen hepatocellular preparation method.
Summary of the invention
The solution of the present invention is the hepatocellular preparation method of a kind of laying hen, mainly comprises the interior perfusion of body and three processes of liver cell washing of laying hen vascular ligation, liver, and it is characterized in that: described laying hen vascular ligation adopts following (1)-(5) process:
(1) good laying hen is put into super clean bench will to sterilize anesthesia, and is middle initial from belly, pushes belly and skin of chest aside;
(2) the first root bone shears is disconnected along the breastbone both sides, exposes liver;
(3) open the abdominal cavity, gently enteron aisle is pulled to body left side, tubal ligation vein, pancreaticoduodenal veins;
(4) under vena mesenterica posterior, wear two cotton threads, ligation distal end cotton thread, the not ligation of proximal part cotton thread;
(5) between two cotton threads, vena mesenterica posterior is cut off an osculum, insert the mouse stomach pin, and fix filling stomach pin with the ligation of proximal part cotton thread along the hepatic vein direction with eye scissors.
The process of perfusion is in the body of described liver:
(1) with the aseptic perfusate A of 40 ℃ of preheatings of the speed of 30 milliliters of per minutes perfusion, treats to cut off when the liver bulging becomes khaki color superior vena cava, continue 20 minutes;
(2) with the aseptic perfusate B of 40 ℃ of preheatings of the speed of 30 milliliters of per minutes perfusion, continue 10 minutes;
(3) treat effusive liquid become clear after, with the aseptic perfusate C of 42 ℃ of preheatings of speed perfusion of 20 milliliters of per minutes, liver subsides gradually that digestion finishes after 15 minutes.
The process of described liver cell washing is:
(1) extracts the liver of irritating good, put into aseptic plate, add 50 milliliters of basic culture solutions, reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion;
(2) tear the liver coating with tweezers, liver cell can be flowed out in a large number, the heavy caliber suction pipe is blown and beaten gently, cross 100 orders (150 microns), 200 orders (75 microns) and 400 orders (30 microns) cell sieve subsequently respectively, the gained hepatocyte suspension cleans with basic culture solution, and with 500 rpms speed centrifugal 5 minutes, cleaning once the centrifugal supernatant of abandoning again with basic culture solution after abandoning supernatant;
(3) resuspended with adherent culture liquid, the trypan blue exclusion method detects cell viability, blood cell counting plate counting;
(4) after the cell counting, be 1 * 10 with the cell suspension dilution with adherent culture liquid
6Every milliliter in individual cell.
Contain among every liter of the above-described aseptic perfusate A: 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, 1.861 gram EDTA disodium salts, surplus is a deionized water, the pH value is 7.4.
Contain among every liter of the above-described aseptic perfusate B: 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, surplus is a deionized water, the pH value is 7.4.
Contain among every liter of the above-described aseptic perfusate C: 0.4g II Collagen Type VI enzyme, 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, 0.6 gram calcium chloride, surplus is a deionized water.
The present invention proposes three step perfusion methods in the body on the basis of existing isolated perfusion separation laying hen liver cell method, effectively raise the quantity and the motility rate of isolating hepatocytes.
Embodiment
For further specifying content of the present invention, below further describe by concrete scheme.
This programme is that example is separated the preparation liver cell with egg-laying peak laying hen liver 30 grams.
At first laying hen fasting 3 hours before operation, the anti-freezing of wing intravenous injection 4-5 milliliter heparin sodium, the anesthesia of abdominal injection 5-10 milliliter vetanarcol.After treating the chicken holonarcosis, with 38 ℃ of bromogeramine solution whole body soaking disinfections, the chicken that disinfects is put into super clean bench, with aseptic engineering line binding wing and leg, lying on the back is fixed on the operating table, middle initial from belly, pushes belly and skin of chest aside; The first root bone shears is disconnected along the breastbone both sides, exposes liver; Open the abdominal cavity, gently enteron aisle is pulled to body left side, tubal ligation vein, pancreaticoduodenal veins; Under vena mesenterica posterior, wear two cotton threads, ligation distal end cotton thread, the not ligation of proximal part cotton thread; Between two cotton threads, vena mesenterica posterior is cut off an osculum, insert the mouse stomach pin, and fix filling stomach pin with the ligation of proximal part cotton thread along the hepatic vein direction with eye scissors.Pour into the aseptic perfusate A of 40 ℃ of preheatings with the speed of 30 milliliters of per minutes, treat to cut off when the liver bulging becomes khaki color superior vena cava, continue 20 minutes; Pour into the aseptic perfusate B of 40 ℃ of preheatings with the speed of 30 milliliters of per minutes, continue 10 minutes; After treating that effusive liquid becomes clearly, pour into the aseptic perfusate C of 42 ℃ of preheatings with the speed of 20 milliliters of per minutes, liver subsides to digest gradually and finishes after 15 minutes.Extract the liver of irritating good, put into aseptic plate, add 50 milliliters of basic culture solutions, reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion; Tear the liver coating with tweezers, liver cell can be flowed out in a large number, the heavy caliber suction pipe is blown and beaten gently, cross 100 orders (150 microns), 200 orders (75 microns) and 400 orders (30 microns) cell sieve subsequently respectively, the gained hepatocyte suspension cleans with basic culture solution, and with 500 rpms speed centrifugal 5 minutes, cleaning once the centrifugal supernatant of abandoning again with basic culture solution after abandoning supernatant; Resuspended with adherent culture liquid, the trypan blue exclusion method detects cell viability, blood cell counting plate counting; After the cell counting, be 1 * 10 with the cell suspension dilution with adherent culture liquid
6Every milliliter in individual cell.
The liver cell of the present invention's preparation is compared as shown in the table with the liver cell of existing isolated perfusion method preparation:
| Fresh separated hepatocyte function state | Present method | The isolated perfusion method |
| Liver cell motility rate (%) | 95-99 | 80-87 |
| The liver cell sum that obtains (individual/milliliter) | 2.05×10 9 | 1.97×10 9 |
As can be seen from the above table, the liver cell that perfusion method obtains in the body of the present invention significantly is better than prior art at motility rate, quantitative aspects.The liver cell of the present invention's preparation can be widely used in hepatocellular research of bird and bird lipid metabolism research.
Claims (3)
1. the hepatocellular preparation method of laying hen comprises that the interior perfusion of body and the liver cell of laying hen vascular ligation, liver washed three processes, and it is characterized in that: described laying hen vascular ligation adopts following (1)-(5) process:
(1) good laying hen is put into super clean bench will to sterilize anesthesia, and is middle initial from belly, pushes belly and skin of chest aside;
(2) the first root bone shears is disconnected along the breastbone both sides, exposes liver;
(3) open the abdominal cavity, gently enteron aisle is pulled to body left side, tubal ligation vein, pancreaticoduodenal veins;
(4) under vena mesenterica posterior, wear two aseptic cotton threads, ligation distal end cotton thread, the not ligation of proximal part cotton thread;
(5) between two cotton threads, vena mesenterica posterior is cut off an osculum, insert aseptic mouse stomach pin, and fix filling stomach pin with the ligation of proximal part cotton thread along the hepatic vein direction with eye scissors.
2. by the hepatocellular preparation method of the described laying hen of claim 1, it is characterized in that: the process of perfusion is in the body of described liver:
(1) with the aseptic perfusate A of 40 ℃ of preheatings of the speed of 30 milliliters of per minutes perfusion, treats to cut off when the liver bulging becomes khaki color superior vena cava, continue 20 minutes; Contain among every liter of the wherein aseptic perfusate A: 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, 1.861 gram EDTA disodium salts, surplus is a deionized water, the pH value is 7.4;
(2) with the aseptic perfusate B of 40 ℃ of preheatings of the speed of 30 milliliters of per minutes perfusion, continue 10 minutes; Contain among every liter of the wherein aseptic perfusate B: 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, surplus is a deionized water, the pH value is 7.4;
(3) treat effusive liquid become clear after, with the aseptic perfusate C of 42 ℃ of preheatings of speed perfusion of 20 milliliters of per minutes, liver subsides gradually that digestion finishes after 15 minutes; Contain among every liter of the wherein aseptic perfusate C: 0.4g II Collagen Type VI enzyme, 2.383 gram HEPES, 8.006 gram sodium-chlor, 0.224 gram Repone K, 1.074 gram disodium hydrogen phosphate dodecahydrates, 0.6 gram calcium chloride, surplus is a deionized water.
3. by the hepatocellular preparation method of the described laying hen of claim 1, it is characterized in that: described liver cell washing process is:
(1) extracts the liver of irritating good, put into aseptic plate, add 50 milliliters of basic culture solutions, reject blood vessel, fat and reticular tissue, cut off the incomplete part of liver periphery digestion;
(2) tear the liver coating with tweezers, liver cell can be flowed out in a large number, the heavy caliber suction pipe is blown and beaten gently, cross 100 orders, 200 orders and 400 order cells sieve subsequently respectively, the gained hepatocyte suspension cleans with basic culture solution, and with 500 rpms speed centrifugal 5 minutes, clean once the centrifugal supernatant of abandoning again with basic culture solution after abandoning supernatant;
(3) resuspended with adherent culture liquid, the trypan blue exclusion method detects cell viability, blood cell counting plate counting;
(4) after the cell counting, be 1 * 10 with the cell suspension dilution with adherent culture liquid
6Every milliliter in individual cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010528886 CN101988048A (en) | 2010-11-03 | 2010-11-03 | Preparation method of liver cells of laying hens |
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| CN 201010528886 CN101988048A (en) | 2010-11-03 | 2010-11-03 | Preparation method of liver cells of laying hens |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525756A (en) * | 2013-10-23 | 2014-01-22 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
| CN109182247A (en) * | 2018-09-04 | 2019-01-11 | 江苏省家禽科学研究所 | A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model |
-
2010
- 2010-11-03 CN CN 201010528886 patent/CN101988048A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525756A (en) * | 2013-10-23 | 2014-01-22 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN103525756B (en) * | 2013-10-23 | 2015-04-08 | 南京农业大学 | Method for separating and culturing primary chicken hepatocytes |
| CN104630130A (en) * | 2014-12-05 | 2015-05-20 | 新乡医学院 | Method for separating rat hepatocytes |
| CN109182247A (en) * | 2018-09-04 | 2019-01-11 | 江苏省家禽科学研究所 | A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model |
| CN109182247B (en) * | 2018-09-04 | 2021-06-04 | 江苏省家禽科学研究所 | In-vitro isolation culture and steatosis model establishment method for chick embryo hepatocytes |
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Application publication date: 20110323 |