CN104027810B - A kind of method building alagille syndrome animal model - Google Patents
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Abstract
The invention discloses a kind of method building alagille syndrome animal model, solve prior art comparatively complicated, costly, inefficient problem.The present invention includes following steps: (1) preparation inhibitors of gamma-secretase corn oil and inhibitors of gamma-secretase phosphate buffer; (2) adult male and female rat is mated, timing from female Mus is conceived, when female pregnant mouse embryo 14 days, to each amniotic cavity injection solution first; Meanwhile, to pregnant tail vein injection solution second; (3) the pregnant Mus of conventional raising produces to it, and tire Mus is alagille syndrome animal model.The present invention is conducive to the research carrying out alagille syndrome, especially the research and development of pathophysiological mechanism research and medicine.
Description
Technical field
The present invention relates to a kind of animal model constructing method of disease, specifically, is a kind of method building alagille syndrome animal model.
Background technology
Congenital diseases are exactly the disease had being born.Mother is at phenolics contact environment harmful factor, as chemicals such as pesticide, organic solvent, heavy metals, or excessive be exposed to various ray under, or take some drugs, or catch some pathogenic bacteria, even some custom hobbies, as Saunas (steam bath) and diet fads, all may cause fetus congenital anomaly, but not belong to genetic diseases.The internal organs that congenital biliary dysplasia syndrome relates to comprise liver, heart, skeleton, eyes and face etc., this disease of external report is about 1/70000 at neonatal sickness rate, therefore the research of its pathophysiological mechanism is particularly important, and the animal model successfully setting up this disease can provide Research foundation for the prevention and therapy of this disease.Gene Knock-Out Animal Model is at present extensively by the method adopted.But in current most of laboratory, be difficult to have ready conditions and set up this animal model, this method is also because its technical sophistication, costly, efficiency is low, is not therefore the best method setting up this animal model in addition.If use additive method induction in addition, can, because a lot of medicine cannot make drug effect reduce by blood vessel placental barrier, induced efficiency be declined.
Summary of the invention
The object of the present invention is to provide a kind of method building alagille syndrome animal model, solve prior art comparatively complicated, costly, inefficient problem.
The present invention is achieved through the following technical solutions:
Build a method for alagille syndrome animal model, comprise the following steps:
(1) inhibitors of gamma-secretase is dissolved in Semen Maydis oil, is mixed with the solution first that concentration is 10mg/ml and (namely refers to that the concentration of inhibitors of gamma-secretase in solution first is 10mg/ml; Such as: containing 10mg inhibitors of gamma-secretase in the solution first of 1ml); Inhibitors of gamma-secretase is dissolved in dimethyl sulfoxide, use phosphate buffer (phosphate buffer adopts concentration to be the phosphate buffer of 0.01M, and concrete concentration is then 0.01mol/L) to be diluted to the solution second that concentration is 5mg/ml again and (namely refer to that the concentration of inhibitors of gamma-secretase in solution second is 5mg/ml; Such as, containing 5mg inhibitors of gamma-secretase in the solution second of 1ml);
(2) adult male and female rat is mated, timing from female Mus is conceived, when female pregnant mouse embryo 14 days, to each amniotic cavity injection solution first; Meanwhile, to pregnant tail vein injection solution second;
(3) the pregnant Mus of conventional raising produces to it, and tire Mus is alagille syndrome animal model.
Further, the injection rate of solution first and solution second is all determine according to pregnant Mus ABW, and solution first is expelled in each amniotic cavity according to the dosage of 5mg/kg, and solution second is expelled in pregnant caudal vein according to the dosage of 1mg/kg.Wherein " 5mg/kg " is if refer to that pregnant Mus body weight is 1kg, so need the inhibitors of gamma-secretase injecting 5mg, that solution first actual amount is then 0.5ml, so the 5mg in " 5mg/kg " measures the amount of the inhibitors of gamma-secretase referred to, instead of the amount of solution first; " 1mg/kg ", if refer to that pregnant Mus body weight is 1kg, so need the inhibitors of gamma-secretase injecting 1mg, so solution second actual amount is then 0.2ml, so the 1mg amount in " 1mg/kg " refers to the amount of inhibitors of gamma-secretase, instead of the amount of solution second.
Again further, the method that described step (2) is concrete was: loaded in same cage by adult male and female Mus and make female Mus conceived, then timing from female Mus is conceived, when female pregnant mouse embryo 14 days, puncture pregnant Mus amniotic cavity under ultrasound guidance, to each intra-amniotic injection solution first; Meanwhile, puncture pregnant caudal vein under ultrasound guidance, injection solution second.
In addition, inhibitors of gamma-secretase of the present invention is the one in DAPT, LY-450139, MK-0752, E2012, BMS-708163, PF-3084014, GSI-953.
The present invention has the following advantages and beneficial effect:
The present invention is conducive to the research carrying out alagille syndrome, especially the research and development of pathophysiological mechanism research and medicine; And the present invention relative to existing technologies, be easy to operation, expense is low, efficiency is high, is applicable to applying.
Accompanying drawing explanation
Fig. 1 is Jagged1 immunohistochemical staining results contrast figure.
Fig. 2 is tire Mus serum bilirubin level results contrast figure.
Fig. 3 is pulmonary artery root internal diameter comparison diagram.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated, but embodiments of the present invention are not limited to this.
Embodiment
Build a method for alagille syndrome animal model, comprise the following steps:
(1) inhibitors of gamma-secretase is dissolved in Semen Maydis oil, is mixed with the solution first that concentration is 10mg/ml; Inhibitors of gamma-secretase is dissolved in dimethyl sulfoxide, then is diluted to phosphate buffer the solution second that concentration is 5mg/ml;
(2) loaded in same cage by adult male and female Mus and make female Mus conceived, then timing from female Mus is conceived, when female pregnant mouse embryo 14 days, punctured pregnant Mus amniotic cavity under ultrasound guidance, to each intra-amniotic injection solution first; Meanwhile, puncture pregnant caudal vein under ultrasound guidance, injection solution second;
(3) the pregnant Mus of postoperative routine raising produces to it, and tire Mus is alagille syndrome animal model.
Wherein, the injection rate of solution first and solution second is all determine according to pregnant Mus ABW, and solution first is expelled in each amniotic cavity according to the dosage of 5mg/kg, and solution second is expelled in pregnant caudal vein according to the dosage of 1mg/kg.
Why inhibitors of gamma-secretase is dissolved in Semen Maydis oil by the present invention, is fat-soluble in order to increase, and is conducive to medicine absorption in vivo, running.The present invention utilizes ultrasonic guidance accurately can navigate to each amniotic cavity to carry out accurate injection, reduces the medicine degraded that causes of metabolism in vivo; In addition, being aided with the administration of tail Intravenous Low-dose simultaneously, controlling the concentration of inhibitors of gamma-secretase in pregnant Mus body, while reducing the mortality rate of pregnant Mus, not weakening the effect that tire Mus congenital stones in intrahepatic bile duct dysplasia model is set up.In addition, due to the incipient stage that embryo is liver stem cells and breaks up to bile duct epithelial cell for 14 days, the effective generation suppressing bile duct epithelial cell of Notch signal path ability is blocked in this stage, finally cause tire Mus generation alagille syndrome, so the present invention selects within 14 days, to carry out drug injection embryo.
As follows to the tissue specimen processing method such as liver, heart, serum of the animal model (tire Mus) that the present invention builds:
1, immunohistochemical method detects the expression (testing result is as Fig. 1) of Jagged1:
(1) use wax embedding machine routine embedding hepatic tissue, embedded rear use paraffin slicing machine and cut into slices to wax stone tissue, paraffin section thickness is 1 μm.
(2) dewax/dehydration: paraffin section is put into dimethylbenzene 5min by A., also can distilled water rinsing (5min, 2 times) after taking-up, and aforesaid operations carries out 3 times altogether repeatedly; B. the tissue slice of dimethylbenzene process is put into dehydrated alcohol 10min, with distilled water rinsing (5min, 2 times) after taking out, aforesaid operations carries out 2 times altogether repeatedly; C. the tissue slice of dehydrated alcohol process is put into 95% ethanol 10min, with distilled water rinsing (5min, 2 times) after taking out, aforesaid operations carries out 2 times repeatedly.
(3) antigen retrieval: the tissue slice completing dewaxing/dehydration is put into the citric acid antigen repair liquid boiled, keeps antigen retrieval buffers to be in critical state (the not making liquid boiling) 10min reaching boiling point, takes out tissue slice and at room temperature cools 30min.
(4) PBS(phosphate buffer is used) tissue slice (5min, 3 times) after rinsing antigen retrieval, immerses 3%H by tissue slice
2o
2middle 10min.
(5) PBS(phosphate buffer is used) rinsing tissue slice (5min, 3 times), used by tissue slice confining liquid at room temperature to close 1h.
(6) discard confining liquid, by the dilution proportion primary antibodie of antibody diluent according to little mouse-anti Jagged1 antibody (1:50, Bo Aosen, Beijing), the primary antibodie after dilution is added on tissue slice, tissue slice is put into wet box, 4 DEG C of overnight incubation (20 ~ 24h).
(7) discard the primary antibodie that tissue slice is hatched, use PBS(phosphate buffer) rinsing tissue slice (5min, 3 times), add respectively in immunohistochemical kit (middle China fir, Beijing) with primary antibodie originate kind corresponding two resist, incubated at room 30min.
(8) discard tissue slice is hatched two resist, use PBS(phosphate buffer) rinsing tissue slice (5min, 3 times), add immunohistochemical kit three resist, incubated at room 15min.
(9) discard three resisting that tissue slice is hatched, use DAB colour reagent box (middle shirt, China) to carry out DAB colour developing (2 ~ 4min), developed the color rear distilled water rinsing tissue slice.
(10) haematoxylin is used to carry out lining dye to tissue slice, with distilled water rinsing tissue slice (5min, 3 times) after lining dye completes.
(11) dewater: 95% ethanol 10min put into by tissue slice by A., with distilled water rinsing (10s, 2 times) after taking out, aforesaid operations carries out 2 times repeatedly; B. the tissue slice that 95% Ethanol Treatment is crossed is put into dehydrated alcohol 10min, with distilled water rinsing (10s, 2 times) after taking out, aforesaid operations carries out 2 times altogether repeatedly; C. the tissue slice of dehydrated alcohol process is put into dimethylbenzene 5min, with distilled water rinsing (10s, 2 times) after taking out, aforesaid operations carries out 2 times altogether repeatedly.
(12) the tissue slice resinene completing dehydration is carried out mounting, after mounting, room temperature is dried under 24h is placed on microscope and is carried out observing and taking pictures.
As can be seen from Figure 1, in model group liver, obviously more than matched group, (all matched groups in the application are all show the Semen Maydis oil of pregnant Mus injection of amnion equal-volume not containing inhibitors of gamma-secretase to biliary system, simultaneously at the phosphate buffer of tail intravenous injection equal-volume not containing inhibitors of gamma-secretase, thus the tire Mus obtained), Jagged1 disappearance can cause congenital stones in intrahepatic bile duct dysplasia simultaneously, immunohistochemical staining is carried out to Jagged1, Jagged1 on the cell membrane of result display model group expresses (brown particle) significantly lower than matched group, pathology meets stones in intrahepatic bile duct dysplasia.Wherein brown is that immunohistochemistry dyes.
2, two groups of tire Mus serum bilirubin levels results contrast (testing result is as Fig. 2):
(1) after tire Mus ether general anaesthesia.Be fixed on operating-table, iodine tincture disinfection skin of abdomen, after 75% de-iodine, keep aseptic condition to open tire Mus abdominal cavity next time, before leaving and taking liver, heart preparation, first use scalp needle puncture ventral aorta, extract whole blood 5ml;
(2) by whole blood centrifugal 10min under 4 DEG C, 4000 revs/min, draw upper serum after layering, automatic clinical chemistry analyzer (COBAs400, Roche Holding Ag, Germany) detects total bilirubin, bilirubin direct, unconjugated bilirubin in serum.
As can be seen from Figure 2, namely there is cholestasis symptom after the model group tire Mus birth that the present invention builds, meet the hypogenetic clinical manifestation of stones in intrahepatic bile duct.
3, vernier caliper measurement pulmonary artery internal diameter (testing result is as Fig. 3): vernier caliper measurement pulmonary artery and heart junction (i.e. pulmonary artery root) internal diameter.
As can be seen from Figure 3, the model group tire Mus pulmonary artery root internal diameter that the present invention builds significantly is less than matched group, the remarkable constriction of model group pulmonary artery root, meets the hypogenetic Cardiovasculai appearance in patient of congenital stones in intrahepatic bile duct.
According to above-described embodiment, just the present invention can be realized well.What deserves to be explained is; under prerequisite based on above-mentioned design, for solving same technical problem, even if some making on the invention are without substantial change or polishing; the essence of the technical scheme adopted is still the same with the present invention, therefore it also should in protection scope of the present invention.
Claims (2)
1. build a method for alagille syndrome animal model, it is characterized in that, comprise the following steps:
(1) inhibitors of gamma-secretase is dissolved in Semen Maydis oil, is mixed with the solution first that concentration is 10mg/ml; Inhibitors of gamma-secretase is dissolved in dimethyl sulfoxide, then is diluted to phosphate buffer the solution second that concentration is 5mg/ml;
(2) adult male and female rat is mated, timing from female Mus is conceived, when female pregnant mouse embryo 14 days, to each amniotic cavity injection solution first; Meanwhile, to pregnant tail vein injection solution second;
(3) the pregnant Mus of conventional raising produces to it, and tire Mus is alagille syndrome animal model;
Wherein, the injection rate of solution first and solution second is all determine according to pregnant Mus ABW, and solution first is expelled in each amniotic cavity according to the dosage of 5mg/kg, and solution second is expelled in pregnant caudal vein according to the dosage of 1mg/kg.
2. a kind of method building alagille syndrome animal model according to claim 1, it is characterized in that, the method that described step (2) is concrete is: loaded in same cage by adult male and female Mus and make female Mus conceived, timing from female Mus is conceived again, when female pregnant mouse embryo 14 days, puncture pregnant Mus amniotic cavity under ultrasound guidance, to each intra-amniotic injection solution first; Meanwhile, puncture pregnant caudal vein under ultrasound guidance, injection solution second.
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Citations (2)
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|---|---|---|---|---|
| CN1630651A (en) * | 2001-08-03 | 2005-06-22 | 先灵公司 | Novel gamma secretase inhibitors |
| CN103237888A (en) * | 2010-07-29 | 2013-08-07 | 荷兰皇家科学院 | Liver organoid, uses thereof and culture method for obtaining them |
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