CN102697812B - Method for extracting exosome from chicken bile and application of exosome in immunology - Google Patents
Method for extracting exosome from chicken bile and application of exosome in immunology Download PDFInfo
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Abstract
The invention belongs to the field of molecular immunology, and in particular relates to an exosome secreted and released from the epidermal cells of a chicken liver, gall bladder and bile duct, and a preparation method of the exosome. The exosome contains hepatogenic proteins, such as C-reactive protein, aspartate aminotransferase, and vitronectin, epithelium-derived mucin and proteins related to an immune reaction, such as immunoglobulin, and can be taken as a biological indicator for studying liver diseases and as an adjuvant for immune reaction for immune-regulation, and can also be taken as a carrier for treating the diseases of livers, gall bladders and intestines.
Description
Technical field
The present invention relates to immunology, be specifically related to a kind of method of the born of the same parents' ectosome that from Fel Gallus domesticus, extracts and the application on immunology thereof.
Background technology
Born of the same parents' ectosome claims " external source body " (Exosomes) again, is a kind of vesicles that derives from the diameter 20-170nm of endosome in late period.1987, last maturation of Johnstone research reticulocyte was in the erythrocytic experiment, will be at the vesicles called after born of the same parents ectosome of the reticulocyte release of the sheep of In vitro culture.Raposo in 1996 etc. find the outer physical ability inducing antigen-specific T lymphocyte reaction of the born of the same parents of B emiocytosis, and Zitvogel etc. successfully induce CD8 with dendritic cell source born of the same parents' ectosome
+The Graft Versus Tumor that the performance of T lymphocyte is very strong, Recent study person has given concern highly to the immunization of born of the same parents' ectosome, becomes the focus of acellular tumor vaccine research.
The source of born of the same parents' ectosome and feature: except reticulocyte, various kinds of cell is all secreted born of the same parents' ectosome, as B cell (Raposo G, 1996), mastocyte (Skokos D, 2001), immature dendritic cell (ne) (Thery C, 2001), platelet, fibroblast, epithelial cell (Wolfers J, 2001), tumor cell, T lymphocyte (Blanchard N, 2002) etc.The molecular composition of born of the same parents' ectosome in various cells source is not quite similar, but generally speaking, born of the same parents' ectosome size of dissimilar emiocytosises is all at 20-170nm, buoyant density is 1.12-1.21g/mL(Smalheiser, 2007), mainly contain following several material: main tissue compatible complex (major histocompatibility complex I, MHC I) class and/or class, heat shock protein, integration element, four transmembrane proteins, cytoskeletal protein and some biological enzymes.
At the beginning of the discovery of born of the same parents' ectosome, it is believed that the effect of born of the same parents' ectosome just removes useless albumen in the erythrocyte maturation, but it seems that now its function is more complicated.The biological function of every kind of born of the same parents' ectosome and its source are closely related.Basis at big quantity research finds that the main effect of born of the same parents' ectosome comprises the following aspects: remove useless albumen; Media as the iuntercellular interchange; Inducing immune tolerance; In immunomodulating, play a significant role, comprise immune activation or immunosuppressant.Born of the same parents' ectosome has easy absorption as from the body composition, advantages such as no rejection, and the carrier as the kinds of tumors medicine is applied in the research of oncotherapy at present.
Born of the same parents' ectosome in the bile: bile is a kind of special body fluid that flows in biliary tract, is produced by hepatic secretion.The generative process of bile is very complicated, and bile is generated by about 75% hepatocyte, and 25% by bile duct cell and the generation of gallbladder epithelial cell.Bile has two effects greatly, and the one, as Digestive system, help fat in digestion and the absorption of enteral; The 2nd, some metabolite is discharged from liver.Fel Gallus domesticus is a kind of aboundresources, extensive, the cheap natural Chinese medicine of effect.Fel Gallus domesticus nature and flavor bitter cold, nontoxic, have heat-clearing and toxic substances removing, relieving cough and asthma, antiinflammatory, function of gallbladder promoting function such as make eye bright.Born of the same parents' ectosome in the bile is by the albumen vesicle that comes together in gallbladder after hepatocyte, epithelial cell secretion and the release, finally is discharged in the small intestinal.Because the surface carries a large amount of antigenic peptides and lymphocyte costimulating factor etc., in body, the born of the same parents' ectosome in the bile plays an important role to stable state and the liver immunoreation of keeping intestinal tract immune system.Born of the same parents' ectosome has easy absorption as from the body composition, advantages such as no rejection, and the carrier as the kinds of tumors medicine is applied in the research of oncotherapy at present.Born of the same parents' ectosome in the bile can orientation in liver and intestinal, can be used as the important tool for the treatment of liver and intestinal tract disease.
The research of present stage and application prospect: at present, the application experiment of the born of the same parents' ectosome that carries out on the muroid has been obtained abundant achievement in research, and provides good reference for the research of people's medicine.At people hospital face, mature dendritic cell secretion born of the same parents ectosome since its effectively the medicine studied on its basis of induction of immunity activating reaction entered the second stage of clinical stage; Born of the same parents' ectosome is as the preparation of carrier and be applied in China and applied for a patent (CN200510082604) in 2005.
The research of fowl source bile born of the same parents ectosome does not at home and abroad appear in the newspapers.But fowl source bile source is abundant, effect is extensive, cheap, has functions such as heat-clearing and toxic substances removing, relieving cough and asthma, antiinflammatory, function of gallbladder promoting make eye bright, and Chinese medicine is listed natural Chinese medicine in China.
If the born of the same parents' ectosome in the Fel Gallus domesticus is applied in the treatment of neoplastic disease of fowl, may be able to change neoplastic disease in case infect the present situation that just must eliminate, this will be the Gospel of aviculture; As anti-stress medicine, can effectively prevent from transporting for long-distance etc. because the gastroenteropathy that stress cause will be retrieved a large amount of losses for aquaculture.Moreover, the animal model in chicken Chang Zuowei tumor, tremulous pulse medicated porridge sample hard generation, the achievement in research of fowl source bile is for the very high reference value of people doctor.
Summary of the invention
In order to improve the extraction efficiency of born of the same parents' ectosome in the Fel Gallus domesticus, and the application of expansion born of the same parents' ectosome on immunology, it is a kind of new for chicken liver that the present inventor provides, gallbladder and bile duct epithelial cell secretion and the born of the same parents' ectosome that discharges, and the method for its preparation is disclosed, contain C-reactive protein (C-reactive protein) in this born of the same parents' ectosome, aspartate aminotransferase (Aspartate aminotransferase), the protein ingredient of vitronectin liver source property such as (Vitronectin), the mucin (Mucin) of epithelial cell source property and immunoglobulin immunoreation associated protein such as (Immunoglobulin), can be used as research hepatic disease Biological indicators and as the immunoreation adjuvant, participate in immunomodulating, can also be applied to treat liver as carrier, the treatment of gallbladder and intestinal tract disease.
The invention provides the bilayer lipid membrane that born of the same parents' ectosome has typical cup-like structure, contain the protein ingredient of liver source property, the protein ingredient of described liver source property is C-reactive protein or aspartate aminotransferase or vitronectin or its mixture; Described born of the same parents' ectosome is the composition of normal presence in the bile, by liver (accounting for 75%), bile duct and gallbladder epithelial cell (accounting for 25%) secretion and release.And this bile is all obtainable bile of birdss such as the broiler, laying hen of each strain.
The present inventor discovers that to fowl source born of the same parents' ectosome protein component its surface carries multiple unique albumen, and C-reactive protein is wherein produced by hepatocyte, is acute phase reactive protein, and is significant as the sign of inflammatory reaction; Vitronectin, its expression difference Chang Zuowei judges the standard of hepatocarcinoma, shows that fowl source born of the same parents' ectosome can be used as judgement; Mucin is produced by epithelial cell, plays the multiple functions such as conduction of lubricated and cell signal, can form chemical barrier; The existence of a large amount of immunoglobulins has illustrated that born of the same parents' ectosome has important function in the transmission of immunne response information.
The born of the same parents' ectosome diameter that obtains is at 30-150nm, and the mass density scope is 1.13-1.19g/cm
3
The antigenic peptides of presenting MHC I molecule or can be combined with MHC I molecule on the outer surface of these born of the same parents, or have the lymphocyte costimulatory molecules, or the above-mentioned substance coexistence.
Wherein above-mentioned MHC I molecule has positive correlation in immunoreation, all be as compared with the past to present MHC II dependency to have remarkable difference, and therefore using in immunology has brand-new breakthrough.
Prepare the method for above-mentioned born of the same parents' ectosome, concrete steps are as follows:
Will be from the bile that extracts in the healthy chicken gallbladder exsomatizes 4h, under 4 ℃ of cryogenic conditions, get supernatant through the centrifugal cell debris that goes step by step, through membrane filtration, 4 ℃ 110,000g is centrifugal, and the 2h precipitation is resuspended with PBS, 4 ℃ 110 then, use the PBS dissolution precipitation behind the centrifugal 90min of 000g, be born of the same parents' ectosome of extraction.
Wherein said centrifugal step by step be 1000～2000g, 15～20min, 3000～4000g, 20～30min, 7000～8000g, 45～50min, 10000～15000g, 40～60min.
Further preferred employing is centrifugal step by step is 2000g15min, 4000g30min, 8000g45min, 15000g 60min.
In order to reach better effect, described through 4 ℃ 110, the precipitation behind the centrifugal 90min of 000g is carried out density gradient centrifugation by the sucrose solution of 30wt%, 45wt%, 60wt%, so just can further isolate born of the same parents' ectosome, improve born of the same parents' ectosome all once.
Concrete grammar is prior art, can adopt in a centrifuge tube and form 3 layers of saccharose gradient that density is not unison by prior art, be 60wt%, 45wt%, 30wt% from top to bottom successively, the superiors are sample precipitations, sample layer separates after centrifugal, penetrate into its density place layer, realize the different separation according to density.
Adopt above-mentioned fractional centrifugation and filter the preparation method that combines, can effectively remove residue and macromolecular substances in the bile, the method for more single centrifugal or filtration, more feasible.There is following superiority:
1. leaching process weak point consuming time is easy to keep the biological activity of born of the same parents' ectosome, and it is good, all once higher that form keeps.
2. the output height of born of the same parents' ectosome, 1ml bile can Fen Li Dao ≧ 14 μ g born of the same parents ectosomes.
In addition, low through its big or small uniformity of product after fractional centrifugation of the present invention and the filtration, may contain some many cell spaces, mainly comprise big vesicle or parcel vesicle (MVB), a plurality of born of the same parents' ectosome individualities may be contained in its inside, for above-mentioned many cell spaces are further gone out, and reach the single vesicle of we required standard 30-150nm, the inventor again in good time carrying out density gradient centrifugation, thereby guarantee the final born of the same parents' ectosome that obtains all once.
Born of the same parents' ectosome by said method obtains except possessing normal born of the same parents' ectosome function, also has following effect, can better application be arranged in immunology and materia medica:
This kind born of the same parents ectosome entrained the variation of special albumen can be used as the Biological indicators of research hepatic disease.Described born of the same parents are contained albumen such as C-reactive protein and aspartate aminotransferase in outer surface.The C-reactive protein is synthetic by hepatocyte, is first phase reaction albumen when being considered to acute, and its blood concentration sharply raises at acute injury and when infecting.Whether aspartate aminotransferase is in the liver function test, generally be used for checking hepatic tissue impaired.Therefore, the composition of entrained special albumen changes the sign can be used as the liver physiology changes of function and is used for detecting hepatic disease on born of the same parents' ectosome.
Application in the immunoreation in vivo and in vitro of this kind born of the same parents ectosome.By discovering, the born of the same parents' ectosome in the bile can specific stimulation CD4
+The lymphocytic increment of T, and present MHC I positive correlation.CD4
+T can promote the maturation of other T cell subsets and B cell to produce antibody, and therefore, the born of the same parents' ectosome in the bile can be by making CD4
+The increment effect of T activates the immunoreation of inside and outside.Perhaps as the immunoreation adjuvant, participate in immunomodulating, as the application of anti-stress class medicine.
Described born of the same parents' ectosome can also be applied to treat the treatment of liver, gallbladder and intestinal tract disease, particularly neoplastic disease as carrier.Born of the same parents' ectosome in the bile has the advantage of easy absorption, nothing repulsion etc. as from the body composition; Because its lipid bilayer membrane structure can be protected medicine, reduce effective ingredient and lose; Born of the same parents' ectosome orientation in the bile is in liver or IL, and this special targeting makes the medicine can orientation.In addition, the content height of the external source body in the bile (more than the 14 μ g/ml), all once good, have a good application prospect as pharmaceutical carrier.
In sum, the extracting method of the born of the same parents' ectosome that the invention provides the secretion of chicken liver cell and epithelial cell and discharge, and method and the application thereof of its preparation are disclosed, for the further research of born of the same parents' ectosome and the application on immunology and materia medica thereof provide new prospect and technology enlightenment.
Description of drawings
Fig. 1 is the transmission electron microscope photo of Fel Gallus domesticus born of the same parents ectosome;
The outer volume morphing of the born of the same parents of Fel Gallus domesticus shown in the arrow is the vesicles with typical cup-like structure of diameter 30-150nm among the figure;
Fig. 2 is SPF chicken liver ultrathin section photo;
The A arrow is depicted as born of the same parents' ectosome of liver Di Shi intracavity among the figure;
The B arrow is depicted as the born of the same parents' ectosome in the little bile duct of liver among the figure;
The C arrow is depicted as many cell spaces and the oozy born of the same parents' ectosome in the hepatocyte among the figure;
D is depicted as the bile duct microvillus among the figure, and the secretion that arrow is depicted as bile duct microvillus epithelial cell born of the same parents ectosome discharges, and has born of the same parents' ectosome to secrete by the microvillus edge;
Fig. 3 is SDS-PAGE result and the high performance liquid chromatogram purification figure of born of the same parents' ectosome in the Fel Gallus domesticus;
A is depicted as the SDS-PAGE result of born of the same parents' ectosome in the Fel Gallus domesticus among the figure;
B is depicted as the high performance liquid chromatogram purification figure of born of the same parents' ectosome in the Fel Gallus domesticus among the figure;
Fig. 4 identifies the major protein kind tabulation of bile born of the same parents ectosome for mass spectral analysis;
Fig. 5 detects surface marker (MHC I/MHC II) the fluidic cell figure of born of the same parents' ectosome in the Fel Gallus domesticus for magnetic bead;
Among the figure, the born of the same parents' ectosome in the bile only connects magnetic bead and shows as figure A, and it is relevant that born of the same parents' ectosome of 95.42% presents the MHC I, and only having 3.78%, to show the MHC II relevant, and therefore, the born of the same parents' ectosome in the bile mainly carries MHC I correlation molecule;
Fig. 6 is the leukocytic fluidic cell figure of born of the same parents' ectosome inoculation liver in the Fel Gallus domesticus;
A is the result of flow cytometry among the figure, behind the statistical analysis as can be known in due course between and appropriate dosage under, the born of the same parents' ectosome in the bile is to liver CD4
+The differentiation of T cell has tangible activation (shown in B), and presents MHC I dependency (shown in C), and visible born of the same parents' ectosome provided by the present invention has tangible CD4
+The T proliferation function.
Fig. 7 is born of the same parents' ectosome inoculation spleen, the leukocytic result of thymus in the Fel Gallus domesticus,
The effect of born of the same parents' ectosome inoculation spleen, thymocyte proliferation is not remarkable in the Fel Gallus domesticus as seen from the figure, (statistical result such as Fig. 7 A﹠amp; Shown in the B), can improve spleen CD4 and add born of the same parents' ectosome
+T, CD8
+The survival rate of T and mononuclear cell and MHC I class cell prolongs the time-to-live of cell (as Fig. 7 C﹠amp; Shown in the D), visible born of the same parents' ectosome provided by the present invention has the survival rate of tangible raising cell and the effect that prolongs the time-to-live.
The specific embodiment:
Test method among the following embodiment if no special instructions, is conventional method.
The separation and purification of born of the same parents' ectosome in embodiment 1, the Fel Gallus domesticus
1. the extraction of born of the same parents' ectosome in the Fel Gallus domesticus: in SPF Fel Gallus domesticus capsule exsomatizes 4h, extract bile, with PBS buffer equivalent mixing, under 4 ℃ of cryogenic conditions through the centrifugal 15min of 2000g, the centrifugal 30min of 4000g, the centrifugal 45min of 8000g, the centrifugal 60min of 15000g removes cell debris, get supernatant and (pass through double-deck filter paper successively through membrane filtration, 5 μ m filter membranes, 1.2 μ m filter membranes, 0.22 μ m filter membrane), 4 ℃ 110, the centrifugal 2h of 000g, precipitation is resuspended with PBS, 4 ℃ 110 then, use the PBS dissolution precipitation behind the centrifugal 90min of 000g, be born of the same parents' ectosome of extraction.
In this method, 4 ℃ 110, the precipitation behind the centrifugal 90min of 000g can further be carried out density gradient centrifugation by 30wt%, 45wt%, 60wt% sucrose solution and further separated, improve born of the same parents' ectosome all once.Concrete grammar is: adopt in a centrifuge tube and form 3 layers of saccharose gradient that density is not unison by prior art, be 60wt%, 45wt%, 30wt% from top to bottom successively, the superiors are sample precipitations, sample layer separates after centrifugal, penetrate into its density place layer, can realize the different separation according to density.
2. protein quantification (BCA method): adopt BCA determination of protein concentration test kit: add an amount of BCA working solution of 1 volume BCA reagent B (50: 1) preparation, fully mixing by 50 volume BCA reagent A; The soluble protein standard substance are got 10 μ L and are diluted to 100 μ L with PBS fully, and making final concentration is 0.5mg/mL; Standard substance are added in the standard substance hole of 96 orifice plates by 0,1,2,4,8,12,16 and 20 μ L, add the standard substance diluent and supply 20 μ L; It is aerial to the sample of 96 orifice plates to add 10 μ L born of the same parents ectosome samples, adds the standard substance diluent to 20 μ L; Each hole adds 200 μ L BCA working solutions, places 30min for 37 ℃; Measure the A562nm wavelength, calculate the protein concentration of born of the same parents' ectosome concentrated solution according to standard curve.
The protein content of the born of the same parents' ectosome in the Fel Gallus domesticus is about more than the 14 μ g/ml.
The evaluation of born of the same parents' ectosome in embodiment 2, the Fel Gallus domesticus
1. born of the same parents' ectosome morphologic observation
Get 10 μ L born of the same parents ectosome stock solutions on the load sample copper mesh, leave standstill 1min in room temperature; Blot liquid from the side with filter paper, drip the about 10 μ L of 1% uranium acetate on copper mesh, room temperature negative staining 1min; Blot negative staining liquid with filter paper, four steam water washing once, dry under electric filament lamp, observe under transmission electron microscope, take a picture.
As shown in Figure 1A ~ C, the outer volume morphing of the born of the same parents in the Fel Gallus domesticus is the vesicles with typical cup-like structure of diameter 30-150nm.
2. the observation of the secretion of born of the same parents' ectosome and release
(1) sample treatment: get the liver organization in the stripped 1min of SPF chicken, be trimmed to the rectangular of 1mm * 1mm * 2mm size.
(2) preceding fixing: fixedly more than the 4h, the 0.1mol/L phosphate buffer cleans 3 times 2.5% glutaraldehyde that places pre-cooling after liver organization taken out immediately, 10min/ time.
(3) back is fixing: 1% osmic acid is 1h fixedly, and the 0.1mol/L phosphate buffer cleans 2 times, 10min/ time.
(4) dehydration: adopt gradient ethanol dehydration method, concentration is 50%, 70%, 90%, 95%, 100%, and 1: 1 liquid of reuse is 100% anhydrous propanone: 100% dehydrated alcohol dehydration 2 times, specimen is the time of staying 15～20min in every grade of concentration.
(5) soak into and embedding: the gradient impregnating method, mix embedding mediums with epoxy resin Epon812 and 815 and soak into sample with 1: 1,2: 1 concentration of acetone, 2h in every kind of concentration, along with the prolongation of time, acetone volatilizees gradually, the increase of embedding medium purity.And then liver organization vertically is embedded in the pure embedding medium 7 ℃ of 12h of polyase 13 in the incubator, 60 ℃ of 24h.
(6) location: with Leica-UC6 type ultramicrotome semithin section, thick 1～2 μ m through the about 10s of Toluidine blue staining, observes the location under the simple microscope.
(7) ultrathin section and dyeing: select the ultramicrotome section for use, section speed 25mm/s, thickness 70～80nm fishes for 4～5 big sections with 100 order band film copper mesh, again through uranium acetate lead citrate double staining, can observe under Electronic Speculum.
The A arrow is depicted as born of the same parents' ectosome of liver Di Shi intracavity among Fig. 2;
The B arrow is depicted as the born of the same parents' ectosome in the little bile duct of liver among Fig. 2;
The C arrow is depicted as many cell spaces and the oozy born of the same parents' ectosome in the hepatocyte among Fig. 2;
D is depicted as the bile duct microvillus among Fig. 2, and the secretion that arrow is depicted as bile duct microvillus epithelial cell born of the same parents ectosome discharges, and has born of the same parents' ectosome to secrete by the microvillus edge;
3.SDS-PAGE electrophoresis
Get born of the same parents' ectosome suspension 10 μ L, add 10 μ L2 * sample-loading buffers, 100 ℃ are boiled 3min, are splined on the polyacrylamide separation gel of 120g/L, and electrophoresis finishes back row Coomassie brilliant blue dyeing, takes pictures after the decolouring.
As shown in Figure 3, the electrophoretic band of born of the same parents' ectosome mainly contains four in the Fel Gallus domesticus, respectively at 26KD, 34KD, 43KD and 72KD.
4. the contained analysis of protein of born of the same parents' ectosome
Send Research Centre for Proteome Analysis(Shanghai) to detect albumen with examining the adhesive tape and the bile born of the same parents ectosome concentrated solution that dye behind the SDS-PAGE electrophoresis.Utilize the ESI-MS mass spectrograph, because MS/MS has the structural analysis function, can obtain the sequence of detected each peptide section.The accurate molecular mass that records according to one-level mass spectrum (MS) and the sequence information of tandem mass spectrum (MS/MS) gained are by the retrieval identification of protein kind of Protein Data Bank.
Be tested and appraised to be divided into and separate out 196 kinds of albumen, main protein ingredient had wherein both comprised the significant albumen of born of the same parents' ectosome as shown in Figure 4, also had the differential protein of bile born of the same parents ectosome, as HSP70, and C-reactive protein, vitronectin, mucin etc. and a large amount of immunoglobulin.The significant albumen of HSP70 protein family Chang Zuowei born of the same parents ectosome is as the foundation of assert born of the same parents' ectosome; The C-reactive protein is produced by hepatocyte, is acute phase reactive protein, and is significant as the sign of inflammatory reaction; Vitronectin, its expression difference Chang Zuowei judges the standard of hepatocarcinoma, shows that fowl source born of the same parents' ectosome can be used as judgement; Mucin is produced by epithelial cell, plays the multiple functions such as conduction of lubricated and cell signal, can form chemical barrier; The existence of a large amount of immunoglobulins has illustrated that born of the same parents' ectosome has important function in the transmission of immunne response information.
1. pre-treatment:
Get born of the same parents' ectosome suspension and place 4mm acetaldehyde/sulfate magnetic bead emulsion (available from Invitrogen company) 100 μ l, hatch 20 ℃ of 15min, add after the PBS buffer on shaking table gentleness again and shake 2h, use 100mmol/L glycine cessation reaction at last.
2. flow cytometry analysis
(1) will connect the centrifugal 2000rpm5min of born of the same parents' ectosome of magnetic bead,
(2) with 1mlFacs Buffer cell piping and druming is scattered, centrifugal 2000rpm5min outwells buffer,
(3) every pipe adds 100 microlitre Facs Buffer
(4) from each pipe, respectively take out a certain amount of sample and form the tee pipe contrast
(5) centrifugal 2000rpm5min,
(6) use chicken antibody CD4
+T-FITC/CD8
+T-PE, 4 ℃ of lucifuge dyeing of antibody such as MHC I-FITC/MON-PE 30min.
(7) 1mlFacs Buffer flushing, centrifugal 2000rpm5min
(8) each pipe adding 200 microlitre paraformaldehyde is fixed
(9) flow cytometer is observed
The result as shown in Figure 5, the born of the same parents' ectosome in the bile only connects magnetic bead and shows as figure A, it is relevant that born of the same parents' ectosome of 95.42% presents the MHC I, only having 3.78%, to show the MHC II relevant.Therefore, the born of the same parents' ectosome in the bile mainly carries MHC I correlation molecule.
1. aseptic taking-up thymus, liver and spleen:
Liver and spleen are got in the aseptic abdominal cavity of opening of healthy SPF chicken (4-6 age in week), cut skin of neck open and get thymus, and weigh.
2. spleen leukocyte separation and Culture:
(1) in ice chest, places the sterilization plate, with a fritter spleen, on the cell sieve, grind gently, with the PBS flushing cell sieve of a small amount of pre-cooling, lapping liquid is collected in the 15ml pipe.
(2) centrifugal back add 1mlPBS resuspended after, the visual cell amount adds erythrocyte cracked liquid, cracking 10min.
(3) it is resuspended with 1640 culture medium to add PBS washing back, and branch is filled in 24 orifice plates, and three groups of negative controls (normal), IgG, born of the same parents' ectosome are set.
(6) place CO
2Incubator takes out after cultivating 3h.
3. thymus leukocyte separation and Culture:
(1) earlier fat related on the thymus is removed totally fully, in ice chest, placed the sterilization plate, each piece thymus is ground on the cell sieve gently, with pre-cooling PBS flushing cell sieve, collect together in the 15ml pipe at last.
(2) the centrifugal 5min of thymocyte cell 1500r of aseptic taking-up abandons supernatant.
(3) add PBS resuspended after, the visual cell amount adds erythrocyte cracked liquid, cracking 10min.
(4) PBS washing back blows precipitation open with 1640 culture medium, and branch is filled in 24 orifice plates, and three groups of negative controls (normal), IgG, born of the same parents' ectosome are set.
(5) place CO
2Incubator takes out after cultivating 3h.
3. the leukocytic separation of liver:
(1) in ice chest, places the sterilization beaker, liver is cut into the about 1cm of size
3Fritter, each fritter liver gone up in cell sieve grinds, with pre-cooling PBS flushing cell sieve, collecting cell liquid is in the 50ml centrifuge tube.
(2) leave standstill 5min after, inclining supernatant, abandons precipitation, and supernatant is contained in the 50ml centrifuge tube.
(3) 4 ℃ of centrifugal 8min of 1500rpm abandon supernatant.
(4) separate liver leukocyte, 20 ℃ of centrifugal 20min of 2500rpm with the chicken lymphocyte separation medium.
(5) get the upper strata, blow precipitation with PBS washing back open with 1640 culture medium, get two 24 orifice plates, blow precipitation open with 1640 culture medium, packing arranges three groups of negative controls (normal), IgG, born of the same parents' ectosome.
(7) place CO
2Incubator takes out after cultivating 3h.
4. flow cytometry analysis pre-treatment:
(1) cultured cells is taken out centrifugal 2000rpm 5min;
(2) with 1mlFacs Buffer cell piping and druming is scattered, centrifugal 2000rpm 5min abandons supernatant;
(3) every pipe adds 100 microlitre Facs Buffer;
(4) from each pipe, respectively take out a certain amount of sample and form the tee pipe contrast;
(5) centrifugal 2000rpm5min;
(6) use chicken antibody CD4
+T-FITC/CD8
+T-PE, 4 ℃ of lucifuge dyeing of antibody such as MHC I-FITC/MON-PE 30min.
(7) 1mlFacs Buffer flushing, centrifugal 2000rpm5min abandons supernatant;
(8) each pipe adding 200 microlitre paraformaldehyde is fixed;
(9) flow cytometer detects.
Show that according to experimental result the born of the same parents' ectosome in the bile is to the cytosis difference of separate sources, the cell in spleen and thymus source not have obviously to promote the effect of breeding, and to liver CD4
+The lymphocytic effect of T is remarkable.The result as shown in Figure 6, in due course between and appropriate dosage under, the born of the same parents' ectosome in the bile is to liver CD4
+The differentiation of T lymphocyte has tangible activation (figure B), and presents MHC I dependency (figure C).As seen born of the same parents' ectosome provided by the present invention has tangible CD4
+The T proliferation function.
Fig. 7 A﹠amp; Shown in the B, bile born of the same parents ectosome is not obvious to the proliferation function of spleen, thymocyte cell, but as Fig. 7 C﹠amp; Shown in the D, add born of the same parents' ectosome and can prolong spleen CD4
+T, CD8
+The time-to-live of T and mononuclear cell and MHC I class cell, (initial concentration that is cell is identical to improve survival rate, compare the lymphocyte amount at each time point that continues to cultivate, it is all higher than the cell concentration that does not add born of the same parents' ectosome group to add born of the same parents' ectosome), visible born of the same parents' ectosome provided by the present invention has the survival rate of tangible raising cell and the effect that prolongs the time-to-live.
Because the lipid bilayer membrane structure of born of the same parents' ectosome is compared as can be known by the routine techniques test, the lipid membrane structure of this bilayer can well be protected medicine, therefore it can be used in the medicine processing process as carrier simultaneously.
Claims (8)
1. born of the same parents' ectosome that from Fel Gallus domesticus, extracts, it is characterized in that: this born of the same parents' ectosome has the bilayer lipid membrane of typical cup-like structure, the protein ingredient that contains liver source property and epithelial cell source property, the protein ingredient of described liver source property are C-reactive protein or vitronectin or its mixture; The protein ingredient of described epithelial cell source property is mucin.
2. born of the same parents' ectosome according to claim 1, it is characterized in that: this born of the same parents' ectosome also carries immunoglobulin or polymeric immunoglobulin receptor or its mixture.
3. born of the same parents' ectosome according to claim 1 and 2, it is characterized in that: described born of the same parents' ectosome diameter is at 30-150nm, and the mass density scope is 1.13-1.19g/cm
3
4. born of the same parents' ectosome according to claim 2 is characterized in that: present MHC I molecule or the antigenic peptides that can be combined with MHC I molecule or above-mentioned substance coexistence on the outer surface of these born of the same parents.
5. prepare the method for born of the same parents' ectosome as claimed in claim 1, it is characterized in that: concrete steps are as follows:
Will be from SPF chicken or other commodity birds gallbladders through routine immunization, the interior bile that extracts of the 4h that exsomatizes, bile is got supernatant through the centrifugal cell debris that goes step by step under 4 ℃ of cryogenic conditions, through membrane filtration, 4 ℃ 110 successively, 000g is centrifugal, and the 2h precipitation is resuspended with PBS, 4 ℃ 110 then, use the PBS dissolution precipitation behind the centrifugal 90min of 000g, namely comprise Fel Gallus domesticus born of the same parents ectosome in the gained solution;
Wherein said centrifugal step by step be 1000～2000g15～20min, 3000～4000g20～30min, 7000～8000g45～50min, 10000～15000g40～60min.
6. preparation method according to claim 5, it is characterized in that: described through 4 ℃ 110, the precipitation behind the centrifugal 90min of 000g is carried out density gradient centrifugation by the sucrose solution of 30wt%, 45wt%, 60wt%.
7. preparation method according to claim 5 is characterized in that: the described centrifugal 2000g15min of being step by step, 4000g30min, 8000g45min, 15000g60min.
8. born of the same parents' ectosome as claimed in claim 1 is preparing the application for the treatment of on liver or gallbladder or the intestinal tract disease medicine as carrier.
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| CN104694466A (en) * | 2013-12-06 | 2015-06-10 | 山西医科大学 | Preparation of mesenchymal stem cells (MSCs) derived exosomes and application of the same in acute lung injury |
| EP3112474A1 (en) * | 2015-06-29 | 2017-01-04 | Evonik Degussa GmbH | Method of detecting avian necrotic enteritis |
| CN104922685A (en) * | 2015-05-28 | 2015-09-23 | 杭州优玛达生物科技有限公司 | Application of naturally-sourced and/or self-assembled biological membrane, closed structure or cell compartment with biological membrane property as medical high polymer material |
| CN104888230A (en) * | 2015-05-28 | 2015-09-09 | 杭州优玛达生物科技有限公司 | Application of biological membrane obtained by natural source and/or self-assembly technology, and closed structure or cellular compartment with biological membrane property as medicine carriers |
| KR20200006108A (en) | 2017-05-12 | 2020-01-17 | 에보니크 오퍼레이션즈 게엠베하 | Seeds from animals. How to Detect Perfringens-Induced Disease |
| US12031991B2 (en) | 2018-03-02 | 2024-07-09 | Evonik Operations Gmbh | In vitro method for detecting intestinal barrier failure in animals |
| CN112129736B (en) * | 2020-09-22 | 2022-08-02 | 四川大学华西医院 | Homogeneous phase visualization/fluorescence POCT detection method for mucin and circulating tumor cells |
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Non-Patent Citations (4)
| Title |
|---|
| "Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia";Anatoliy I. Masyuk, et al.;《American Joural of Physiology Gastrointestinal and Liver Physiology》;20100715;第299卷;第G991页左列最后1段，第G993页左列第3-4段和右列全部 * |
| "Exosomes: immune properties and potential clinical implementations";Nathalie Chaput, et al.;《Seminars of Immunopathology》;20101221;第33卷;全文 * |
| Anatoliy I. Masyuk, et al.."Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia".《American Joural of Physiology Gastrointestinal and Liver Physiology》.2010,第299卷第G990-G999页. |
| Nathalie Chaput, et al.."Exosomes: immune properties and potential clinical implementations".《Seminars of Immunopathology》.2010,第33卷第419-440页. |
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