CN1322115C - Ectobody loaded with exogenous ligand molecule and its preparation method and application - Google Patents
Ectobody loaded with exogenous ligand molecule and its preparation method and application Download PDFInfo
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- CN1322115C CN1322115C CNB2005100826046A CN200510082604A CN1322115C CN 1322115 C CN1322115 C CN 1322115C CN B2005100826046 A CNB2005100826046 A CN B2005100826046A CN 200510082604 A CN200510082604 A CN 200510082604A CN 1322115 C CN1322115 C CN 1322115C
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Abstract
The present invention discloses an exosome loaded with an exogenous ligand molecule, and the preparation method and the application of the exosome. The exosome loded with an exogenous ligand molecule is composed of an exogenous ligand molecule and an exosome, wherein the exogenous ligand molecule joints at the surface of the membrane of the exosome or is wrapped in the cavity of the exosome, or exists at both places. The exogenous ligand molecule is albumen or a molecule which are synthesized by a nontarget cell, or the exogenous ligand molecule can directly act on a target cell or can be absorbed by the target cell via endocytosis. The exosome loaded with an exogenous ligand molecule of the present invention can transfer the exogenous ligand molecule with biological activity (exogenous protein, exogenous toxin or exogenous medicine molecules) between isogenous cells or allogenous cells in a specificity mode; an active molecule is led to the target cell. The exosome loaded with an exogenous ligand molecule of the present invention can be used for regulating the activity of the isogenous target cells or the allogenous target cells, and can be used for preparing medicines (such as a medicine for treating tumors).
Description
Technical field
The present invention relates to be loaded with born of the same parents' ectosome and preparation method thereof and application of exogenous ligand molecule.
Background technology
A lot of zooblasts comprise kinds of tumor cells, can secrete a kind of little membrane vesicle of endocytosis origin, are called " born of the same parents' ectosome " (exosome).It is made of jointly the material of phosphatide bilayer capsule and parcel thereof, and diameter is 30-100nm, and buoyant density is about 1.1-1.2g/ml, be loaded with some characteristic molecules, as transferrinreceptor, flotillin, tsg101, rab5 etc. (all or wherein a part).Born of the same parents' ectosome is by (the multivesicular body of the multivesicular body in the cell, MVB) merge with cytoplasmic membrane and generate, specifically forming process is as shown in Figure 1: 1. cell absorbs the outer material of born of the same parents by endocytosis, and endocytic vesicle in forming (endocyticvesicle, EV).The early stage endocytosis body of 2. interior endocytic vesicle further fusion becoming (early endosome, EE).3. early stage endocytosis body is in ripening process, and its restriction film caves inward and sprouts, and forms the little membrane vesicle in the chamber; The organoid with multi-layer film structure that generate this moment is called multivesicular body (MVB).4. (plasma membrane PM) merges, and the membrane vesicle in the chamber is discharged in born of the same parents' external environment, forms born of the same parents' ectosomes (exosomes) for sophisticated MVB and cytoplasmic membrane.
Born of the same parents' ectosome is loaded with specific film integral protein or embrane-associated protein from cytoplasmic membrane or endocytosis body, and some are from cytoplasmic soluble proteins.The born of the same parents' ectosome that is secreted in the cell matrix can be in contiguous iuntercellular transmission, and on the homology or allos cell around optionally being attached to, thereby membrane component between mediated cell and cytoplasm fraction exchange perhaps activate specific signal reaction (Stoorvogel, W., Kleijmeer, M.J., Geuze, H.J., and Raposo, G. (2002) .The biogenesis and functions ofexosomes.Traffic 3,321-330; Thery, C., Zitvogel, L., and Amigorena, S. (2002) .Exosomes:composition, biogenesis and function.Nat Rev Immunol2,569-579).
Trichosanthin (Trichosanthin is isolated a kind of toxin protein from the piece root of Chinese medicinal plant snakegourd TCS), and it has RNA N-glycosidase activity, belong to I type ribosome inactivating protein (ribosome-inactivating proteins, RIPs).After target cell combined, TCS can invade in the tenuigenin, the inactivation large ribosomal subunit, and the biosynthesizing of blocking protein, thus cause the death of target cell.TCS mainly acts on the fit germinal layer cell, some tumour cells-as people uterus villioma epithelial cell (jar cell), leukemia cell's (K562 cell), melanoma cells etc. grown, the scavenger cell of and the immunity-associated cell of virus infection-infect as HIV and lymphocyte etc.Therefore, TCS and deutero-immunotoxin thereof are widely used in the treatment of induction of labour in second trimester, antitumor and anti-HIV.
Summary of the invention
An object of the present invention is to provide a kind of born of the same parents' ectosome that is loaded with exogenous ligand molecule.
(cargo-loaded exosome CLE), is made up of exogenous ligand molecule and born of the same parents' ectosome the born of the same parents' ectosome that is loaded with exogenous ligand molecule provided by the present invention; Described exogenous ligand molecule is combined in the film surface of born of the same parents' ectosome, perhaps is wrapped in the chamber of born of the same parents' ectosome, perhaps all exists two positions; Described exogenous ligand molecule is non-target cell self synthetic albumen or molecule, can directly act on target cell or is absorbed by endocytosis by target cell.
Described exogenous ligand molecule specifically can be albumen, toxin or the drug molecule of external source, as Trichosanthin or trichosanthin protein derived thing; Described target cell can be the fit immunity-associated cell of growing germinal layer cell, tumour cell or virus infection.
Described tumour cell can be people uterus villioma epithelial cell, leukemia cell or melanoma cells etc.
Described immunity-associated cell comprises lymphocyte, hemopoietic stem cell, monocyte and phagocytic cell.
Another object of the present invention provides two kinds of methods that prepare the born of the same parents' ectosome that is loaded with exogenous ligand molecule, and wherein, a kind of is the natural born of the same parents' ectosome that is loaded with exogenous ligand molecule of preparation, and another kind is the born of the same parents' ectosome that is loaded with exogenous ligand molecule of preparation reorganization.
The method of the born of the same parents' ectosome that is loaded with exogenous ligand molecule that preparation provided by the present invention is natural, be that target cell was cultivated 4-12 hour in containing the substratum of exogenous ligand molecule, collect culturing mixt, centrifugal cell and the cell debris removed, it is the filtration of 0.2 μ M that the supernatant liquor that obtains is filtered the footpath, at the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule with the filtrate that obtains.
In this method, the available PBS damping fluid of the precipitation rinsing that obtained in the centrifugal 1-2 of 60000g-100000g hour obtains the born of the same parents' ectosome that is loaded with exogenous ligand molecule of purifying.
Described exogenous ligand molecule can be TCS; Described target cell can be K562 cell, jar cell; The ratio of described TCS and target cell can be 0.5-1 * 10
7Individual cell adds 0.2-1 μ M TCS/8ml substratum.
The method of the born of the same parents' ectosome that is loaded with exogenous ligand molecule of preparation reorganization provided by the present invention, be to extract target cell excretory born of the same parents ectosome earlier, born of the same parents' ectosome was hatched in the substratum of pH4.5-5.5 30-50 minute, in substratum, add exogenous ligand molecule again, hatched again 2-6 hour, the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule.
Described born of the same parents' ectosome was hatched in the substratum that is grouped into by following one-tenth 30-50 minute: pH5.0 RPMI 1640 substratum, contain 10mM Hepes, 50mM NaAc, 10% (quality percentage composition) bovine serum.
In this method, the available PBS damping fluid of the precipitation rinsing that obtained in the centrifugal 1-2 of 60000g-100000g hour obtains the born of the same parents' ectosome that is loaded with exogenous ligand molecule of purifying.
Described exogenous ligand molecule can be TCS; Described target cell can be the K562 cell; The concentration of the Trichosanthin that is added can be 0.5-2.0 μ M.
The Its Mechanisms result of TCS in jar cell and K562 cell shown: 1, can be enriched in MVB in large quantities by the TCS molecule of jar cell or K562 cell endocytic, and with toxin bonded born of the same parents ectosome (TCS-loaded exosome, TLE) form is secreted into the extracellular.2, excretory TLE can be in contiguous iuntercellular transmission, and this transmission is optionally, and the TLE that jar cell and K562 cell produce can transmit between homologous cell; And jar cell and K562 iuntercellular also can carry out the exchange of allos TLE, and in addition, the TLE that the K562 cell produces can also combine with the Hela cell, but does not combine with 293T cell and SP2/0 cell.3, TLE can be transported to the TCS molecule that carries in the target cell, and the latter brings into play toxic action to target cell, and it is killed.4, separation and purification the culture supernatant of the tumour cell that can handle from TCS of the TLE of K562 emiocytosis.5, the TLE of separation and purification has kept the selective binding ability to homologous K562 cell or allogenic jar cell.6, the TLE of purifying can import the TCS molecule that carries in the tenuigenin of target cell effectively, causes the death of cell.TLE to the direct toxicity of target cell be equal amts free TCS molecule to the toxic 3-4 of target cell doubly.7, the environment among the MVB in the analog cell is hatched the TLE that can obtain recombinating with the cell-derived born of the same parents' ectosome of the normal K562 of TCS and purifying in the acellular system.The TLE of reorganization has the character similar to the TLE of emiocytosis.
Above-mentioned result of study shows that born of the same parents' ectosome of emiocytosis can be used as the carrier of a kind of " natural ", transmit soluble ligand molecule specifically in homology or allos iuntercellular with biologic activity, and effectively it is imported in the target cell, regulate effect to bring into play corresponding cell.Ining contrast to that ligand molecular is intercellular freely to spread, is the method for transporting of carrier with artificial liposome perhaps, utilizes born of the same parents' ectosome to transmit soluble bioactive molecule at iuntercellular and has multiple superiority:
1. born of the same parents' ectosome can be protected bioactive molecule, prevents that it from being decomposed or destruction by the enzyme material in the environment in transmittance process.
2. a lot of external source bioactive molecules have stronger immunogenicity, can cause allergic reaction.With the delivery of born of the same parents' ectosome, bioactive molecule can directly not be free in born of the same parents' external environment, can avoid contacting with immune.
3. with respect to artificial liposome, born of the same parents' ectosome is by cell self excretory " organoid ", can not cause the rejection of body.
4. with respect to liposome, born of the same parents' ectosome has better self stability as a kind of organoid, and intercellular circulation, is difficult for impaired.
5. born of the same parents' ectosome has natural specificity to the identification of target cell, can directionally bioactive molecule be transported in the specific cell, and to the not injury of other cells.
6. utilize born of the same parents' ectosome of different sorts cell generation, can prepare the transport vehicle of difference " target ".
7. born of the same parents' ectosome can pass through the cytolemma barrier efficiently as a kind of film parcel organoid, the bioactive molecule that carries is transported to the target site of performance biological action.
8. born of the same parents' ectosome of carrying active molecule can directly extract from cell culture or obtain in vitro recombination, and is simple.
The born of the same parents' ectosome that is loaded with exogenous ligand molecule of the present invention can transmit ligand molecular external source, biologically active (albumen of external source, toxin or drug molecule) in homology or allos iuntercellular specificity, and bioactive molecule is imported target cell.The born of the same parents' ectosome that is loaded with exogenous ligand molecule of the present invention can be used for regulating homology or the allos target cell is active and preparation medicine (as the medicine of treatment tumour).Be widely used in fields such as scientific research, medication preparation and treatment of diseases prospect and exploitation of the born of the same parents' ectosome that is loaded with exogenous ligand molecule of the present invention is worth.
Description of drawings
Fig. 1 is the production process synoptic diagram of born of the same parents' ectosome
Fig. 2 is the dynamic production process of TLE in jar cell
Fig. 3 is the dynamic production process of TLE in the K562 cell
Fig. 4 A is the immuno-electron microscope photo of multivesicular body that contains the TCS molecule of endocytosis in the jar cell
Fig. 4 B is the immuno-electron microscope photo of jar cell secretion TLE
Fig. 5 A is the immuno-electron microscope photo of multivesicular body that contains the TCS molecule of endocytosis in the K562 cell
Fig. 5 B is the immuno-electron microscope photo of K562 emiocytosis TLE
Fig. 6 is the fluorescence microscope of TLE of the K562 emiocytosis of purifying
Fig. 7 is the immune electron microscopy of TLE of the K562 emiocytosis of purifying
Fig. 8 is the continuous density gradient centrifugal analysis of the TLE of K562 emiocytosis
Fig. 9 is the albumen compositional analysis of the TLE of K562 emiocytosis
Figure 10 is the distribution of TCS molecule in the TLE of K562 emiocytosis
Figure 11 is the trysinization analysis of the TLE of K562 emiocytosis
Figure 12 analyzes for the Western blotting of reorganization TLE
Figure 13 is the fluorescent microscope photo of reorganization TLE
Figure 14 A and Figure 14 B are the distribution of TCS in reorganization TLE
Figure 15 A is the transmission of TLE mediation TCS at jar cell
Figure 15 B is the transmission of TLE mediation TCS at the K562 cell
Figure 16 mediates TCS in the intercellular transmission of allos for TTLE
The selectivity that Figure 17 transmits for TLE mediation intercellular molecule
Figure 18 is the TLE of reorganization and the selective binding of target cell
Figure 19 imports the TCS molecule that carries in the target cell for TLE
Figure 20 is the endocytosis of TLE in target cell and the electron microscopic observation of fusion
Figure 21 is the direct toxicity of the TLE of purifying to target cell
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The natural born of the same parents' ectosome that is loaded with TCS of jar cell that embodiment 1, TCS handle and K562 emiocytosis (TCS-loaded exosome, TLE)
1, the preparation of FITC-TCS
TCS is dissolved in 100-500 μ l pH9.5,0.1M Na
2CO
3-NaHCO
3Damping fluid, final concentration are 2-10mg/ml; Add FITC (sigma company), make the mol ratio 1 of TCS and FITC: 5-1: 10, mix, hatch at 37 ℃ and hatched 12-16 hour in 4-6 hour or 4 ℃; Remove free F ITC with the dialysis of 2-4L PBS damping fluid, obtain FITC-TCS.
2, the production process of fluorescence microscope TLE
Add 0.2 μ MFITC-TCS in jar cell in being grown in the RPMI-1640 substratum or the K562 cell, hatch 10min or 30min, make the FITC-TCS molecule by cell endocytic at 37 ℃.Remove the substratum that contains TCS then, add fresh substratum, cell is continued to hatch 0-120min at 37 ℃.The result as shown in Figures 2 and 3, after target cell combined, TCS can promptly be absorbed endocytic vesicle (Fig. 2 A and Fig. 3 A) in cell edges elementary, and and then was transported to (Fig. 2 B and Fig. 3 B) in the early stage endocytosis body.To 90min, a large amount of TCS molecules are accumulated among the MVB of pomegranate sample at endocytosis 60.There, the TCS molecule fully is attached on the endometrial cavity structure of MVB (Fig. 2 C and Fig. 3 C).Along with the prolongation of incubation time, the TCS molecule is further assembled to the endometrial cavity vesica of MVB; Simultaneously, sophisticated MVB shifts to cell surface (Fig. 2 D and Fig. 3 D).About 120-150min, part MVB and cytoplasmic membrane merge, and the chamber intracellular vesicle that combines the TCS molecule is discharged in born of the same parents' external environment, form TLE (TCS-loaded exosome, TLE are loaded with born of the same parents' ectosome of TCS) (Fig. 2 E and Fig. 3 E).Among Fig. 2 and Fig. 3,10 ', 30 ' respectively represent that cell and FITC-TCS hatch 10min or 30min jointly; 30 '+30 ', 30 '+60 ', 30 '+90 ', 30 '+120 ' respectively represent that cell and FITC-TCS are hatched 30min jointly after, add fresh culture and continued again to hatch 0 minute, 30 minutes, 60 minutes, 90 minutes, 120 minutes; Image to left is a fluorescence photo, and Image to right is the photo after fluorescence photo and the stack of differential interference difference photo.
3, the generation of immune electron microscopy TLE
JAR and K562 cell and the of short duration 30min of hatching of 0.2 μ MTCS is the same, and used minimum medium is RpMI 1640.Remove the substratum that contains TCS, add fresh substratum, cell is continued to hatch 60 (Fig. 4 A and Fig. 5 A) or 120min (Fig. 4 B and Fig. 5 B) at 37 ℃.Collecting cell after the PBS rinsing, carries out conventional electron microscopic sample embedding operation.Concise and to the point step is that cell is fixed with 4% Paraformaldehyde 96,2.5% glutaraldehyde, 1% osmium tetroxide successively; Carry out processed with 30%, 40%, 50%, 70%, 80%, 90%, 100% acetone successively then, use Resins, epoxy Epon812 embedding at last.The embedding cell sample carries out ultrathin section(ing), gets the section of thick about 70nm, and drags on the nickel screen that is coated with formvar film (polyvinyl formal film).Section is after the hydrogen peroxide etching, with (the preparation according to a conventional method of the anti-TCS polyclonal antibody of rabbit, be the antigen immune rabbit promptly, get antiserum(antisera), separate the polyclonal antibody that obtains TCS through the protein-G affinity purification with purifying TCS) hatch at 37 ℃ and carried out mark in 4-6 hour; Sample is through fully hatching with gold mark goat-anti rabbit two anti-(the 5nm Radioactive colloidal gold is purchased the company in Jackson ImmunoResearch) after the rinsing.The sample that mark is good with acetic acid uranium and lead citrate dyeing, dries the back and observes after abundant rinsing.For TCS and the two standard specimen product of flotillin, then section is hatched at 37 ℃ with goat-anti flotillin polyclonal antibody (purchasing the company in Santa Cruz Biotechnology) earlier and was carried out mark in 4-6 hour; Mark protein A (the 10nm Radioactive colloidal gold is purchased the company in sigma) mark with gold after the rinsing; And then the mark that carries out TCS is the same.
Under Electronic Speculum, can observe the particular procedures that TLE produces.Behind TCS endocytosis 60-90min, visible a considerable amount of TCS molecules are combined on the little membrane vesicle of the 30-100nm in the MVB chamber.On these vesicles, also contain body protein outside flotillin-common born of the same parents.After MVB and cytoplasmic membrane fusion, TCS and flotillin molecule are along with the chamber intracellular vesicle of MVB is released to born of the same parents outer (Fig. 4 A, Fig. 4 B, Fig. 5 A and Fig. 5 B).Fig. 4 A and Fig. 5 A show the multivesicular body of the TCS molecule that contains endocytosis, and TCS molecule (the 5nm gold grain is immune labeled) and flotillin molecule (the 10nm gold grain is immune labeled) are combined on the endometrial cavity vesica of MVB; Fig. 4 B and Fig. 5 B show the secretion process of TLE, and the MVB and the cytoplasmic membrane that contain TCS merge, and the endometrial cavity vesica that combines the TCS molecule is discharged into outside the born of the same parents.Fig. 4 A, Fig. 4 B, the scale among Fig. 5 A and Fig. 5 B is 100nm.
The separation and purification of the TLE of embodiment 2, K562 emiocytosis and evaluation
One, the separation and purification of TLE
1. with 2 * 10
8Individual K562 cell places fresh 100ml RPMI-1640 substratum, adds 0.2 μ MFITC-TCS (being used for fluorescent microscope detects) or TCS (being used for other detections), hatches 1h at 37 ℃.Then, remove the substratum that contains TCS, add fresh RPMI 1640 substratum, cell is continued to hatch 2h at 37 ℃.
2. the centrifugal 10min of culturing mixt 300g after will hatching removes cell, gets supernatant 100ml.
Supernatant carry out successively 3 times centrifugal, the centrifugal 15min of preceding twice 800g, the centrifugal 30min of 10000g removes the cell relic for the third time.
4. centrifugal back supernatant is through 0.2 μ M membrane filtration, and ultrafiltration and concentration is to 20ml.
5. concentrate the back supernatant through centrifugal 2 hours of hypervelocity 60000g-100000g, the film precipitation.
6. the film precipitation with the PBS rinsing once suspends again with an amount of PBS (0.5-1ml), is TLE.
Two, the evaluation of TLE
To from the cell sample that FITC-TCS handles, directly place observation under the fluorescent microscope by the TLE of purifying, can find that TLE is the vesicles (Fig. 6) that is loaded with lot of F ITC-TCS molecule.Image to left is a fluorescence photo, and Image to right is a differential interference difference photo, and scale is 1 μ M.
With resuspended TLE drip in Electronic Speculum with nickel screen on, carry out immune-gold labeledly, method is with embodiment 1, is about to sample TCS, or flotillin, or the anti-mark of tsg101, resists or protein A marks with golden target two then.The anti-method preparation according to embodiment 1 of TCS, the antibody of flotillin and tsg101 is purchased the company in SantaCruz Biotechnology.Sample is through acetic acid uranium negative staining, electron microscopic observation behind the mark.The result shows that the centrifugal TLE that obtains is the little membrane vesicle of diameter 30-100nm, is loaded with a considerable amount of TCS molecules on it, and contains flotillin and the outer body protein (Fig. 7) of two kinds of common born of the same parents of tsg101-simultaneously.Among Fig. 7, A is the immune labeled flotillin of 10nm gold grain (Flo); B is the immune labeled tsg101 of 10nm gold grain (Tsg); C is 5nm gold grain and 10nm gold grain immunity double-tagging TCS and flotillin; D is 5nm gold grain and 10nm gold grain immunity double-tagging TCS and tsg101; Scale is 100nm.
The TLE that separation is obtained carries out continuous sucrose density gradient centrifugation, the result shows that excretory TLE mainly is distributed in low buoyant density equilibrium region, buoyant density is in the scope of 1.1-1.2g/ml, in contrast, free TCS molecule has higher buoyant density, mainly be distributed in the gradient bottom, corresponding density value is about 1.3g/ml (Fig. 8).
TLE and the full cell pyrolysis liquid of K562 (Cell) that separation is obtained carry out 12.5%SDS-PAGE and Western blotting.Wherein, used one anti-being respectively among the Western blotting: TCS antibody, according to the method preparation of embodiment 1; TfR, flotillin, tsg101, rab5, the antibody of calnexin and rab6 purchase the company in Santa Cruz Biotechnology.Two anti-are the antibody of horseradish peroxidase-labeled, purchase China fir bio tech ltd in Beijing.The result shows that isolating TLE forms spectrum with K562 cell pyrolysis liquid (Cell) by different albumen as shown in Figure 9; Among the TLE except being loaded with a large amount of TCS molecules, also contain the distinctive molecule of multiple born of the same parents' ectosome, as above-mentioned flotillin and tsg101 and transferrin receptor (TfR) and rab5, but do not contain rab6 and calnexin-among the isolating TLE and be present in specificity marker molecule among Golgi complex body and the ER respectively, show the pollution (Fig. 9) of in purge process, having avoided cell debris and other membrane structures preferably.
The TLE that separation is obtained is with 0.1%Triton X-100 incubated at room 30min.Supernatant (S) and membrane component (P) after the centrifugal 1-2h separation of 60000-80000g is penetrating.Supernatant (S) and membrane component (P) are carried out 12.5%SDS-PAGE and Western blotting, wherein used one anti-being respectively among the Western blotting: the antibody of TCS, according to the method preparation of embodiment 1; TfR, the antibody of flotillin purchase the company in Santa Cruz Biotechnology, and the two anti-antibody that are horseradish peroxidase-labeled are purchased China fir bio tech ltd in Beijing.The result as shown in figure 10, film integral protein transferrin receptor (TfR) and flotillin are distributed in the penetrating caudacoria precipitation, most of TCS also is combined on the film of born of the same parents' ectosome, and a spot of TCS molecule appears in the supernatant of penetrating back, shows that part TCS is positioned at the chamber of born of the same parents' ectosome.Non is without the film precipitation of centrifugation the culture supernatant of the penetrating K562 cell of handling from TCS.
The TLE that separation is obtained digests at 37 ℃ with the 1mg/ml pancreatin, damping fluid is PBS damping fluid or the PBS damping fluid that contains 0.1%Triton X-100, utilize Western blotting to detect the quantity of TCS remaining behind the digestion different time, wherein used one anti-ly be the antibody of TCS among the Western blotting, prepare according to the method for embodiment 1; Two anti-are the antibody of horseradish peroxidase-labeled, purchase China fir bio tech ltd in Beijing.(Control) in contrast carries out same digestion process with the 1mg/ml pancreatin at 37 ℃ with equivalent free TCS, and damping fluid is PBS damping fluid or the PBS damping fluid that contains 0.1%Triton X-100.The result shows that no matter whether add Triton X-100 free TCS can degrade fully as shown in figure 11 in 15min; And most of TCS molecule that TLE carries also can be degraded in 15min, shows that it is exposed to the surface of TLE.And a small amount of TCS molecule (account for greatly total amount 1/10), still residual behind digestion 60min, only after 0.1%Triton X-100 was penetrating with born of the same parents' ectosome, this part TCS molecule could be digested degraded, and show that it is positioned at the chamber of TLE.Non cuts the sample of processing without enzyme.
The penetrating experiment of 0.1%Triton X-100 and pancreatin enzyme are tested conscientiously and are shown that most of TCS molecule is combined in the film surface of born of the same parents' ectosome, and a small amount of TCS molecule is wrapped in the chamber of born of the same parents' ectosome, and the quantity ratio that is in the TCS molecule of these two kinds of distribution modes is approximately 9: 1.
Environment in the analog cell among the MVB can prepare the TLE of reorganization in the acellular system.Concrete operations are as follows:
1. born of the same parents' ectosome of normal K562 emiocytosis extracts from the 12h cells and supernatant, and concrete steps are as follows:
(1) with 2 * 10
8The K562 cell in the RPMI-1640 substratum 37 ℃ cultivated 12-24 hour, the centrifugal 10min of culturing mixt 300g removes cell, gets honest and upright and thrifty 100ml.
(2) supernatant carry out successively 3 times centrifugal, the centrifugal 15min of preceding twice 800g, the centrifugal 30min of 10000g removes the cell relic for the third time.
(3) centrifugal back supernatant is through 0.2 μ M membrane filtration, and ultrafiltration and concentration is to 20ml, concentrates the back supernatant through centrifugal 2 hours of hypervelocity 60000g-100000g, the film precipitation.The film precipitation with the PBS rinsing once obtains born of the same parents' ectosome.
2. get K562 born of the same parents' ectosome of purifying, be positioned over pH5.0 RPMI-1640 substratum (10mM Hepes, 50mM NaAc, 10% bovine serum), hatch 40min for 37 ℃.
3. in above-mentioned born of the same parents' ectosome, add the TCS (or FITC-TCS is used for fluorescent microscope and detects) of different concns (0.1,0.2,0.5 or 1 μ M), hatch 4h for 37 ℃.
4.100000g centrifugal 2h obtains born of the same parents' ectosome precipitation (P) and supernatant liquor (S).
5. with the outer body and function PBS rinsing of sedimentary born of the same parents once.
Born of the same parents' ectosome precipitation (P) that supernatant liquor (S) that step 4 is obtained and step 5 obtain is carried out 12.5%SDS-PAGE and Western blotting, wherein used one anti-ly be the antibody of TCS among the Western blotting, prepares according to the method for embodiment 1; Two anti-are the antibody of horseradish peroxidase-labeled, purchase China fir bio tech ltd in Beijing, and the result as shown in figure 12, under lower concentration, TCS is very faint with combining of born of the same parents' ectosome, when concentration is brought up to 0.5 μ M or when above, the TCS molecule can be attached on born of the same parents' ectosome fully.
Born of the same parents' ectosome that FITC-TCS is handled places observation under the fluorescent microscope, and the result shows to combine a considerable amount of FITC-TCS molecules on most born of the same parents' ectosomes as shown in figure 13.Among Figure 13, scale is 1 μ M; Image to left is differential interference poor (DIC) photo, and intermediate picture is a fluorescence photo, and Image to right is the photo after differential interference difference photo and the fluorescence photo stack.
Western blotting and fluorescence microscope result show that when the middle mutually TCS concentration of body was higher than 0.5 μ M, the TCS molecule can fully, spontaneously be attached on the normal K562 cell born of the same parents ectosome.
Method according to embodiment 2 is carried out the penetrating experiment of 0.1%Triton X-100 and trysinization experiment and detection to the TLE that recombinates, show in reorganization TLE, the TCS molecule also is distributed in two different positions: most of molecule is combined in the film surface of born of the same parents' ectosome, and small number of molecules is wrapped in the chamber of born of the same parents' ectosome, and the quantity that is in the TCS of these two kinds of distributions is approximately 9: 1 than also, with the natural TLE of emiocytosis very similar (Figure 14 A and Figure 14 B).Figure 14 A, it is the same that reorganization TLE carries out penetrating processing with 0.1%Triton X-100, penetrating back centrifugation supernatant (S1) and film precipitation (P1).Non is the reorganization TLE without penetrating processing.Figure 14 B recombinates TLE and 1mg/ml pancreatin in PBS (buffer), or contains among the PBS of 0.1%Triton X-100,37 ℃ digest 60min down, the result shows that most of TCS molecular distribution are in the film surface of reorganization TLE, and small number of molecules is wrapped in the chamber of TLE; Non is for to cut the reorganization TLE of processing without enzyme.
The intercellular transmission of embodiment 4, TLE mediation TCS
One, the transmission between homologous cell
1. get two groups of jar cells of A, B or K562 cell, add 0.2 μ M FITC-TCS (preparation method is with embodiment 1) in the A group, add 0.2 μ M TRITC-TCS (the same FITC-TCS of preparation method in the B group, change FITC into TRITC during mark), hatch 30min at 37 ℃, used substratum is RPMI 1640.
2. remove the substratum that contains TCS, add fresh substratum, continue to cultivate 120min.
3. after cell being used the abundant rinsing of PBS of precooling, get two groups of cytomixis of A, B of equivalent, co-cultivation 30-60min in fresh RPMI 1640 substratum.
The result is shown in Figure 15 A and Figure 15 B, show that the TLE after the secretion can be in contiguous iuntercellular transmission, cause respectively being transported to (illustration amplifies demonstration) on the same cell from the TLE that contains FITC-TCS of A group and B group cell and the TLE that contains the TRITC-TCS molecule.Among Figure 15 A and Figure 15 B, long arrow shows the TLE that contains FITC-TCS, and short arrow shows the TLE that contains TRITC-TCS, and scale is 3 μ M, and Image to left is a fluorescence photo, and Image to right is a differential interference difference photo.
Two, the intercellular transmission of allos
According to the operation of step 1, the jar cell of FITC-TCS processing and the K562 cytomixis of TRITC-TCS processing are cultivated.The result shows that the TLE that the jar cell excretory carries the FITC-TCS molecule can be delivered on the K562 cell (row down) as shown in figure 16; Simultaneously, the TLE that carries the TRITC-TCS molecule of K562 emiocytosis can be delivered on the jar cell (row of going up).Among Figure 16, long arrow shows the TLE (by the JAR secretion) that contains FITC-TCS, and short arrow shows the TLE (by K562 emiocytosis) that contains TRITC-TCS, and scale is 3 μ m, and illustration amplifies demonstration, and Image to left is a fluorescence photo, and Image to right is a differential interference difference photo.
Three, TLE is in the selectivity of iuntercellular transmission
According to the operation of step 1, the K562 cell that the FITC-TCS of same quantity handles is hatched jointly with jar cell, Hela cell, 293T cell and sp2/0 cell respectively.The result shows that the TLE of K562 emiocytosis can be delivered on the jar cell effectively as shown in figure 17, also can partly be delivered on the Hela cell, but then significantly not interact with 293T cell and sp2/0 cell.Among Figure 17, scale is 3 μ m, on to arrange picture be fluorescence photo, following row's picture is a differential interference difference photo.
The natural TLE of embodiment 5, separation and purification and the TLE of vitro recombination keep the selective binding ability with target cell
According to the operation among the embodiment 2, separation and purification obtains the natural TLE that contains FITC-TCS the culture supernatant of the K562 cell of handling from FITC-TCS.The TLE of equivalent purifying is added to respectively in K562 cell, jar cell, 293T cell or the sp2/0 cell, in the RPMI-1640 substratum, hatches 10-30min for 37 ℃.The result shows that TLE can optionally be attached to homologous K562 cell or allogenic jar cell surface (Figure 19 A and D) as shown in figure 19, but does not significantly interact with 293T and sp2/0 cell.
Similarly,,, and itself and above-mentioned cell hatched jointly, obtain similar result at the TLE that contains FITC-TCS of external preparation reorganization according to the operation among the embodiment 3.The TLE of reorganization optionally is attached to K562 cell and jar cell surface, and does not combine (Figure 18) with 293T and sp2/0 cell.Among Figure 18, first row's picture is a fluorescence photo, and second row's picture is a differential interference difference photo, and scale is 3 μ m.
Embodiment 6, TLE import the TCS molecule that carries in the target cell effectively
According to the operation among the embodiment 2, separation and purification obtains the natural TLE that contains FITC-TCS the culture supernatant of the K562 cell of handling from FITC-TCS.Separation and purification TLE is joined in K562 cell or the jar cell, hatch 10min at 37 ℃, TLE is combined with target cell, used substratum is RPMI-1640.To not remove, add fresh substratum, cell is continued to cultivate 60-120min at 37 ℃ with cell bonded TLE.Cell sample is with fluorescence microscope (Figure 19) or prepare ultrathin section(ing) and carry out immune-gold labeled (as method among the embodiment 1) with electron microscopic observation (Figure 20)
The result shows that TLE can be attached to cell surface rapidly, and is caught in the endocytosis depression (Figure 19 A and D, Figure 20 A and D).Through hatching of 60min, a large amount of bonded TLE are absorbed in the born of the same parents by target cell, appear near (Figure 19 B and E, Figure 20 B and E) in the nuclear endocytosis organoid.The TLE of endocytosis can merge with the restriction film of organoid, the TCS molecule that carries is discharged in the tenuigenin of target cell (Figure 20 C and E, arrow).After hatching general 120min, almost all the TLE of endocytosis merges with target cell, only can observe the TLE (Figure 19 C and F) of micro residue this moment in cell.The TCS molecule that discharges from TLE can be spread to cytoplasmic each position, especially near nuclear position, the there rough surfaced endoplasmic reticulum that distributing, above the target site attacked in conjunction with a large amount of rrna-TCS.
Among Figure 19,10 ', 10 '+60 ', 10 '+120 ' respectively represent that cell and TLE are hatched 10min jointly after, add the photo that continued behind the fresh culture to hatch 0 minute, 60 minutes, 120 minutes; First row and the 3rd row's picture are fluorescence photo, and second row and the 4th row's picture are the photo after fluorescence photo and the stack of differential interference difference photo, and picture A-C is a jar cell, and picture D-F is the K562 cell, and scale is 3 μ m, and Nu is a nucleus.
Among Figure 20, A-C is a jar cell, and D-E is the K562 cell, scale, and 200nm, Nu are nucleus, PM is a cytoplasmic membrane.TLE is absorbed in the endocytosis depression (A and D), subsequently with after target cell combines, TLE is by endocytosis (B and E) in the organoid of adjacent cells nuclear, the TLE of endocytosis can merge with the restriction film of organoid, the TCS molecule that carries is discharged in the tenuigenin of target cell (C and E, arrow).
TLE can weigh the cytoplasmic efficient that the TCS molecule that carries imports target cell by its direct killing to target cell, because after the TCS molecule enters tenuigenin, can attack rrna and causes the death of target cell.According to method among the embodiment 2, separation and purification TLE the culture supernatant of the K562 cell of handling from TCS.Hatch 48h jointly with being loaded with the purifying TLE of 85ng TCS and jar cell and K562 cell, can cause about 19% jar cell death, and about 16% K562 necrocytosis.The free TCS molecule of its cytotoxicity and 675ng is (effect 48h can cause about 22%JAR necrocytosis, 18%K562 necrocytosis) quite.By contrast, the cytotoxicity of the free TCS of 85ng only is about the 1/3-1/4 of TLE (Figure 21).This result has proved fully that also TLE can be transported to the TCS molecule that carries in the tenuigenin of target cell effectively.
Claims (10)
1, is loaded with born of the same parents' ectosome of exogenous ligand molecule, forms by exogenous ligand molecule and born of the same parents' ectosome; Described exogenous ligand molecule is combined in the film surface of born of the same parents' ectosome, perhaps is wrapped in the chamber of born of the same parents' ectosome, perhaps all exists two positions;
Described exogenous ligand molecule is non-target cell self synthetic albumen or molecule, can directly act on target cell or is absorbed by endocytosis by target cell.
2, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 1 is characterized in that: described exogenous ligand molecule is albumen, toxin or the drug molecule of external source; Described target cell is the fit immunity-associated cell of growing germinal layer cell, tumour cell or virus infection.
3, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 1 and 2, it is characterized in that: described exogenous ligand molecule is a Trichosanthin.
4, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 2 is characterized in that: described tumour cell behaviour uterus villioma epithelial cell, leukemia cell or melanoma cells.
5, the born of the same parents' ectosome that is loaded with exogenous ligand molecule according to claim 2, it is characterized in that: described immunity-associated cell is lymphocyte, hemopoietic stem cell, monocyte or phagocytic cell.
6, a kind of described method that is loaded with born of the same parents' ectosome of exogenous ligand molecule of claim 1 for preparing, be that target cell was cultivated 4-12 hour in containing the substratum of exogenous ligand molecule, collect culturing mixt, centrifugal cell and the cell debris removed, it is the filtration of 0.2 μ M that the supernatant liquor that obtains is filtered the footpath, at the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule with the filtrate that obtains.
7, method according to claim 6 is characterized in that: described exogenous ligand molecule is a Trichosanthin; Described target cell is K562 cell or jar cell; The ratio of described Trichosanthin and target cell is 0.5-1 * 10
7Individual cell 0.2-1 μ M Trichosanthin/8ml substratum.
8, a kind of described method that is loaded with born of the same parents' ectosome of exogenous ligand molecule of claim 1 for preparing, be to extract target cell excretory born of the same parents ectosome earlier, born of the same parents' ectosome was hatched in the substratum of pH4.5-5.5 30-50 minute, in substratum, add exogenous ligand molecule again, hatched again 2-6 hour, the centrifugal 1-2 of 60000g-100000g hour, the precipitation that obtains was the born of the same parents' ectosome that is loaded with exogenous ligand molecule.
9, method according to claim 8 is characterized in that: described exogenous ligand molecule is a Trichosanthin; Described target cell is K562 cell or jar cell; The concentration of the Trichosanthin that is added is 0.5-2.0 μ M.
10, the described born of the same parents' ectosome that is loaded with exogenous ligand molecule of claim 1 is being regulated homology or allos target cell activity and the application in the preparation medicine.
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| CN1265038A (en) * | 1997-07-16 | 2000-08-30 | 法国国家卫生及研究医学协会 | Cellular vesicle called 'exosome', prepn. and use thereof in immune stimulation |
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