CN108841775A - The method of cell membrane dynamic function - Google Patents
The method of cell membrane dynamic function Download PDFInfo
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- CN108841775A CN108841775A CN201810553009.3A CN201810553009A CN108841775A CN 108841775 A CN108841775 A CN 108841775A CN 201810553009 A CN201810553009 A CN 201810553009A CN 108841775 A CN108841775 A CN 108841775A
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Abstract
The present invention relates to a kind of method of cell membrane dynamic function, with cell membrane surface sulfydryl exchange reaction occurs for the high curing substance of reactivity under physiological environment, carries out dynamic function to cell membrane, obtains cell membrane by materials engineering cell.Compared with prior art, the present invention carries out functionalization process to cell membrane and is suitable for different cell types, whole process has very high cell compatibility, and the high curing substance of used reactivity can voluntarily degrade over time, effectively avoid the long-term influence on cell function.The technology can temporarily regulating cell function as be proliferated, the period, endocytosis efficiency, on cell fate and function controlling have wide application prospect.
Description
Technical field
The invention belongs to technological field of biochemistry, more particularly, to a kind of method of cell membrane dynamic function.
Background technique
Cell membrane is as the boundary in eucaryotic cell structure --- by phospholipid bilayer and integral protein texture at being not only to tie up
The natural barrier of cell Yu the relatively independent environment of surrounding is held, and guarantees that cell and ambient enviroment carry out complex material, energy, letter
Cease the intelligent door of exchange.Inherently one, cell membrane is difficult to the complexity to match in excellence or beauty, precision, dynamic, orderly and unordered molecule
Engineering.The dynamic and biological function characteristic of membrane structure are that the challenge that cell membrane macromolecular engineering proposes is the key that general character
Technical problem.Since eucaryotic cell structure is in dynamic change always, construct be not enough be easy by the direct endocytosis of cell, excessive structure
It builds and is easy to cause unexpected cell function injury or even apoptosis.Nevertheless, the effort of researcher constantly overcomes
Difficulty has developed serial strategy.
Recently, Langer predicts that " Living Biomaterials " will become future thrust in perspective summary.
In addition, cell membrane macromolecular engineering is expected to answer all many cells application the relevant technologies bottleneck problems.Therefore, no matter from basis or
From the point of view of application study, the marriage of Nature and Man work engineering highlight great science and technology value.Currently, cell membrane engineering
In regulating cell epidemic focus, migration, adhesiveness, improve cell anti-adverse environment ability, control cell metabolism, assembly manually by
Body, assembles cell mass, and secondary stimulus cell function removes environment heavy metal, differentiation of stem cells, the side such as functional molecular delivering
Face obtains greater advance.Cell membrane engineering main thought is the physico-chemical attributes using cell membrane at present, mainly includes cell membrane
Elecrtonegativity, carboxyl that albumen is rich in, amino, sulfydryl carry out structural modification to cell membrane, to achieve the purpose that regulation behavior
(Exp Biol Med.2016；241:1098-106).Electrostatic interaction based on cation high molecular and bear electricity cell membrane carries out
The method of cladding is relatively mature using more.Such as based on Electrostatic Absorption, polylysine and Vitamin C are deposited in cell surface
Sour macromolecule layer and gelatin and sodium alginate layer (Chemnanomat.2016；2:376-84.).The strategy is high-efficient, can be accurate
The toxicity for controlling the macromolecule number of plies, but being limited to cation high molecular itself, it is difficult to play bigger influence.It is contracted based on carboxyamino
Reaction is closed, the addition reaction of sulfydryl and maleimide base group, which is chemically modified, to be also widely used.For example, thin using pancreas islet
Cellular surface amino reacts PEG layers upper in islet cell surface modification with active ester, can help to inhibit its immune response, be conducive to pancreas
The success of island transplanting.
To cope with some extreme environments (heat, drying, radiation, chemistry etc.), bacillus can be temporarily formed one layer it is firm
Protectiveness gemma, is mainly made of peptide glycan.Gemma can disintegrate later, and bacterium comes back to normal physiological condition.(Accounts
Chem Res.2016；49:792-800) cell membrane macromolecular engineering can also play similar protective effect, however, less research pair
How carry out targeted design is disintegrated.This relationship completes " dealing with problems arising from an accident " problem after engineering mission --- it avoids manually tying on cell membrane
Structure continues existing influence.This is most important in many instances, because regulation is temporary most of the time.For example, passing through
The regulation stem cell differentiation of stem cell surface engineering carries the introduced material of engineering after being not intended to differentiation.In targeted delivery, carefully
Also wish that functional material that surface carries falls off after born of the same parents' chemotactic target tissue to penetrate into tissue and play a role.
Summary of the invention
Disintegrate carry out targeted design, a kind of method that the present invention proposes cell membrane dynamic function, this hair for above-mentioned
It is bright to be based on disulfide bond and cell membrane surface covalent bonding, dynamic function is carried out to its cell membrane.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of cell membrane dynamic function, the high curing substance of reactivity, under physiological environment, with cell
Efficient exchange reaction occurs for film surface sulfydryl, carries out dynamic function to cell membrane, obtains cell membrane by materials engineering
Cell.
The high curing substance of the reactivity selects one of following substance or a variety of：
(1) after the substance for containing only free sulfhydryl group, with the substance priming reaction with activated thiol groups, the reactivity of formation
High curing substance；
(2) free sulfhydryl group but the substance containing disulfide bond are free of, the substance that can be interrupted disulfide bond interrupts disulfide bond, obtains
Free sulfhydryl group, then with after the substance priming reaction with activated thiol groups, the high curing substance of the reactivity of formation；
(3) it simultaneously after the substance containing free sulfhydryl group and disulfide bond, with the substance priming reaction with activated thiol groups, is formed
The high curing substance of reactivity.
The substance containing free sulfhydryl group includes macromolecular compound or small molecule compound；
It is described without free sulfhydryl group but the substance containing disulfide bond includes macromolecular compound or small molecule compound；
The substance simultaneously containing free sulfhydryl group and disulfide bond includes macromolecular compound or small molecule compound；
Macromolecular compound includes the substances such as protein.Macromolecular compound or small molecule compound can for it is some containing
Functional compound.
The substance for containing only free sulfhydryl group refers to the substance containing 2 or 2 or more free sulfhydryl groups.
The substance for interrupting disulfide bond includes but is not limited to dithiothreitol (DTT) (DTT) or glutathione etc..
The substance with activated thiol groups includes bis- thiobis of 5,5'- (2- nitrobenzoic acid) etc..
The cell membrane is rich in tumour cell, stem cell, immunocyte or the leaching of sulfydryl from the surface of human or animal
Bar cell etc..
The progress time of exchange reaction can complete within 2 hours, not use any poisonous and harmful substances, rapidly and efficiently.
It further include the process for removing the high curing substance of reactivity after swapping reaction.
The technical program has good biocompatibility, does not influence cell normal physiological function.
The high curing substance of reactivity used in the present invention, can be within a couple of days, by cell absorbed themselves, therefore can
Avoid the long-term influence on cell function.
While the present invention can keep cell normal physiological function, multilevel regulation, including proliferation, week are carried out to cell
Phase, endocytosis efficiency etc..
The invention further relates to the cells using cell membrane obtained by the above method engineering.
It is described in detail for using protein material：By carrying out three-dimensional structure regulation, reduction to protein
Exposure sulfydryl is handled, and modifies the stronger functional group of leaving capability on sulfydryl, obtains sulfhydryl activated protein.Activation of protein
Efficiently cell surface can be modified by forming disulfide bond with sulfydryl on various kinds of cell film, and using remaining activated group
Disulfide bond continuous several times packing engineering is formed with thiolated protein matter.The feasibility of entire method is confirmed, and process is green
Color, mild condition, rapid reaction, engineering are high-efficient.
Compared with prior art, the present invention carries out functionalization process suitable for different cell types, entire mistake to cell membrane
Journey has very high cell compatibility, and the high curing substance of used reactivity can voluntarily drop over time
Solution, effectively avoids the long-term influence on cell function.The technology can temporarily regulating cell function as be proliferated, the period, endocytosis effect
Rate has wide application prospect on cell fate and function controlling.
Detailed description of the invention
Fig. 1 is the scanning electron microscopic picture that bovine serum albumin(BSA) used in embodiment is engineered (a) (b) afterwards before B16 cell.
Fig. 2 is after being engineered after using FITC tagged albumin in embodiment to B16 cell membrane different time hatching membrane
Laser co-focusing picture, nucleus are dyed with DAPI.
Fig. 3 is that application project technology promotes what difficulty entered born of the same parents' substance (glucan) to enter born of the same parents' efficiency in embodiment.Using streaming
Technology quantifies glucan analysis and enters born of the same parents' amount.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
1. the cow's serum containing 1 free sulfhydryl group and 17 disulfide bond by the way that a certain amount of SDS is added, is promoted albumen
Exposure hydrophobic region, suitable DTT, which is then added, will interrupt disulfide bond, obtain the albumin containing free sulfhydryl group.
2. the albumen activated in right amount and target cell-B16 cell are interacted 20min~a few hours.
3. centrifugation removes excessive material, after washing several times, the cell of cell membrane engineering is obtained.
Bovine serum albumin(BSA) used in the present embodiment is engineered the forward and backward scanning electron microscopic picture such as Fig. 1 institute of B16 cell
Show, it can be clearly seen that the cell membrane of B16 cell has significant change.
4. proliferative capacity is analyzed：After CFSE marks cell membrane, when cell carries out division growth, the cytoplasm egg with fluorescence
White be averaged is assigned in second generation cell, and in this way compared with first generation cell, fluorescence intensity will weaken to half；With this
Analogize, the fluorescence intensity of the third generation cell divided will weaken again than second generation cell.This phenomenon can be
It under the exciting light of 488nm, is analyzed, is reduced by detecting cell fluorescence intensity constantly, into one using flow cytomery
Step analysis obtains the case where cell division proliferation, as a result as shown in Figure 2.The principle can be used to detect the proliferative conditions of cell.
5. the cell engineered technology of flow cytometric analysis promotes cell to swallow efficiency.By taking DC2.4 as an example, by logarithm
The DC2.4 cell in growth period is centrifuged digestion, and cell concentration is configured to 5 × 104A/ml.According to every hole 5 × 104A/ml cell
Concentration spreads cell into 24 orifice plates, every hole 1ml.Cladding processing is carried out to cell by aforementioned condition.It is poly- that fluorescent marker Portugal is added
Sugar is incubated for different time, and with FACSCalibur flow cytometer, sampling analysis testing result calculates the flat of each group different time
Equal fluorescence intensity, as a result as shown in Figure 3.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of method of cell membrane dynamic function, which is characterized in that the high curing substance of reactivity, in physiological environment
Under, exchange reaction occurs with cell membrane surface sulfydryl, dynamic function is carried out to cell membrane, it is materials engineering to obtain cell membrane
Cell.
2. a kind of method of cell membrane dynamic function according to claim 1, which is characterized in that the reactivity is high
Curing substance select one of following substance or a variety of：
(1) after the substance for containing only free sulfhydryl group, with the substance priming reaction with activated thiol groups, the reactivity of formation is high
Curing substance；
(2) free sulfhydryl group but the substance containing disulfide bond are free of, the substance that can be interrupted disulfide bond interrupts disulfide bond, gains freedom
Sulfydryl, then with after the substance priming reaction with activated thiol groups, the high curing substance of the reactivity of formation；
(3) simultaneously after the substance containing free sulfhydryl group and disulfide bond, with the substance priming reaction with activated thiol groups, formation it is anti-
The curing substance for answering activity high.
3. a kind of method of cell membrane dynamic function according to claim 2, which is characterized in that described to contain free mercapto
The substance of base includes macromolecular compound or small molecule compound；
It is described without free sulfhydryl group but the substance containing disulfide bond includes macromolecular compound or small molecule compound；
The substance simultaneously containing free sulfhydryl group and disulfide bond includes macromolecular compound or small molecule compound.
4. a kind of method of cell membrane dynamic function according to claim 2, which is characterized in that described to contain only freedom
The substance of sulfydryl refers to the substance containing 2 or 2 or more free sulfhydryl groups.
5. a kind of method of cell membrane dynamic function according to claim 2, which is characterized in that described to interrupt two sulphur
The substance of key includes but is not limited to dithiothreitol (DTT) or glutathione.
6. a kind of method of cell membrane dynamic function according to claim 2, which is characterized in that described that there is activation mercapto
The substance of base includes bis- thiobis of 5,5'- (2- nitrobenzoic acid).
7. a kind of method of cell membrane dynamic function according to claim 1, which is characterized in that the cell membrane comes from
Tumour cell, stem cell, immunocyte or the lymphocyte of sulfydryl are rich in the surface of human or animal.
8. a kind of method of cell membrane dynamic function according to claim 1, which is characterized in that the progress of exchange reaction
Time completes within 2 hours.
9. a kind of method of cell membrane dynamic function according to claim 1, which is characterized in that swap reaction
It afterwards, further include the process for removing the high curing substance of reactivity.
10. using the cell for the cell membrane engineering that any one of claim 1-9 the method obtains.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111094545A (en) * | 2019-03-26 | 2020-05-01 | 广东石油化工学院 | Method for cell surface modification |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245104A (en) * | 2017-06-13 | 2017-10-13 | 厦门大学 | A kind of raising compound or material enter method and the application of cell efficiency |
| CN107903337A (en) * | 2017-11-10 | 2018-04-13 | 中国药科大学 | A kind of chitosan derivatives with mucosa-adherent and Pept-1 targetings and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245104A (en) * | 2017-06-13 | 2017-10-13 | 厦门大学 | A kind of raising compound or material enter method and the application of cell efficiency |
| CN107903337A (en) * | 2017-11-10 | 2018-04-13 | 中国药科大学 | A kind of chitosan derivatives with mucosa-adherent and Pept-1 targetings and preparation method thereof |
Non-Patent Citations (9)
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111094545A (en) * | 2019-03-26 | 2020-05-01 | 广东石油化工学院 | Method for cell surface modification |
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