CN101782569B - Microfluidic chip group used for screening formyl peptide receptor agonist and screening method - Google Patents
Microfluidic chip group used for screening formyl peptide receptor agonist and screening method Download PDFInfo
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- CN101782569B CN101782569B CN 200910010225 CN200910010225A CN101782569B CN 101782569 B CN101782569 B CN 101782569B CN 200910010225 CN200910010225 CN 200910010225 CN 200910010225 A CN200910010225 A CN 200910010225A CN 101782569 B CN101782569 B CN 101782569B
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Abstract
The invention provides a microfluidic chip group used for screening a formyl peptide receptor agonist and a screening method. The microfluidic chip group consists of three microfluidic chip units, i.e. a cell chemotaxis detection unit, a receptor endocytosis detection unit and a calcium ion transient flow process monitoring unit. The invention designs a group of chips by aiming to the characteristics of different cell function analytical experiments, can respectively carry out cell chemotaxis, receptor endocytosis detection and calcium ion transient flow (release) process monitoring, and judges biologic activity thereof by comprehensively evaluating influence on the formyl peptide receptor signal conduction path by an alternative compound. Compared with the traditional technology, the invention realizes real-time monitoring of the intracellular calcium ion releasing process and saves a series of complex operations, such as film laying, film obtaining, cell doctoring, fixing, dying and the like, thereby greatly saving the analysis time and lowering the detection cost.
Description
Technical field
The present invention relates to the cellular level drug screening technology, a kind of used for screening formyl peptide receptor agonist micro-fluidic chip group and screening technique are provided especially.
Background technology
Formyl peptide receptor (formyl peptide receptor, FPR) plays a significant role in the process of the exogenous cause pathogeny imcrobe infection of body antagonism.When pathogenic microorganism invasion body, its secretion N-formyl peptides can be raised phagocytosis property leukoplania and be gathered in affected area after FPR is combined in inflammation and immune emergency reaction, resist and remove pathogenic microorganism.Research in recent years confirms, FPR not only expresses in the leucocyte that the phagocytosis function is arranged, and in liver cell, dendritic cell, sternzellen and microglia cell etc., expression being arranged all also, the FPR activator also is included in the early stage non-N-formyl peptide matters such as endogenous polypeptide that occur of inflammation.Therefore, the function of FPR also not only is confined to leukocyte activation, also participate in the aspects such as central nervous system pathological change and immunoregulation effect, find that new FPR activator and inhibitor might provide potential drug for mankind's major diseases such as treatment nerve retrograde affection, acquired immune deficiency syndrome (AIDS).Activator is after the FPR that is positioned at cell surface is combined, FPR signal transduction pathway in active cell, cell generation acceptor endocytosis, calcium ion discharge, take off a series of response events such as grain effect, Chemotaxis and inflammatory mediator release, by investigating these cell functions, can realize FPR activator and inhibitor screening.
At present, the cellular response event that FPR is mediated detects, still take the array microwell plate as the major technique platform.Because the orifice plate size is fixed, supporting automatic operation equipment and detection system pattern are single with it, can't for the characteristics flexible transformation of different cell function test experience, be difficult to more realize that cell is to the Real-Time Monitoring of medicine response process.In addition, cell chemotaxis detects and usually adopts Boyden cecum cell method.Although the method sensitivity is higher, experimentation is loaded down with trivial details, especially plastic film mulch and to get membrane process be manual operations fully and need certain experiences.In sum, if the micro-fluidic chip technology is introduced in this class drug screening research, index according to the required detection of difference screening system, to various functional units on chip carry out flexible combination, scale is integrated, realize that cell analyzes simultaneously to multiparameter, many indexs of medicine response, will solve to a great extent the problem that prior art faces.
Summary of the invention
The purpose of this invention is to provide a kind of used for screening formyl peptide receptor agonist micro-fluidic chip group and screening technique, used special-purpose functionalization micro-flow control chip group to carry out screening formyl peptide receptor agonist at cellular level.
The invention provides a kind of used for screening formyl peptide receptor agonist micro-fluidic chip group, this micro-fluidic chip group is comprised of three functions micro-fluidic chip unit:
First functionalization micro-flow control chip unit is the cell chemotaxis detecting unit;
Second functionalization micro-flow control chip unit is acceptor endocytosis detecting unit;
Three functions micro-fluidic chip unit is calcium ion mobile process monitoring of moment unit;
Described cell chemotaxis detecting unit is comprised of 2～99 identical structural units, is connected by a public sample cell between these structural units; Each structural unit is comprised of a short and thin gel channels, up-thin-low-thick cell perfusion channel and cell migration passage; The cell migration passage is between gel channels and cell perfusion channel;
Described acceptor endocytosis detecting unit is comprised of the cell culture array that is positioned at chip center, the cell perfusion channel that is positioned at the chip surrounding, compound-quantum dot perfusion channel and washing fluid perfusion channel;
Described calcium ion moment flow the process monitoring unit by the cell culture chamber that is positioned at chip center, be positioned at the chip surrounding and the cell perfusion channel, compound perfusion channel and the dyestuff that are connected with cell culture chamber/washing fluid perfusion channel forms.
Used for screening formyl peptide receptor agonist micro-fluidic chip group provided by the invention, cell culture array in described acceptor endocytosis detecting unit is connected as row mutually by 2～99 cell culture chambers, 2～99 row forming array formula media to cell culture modules parallel with one another, the entrance and exit of every two row all interconnects step by step, finally connects into an entrance and an exit passageway; The end of access road has two branches, is connected with passage of irrigation fluid with compound-quantum dot passage respectively, and the end of exit passageway is connected with the cell perfusion channel.
Used for screening formyl peptide receptor agonist micro-fluidic chip group provided by the invention, the number of each row cell culture chamber of described cell culture array is identical, and every two row cell culture chambers are connected by the microchannel, these microchannels more every two be connected to each other, finally connect into a passage.
The invention provides a kind of screening formyl peptide receptor agonist method based on the micro-fluidic chip group, procedure is:
carry out the cell chemotaxis analysis: at first slowly inject liquid basement membrane-like material (BME) in gel channels, when seeing that at microscopically BME soon is full of whole gel channels, stop immediately injecting, after standing several minutes of chip, add successively 50% ethanol (v/v), phosphate buffer PBS and cell culture medium, chip is placed in 37 ℃ of numbers minute, BME is solidified fully, utilize in the liquid storage tank of cell perfusion channel two ends the poor static pressure that produces of liquid level that cell is introduced perfusion channel, chip is put into cell culture incubator, after cell attachment, the taking-up chip is taken pictures, record initial cell number in migrating channels, basad film/compound entrance adds compound candidate, chip is taken pictures after standing a few hours in incubator, record end of a period number of cells in migrating channels,
Carry out the analysis of formyl peptide receptor endocytosis: cell suspension is injected chip by the cell entrance, after cell evenly enters each little culturing room and sedimentation, chip is put into incubator to spend the night, the abundant adherent and stretching, extension of cell, with mark the lead compound solution of quantum dot introduce chip, cell is stimulated, cleansing solution is introduced chip cell is rinsed, at last chip is placed in imaging under fluorescent microscope;
carry out calcium ion mobile (release) process monitoring of moment: cell suspension injects chip by the cell entrance, after cell settlement, chip being put into incubator spends the night, the abundant adherent and stretching, extension of cell, inject Hank ' s balanced salt saline (HBSS) solution washing cell twice to chip, add the calcium ion fluorescent dye to hatch a period of time, inject HBSS solution washing cell twice to chip again, chip is placed under fluorescent microscope, at once cell is applied the compound stimulation after opening excitation source, and unlatching CCD shutter, record intracellular calcium moment flow process.
The present invention has following advantage:
1. cell chemotaxis of the present invention is analyzed micro-fluidic chip and is only relied on diffusion, and just can form the compound concentration gradient in the cell migration passage without any need for other utility appliance.
2. the compound concentration gradient that forms in cell chemotaxis analysis micro-fluidic chip cell migration passage of the present invention is a kind of static gradient, make cell accept stimulation under the solution static situation, when having avoided must relying in original method flow of solution to form concentration gradient, the impact that hydrodynamic shear brings cell.
Calcium ion moment mobile monitoring micro-fluidic chip of the present invention can be after on unicellular level of resolution, Real-Time Monitoring be excited intracellular calcium moment flow process.
4. creativeness of the present invention is to have designed a core assembly sheet for the characteristics that different cell function analyses are tested, can carry out respectively that cell chemotaxis, acceptor endocytosis detect and (release) process monitoring of flowing of calcium ion moment, by the comprehensive evaluation compound candidate, the impact of formyl peptide receptor signal transduction pathway be judged its biologic activity.
5. compare with conventional art, micro-fluidic chip has been realized the Real-Time Monitoring of intracellular calcium dispose procedure, and saved plastic film mulch in cell chemotaxis experiment, got film, a series of troublesome operation such as cell strikes off, fix, dyeing, greatly save analysis time, the reduction testing cost.
Description of drawings
Fig. 1 is that cell chemotaxis detects the global design figure (a) of micro-fluidic chip and the partial enlarged drawing (b) of one of them structural unit, wherein: (1) is gel channels, (2) be the cell perfusion channel, (3) be the cell migration passage, (4) be the gel entrance, (5) be the cell entrance, waste liquid pool centered by (6), (7) are the cell waste liquid pool;
Fig. 2 is that the formyl peptide receptor endocytosis detects the micro-fluidic chip design drawing, and wherein: (1) is compound-quantum dot entrance, and (2) are the washing fluid entrance, and (3) are cell culture array, and (4) are the cell entrance;
Fig. 3 is calcium ion mobile process monitoring micro-fluidic chip design drawing of moment, and wherein: (1) is the cell entrance, and (2) are dyestuff/washing fluid entrance, and (3) are the activator entrance, and (4) are waste liquid pool;
Fig. 4 is that typical fMLF and GSH induce RBL-FPR cell chemotaxis result;
Fig. 5 is the dosage effect result that typical fMLF induces RBL-FPR cell generation Chemotaxis;
Fig. 6 is the dosage effect result that typical GSH induces RBL-FPR cell generation Chemotaxis;
Fig. 7 is typical fMLF-QD effect RBL-FPR cell result;
Fig. 8 is typical GSH-QD effect RBL-FPR cell result;
Fig. 9 is the dose-effect relationship that fMLF induces RBL-FPR cell generation calcium ion to discharge;
Figure 10 is the Time-effect relationship that fMLF induces RBL-FPR cell generation calcium ion to discharge.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
BME takes out from-20 ℃ of refrigerators and is placed on ice, when dissolving into liquid state, it draws 0.1 μ L with pipettor, chip shown in Figure 1 is placed under optical microscope, suction nozzle is aimed at basilar memebrane feeder connection place and is slowly injected liquid BME in passage, when seeing that at microscopically liquid soon is full of whole basilar memebrane passage, remove immediately suction nozzle, the standing 5min of chip, add 50% ethanol (v/v), PBS and cell culture medium successively in chip channel, chip is placed in 37 ℃ of incubator 15min, and BME solidifies fully.Chip is placed under optical microscope, add 7 μ L nutrient culture media in each sample cell, extract 1 μ L nutrient culture media from the cell waste liquid pool of outer ring, and inject 1 μ L cell suspension to the cell entrance immediately, overwhelming majority cell enters perfusion channel and stops, only have the only a few cell to enter migrating channels, chip is put into incubator.After cell attachment, take out chip and take pictures, record initial cell number in migrating channels.Dripping concentration in the gel sample pond of four structural units respectively is fMLF or the GSH of 0nM, 4nM, 40nM and 400nM, and chip is placed in cell culture incubator.Because N-formyl peptides material (fMLF) and reduced glutathione (GSH) are all little peptides by three Amino acid profiles, under static state, this micromolecular medicine can see through the BME gel to the diffusion of cell inlet end, forms concentration gradient in migrating channels.In case wherein the FPR of a kind of material on cell membrane is combined, FPR signal transduction pathway in will active cell, cell generation Chemotaxis, infall by perfusion channel and migrating channels enters migrating channels and moves to the high direction of drug concentration, after drug effect 4h, record end of a period number of cells in migrating channels.Fig. 4 is the result after the rat basophilic grain leukaemia (RBL-FPR) of people's formyl peptide receptor is expressed in typical fMLF and GSH effect, can find out, compared with 0 hour, the fMLF effect is migrating channels inner cell quantity showed increased after 4 hours, yet the cell of GSH effect is but without this phenomenon.Calculating the chemotactic index of compound candidate finds, compare with control group, obvious chemotaxis has all occured in fMLF experimental group cell, and is especially obvious with the 40nM effect, 4nM and 400nM take second place, and after the GSH effect, chemotactic index does not strengthen (seeing Fig. 5 and Fig. 6).
The RBL-FPR cell suspension injects chip shown in Figure 2 by the cell entrance, after cell evenly enters each little culturing room and sedimentation, chip is put into incubator spend the night, the abundant adherent and stretching, extension of cell.With mark the compound solution (fMLF-QD and GSH-QD) of quantum dot (quantum dot, QD) introduce chip, cell is stimulated, chip is placed in 37 ℃ of CO
2In incubator after 45min, cleansing solution is introduced chip, rinse approximately 45s of cell, at last chip is placed in imaging under fluorescent microscope, detect formyl peptide receptor endocytosis situation, the mercury lamp excitation wavelength is made as 330-385nm, detects wavelength and is made as greater than 420nm, takes fluorescence photo and light field photo in same visual field.Compound-quantum dot and RBL-FPR cytosis time, activity and wash time have been optimized respectively.Found that, compound-40 times of quantum dot solution dilutions are hatched 45min with cell at 37 ℃, then rinse approximately 45s with the PBS damping fluid, and effect is better.Can be found out by Fig. 7, Fig. 8, most cells of hatching with fMLF-QD all combine quantum dot, and part fMLF-QD has been positioned at the cell membrane inboard, yet the cell surface of hatching with GSH-QD is not but in conjunction with quantum dot.This explanation fMLF-QD can with cell membrane on the FPR receptors bind, and enter in cell with the acceptor that endocytosis occurs, and GSH-QD is owing to not being rinsed with the FPR receptors bind.
The RBL-FPR cell suspension injects chip shown in Figure 3 by the cell entrance, after cell settlement, chip is put into incubator and spends the night, the abundant adherent and stretching, extension of cell.drain the nutrient culture media in sample cell, inject HBSS solution washing cell twice to chip, add the calcium ion fluorescent dye Fluo-4AM that is diluted in advance 5 μ g/mL with HBSS, chip is put into incubator and is hatched 45min, drain the dyestuff in sample cell, again inject HBSS solution washing cell twice to chip, record with fluorescent microscope the calcium ion moment flow process that cell is excited to cause, the mercury lamp excitation wavelength is made as 470-495nm, detect wavelength and be made as 510-550nm, apply at once the compound stimulation after opening excitation source, and unlatching CCD shutter, take continuously 50 photos with the speed that 1s/ opens.Investigated concentration and be respectively 0nM, calcium ion mobile action effect of moment in 4nM, the fMLF of 40nM and 400nM or GSH inducing cell.Result shows, compares with control group, and fMLF effect group cell fluorescence intensity obviously raises, and GSH effect group cell has no the fluorescence intensity increase.Because the fluorescence intensity of cell is directly proportional to intracellular free calcium level, illustrate that fMLF effect group intracellular calcium concentration obviously increases, fMLF induces RBL-FPR that calcium ion moment occured to flow, and GSH does not have inducing cell generation calcium ion moment to flow.Further investigate dosage effect and the time effect of fMLF effect RBL-FPR cell, as can be seen from Figure 9, calcium ion transient flow fatigue resistance raises with the fMLF activity and strengthens, and the speed that variable concentrations fMLF inducing cell generation calcium ion moment flows is also different, fMLF concentration is higher, and intracellular calcium concentration reaches peak value required time shorter (Figure 10).
Claims (8)
1. used for screening formyl peptide receptor agonist micro-fluidic chip group is characterized in that: this micro-fluidic chip group is comprised of three functions micro-fluidic chip unit:
First functionalization micro-flow control chip unit is the cell chemotaxis detecting unit;
Second functionalization micro-flow control chip unit is acceptor endocytosis detecting unit;
Three functions micro-fluidic chip unit is calcium ion mobile process monitoring of moment unit;
Described cell chemotaxis detecting unit is comprised of 2～99 identical structural units, is connected by a public sample cell between these structural units; Each structural unit is comprised of gel channels, cell perfusion channel and cell migration passage;
Described acceptor endocytosis detecting unit is comprised of cell culture array, cell perfusion channel, compound-quantum dot perfusion channel and washing fluid perfusion channel;
Described calcium ion mobile process monitoring of moment unit is comprised of cell culture chamber, cell perfusion channel, compound perfusion channel and dyestuff/washing fluid perfusion channel.
2. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 group, it is characterized in that: the cell migration passage in described cell chemotaxis detecting unit is between gel channels and cell perfusion channel.
3. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 group, it is characterized in that: the cell culture array in described acceptor endocytosis detecting unit is positioned at the center of acceptor endocytosis detecting unit.
4. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 group, it is characterized in that: the cell perfusion channel in described acceptor endocytosis detecting unit is positioned at the surrounding of acceptor endocytosis detecting unit.
5. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 group, it is characterized in that: the cell culture array in described acceptor endocytosis detecting unit is connected as row mutually by 2～99 cell culture chambers, 2～99 row forming array formula media to cell culture modules parallel with one another, the entrance and exit of every two row all interconnects step by step, finally connects into an entrance and an exit passageway; The end of access road has two branches, is connected with passage of irrigation fluid with compound-quantum dot passage respectively, and the end of exit passageway is connected with the cell perfusion channel.
6. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 or 5 group, it is characterized in that: the number of each row cell culture chamber of described cell culture array is identical, and every two row cell culture chambers are connected by the microchannel, these microchannels more every two be connected to each other, finally connect into a passage.
7. according to the described used for screening formyl peptide receptor agonist micro-fluidic chip of claim 1 group, it is characterized in that: the cell culture chamber that described calcium ion moment flows in the process monitoring unit is connected with the cell perfusion channel.
8. screening formyl peptide receptor agonist method based on the described micro-fluidic chip group of claim 1 is characterized in that procedure is:
carry out the cell chemotaxis analysis: at first slowly inject liquid basement membrane-like material BME in gel channels, when seeing that at microscopically BME soon is full of whole gel channels, stop immediately injecting, after the cell chemotaxis detecting unit is standing, add successively 50% ethanol, phosphate buffer PBS and cell culture medium, the cell chemotaxis detecting unit is placed in 37 ℃, BME is solidified fully, utilize in the liquid storage tank of cell perfusion channel two ends the poor static pressure that produces of liquid level that cell is introduced perfusion channel, the cell chemotaxis detecting unit is put into cell culture incubator, after cell attachment, taking out the cell chemotaxis detecting unit takes pictures, record initial cell number in migrating channels, basad film/compound entrance adds compound candidate, take pictures after the cell chemotaxis detecting unit is standing in incubator, record end of a period number of cells in migrating channels,
Carry out the analysis of formyl peptide receptor endocytosis: cell suspension is injected acceptor endocytosis detecting unit by the cell entrance, after cell evenly enters each little culturing room and sedimentation, acceptor endocytosis detecting unit is put into incubator to spend the night, the abundant adherent and stretching, extension of cell, with mark the lead compound solution of quantum dot introduce acceptor endocytosis detecting unit, cell is stimulated, cleansing solution is introduced acceptor endocytosis detecting unit cell is rinsed, at last acceptor endocytosis detecting unit is placed in imaging under fluorescent microscope;
carry out calcium ion mobile process monitoring of moment: cell suspension is by cell entrance injection calcium ion mobile process monitoring of moment unit, after cell settlement, calcium ion moment is flowed process monitoring unit is put into incubator and is spent the night, the abundant adherent and stretching, extension of cell, to calcium ion moment flow process monitoring unit inject Hank ' s balanced salt saline solution washing cell twice, add the calcium ion fluorescent dye to hatch, again to calcium ion moment flow process monitoring unit inject HBSS solution washing cell twice, calcium ion mobile process monitoring of moment unit is placed under fluorescent microscope, at once cell is applied the compound stimulation after opening excitation source, and unlatching CCD shutter, record intracellular calcium moment flow process.
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| CN102816682B (en) * | 2011-06-08 | 2014-06-18 | 大连医科大学 | Micro-fluidic chip and method for studying exosmosis of tumor cell cluster |
| CN102978111A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment method of diabetic vasculopathy model based on microfluidic chip |
| CN103952300B (en) * | 2014-03-31 | 2017-07-28 | 大连医科大学 | A kind of micro-fluidic chip and cell chemotaxis motion study method |
| CN107121421A (en) * | 2017-06-20 | 2017-09-01 | 内江师范学院 | Portable range estimation luminoscope and method for heavy metal ion in Site Detection water sample |
| CN114317669A (en) * | 2022-01-04 | 2022-04-12 | 北京大学 | Method for screening drugs based on intracellular protein kinase C activation state and high-throughput screening device |
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| CN1894580A (en) * | 2003-11-07 | 2007-01-10 | 阿卡蒂亚药品公司 | The lipoxin receptor, FPRL1, as a tool for identifying compounds effective in the treatment of pain and inflammation |
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