CN107267439A - The construction method of three-dimensional lung micro-group organization model and application - Google Patents
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Abstract
The present invention relates to a kind of construction method of three-dimensional lung micro-group organization model, comprise the following steps：Label L C 3 pulmonary macrophage and pulmonary epithelial cells is distinguished using fluorescin, is then blended in the aqueous solution of polyanion polyelectrolyte, obtains cell blended liquid；Cell blended liquid is instilled in the coagulation bath containing bivalent metal ion using electrostatic drop generation, is formed and carries Cellular gels microballoon；Cellular gels microballoon culture 14 28 days will be carried, three-dimensional lung micro-group organization model is obtained.Present invention also offers using application of the three-dimensional lung micro-group organization model constructed by the above method as vitro detection and evaluation lung autophagy effect model.The present invention constructs three-dimensional lung micro-group organization model in vitro, than two dimensional model closer to internal situation；Constructed threedimensional model, can visual inspection external substance trigger autophagy effect situation.
Description
Technical field
The present invention relates to construction method and the application of biomedical sector, more particularly to a kind of three-dimensional lung micro-group organization model.
Background technology
Autophagy is highly important biological phenomena as Apoptosis, cell ageing, participates in developing, giving birth to for biology
The various procedures such as long.The exception of cell autophagy can cause the appearance of cancer cell.And some allogenic materials, such as nanoparticle, environment dirt
Dye thing, medicine etc. can trigger cell autophagy different degrees of in human body.
The detection of cell autophagy is triggered to be all based on the detection of two-dimentional single cell to allogenic material at present, but external two
Cell is tieed up due to polarization, lacks the interaction between cell-ECM and cellular matrix, its evaluation result tends not to extrapolation
It is associated with vivo, the in vitro study of cell autophagy can not be still realized in tissue aspect.Therefore one external class group of searching is needed badly
Organization model, under three-dimensional state, the autophagy effect that quick effectively observation determinand triggers.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of construction method of three-dimensional lung micro-group organization model and
Using, using the present invention method constructed by threedimensional model, can visual inspection external substance trigger autophagy effect situation.
The invention provides a kind of construction method of three-dimensional lung micro-group organization model, comprise the following steps：
(1) label L C-3 pulmonary macrophage and pulmonary epithelial cells is distinguished using fluorescin, then homogeneous blend is in poly-
In the aqueous solution of anionic polyelectrolyte, cell blended liquid is obtained；
(2) the cell blended liquid for being obtained step (1) using electrostatic drop generation instills the coagulation bath containing bivalent metal ion
In, formed and carry Cellular gels microballoon；
(3) the load Cellular gels microballoon culture for obtaining step (2) 14-28 days, obtain three-dimensional lung micro-group organization model.
Further, in step (1), LC-3 pulmonary macrophage and pulmonary epithelial cells is after fluorescent protein labeling, lung
Macrophage is different colours with pulmonary epithelial cells.Pulmonary macrophage and pulmonary epithelial cells can be distinguished with two kinds of colors.LC-3 is
Autophagy Research of predicting markers.
Further, it is glimmering using red using Green Fluorescent Protein LC-3 pulmonary macrophage in step (1)
Photoprotein label L C-3 pulmonary epithelial cells.
Further, green fluorescent protein is GFP, EGFP or sfGFP albumen；Red fluorescent protein be RFP, mRFP or
Mcherry albumen.
Further, in step (1), pulmonary macrophage is THP-1, RAW264.7 or U937 cell, pulmonary epithelial cells
For BEAS-2B, A549, NCI-H441 or TC-1 cell.
Further, in step (1), the number ratio of pulmonary macrophage and pulmonary epithelial cells is 1:2-2:1.
Further, in step (1), the cell density in cell blended liquid is 2 × 105-1×107cells/mL。
Further, in step (1), polyanion polyelectrolyte be sodium alginate, hyaluronic acid, agar, gelatin and
One or more in pectin.
Further, in step (1), the concentration of the aqueous solution of polyanion polyelectrolyte is 1%w/v-1.5%w/v.
Further, in step (2), the coagulation bath containing bivalent metal ion is that the sodium chloride containing bivalent metal ion is molten
Liquid, metal ion is the one or more in calcium ion, barium ions, magnesium ion and iron ion.
In step (2), cell blended liquid is instilled after coagulation bath, and bivalent metal ion can chelate the moon in polyelectrolyte
Ion, whole reaction system is changed into gel state from solution state, thus forms load Cellular gels microballoon, uses this method, institute
The load Cellular gels microballoon size of formation is homogeneous, uniform particle sizes, sphericity are high, pattern is controlled.
In step (3), three-dimensional lung micro-group organization model is the pulmonary macrophage and pulmonary epithelial cells of fluorescent protein labeling
Three-dimensional cell aggregate structure.
Present invention also offers the three-dimensional lung micro-group organization model constructed by a kind of use above method as vitro detection and
Evaluate the application of lung autophagy effect model.
Further, the construction method of three-dimensional lung micro-group organization model, comprises the following steps：
(1) label L C-3 pulmonary macrophage and pulmonary epithelial cells is distinguished using fluorescin, then homogeneous blend is in poly-
In the aqueous solution of anionic polyelectrolyte, cell blended liquid is obtained；
(2) the cell blended liquid for being obtained step (1) using electrostatic drop generation instills the coagulation bath containing bivalent metal ion
In, formed and carry Cellular gels microballoon；
(3) the load Cellular gels microballoon culture for obtaining step (2) 14-28 days, obtain three-dimensional lung micro-group organization model.
Further, using when determinand is added in three-dimensional lung micro-group organization model, qualitative or quantitative detection LC-3II exists
Pulmonary macrophage and the expression in pulmonary epithelial cells, to judge the lung autophagy effect type of determinand initiation.
Further, above-mentioned determinand is various human body allogenic materials, as with different chemical compositions and physicochemical property
Nanoparticle, medicine, environmental contaminants etc..
Further, due to using fluorescence labeling mark pulmonary macrophage and the pulmonary epithelial cells of different colours respectively,
Therefore distribution and the number of different colours fluorescent spot can be observed under laser confocal microscope, it is carried out qualitatively or quantitatively
Analysis, judge determinand initiation is the autophagy throat floater of pulmonary epithelial cells or pulmonary macrophage, and judges determinand with this
The lung autophagy effect type of initiation.
By such scheme, the present invention at least has advantages below：
1st, the present invention builds the three-dimensional lung micro-group organization model of many cells co-cultivation, its property using simpler method in vitro
Matter is than two dimensional model closer in vivo.
2nd, in the three-dimensional micro-group organization model that the present invention is built, using different fluorescence labeling LC-3 pneumonocyte, can intuitively it examine
Survey the autophagy effect of determinand, and the distribution situation in various pneumonocytes.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the light microscopic photo that the present invention carries Cellular gels microballoon；
Fig. 2 illustrates the autophagy abnormal conditions of the three-dimensional lung micro-group organization model that allogenic material triggers in embodiment 2；
Fig. 3 is to trigger the abnormal schematic diagram of cell autophagy the present invention relates to nanoparticle.
Embodiment
With reference to the accompanying drawings and examples, the embodiment to the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Embodiment 1
Configuration concentration is the 1.5%w/v sterile sodium alginate aqueous solution, by Green Fluorescent Protein LC-3 lung macrophage
After cell THP-1 and red fluorescent protein marker LC-3 pulmonary epithelial cellses A549 centrifugations, with 1:1 cell proportion homogeneous blend in
In sodium alginate aqueous solution, cell blended liquid is obtained, wherein cell density is 2 × 106cells/mL。
Cell blended liquid is instilled using electrostatic drop generation and contains Ca2+Coagulation bath in (coagulation bath is water-soluble for sodium chloride
Liquid), formation size is homogeneous, uniform particle sizes load Cellular gels microballoons.The concrete operation step of electrostatic drop generation is referring to document
“Investigation of spherical hydrogel surface with optical interferometer,
Colloids and Surfaces A:Physicochemical and Engineering Aspects,484(2015)：
Method in 457-462 ".
Characterized to carrying cell microsphere, test result is as shown in figure 1, it can be seen that the load cell of the present invention
The uniform particle diameter of gel micro-ball, and cell is evenly distributed in gel particle.
Then in the DMEM culture mediums of 10% serum, cell microsphere will be carried and co-cultured, incubation time is 28 days, is carried
Two kinds of cells formation three-dimensional cell aggregate structure in cell microsphere, i.e., three-dimensional lung micro-group organization model.
25 μ g/mL chloroquines are added in three-dimensional lung micro-group organization model, red fluorescence is observed under laser confocal microscope
The distribution of spot and green fluorescence spot and number.Then qualitative and quantitative analysis red fluorescence spot and green fluorescence spot, judge chloroquine
Trigger the autophagy throat floater situation of THP-1 and A549 cells in three-dimensional lung micro-assembly robot.As a result show, three-dimensional lung is handled using chloroquine
After micro-group organization model, stronger red fluorescence spot and green fluorescence spot can be triggered, therefore under chloroquine effect, pulmonary epithelial cells
It is abnormal that A549 and pulmonary macrophage THP-1 can produce strong autophagy.
Embodiment 2
Configuration concentration is the 1.5%w/v sterile sodium alginate aqueous solution, by Green Fluorescent Protein LC-3 THP-1
After red fluorescent protein marker LC-3 BEAS-2B centrifugations, with 1:1 cell proportion homogeneous blend is in sodium alginate aqueous solution
In, cell blended liquid is obtained, wherein cell density is 2 × 106cells/mL。
Cell blended liquid is instilled using electrostatic drop generation and contains Ca2+Coagulation bath in (coagulation bath is water-soluble for sodium chloride
Liquid), formation size is homogeneous, uniform particle sizes load Cellular gels microballoons.Concrete operation step is same as Example 1.
With reference to the method for embodiment 1, cell microsphere will be carried and co-cultured, incubation time is 28 days, is carried in cell microsphere
Two kinds of cells formation three-dimensional cell aggregate structure, i.e. three-dimensional lung micro-group organization model.
By 50 μ g/mL La2O3Nanoparticle is added in three-dimensional lung micro-group organization model, observes red under laser confocal microscope
The distribution of color fluorescent spot and green fluorescence spot and number.Then qualitative and quantitative analysis red fluorescence spot and green fluorescence spot, sentence
Disconnected La2O3Nanoparticle triggers the autophagy throat floater situation of THP-1 and A549 cells in three-dimensional lung micro-assembly robot.As a result as shown in Fig. 2
Fig. 2 (A) is green fluorescence spot, that is, triggers the autophagy of pulmonary macrophage abnormal；Fig. 2 (B) is red fluorescence spot, that is, triggers lung epithelial
The autophagy of cell is abnormal, and comparison diagram 2 (A) (B) can be found, use La2O3After the three-dimensional lung micro-group organization model of nanoparticle processing, trigger
Green fluorescence spot be significantly more than red fluorescence spot, therefore in La2O3Under nanoparticle effect, pulmonary macrophage THP-1 can be produced more
Strong autophagy throat floater.
Embodiment 3
Configuration concentration is the 1%w/v sterile sodium alginate aqueous solution, by Green Fluorescent Protein LC-3 U937 and red
After color fluorescent protein labeling LC-3 pulmonary epithelial cellses BEAS-2B centrifugations, with 1:2 cell proportion homogeneous blend is in sodium alginate water
In solution, cell blended liquid is obtained, wherein cell density is 5 × 106cells/mL。
Cell blended liquid is instilled using electrostatic drop generation and contains Ba2+Coagulation bath in (coagulation bath is water-soluble for sodium chloride
Liquid), formation size is homogeneous, uniform particle sizes load Cellular gels microballoons.Concrete operation step is same as Example 1.
With reference to the method for embodiment 1, cell microsphere will be carried and co-cultured, incubation time is 14 days, is carried in cell microsphere
Two kinds of cells formation three-dimensional cell aggregate structure, i.e. three-dimensional lung micro-group organization model.
50 μ g/mL graphene nanos grains are added in three-dimensional lung micro-group organization model, observed under laser confocal microscope
The distribution of red fluorescence spot and green fluorescence spot and number.Then qualitative and quantitative analysis red fluorescence spot and green fluorescence spot,
Judge that graphene nano grain triggers the autophagy throat floater situation of cell in three-dimensional lung micro-assembly robot.
Fig. 3 is the schematic diagram of foundation of the present invention, and external source toxicant (such as nanoparticle) enters cell by endocytosis, can caused
Lyase bulk damage, so as to block autophagy logical circulation road, autophagosome is largely accumulated in cell, causes cell autophagy throat floater.Tool
Body principle and document " Interference in Autophagosome Fusion by Rare Earth Nanoparticles
Disrupts Autophagic Flux and Regulation of an Interleukin-1βProducing
Inflammasome,ACS nano,8(2014)：It is identical in 10280-10292 ".The present invention sets up three-dimensional lung according to the principle
Micro-assembly robot, evaluates the cell autophagy effect of test substance under three-dimensional system first.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of construction method of three-dimensional lung micro-group organization model, it is characterised in that comprise the following steps：
(1) label L C-3 pulmonary macrophage and pulmonary epithelial cells is distinguished using fluorescin, is then blended poly- in polyanion
In the aqueous solution of electrolyte, cell blended liquid is obtained；
(2) the cell blended liquid for being obtained step (1) using electrostatic drop generation instills the coagulation bath containing bivalent metal ion
In, formed and carry Cellular gels microballoon；
(3) the load Cellular gels microballoon culture for obtaining step (2) 14-28 days, obtain the three-dimensional lung micro-group organization model.
2. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1),
LC-3 pulmonary macrophage is with pulmonary epithelial cells after fluorescent protein labeling, and the two is different colours.
3. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1), lung
Macrophage is THP-1, RAW264.7 or U937 cell, and pulmonary epithelial cells is that BEAS-2B, A549, NCI-H441 or TC-1 are thin
Born of the same parents.
4. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1), institute
The number ratio for stating pulmonary macrophage and pulmonary epithelial cells is 1:2-2:1.
5. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1), institute
It is 2 × 10 to state the cell density in cell blended liquid5-1×107cells/mL。
6. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1), institute
It is the one or more in sodium alginate, hyaluronic acid, agar, gelatin and pectin to state polyanion polyelectrolyte.
7. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (1), institute
The concentration for stating the aqueous solution of polyanion polyelectrolyte is 1%w/v-1.5%w/v.
8. the construction method of three-dimensional lung micro-group organization model according to claim 1, it is characterised in that：In step (2), institute
The coagulation bath containing bivalent metal ion is stated for the sodium chloride solution containing bivalent metal ion, the metal ion is calcium ion, barium
One or more in ion, magnesium ion and iron ion.
9. three-dimensional lung micro-group organization model constructed by the method according to any one of claim 1-8 as vitro detection and
Evaluate the application of lung autophagy effect model.
10. application according to claim 9, it is characterised in that comprise the following steps：
Determinand is added in the three-dimensional lung micro-group organization model, qualitative or quantitative detection LC-3II is on pulmonary macrophage and lung
Expression in chrotoplast, to judge the lung autophagy effect type of the determinand initiation.
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