CN103255097B - Establishment of humanized three-dimensional intestinal mucosa model and application thereof - Google Patents
Establishment of humanized three-dimensional intestinal mucosa model and application thereof Download PDFInfo
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Abstract
The invention provides an establishment of humanized three-dimensional intestinal mucosa model and an application thereof. The model improves a traditional Caco-2 monolayer cell model mainly based on normal physiological structure characteristics of small intestinal epithelium, wherein on basis of adding extracellular matrix, the Caco-2 cells are co-cultured with mucus-secreting cells, interstitial cells and immune cells for a period of time to form a three-dimensional intestinal mucosa model being similar with structure and function of the small intestinal epithelium in vivo. By using the model, a membrane permeating capability at small intestine epithelium as well as a metabolism degree of oral drug molecules can be predicted. The model is mainly used for researching transmembrane transportation and absorption, and metabolism process of medicament molecules at the small intestine in vitro.
Description
Technical field
The invention belongs to medical art, be specifically related to foundation and the application thereof of the three-dimensional intestinal mucosa model of a kind of humanization.
Background technology
Oral administration as a kind of traditional administering mode, the accessibility relative with it and security and accepted extensively by people.Absorption process is one of key factor determining oral drug bioavailability.And small intestine is the main portions of oral drug absorption, so mainly utilize at present both at home and abroad various external and in situ intestinal absorptive model drugs in the transmembrane transport process of small intestine site, thus predict its oral absorption situation.Wherein, Caco-2 monolayer cell model is widely used in one of the microbial film permeability of drugs molecule and the external model of transmembrane transport mechanism at present.This model system compositing factor is clearly single, and in cell phenotype and function and body, intestinal epithelial cells exists part similarity, so it demonstrates certain advantage in the absorption characteristic research of medicine.Current Caco-2 cell model has become the representative in vitro study system of drugs Absorption Characteristics.But it may be noted that: this model remains in a lot of defect.It is poor that large quantity research shows its dependency, and predicting the outcome between different experiments room differs greatly.This model not only lacks rete malpighii and the part metabolic enzyme (as CYP3A) of intestines wall, thus affects the absorption prediction of some compounds (as metabolic enzyme substrate), and lacks extracellular matrix (ECM) and mesenchymal cell.In fact, the existence of ECM and mesenchymal cell and interaction complicated between they and epithelial cell are maintaining the phenotype of intestinal epithelial cells and function aspects is vital.Therefore, the improvement on Caco-2 monolayer cell model basis just seems extremely important.There is relevant report abroad recently: adopt Caco-2 cell and other cell co-cultivations (comprising HT29-MTX cell, people Burkitt ' s lymphoma cell and PC-12 cell etc.), thus set up two kinds of cell co-culture models to simulate intestinal absorption metabolic process.But these models are only limitted to a kind of mesenchymal cell Dual culture, and lack ECM, and model stability and circulation ratio all poor.Under contrast, although animal model has the complex physiologic structure and function of normal intestinal epithelial, overcome the defect existing for In vitro cell model, the existence of species variation greatly limit the extrapolation of its data at human body.In addition, experimentation on animals itself is difficult to Quality Control of realizing ideal, and lacks the advantage of high flux screening; The proposition of relevant ethics dispute and " 3R " principle also constrains the application of animal model in drug absorption metabolism research all greatly.In recent years, the development of regeneration medicine technology makes the external structure of three-dimensional tissue's model become possibility.Dimensional culture system can provide and similar microenvironment in body, make cell and cell, set up between cell and extracellular matrix and be closely connected and carry out signal transmission, thus make the genetic expression of cell in this system, matrix secretion and physiological function more similar at somatocyte.Up to now, have the model construction success of multiple three-dimensional tissue and be applied in research.But by literature search, do not find the relevant report of the three-dimensional intestinal mucosa model building the metabolism of drugs intestinal absorption at present yet.
Based on above reason, the present invention utilizes intestinal epithelial cells, multiple mesenchymal cell and extracellular matrix to build three-dimensional intestinal mucosa model.This culture system can provide with in the similar microenvironment of body tissue, make the growth of intestinal epithelial cells closer to three dimensional growth mode in body, simulate to greatest extent at somatocyte or tissue in cellular form and functional character, thus play it transports metabolism physiological function to external source material absorbing.This three-dimensional model can as a bridge of traditional external monolayer cell model research and experiment in vivo, thus more convenient, to evaluate oral pharmaceutical more accurately permeable membrane ability and bioavailability thereof.
Summary of the invention
The object of this invention is to provide the three-dimensional intestinal mucosa model of a kind of humanization and application thereof, this model can be widely used in the permeable membrane ability and bioavailability thereof of predicting oral pharmaceutical in medicine early discovery and new drug development.
Small intestine epithelium is the main place that exogenous material (mainly comprising nutritive substance and medicine) absorbs, and it is the physiologic barrier that one deck is formed jointly primarily of the immunocyte under intestinal epithelial cells, goblet cell slimy, inoblast, endocrine cell, neurocyte, M cell and mucous membrane.Intestinal epithelial cells is polar cell, is divided into enteric cavity side and base side.
The invention provides the foundation of the three-dimensional intestinal mucosa model of a kind of humanization, step be by mesenchymal cell miscegenation in extracellular matrix, hatch 2 hours later cell epimatrixs to solidify, then last layer mixed epithelium cell is planted in the above, Dual culture is after two weeks, and epithelium layer reaches complete densifie state, and (cross-film resistance value is greater than 250 Ω cm
2), then inoculate one deck immunocyte in Transwell base side, above-mentioned four kinds of co-culture of cells, after one week, obtain breaking up three-dimensional intestinal mucosa model completely.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, described mesenchymal cell comprises the mesenchymal cell existed in all intestinal mucosas of inoblast, is preferably inoblast.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, described extracellular matrix comprises the extracellular matrix of collagen, all natural classes of matrigel and synthesis class, is preferably collagen.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, described mixed epithelium cell is mucilage secretion cell and Caco-2 (adenocarcinoma of colon) cell, and the inoculative proportion of mucilage secretion cell and Caco-2 cell is 1: 9 ~ 1: 3.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, described mucilage secretion cell comprises the adenocarcinoma of colon HT29-MTX cell of resistance to methotrexate, is adapted to all cells with mucilage secretion function of the adenocarcinoma of colon HT29-H cell of semi-lactosi substratum, is preferably HT29-MTX cell.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, described immunocyte comprises the immunocyte with immunoloregulation function existed in scavenger cell, all intestinal mucosas of lymphocyte, is preferably the scavenger cell of THP-1 (people's monocytic leukemic) cell derived.
The foundation of the three-dimensional intestinal mucosa model of humanization provided by the invention, substratum used in described Dual culture is the co-culture of cells substratum optimized containing Multiple components such as FBS (foetal calf serum), non-essential amino acid, L-glutaminate and microbiotic.
The three-dimensional intestinal mucosa model of humanization provided by the invention is applicable to the oral pharmaceutical for human body in natural product, chemical synthetic drug, all sources of bio-pharmaceutical.
The present invention compares tool with existing Caco-2 monolayer cell model and has the following advantages:
1. this three-dimensional model has the rete malpighii similar with intestinal mucosa in body, and itself can as the barrier of some drugs molecule through enteron aisle.
2. the expression amount of the intercellular connection albumen of this model is lower, intercellular is connected more loose, closer to real intestinal epithelial cell intercellular connection state.
3. the translocator level of this three-dimensional model is closer to the expression level of real intestinal epithelial cells.
4. the metabolic enzyme expression level (Procaine esterase) of this three-dimensional model is higher, compensate for the shortcoming that Caco-2 cellular metabolism expression of enzymes is lower.
5. this three-dimensional model to provide between cell and cell and interaction complicated between cell and extracellular matrix, forms the microenvironment similar with small intestine epithelium position in body, makes the growth of intestinal epithelial cells closer to three dimensional growth mode in body.
The present invention mainly utilizes Transwell Establishing and evaluates three-dimensional intestinal mucosa model, this dimensional culture system mainly comprises human intestinal epithelial cells's (Caco-2 cell and mucilage secretion cell), mesenchymal cell, immunocyte and extracellular matrix, thus normal bowel mucous membrane physiological structure in analogue body better.The three-dimensional intestinal mucosa model that the present invention sets up can be widely used in the oral drug absorption metabolism research with different physico-chemical property.This model utilizes various kinds of cell and iuntercellular, interaction direct or indirect between cell and extracellular matrix, set up a kind of physiological structure and the function outer Absorption And Metabolism model of said three-dimensional body closer to internal milieu, can ensure that this model has desirable stability and repeatability simultaneously, thus the permeable membrane ability of unknown compound can be predicted more exactly, and understand their transmembrane transport mechanism in vivo in depth, for the screening of class drug compound and the exploitation of new drug are provided convenience reliable instrument.
Accompanying drawing explanation
The three-dimensional intestinal mucosa model schematic of Fig. 1;
Fig. 2 sets up three-dimensional intestinal mucosa model flow figure;
Periodic acid-Schiffreagent mucus quantitative analysis in Fig. 3 Caco-2 monolayer cell model and three-dimensional intestinal mucosa model;
Fig. 4 Caco-2 monolayer cell model changes with three-dimensional intestinal mucosa model different incubation time cross-film resistance value (TEER);
The marker compounds of the different transporting pathway of Fig. 5 is at the permeable membrane coefficients comparison of Caco-2 monolayer cell model and three-dimensional intestinal mucosa model;
The marker compounds of the different permeable membrane ability of Fig. 6 is at the permeable membrane coefficients comparison of Caco-2 monolayer cell model and three-dimensional intestinal mucosa model;
Fig. 7 esterase substrate (enalapril) is at the permeable membrane coefficients comparison of Caco-2 monolayer cell model and three-dimensional intestinal mucosa model;
The concentration ratio that Fig. 8 esterase meta-bolites (enalaprilat) and substrate (enalapril) thereof are accumulated in Caco-2 cell.
Embodiment
The following example is to further illustrate the present invention, instead of will limit its scope.
Embodiment 1 utilizes Transwell system construction to have the three-dimensional intestinal mucosa model of polarity
Be illustrated in fig. 1 shown below, mesenchymal cell (as inoblast) is inoculated in extracellular matrix (as collagen), hatch extracellular matrix after 2 hours to solidify, plant last layer mixed epithelium cell in the above, i.e. a certain proportion of Caco-2 cell and mucilage secretion cell (as HT29-MTX cell), Dual culture after two weeks, then inoculates one deck immunocyte (scavenger cell as THP-1 cell derived) in the base side of Transwell.Above-mentioned four kinds of co-culture of cells are after one week, and the differentiation of this model completely, after utilizing resistivity meter mensuration cross-film resistance value (TEER) to determine epithelium layer compactness and integrity, namely can be used for the transmembrane transport experiment of medicine.
The substratum of co-culture model adopts containing FBS, and the co-culture of cells substratum that L-glutaminate and microbiotic etc. are optimized, the foundation of model designs according to the time of Fig. 2 completely.
Can find out that this model can secrete a large amount of mucus by periodic acid-Schiffreagent histochemical stain experimental result (Fig. 3), make intestinal epithelial cells surface form one deck rete malpighii, compensate for the deficiency that Caco-2 monolayer cell model lacks rete malpighii; The activity that active coloring experimental result shows four kinds of cells in this model is all better, proves that mass transfer is better in whole dimensional culture system; To utilize in Picogreen fluorescence dye Accurate Measurement 21 days STb gene content in different time point three-dimensional model, result shows that ability of cell proliferation total in this model is stronger; The cross-film resistance value (Fig. 4) that ZO-1 tight junction protein immunofluorescence experiment and different incubation time record shows that in this model, epithelial cell intercellular connects more loose, overcomes Caco-2 monolayer cell model drawback too closely; P-gp immunofluorescence experiment shows that the expression of P-gp in this three-dimensional model slightly declines, thus optimizes the characteristic of P-gp process LAN in Caco-2 monolayer cell model; Meanwhile, RT-PCR experimental result shows this model P-gp expression contents compared with Caco-2 monolayer cell model and slightly lowers, and MRP2 and BCRP expression contents increases, show that the expression amount of translocator in this model is closer to the intestinal epithelial cells expression level in body.
Embodiment 2 utilizes the permeable membrane ability of the passive born of the same parents of this three-dimensional model studying other transhipment marker compounds
Adopt fluorescent yellow as the marker compounds of the other transhipment of born of the same parents, Caco-2 monolayer cell model in contrast, three-dimensional model is pressed preceding method and is set up, every other day liquid is changed between incubation period, until hatch about 21 days, completely, detect compactness and the integrity of epithelium layer by measuring cross-film resistance (TEER), TEER value reaches 250 Ω cm simultaneously in epithelial cell differentiation
2three-dimensional model may be used for transport experiment.At cellular layer top side (villous surface, A face) add the fluorescent yellow liquid of 0.5ml 20 μ g/ml in nutrient solution, simultaneously at bottom side (basal surface, B face) add the blank buffered soln of 1.5ml, start transport experiment, get 100 μ l liquids at 30min, 60min, 90min, 120min in bottom side respectively, and supply the blank buffered soln of 100 μ l simultaneously.Utilize fluorescent spectrophotometer assay fluorescent yellow content, excitation wavelength and emission wavelength are 490nm and 514nm respectively.Utilize formula: P
app=Δ Q/ (Δ tAC
0) calculate apparent permeability coefficients (apparent permeability coefficients, the P of fluorescent yellow
app), wherein Δ Q is the transhipment amount in Δ t, and A is membrane area, C
0for the starting point concentration of medicine, according to the P calculated
appvalue evaluates its permeable membrane capacity variation, result (Fig. 5) shows fluorescent yellow apparent permeability coefficients increase in the three-dimensional model, namely permeability increases, this reduces with the TEER value recorded and ZO-1 immunofluorescence experiment intercellular connects that to become loose result be consistent, illustrate that this three-dimensional model improves Caco-2 monolayer cell model not enough too closely, it can predict the permeable membrane ability of born of the same parents' other transhipment medicine more exactly.
Embodiment 3 utilizes the permeable membrane ability of the passive transcellular transport marker compounds of this three-dimensional model studying
Adopt Proprasylyte as the marker compounds of passive transcellular transport, Caco-2 monolayer cell model in contrast, three-dimensional model is pressed preceding method and is set up, every other day liquid is changed between incubation period, until hatch about 21 days, completely, detect compactness and the integrity of epithelium layer by measuring cross-film resistance (TEER), TEER value reaches 250 Ω cm simultaneously in epithelial cell differentiation
2three-dimensional model may be used for transport experiment.At cellular layer top side (villous surface, A face) add the Proprasylyte liquid of 0.5ml 50 μMs in nutrient solution, simultaneously at bottom side (basal surface, B face) add the blank buffered soln of 1.5ml, start transport experiment, get 100 μ l liquids at 30min, 60min, 90min, 120min in bottom side respectively, and supply the blank buffered soln of 100 μ l simultaneously.Utilize UFLC to measure Proprasylyte content, determined wavelength is 290nm respectively.Utilize formula: P
app=Δ Q/ (Δ tAC
0) calculate the P of Proprasylyte
appvalue, result (Fig. 5) show Proprasylyte in the three-dimensional model apparent permeability coefficients also increase to some extent, namely permeability strengthen.
Embodiment 4 utilizes the permeable membrane ability of this three-dimensional model studying translocator mediate transport marker compounds
Three-dimensional intestinal mucosa model is set up equally according to preceding method, adopt digoxin as the marker compounds of P-gp mediate transport, methotrexate is as the marker compounds of BCRP mediate transport, Etoposide is as the marker compounds of MRP2 mediate transport, Caco-2 monolayer cell model in contrast, the change of above three marker compounds permeable membrane ability in three-dimensional intestinal mucosa model of research respectively, and then understand the changes of function of three kinds of translocators at three-dimensional model.Utilize UFLC to carry out assay to three kinds of marker compounds equally, the determined wavelength of digoxin, methotrexate and Etoposide is respectively 220nm, 302nm and 254nm.Result (Fig. 5) shows that the permeable membrane coefficient of digoxin increases to some extent, and show that outer row's transhipment that P-gp mediates reduces to some extent, namely P-gp function slightly declines, and this and P-gp immunofluorescence experiment result have consistence; And the permeable membrane coefficient of methotrexate and Etoposide reduces to some extent, show that outer row's transhipment that BCRP and MRP2 mediates increases to some extent, namely in three-dimensional model, the function of BCRP and MRP2 slightly strengthens.These results suggest that the function of different translocator in three-dimensional intestinal mucosa model changes to some extent, improve P-gp function in Caco-2 single-layer model excessively strong, and the deficiency that BCRP and MRP2 function is relatively weak.
Embodiment 5 utilizes the permeable membrane ability of this three-dimensional model studying Thief zone marker compounds
Set up three-dimensional intestinal mucosa model according to preceding method equally, choose the marker compounds of three kinds of Thief zone, i.e. caffeine, quinizine and Ketoprofen, study the change of their permeabilities in Caco-2 single-layer model and three-dimensional model.Utilize UFLC to carry out assay to caffeine, quinizine and Ketoprofen, their determined wavelength is respectively 275nm, 241nm and 256nm.Result (Fig. 6) shows the permeable membrane coefficient all decreases to some degree in the three-dimensional model of three kinds of Thief zone marker compounds, and this shows that the extracellular matrix in three-dimensional model and mesenchymal cell wherein may play certain barrier action to Thief zone compound.
Embodiment 6 utilizes in this three-dimensional model studying, the permeable membrane ability of hyposmosis marker compounds
Set up three-dimensional intestinal mucosa model according to preceding method equally, choose in two kinds, the marker compounds of hyposmosis, i.e. Furosemide and Ranitidine HCL, studies the change of their permeabilities in Caco-2 single-layer model and three-dimensional model.Utilize UFLC to carry out assay to them, the determined wavelength of Furosemide and Ranitidine HCL is respectively 271nm and 314nm.Result (Fig. 6) shows in two kinds, hyposmosis marker compounds permeable membrane coefficient in the three-dimensional model slightly increases.
Embodiment 7 utilizes permeable membrane ability and the extent of metabolism of this three-dimensional model studying carboxylester substrates
Three-dimensional intestinal mucosa model is set up equally according to preceding method, choose the positive substrate of enalapril as esterase, study the change of its permeability in Caco-2 single-layer model and three-dimensional model, investigate the extent of metabolism of enalapril in this model simultaneously, namely extract the enalapril of accumulation in epithelial cell and meta-bolites (enalaprilat) thereof and utilize UFLC to carry out assay to them, determined wavelength is 215nm.Result shows that enalapril permeable membrane coefficient in the three-dimensional model reduces (Fig. 7), in epithelial cell, the concentration ratio of enalaprilat and enalapril increases (Fig. 8) simultaneously, and above result confirms that the esterase activity in three-dimensional model is significantly improved.
Claims (3)
1. the establishment method of the three-dimensional intestinal mucosa model of humanization, it is characterized in that: by a kind of mesenchymal cell miscegenation in extracellular matrix, hatch rear extracellular matrix to solidify, then two kinds of mixed epithelium cells are planted in the above, Dual culture is after two weeks, inoculate one deck immunocyte in Transwell base side again, above-mentioned four kinds of co-culture of cells, after one week, obtain breaking up three-dimensional intestinal mucosa model completely;
Described mesenchymal cell is inoblast;
Described extracellular matrix is collagen;
Described mixed epithelium cell is mucilage secretion cell and adenocarcinoma of colon Caco-2 cell, and the inoculative proportion of mucilage secretion cell and Caco-2 cell is 1:9 ~ 1:3; Described mucilage secretion cell is HT29-MTX cell;
The scavenger cell of described immunocyte behaviour monocytic leukemic THP-1 cell derived.
2. according to the establishment method of the three-dimensional intestinal mucosa model of humanization described in claim 1, it is characterized in that: substratum used in described Dual culture is for containing foetal calf serum FBS, non-essential amino acid, L-glutaminate and antibiotic co-culture of cells substratum.
3. the three-dimensional intestinal mucosa model of the humanization of method establishment described in claim 1 is used for the oral pharmaceutical of researching human body.
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| CN103589637A (en) * | 2013-11-08 | 2014-02-19 | 王海涛 | Three-dimensional culture system for cells in vitro |
| CN105441062B (en) * | 2014-09-29 | 2017-12-01 | 中国科学院大连化学物理研究所 | A kind of fluorescence probe substrate of people's enteron aisle Carboxylesterase Activity detection and its application |
| CN105567564A (en) * | 2016-01-22 | 2016-05-11 | 广西大学 | Joint evaluation model for human-body bioavailability and toxicity of lead in food |
| CN110191947A (en) * | 2016-11-10 | 2019-08-30 | 奥加诺沃公司 | It is engineered intestinal tissue and its purposes |
| CN107083364A (en) * | 2017-01-13 | 2017-08-22 | 江苏省农业科学院 | A kind of structure of Caco-2/HUVEC cells co-culture system |
| CN109112096A (en) * | 2018-09-07 | 2019-01-01 | 程辉 | A kind of engineered external three-dimensional mucous membrane of mouth experimental model |
| CN112342183A (en) * | 2020-10-30 | 2021-02-09 | 广东省农业科学院动物科学研究所 | Method for establishing piglet gastrointestinal tract in-vitro environment model and application |
| CN115141793B (en) * | 2022-07-20 | 2024-04-16 | 微康益生菌(苏州)股份有限公司 | Method for evaluating and regulating intestinal barrier function and intestinal immunity |
| CN116994662B (en) * | 2023-09-06 | 2024-02-23 | 北京晶泰科技有限公司 | Method, device and equipment for predicting PBPK model for small molecule attribute prediction |
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