CN104694456B - Hepatic lineage after the method for in vitro culture hepatic lineage and the optimization of use this method culture - Google Patents
Hepatic lineage after the method for in vitro culture hepatic lineage and the optimization of use this method culture Download PDFInfo
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Abstract
The invention belongs to biological technical fields, and in particular to it is a kind of optimized after hepatic lineage extracorporeal culturing method and using this method obtain optimization after hepatic lineage.The extracorporeal culturing method is included using sandwich cell culture method, free serum culture and inducing culture.For hepatic lineage close to the 26S Proteasome Structure and Function of primary liver cell, the expression of drug transporters, bile acid transporter and/or cholic acid synzyme is significantly higher than traditional hepatic lineage after the optimization of acquisition；Its drug choleresis index (BEI) and inherent bile clearance rate (CLb,int) it is significantly higher than traditional hepatic lineage；There is the polarization expression of drug transporters in it.
Description
Technical field
The invention belongs to biological technical fields, and in particular to a kind of method of in vitro culture hepatic lineage and using the party
Hepatic lineage after the optimization of method culture.
Background technology
Liver is to determine drug metabolism and the major organs of toxic action and a variety of virus hepatitis, liver neoplasm disease
The target organ of disease.At present, in pharmaceuticals industry circle, primary hepatocyte (including humans and animals primary hepatocyte) is because of its drug metabolic enzyme
Expression and new drug development and clinical picture with drug transporters have relatively good correlation, are referred to as pharmacokinetic study
Golden standard, while be also the precious resources (Sinz, Wallace et al.2008) of liver transfer operation and bioreactor.At present
The international market conservative estimation of primary hepatocyte is at 2,000,000,000 dollars.But primary hepatocyte cannot pass on, multiple proteins expression
Decline, in addition expensive and donor dependence, limits its further use in scientific research and clinically.Therefore, either
In scientific research or pharmaceuticals industry practice there is an urgent need to a kind of function it is similar with liver cell, passage, reliably and the work(that is easy to get can be stablized
It can property liver cell.
The FDA white paper of 2010 points out that drug transporters have in new drug development and drug metabolic enzyme is equal for the first time
Consequence, and define 7 kinds of drug transporters that must be investigated, wherein have 5 kinds of drug transporters liver metabolism,
Significant role (International Transporter, Giacomini et al.2010) is played in removing toxic substances.This content exists
It is determined in FDA drug interaction guides in 2012.
But the cell line for the similar liver cell being currently available has various shortcomings, it is suitable particular without possessing
Drug transporters expression, be not used to the distribution of liver relevant drug liver and gall and metabolism, toxicity research.It is for example, most general
It is expressed above liver derived cell strain HepG2 almost without important drug transporters, needless to say bile duct similar structures.Mesh
The preceding cell line HepaRG generally acknowledged closest to primary hepatocyte, although drug metabolic enzyme CYP3A4 expression is compared with horn of plenty and can be with
Induction, but the expression of its drug transporters, which only has Pgp, to gain recognition, and shown by immunohistochemical assay, it is impossible to reach
Polarization expression (Andersson, Kanebratt et al.2012) (referring to attached drawing 4).
In recent years, many in vitro tests confirm embryonic stem cell or induction versatile stem cell are divided into liver sample
Cell, still, these technologies are by donor source (the ES cells for coming from embryo) and the high cost of induction generation process and complexity
(deriving from the iPS cells of human epithelial cell, it is necessary to carry out transdifferentiation trilogy) is limited, and can not provide suitable hepatic lineage
For medicament research and development (Jensen, Hyllner et al.2009).Although there are more document report these cells to possess centainly
Drug metabolism expression of enzymes, but be rarely reported for the expression of drug transporters, and unsatisfactory.At present in the world
L cell can be divided into mouse source induction hepatic lineage (induced by newest direct transdifferentiation technology
hepatocyte-like cell,iHep).Although iHep has been compared much in form, function and application with existing cell line
Advantage, still, iHep are not significantly increased (Fig. 1) in terms of the expression of transporter.In drug transporters and cholic acid synthesis point
Secrete aspect and immature, the cholic acid synthesis secretion capacity that does not form the polarization expression of drug transporters and can detect.It is true
On, the synthesis of cholic acid is secreted and the detoxification ability of drug is the important symbol of mature hepatocytes.Experimental data is shown, existing
In iHep cells, drug transporters and the expression quantity of the relevant synzyme of cholic acid and transporter are with it in mouse primary hepatocytes
Expression quantity compared to extremely low, be only 5-10% (Fig. 1).It similarly, can also be thin by the non-liver in people source using direct transdifferentiation technology
Born of the same parents obtain people source induction hepatic lineage (human induced hepatocyte-like cell, HiHep), but the cell is also same
Sample has drawbacks described above.
The content of the invention
Technical problem
It 2011, publishes thesis on Nature, epigenetics means can be utilized in the world by being put forward for the first time, and directly will
The rat-tail fibrocyte transdifferentiation of mouse is into hepatic lineage（Huang PY,He ZY,Ji SY,Sun HW,Xiang D,et al.
(2011)Induction of functional hepatocyte-like cells from mouse fibroblasts by
defined factors.Nature475:386-U142.）.Compared with primary hepatocyte, pass through above-mentioned direct transdifferentiation technology
The source of the hepatic lineage of generation theoretically can infinitely pass from the limitation of donor resource on the basis of inhereditary feature is kept
Generation.They not only have the function of the multinomial physiological characteristic of liver cell and, but also with clear genetic background and certain medicine
Object is metabolized expression of enzymes.Therefore with substitution primary hepatocyte, become for medicament research and development and the neoblast model of clinical practice
Potentiality.
But the cell line only possesses preliminary drug transporters and cholic acid metabolism expression of enzymes, and various related proteins
There is no the polarization distributions on cell membrane.It is explicitly pointed out on the drug interaction guide of FDA2012 issues, only drug turns
Fortune body polarize expression on liver plasma membrane, cell be only possible to possess cholate polarization absorb, secretion capacity, this is that evaluate liver sample thin
The key property of born of the same parents.
Therefore, in the prior art, for example, embryonic stem cell or induction versatile stem cell either are divided into liver
The polarization that the hepatic lineage that like cell is still obtained using for example direct transdifferentiation technology does not form drug transporters is expressed
And/or the cholic acid synthesis secretion capacity that can be detected.In this application, the prior art will be used (including direct transdifferentiation technology
With embryonic stem cell or versatile stem cell induction differentiation technique) the non-liver cell of the non-liver cell of animal sources or people source is obtained
Hepatic lineage be defined as traditional hepatic lineage, do not form the polarization expression of drug transporters and/or the courage that can detect
Acid synthesis secretion capacity, has marked difference in function and form with primary hepatocyte.
To improve the prior art（Including direct transdifferentiation technology and embryonic stem cell or versatile stem cell induction differentiation skill
Art）Cholic acid synzyme, the expression of bile acid transporter and drug transporters and function in traditional hepatic lineage of generation, the application's
Inventor is generated by test of many times suitable for the prior art（Including direct transdifferentiation technology and embryonic stem cell or more work(
It can stem cell induction differentiation technique）The special extracorporeal culturing method of traditional hepatic lineage of generation.This method effective stimulus is by existing
There is technology（Including direct transdifferentiation technology and embryonic stem cell or versatile stem cell induction differentiation technique）Traditional liver of generation
Further differentiation, cholic acid synthesis expression of enzymes and the related drugs transporter polarization distribution of like cell, so that traditional hepatic lineage
Possess the bile intake similar with primary hepatocyte, secretion capacity, drug transporters polarization expression, so as to be used for related section
Grind work.
Therefore, one object of the present invention is to provide a kind of traditional hepatic lineage to obtaining using the prior art to carry out body
The method of outer culture.Specifically, the method can further promote the growth of traditional hepatic lineage and differentiation and improve drug
Transporter, the expression of bile acid transporter and/or cholic acid synzyme and polarization expression.
Another object of the present invention is provides hepatic lineage after a kind of optimization, and cholic acid closes in hepatic lineage after the optimization
Expression into enzyme, bile acid transporter and drug transporters increases substantially, and function is close to primary hepatocyte.
Technical solution
The present invention provides a kind of extracorporeal culturing method of traditional hepatic lineage, including：
Step 1, using sandwich（sandwich）Cell culture method culture tradition hepatic lineage, i.e.,：Traditional liver sample is thin
Born of the same parents are seeded on 24 orifice plates containing Collagen type-I I, in addition ordinary culture medium, when culture cell 24-48 is small after, spread matrigel,
Formed sandwich culture structure, continue cultivate 24-48 it is small when；
Step 2 removes original ordinary culture medium, uses inducing culture instead and continues culture 3-5 days, liver sample is thin after being optimized
Born of the same parents；
Wherein, the inducing culture to add derivant on the basis of serum free medium, i.e. remove by ordinary culture medium
1% hyclone, and add cAMP (cyclic adenosine monophosphate), 3MC (3-MECA), PB (phenobarbital), TCA (cow-bezoar courages
Acid) and VPA（Valproic acid）At least one of nuclear receptors agonists, above-mentioned each nuclear receptors agonists are in the inducing culture
Concentration range be respectively cAMP1-10 μM, 3MC1-10 μM, PB10-200 μM, TCA5-50 μM and VPA0.2-4 μM；
Preferably, the inducing culture contains two or three in cAMP, 3MC, PB and TCA, further excellent
Selection of land, the inducing culture contain the cAMP that concentration is 2 μM and the PB that concentration is 50 μM or are 2 μM containing concentration
CAMP, concentration are 2 μM of 3MC and concentration is 50 μM PB contains the cAMP that concentration the is 2 μM, 3MC that concentration is 2 μM, dense
The PB and concentration that spend for 50 μM are 20 μM of TCA.
It is highly preferred that it is 20 μM that the inducing culture, which contains the cAMP that concentration is 2 μM, the PB that concentration is 50 μM and concentration,
TCA.
Wherein, traditional hepatic lineage does not form the polarization expression of drug transporters and/or the cholic acid that can be detected
Synthesize secretion capacity.
Preferably, traditional hepatic lineage has transfected hepatocyte nuclear factor.
Preferably, traditional hepatic lineage is thin by the liver sample of embryonic stem cell or induction versatile stem cell differentiation
Born of the same parents；Or traditional hepatic lineage is to utilize direct transdifferentiation technology, the hepatic lineage obtained by the non-liver cell of animal sources,
For example, the mouse source hepatic lineage obtained by rat-tail into fiber (TFF) cell or rat embryo fibroblast (MEF) cell；Alternatively, institute
It is by the non-liver cell in people source using direct transdifferentiation technology to state traditional hepatic lineage, and preferably human embryos fibroblast obtains
People source hepatic lineage；（Referring for example to Huang PY, He ZY, Ji SY, Sun HW, Xiang D, et al. (2011)
Induction of functional hepatocyte-like cells from mouse fibroblasts by
defined factors.Nature475:386-U142. the method recorded is formed）.
It is highly preferred that mouse source hepatic lineage has transfected Hepatocyte nuclear factor 4 α (Hnf4 α)；People source hepatic lineage
Hepatocyte nuclear factor 4 A (HNF4A) is transfected.
On the other hand, the present invention provides hepatic lineage after a kind of optimization, wherein, hepatic lineage passes through upper after the optimization
State extracorporeal culturing method acquisition.
The expression of the drug transporters of hepatic lineage is one times or more of traditional hepatic lineage after the optimization；
The expression of the bile acid transporter of hepatic lineage is significantly higher than traditional hepatic lineage after the optimization；
The expression of the cholic acid synzyme of hepatic lineage is significantly higher than traditional hepatic lineage after the optimization；
The drug choleresis index (BEI) of hepatic lineage and inherent bile clearance rate (CL after the optimizationb,int) significantly
Higher than traditional hepatic lineage；
There is drug transporters polarization expression in hepatic lineage after the optimization.
Wherein, the bile acid transporter is Bsep, Mrp2 and/or Ntcp；The drug transporters for P-gp, Bcrp,
Oatp1b2, Oatp1a1 and/or Oatp1a4；The cholic acid synzyme is Cyp7a1.
Advantageous effect
To improve traditional hepatic lineage liner acid enzyme, bile acid transporter and the drug transporters that direct transdifferentiation generates
Expression and function, the application employ the extracorporeal culturing method for including following feature：Hnf4 α/HNF4A are being transfected into traditional liver
Like cell and express after, cultivated using sandwich cultivation, afterwards using serum free medium and add in nuclear receptor induction
The activation of factor pair tradition hepatic lineage, optimization.
Hepatic lineage has the advantage that feature after the optimization obtained using herein described extracorporeal culturing method：(1) medicine
Object transporter and the mRNA level in-site of endogenous bile transport body and its relevant nuclear receptor with they primary hepatocyte mRNA water
It is flat substantially close to；(2) cholangiole can be formed in vitro and with secreting function；(3) hepatic lineage and primary liver after optimizing
Cell is fine for the correlation of the choleresis rate of drug.
Hnf4 α/HNF4A significantly increase the mRNA of Bsep, Mrp2, Ntcp, Cyp7a1 in mouse source/people source hepatic lineage
Expression quantity.
Different with the cultivation of traditional hepatic lineage, extracorporeal culturing method described herein uses free serum culture,
So that in hepatic lineage the mRNA of liver drug transporter expression be improved significantly, especially cholic acid intake transporter
The expression of Ntcp and outer row's transporter Bcrp significantly improve.
Extracorporeal culturing method described herein uses Three-dimensional cell culture method (sandwich cell culture method) so that liver
The expression of the mRNA of liver drug transporter significantly improves in like cell.
CAMP, PB and/or TCA etc. are added in serum free medium and greatly enhances liver main cholic acid turn
Transport the level of the mRNA of body Bsep, Mrp2 and Ntcp.In addition, cAMP, PB and/or TCA etc. are to the drug transporters (P- in liver
Gp, Bcrp, Oatp1a1/4 and Oatp1b2) the mRNA level in-site effect of improving a lot.The mRNA of cholic acid synzyme Cyp7a1
Level can be enhanced 4 times under the induction of cAMP.The increase of these drug transporters, bile acid transporter and cholic acid synthesis expression of enzymes
It is the important symbol that hepatic lineage is improved for drug and endogenous material metabolism distribution capability.
This work will lay the foundation for the practical application of epigenetics and direct transdifferentiation technology, make novel liver sample thin
Born of the same parents are possibly realized for new drug development.
Description of the drawings
Figure 1A-Fig. 1 F show that different experimental conditions and different inducible factors synthesize cholic acid in the tradition hepatic lineage of mouse source
The influence of the gene expression dose of enzyme (Cyp7a1) and a variety of bile acid transporters.
Fig. 2A-Fig. 2 G show different experimental conditions and different inducible factors to multi-medicament in the tradition hepatic lineage of mouse source
The gene expression dose of transporter or the influence to BEI (%).
Fig. 3 A- Fig. 3 B are shown to the drug efflux transporter in hepatic lineage after the mouse source optimization that is obtained in embodiment 1
With intake transporter（3A）And related nuclear receptor（3B）Gene expression dose influence.Wherein, with rat-tail fibrocyte and
Mouse liver cell is control.
Fig. 4 is the image to after HepaRG cells progress immunohistochemical staining, being obtained under Electronic Speculum.HepaRG cells are mesh
The preceding hepatic lineage strain uniquely recognized by FDA can be used for liver drug metabolism enzyme induction research.It but can from Fig. 4
Go out, the cell line drug metabolic enzyme (CYP3A4, Fig. 4 C) expression is fine, but important drug and bile acid transporter (Fig. 4 A：P-
Gp, Fig. 4 B, MRP2) expression it is very insufficient, only in part, cell has expression, and uneven.
Fig. 5 be after the optimization that mouse liver cell, traditional hepatic lineage and embodiment 1 obtain hepatic lineage in fluorescence microscopy
Microscopic observation whether there is the image that CDF collects in the secretion of the bile duct region of formation.As can be seen that mouse liver cell and embodiment 1
Hepatic lineage has CDF to collect in the bile duct region of formation after the optimization of acquisition, and traditional hepatic lineage does not observe this phenomenon.
Fig. 6 is confocal microscope photo.First row and the photo that the 3rd row is mouse liver cell, wherein, weight
Cholic acid and drug transporters Bsep, Mrp2, Pgp and Bcrp is wanted to polarize along gallbladder tube structure to express；Second row and the 4th row are real
Apply example 1 acquisition optimization after hepatic lineage photo, wherein, Bsep, Mrp2, Pgp and Bcrp equally occur polarization expression.It can be with
Find out, the photo form of hepatic lineage is sufficiently close to after the optimization of mouse liver cell and the acquisition of embodiment 1.
Fig. 7 is DPDPE（Enkephalins）、d8-TCA（Isotope marks taurocholate）, Rosuvastatin and methotrexate (MTX) four
In the correlation of bile clearance rate in hepatic lineage and mouse primary hepatocytes after planting compound before optimization.
The gene expression dose (Fig. 8 a) of the drug transporters of hepatic lineage after people's source optimization that Fig. 8 obtains for embodiment 2
With BEI (%) (Fig. 8 b).
Specific embodiment
By the following specific examples further illustrate the invention.It is to be understood that following embodiment is merely to illustrate this hair
Bright rather than restriction the scope of the present invention.
The acquisition of hepatic lineage after 1 mouse source optimization of embodiment
Step 1 generates traditional mouse source hepatic lineage using direct transdifferentiation technology
1) generation of molecular cloning and slow virus
The carrier of target gene is built first：Multiple cloning sites are inserted into the slow virus carrier with reporter gene GFP
The PmeI restriction enzyme sites of pWPI, target gene Gata, Hnf1 α, the cDNA of Foxa3 these three transcription factors are connected respectively
Onto so carrier for having modified, three pWPI carriers containing transcription factor building afterwards respectively with two packaging plasmids
PsPAX2, pMD2.G are added in 293T cells.48 it is small when after, collect the supernatant containing slow virus and with 0.45μm filter
Filtering, is dispensed into EP pipes, -80 DEG C freeze.
2) rat-tail is into the culture of fiber (TFF) cell or rat embryo fibroblast (MEF) cell：
Rat-tail fibroblast is to cut 5cm tails with big mouse from 2 months first, removes corium, shreds to 1cm
Left and right, is put into the 6cm tablets for be covered with Collagen I and grows, and culture medium is the DMEM containing 10%FBS, volume 5ml.After 5 days, into
Fibrocyte is transferred to new be covered in the 6cm tablets of Collagen I.General rat-tail fibroblast the 7 to 9th is alternative in slow virus
Transfection.
Rat embryo fibroblast cell is the mouse embryo of acquisition 13.5 days first, head and internal organ removal, and residue tissue shreds,
0.25% trypsase digests 15min under conditions of 37 DEG C, and cell is transferred in the 6cm tablets for be covered with Collagen I and grows afterwards,
Culture medium is still the DMEM containing 10%FBS, volume 5ml.The 3rd alternative transfection in slow virus of general rat embryo fibroblast cell.
3) transfection of slow virus and the acquisition of ripe traditional hepatic lineage
First day on the 6cm tablets upper berth 1.5 × 10 for being covered with Collagen I5A rat-tail fibroblast or mouse embryo are into fibre
Cell is tieed up, the virus titer surveyed according to flow cytometer adds in three transcription factors Gata, Hnf1 α, Foxa3 within second day, and same
When add in polybrene (polybrene, final concentration reach 8 μ g/ml or 4 μ g/ml), change fresh culture within the 3rd day, culture medium is still
For the DMEM containing 10%FBS, fresh culture is changed within the 4th day, culture medium is ordinary culture medium, changes a Nostoc commune Vanch within two days afterwards
Base transfects 14-21 days, obtains ripe traditional hepatic lineage.Note：Nostoc commune Vanch based formulas：DMEM/F121:1 basal medium
（Hyclone）Containing 0.1 μM of dexamethasone, 20 μ g/LTGF- α, 10 μ g/L EGF, 4.2mg/L insulin, 3.8mg/L people turns iron egg
In vain, 5 μ g/L selenites and 1% hyclone.
Step 2,4 α of transfected hepatocytes nuclear factor (Hnf4 α)
The cDNA of target gene Hepatocyte nuclear factor 4 α (Hnf4 α) is connected to the slow virus with reporter gene GFP to carry
In body pWPI, the pWPI carriers and packaging plasmid psPAX2, pMD2.G of the α containing Hnf4 built are infected into 293T cells.48 is small
Shi Hou collects the supernatant containing slow virus and is filtered with 0.45 μm of filter, is dispensed into EP pipes.It is examined using flow cytometer
Virus titer has been surveyed, has finally carried out traditional hepatic lineage that transfection procedure 1 obtains.
Step 3, the traditional hepatic lineage for having transfected Hnf4 α obtained using sandwich cell culture method incubation step 2.
Traditional hepatic lineage of formation is digested into separation first, then traditional hepatic lineage is seeded in containing Collagen type-I I
24 orifice plates on, density be 1 × 106A/cm2, in addition 500 μ l ordinary culture mediums, when culture cell 24 is small after, spread matrigel,
Formed sandwich culture structure, continue culture 24 it is small when.The formula of ordinary culture medium：DMEM/F121:1 basal medium contains 0.1
μM dexamethasone, 20 μ g/L TGF- α, 10 μ g/L EGF, 4.2mg/L insulin, 3.8mg/L human transferrins, 5 μ g/L Asias selenium
Hydrochlorate and 1% hyclone.
Hepatic lineage (mouse source) after step 4, optimization culture are optimized
After step 3, original ordinary culture medium is removed, uses inducing culture instead, i.e., removes 1% tire ox blood in ordinary culture medium
On the basis of clear, in addition nuclear receptors agonists：CAMP (2 μM), PB (50 μM) and TCA (20 μM) continue culture 3-5 days, obtain
Hepatic lineage after optimization.
The acquisition of hepatic lineage after 2 people's source optimization of embodiment
Except with mankind's embryo fibroblast（Source pregnant women placental sample）Instead of rat-tail into fiber (TFF) cell or
Rat embryo fibroblast (MEF) cell, and transfect mankind's embryo fibroblast using Hepatocyte nuclear factor 4 A (HNF4A)（Source
Pregnant women placental sample）Outside, using hepatic lineage after method acquisition people's source optimization similar to Example 1.
Embodiment 3 carries out experimental study to various concentration and the combination of different types of nuclear receptors agonists.
It is obtained first on the basis of based on traditional hepatic lineage and has transfected the hepatic lineage of Hnf4 α (step is the same as embodiment 1
In step 1-3).Then use the inducing culture containing various concentration and various combination nuclear receptors agonists instead and optimize choosing
It selects, has attempted a kind of single nuclear receptor and its concentration, a variety of nuclear receptors agonists combinations and its concentration respectively, finally obtained optimal
The combination of the nuclear receptors agonists of choosing and concentration.Concrete outcome is as shown in the following table 1,2,3.
Table 1：A kind of single nuclear receptors agonists and its concentration range
Table 2：A variety of nuclear receptors agonists combine and its using concentration
3 most preferred nuclear receptors agonists of table combine and its using concentration
EXPERIMENTAL EXAMPLE function assessment and gene expression are tested to confirm that cell optimizes successfully
(it is generally necessary to 3-5 days time) after cell optimization is completed, verified respectively from expression and function of genes angle
Whether optimization process succeeds：
First, the expression of drug transporters, bile acid transporter, cholic acid synzyme is measured by quantitative PCR (QPCR) method
First with the total serum IgE of Trizol extracting cells, the kit extracting RNA extracted according to RNA.By the total serum IgE of extraction
After diluting certain multiple, the absorbance value at two wavelength of 260nm and 280nm is measured with ultraviolet specrophotometer, is then pressed
The following formula extracts the concentration of obtained total serum IgE to calculate：[RNA concentration (μ g/ml)]=A260 × 40 × extension rate, 260nm
With the ratio (A260/A280) of reading at 280nm two, it can reflect the purity of nucleic acid, general A260/A280 is between 1.8-2.0.
2 μ g RNA react generation cDNA by reverse transcription system, and reverse transcription reaction condition is：37 DEG C of 15min, 85 DEG C of 5s, keep afterwards
At 10 DEG C.Quantitative PCR operation, Pgp, Bcrp, Mrp2, Ntcp, Bsep etc. is carried out by SYBR Premix Ex Taq kits
Primer is designed synthesis as requested, is measured afterwards into instrument ABI7500 quantitative PCR systems.
Whether the expression of measure important drugs transporter, bile acid transporter etc. is compared with conventional cell is significantly increased, and 2/3
It is qualified that measure object expression, which is improved more than 50%,.
I. referring to Figure 1A-Fig. 1 F, in every width coordinate diagram, first group of Nogata icon (the Nogata icon of the leftmost side three) represents
Rat-tail fibrocyte (TTF), primary hepatocyte (PriHep) and the mouse source tradition hepatic lineage obtained using 1 step 1 of embodiment
Cholic acid synzyme (Cyp7a1) and a variety of bile acid transporters gene expression dose；Second group of (intermediate three Nogata icons) table
Show that the mouse source tradition hepatic lineage obtained using 1 step 1 of embodiment carries out sandwich culture respectively（That is, embodiment 1 is only carried out
Step 1 and step 3）, free serum culture（That is, after carrying out 1 step 1 of embodiment, using the tire ox blood that 1% is removed in ordinary culture medium
Clear serum free medium, when culture 48 is small）And the long-term Nostoc commune Vanch of 6 weeks is carried out after traditional hepatic lineage maturation again（That is,
After carrying out 1 step 1 of embodiment, it is further cultured for 6 weeks using ordinary culture medium）.Then measure respectively under these three condition of culture
The gene expression dose of cholic acid synzyme (Cyp7a1) and a variety of bile acid transporters；3rd group of (the Nogata icon of the rightmost side 6) table
Show the mouse source tradition hepatic lineage obtained using 1 step 1 of embodiment in inducing culture（It is separately added into serum free medium
Each 2 μM of cAMP of nuclear receptors agonists, 2 μM of 3MC, 50 μM of PB, 20 μM of TCA or 1mM VPA）It is cultivated after either transfection Hnf4 α
3-5 days time measured the gene expression dose of cholic acid synzyme (Cyp7a1) and a variety of bile acid transporters.
It can be seen that from the result of Fig. 1：
(1) for traditional hepatic lineage, such as the culture item of the long-term cultivation of sandwich culture, free serum culture or 6 weeks
Part can improve the mRNA expression (figures of its liver bile acid transporter (Bsep, Mrp2, Ntcp) and cholic acid synzyme (Cyp7a1)
1A- Fig. 1 D)；
(2) when addition such as cAMP in inducing culture（2μM）、3MC（2μM）、PB（50μM）、TCA（20μM）Or VPA
（1mM）Nuclear receptor coactivator agent when, bile acid transporter Bsep, Mrp2, Ntcp expression all improve (Figure 1A-Fig. 1 C), and cholic acid close
4 times or so are improved under cAMP inductions and the inducing action of PB or TCA is used alone not into enzyme (Cyp7a1) mRNA expression
Substantially (Fig. 1 D).
(3) in BEI experiments (Fig. 1 E and Fig. 1 F), such as the long-term cultivation of sandwich culture, free serum culture or 6 weeks
The induction of condition of culture and cAMP or TCA etc. and the transfection expression of transcription factor Hnf4 α, can significantly improve CLF
BEI (%) value of (Bsep substrates), D8-TCA (Bsep and Mrp2 substrates), illustrating the outer row of CLF, d8-TCA increases, so as into one
Step illustrates the gene expression water of different condition of culture and different inducible factors to a variety of bile acid transporters in traditional hepatic lineage
Flat influence.
II. it is similar with Fig. 1 referring to Fig. 2A-Fig. 2 E, in every width coordinate diagram of Fig. 2, first group of Nogata icon (leftmost side
Three Nogata icons) represent rat-tail fibrocyte (TTF), primary hepatocyte (PriHep) and using the acquisition of 1 step 1 of embodiment
The gene expression dose of the drug transporters of mouse source tradition hepatic lineage；Second group (intermediate three Nogata icons) are represented using real
Apply 1 step 1 of example acquisition mouse source tradition hepatic lineage carry out respectively sandwich culture (that is, only carry out embodiment 1 step 1 and
Step 3), free serum culture are (that is, after carrying out 1 step 1 of embodiment, using the nothing for the hyclone that 1% is removed in ordinary culture medium
Blood serum medium, when culture 48 is small) or traditional hepatic lineage maturation after carry out the long-term Nostoc commune Vanch of 6 weeks again（That is, implemented
After 1 step 1 of example, it is further cultured for 6 weeks using ordinary culture medium）, then measure drug transporters gene expression dose；3rd group
The mouse source tradition hepatic lineage that (the Nogata icon of the rightmost side 6) represents to obtain using 1 step 1 of embodiment is in inducing culture（It is general
Each 2 μM of cAMP of nuclear receptors agonists, 2 μM of 3MC, 50 μM of PB, 20 μM of TCA or 1mM VPA are separately added into logical culture medium）Or
Person cultivates 3-5 days after transfecting Hnf4 α, measures the gene expression dose of drug transporters.
It can be seen that from the result of Fig. 2：
(1) for traditional hepatic lineage, sandwich culture and free serum culture can significantly improve outer row's transporter Pgp,
The mRNA expression of Bcrp and intake transporter Oatp1b2, Oatp1a1/4.Derivant cAMP, PB, TCA can improve Pgp,
The mRNA expression of Bcrp, Oatp1b2, Oatp1a1/4, but VPA and 3-MC influences the gene expression dose of each drug transporters
Unobvious.
(2) transcription factor Hnf4 α can also be greatly improved outer row and absorb the mRNA expression of transporter.In addition, BEI experiment knots
Fruit (Fig. 2 F and Fig. 2 G) shows that sandwich culture and free serum culture and derivant cAMP, PB, TCA and Hnf4 α can be improved
Rosuvastatin (Bcrp substrates) and BEI (5) value of methotrexate (MTX) (Bcrp substrates).
III. referring to Fig. 3 A results as it can be seen that from the point of view of gene expression dose, hepatic lineage after the optimization that embodiment 1 obtains
In, the important drug transporters of various surveyed livers, the expression of bile acid transporter are greatly improved and primary hepatocyte tool
There is comparativity.Referring to Fig. 3 B results as it can be seen that the nuclear receptor and outer row surveyed also are significantly improved with intake transporter mRNA expression.
2nd, drug transporters, bile acid transporter common location research
Hepatic lineage after the optimization that experimental subjects obtains for embodiment 1.
Experimental procedure：Under room temperature, 4% formaldehyde fixes cell 1h；PBS is washed three times, each 5min；0.25%Triton
(being dissolved in 3%BSA-PBS solution) permeabilized cells, room temperature 15min；PBS is washed three times, each 5min；3%BSA-PBS is closed, room
Warm 1h；Sop up confining liquid, add primary antibody P-19 (mouse anti-Bsep, 1:100) and M2- III -6 (mouse anti-Mrp2,1:40), 16 are incubated for 4 DEG C
Hour is stayed overnight；PBS is washed three times, each 5min；After sopping up PBS, add the anti-mouse lgG (1 of secondary antibody FITC-:300) resist with TRITC-
Mouse lgG (1:400), it is protected from light incubation at room temperature 1h；PBS is washed three times, each 5min；PBS is sopped up, the diazabicylo for adding 2.5% is pungent
Alkane (9:1 glycerine/PBS is prepared) sealing and covered are closed, it is total in the micro- Microscopic observation Bsep and Mrp2 of confocal laser
Positioning scenarios.For the common location of P-gp and Bcrp, steps flow chart is similar with Bsep, Mrp2 common location, and simply primary antibody is CD44
(mouse anti-P-gp, 1:And mouse anti-CD 33 8 (1 200):100).After the results show optimization in hepatic lineage transporter have it is certain fixed altogether
Position.
Specifically, hepatic lineage possesses similar with primary hepatocyte for the first time after the optimization obtained referring to Fig. 6, embodiment 1
Important drugs transporter polarization expression (for example, outer row's type drug transporters Bsep, Mrp2 are expressed in cholangiole side), this
Feature just has embodiment in primary hepatocyte.And all do not find that this is special in hepatic lineage model HepaRG best at present
Sign is (referring to Fig. 4).By comparison, it is apparent that using liver sample after the optimization of extracorporeal culturing method described herein acquisition
Cell is substantially better than existing best hepatic lineage model HepaRG and primary hepatocyte in-vitro culture model possesses comparativity.
3rd, the functional examination of hepatic lineage after optimizing
Hepatic lineage after the optimization that experimental subjects obtains for embodiment 1.
I. whether hepatic lineage forms functional gallbladder tube structure after inspection optimization.
Experimental procedure：After the step 4 of embodiment 1, with the 6th day of inducing culture culture, triplicate cell was used
Warm plus Ca per 1 milliliter of hole2+Or it is not added with Ca2+HBSS wash twice.After washing, 1 milliliter of addition adds Ca2+Or it is not added with Ca2+
HBSS, cell incubates 15 minutes at 37 DEG C.Culture solution is suctioned out from each hole, and contains (5 μM of substrate with 0.5 milliliter
CDF, 2.5 μM of CLF and 2.5 μM of d8TCA) culture solution at 37 DEG C Incubate cells 10 minutes.After incubation, culture is discarded
Liquid washs cell 3 times with ice-cold conventional PBS.By cell plates in -80 DEG C of storages.CDF(5-(and-6)-carboxy-2’,
7 '-dichloro-fluorescein, fluorescein) for detecting the formation of bile duct, after cell optimization is completed, the shape of cell
State and CDF can be assessed in the aggregation of bile duct by phase contrast microscope and fluorescence microscope respectively.
Fig. 5 is that the image of CDF aggregations is whether there is in fluorescence microscopy Microscopic observation, wherein, mouse liver cell has apparent CDF
Aggregation, traditional hepatic lineage are assembled without apparent CDF, and obvious CDF aggregations occurs in hepatic lineage after optimization, the results showed that adopts
Hepatic lineage can be formed similar with primary hepatocyte in vitro after the optimization obtained with extracorporeal culturing method described herein
Gallbladder tube structure.
II. it is interior in bile clearance rate（CLb,int）Measure
After the optimization obtained after 1 step 4 of traditional hepatic lineage and embodiment that experimental subjects obtains for 1 step 1 of embodiment
Hepatic lineage.
Experimental procedure：After the step 4 of embodiment 1, with the 6th day of inducing culture culture, triplicate cell was used
Warm plus Ca per 1 milliliter of hole2+Or it is not added with Ca2+HBSS wash twice.After washing, 1 milliliter of addition adds Ca2+Or it is not added with Ca2+
HBSS, cell incubates 15 minutes at 37 DEG C.Culture solution is suctioned out from each hole, and contains (2.5 μM of substrate with 0.5 milliliter
DPDPE, 2.5 μM of d8-TCA, 2.5 μM of Rosuvastatins and 2.5 μM of methotrexate (MTX)s) culture solution at 37 DEG C Incubate cells 10
Minute, it collects 0 minute and 10 minutes culture solutions and freezes in -80 DEG C of refrigerators, and washed immediately with ice-cold conventional PBS after incubation
It washs cell 3 times, cell plates is placed on -80 DEG C of storages.
Note：Inherent bile clearance rate（CLb,int）Calculation formula：
CLb,int=（Aplus_Ca++-Aminus_Ca++）/AUC0-10min
Wherein Aplus_Ca++It represents surveyed compound and is adding Ca2+Cumulant in the cell of processing, Aminus_Ca++It represents and is surveyed
Compound is being not added with Ca2+Cumulant in the cell of processing, AUC0-10min=incubative time × (Cm,0min+Cm,10min)/2, Cm,0min
And Cm,10minSurveyed compound was represented respectively at 0 minute（Incubate starting）With 10 minutes（Incubative time）Concentration.
Referring to Fig. 7, hepatic lineage and primary hepatocyte are removed for the inherent bile of drug after the results show optimization
Rate（CLb,int）There is good correlation, hepatic lineage is thinner more close to primary liver than the hepatic lineage being not optimised after illustrating optimization
Born of the same parents.
III. choleresis index BEI (%) and inherent bile clearance rate (CLb,int) measure.
Present inventor also selects important tool drug, passes through isotope method or liquid phase Tandem Mass Spectrometry Analysis drug
Accumulation situation, calculate selected drug choleresis index BEI (%) [calculate the formula of BEI for BEI (%)=（Aplus_Ca++-
Aminus_Ca++）/Aplus_Ca++× 100%, wherein Aplus_Ca++It represents surveyed compound and is adding Ca2+Cumulant in the cell of processing,
Aminus_Ca++It represents surveyed compound and is being not added with Ca2+Cumulant in the cell of processing] and inherent bile clearance rate (CLb,int)。
(result is referring to the following table 4)
4 important tool drug of table choleresis ability on different cell models compares
Wherein, traditional hepatic lineage is what is obtained after the step 1 of embodiment 1, and hepatic lineage is 1 step of embodiment after optimization
It is obtained after 4；
With traditional hepatic lineage group as a control group, statistical analysis (ANOVA, SPSS) is carried out to experimental result；
*, which is represented, has extremely significant difference (p<0.01)
4th, to the detection of hepatic lineage after people's source optimization
Drug transport is carried out to the optimization descendant source hepatic lineage that embodiment 2 obtains using experimental implementation similar to the above
The measure of body gene expression dose and BEI (%).
It can be seen that from the result of Fig. 8：
(1) the people source hepatic lineage that embodiment 2 obtains can express a variety of important drugs transporters, such as P-gp, MRP2,
MRP3, BSEP, NTCP, OATP etc., the mRNA expressions and human primary hepatocyte (PHH) of these transporters it is close (Fig. 8 a,
MRNA level in-site is 80% or so of human liver cell).
(2) the people source hepatic lineage that embodiment 2 obtains possesses choleresis ability.Compound DPDPE, TCA and CLF are in people
BEI in the hepatic lineage of source reaches more than 50% (Fig. 8 b) of primary human liver cell.
Therefore, either obtained from gene expression or functional perspective using extracorporeal culturing method described herein optimization
The people source hepatic lineage and human primary hepatocyte obtained is all comparable, and is expected to become the cell line for substituting human primary hepatocyte.
Based on above-mentioned experimental result, it can be deduced that such conclusion is obtained using extracorporeal culturing method described herein
Optimization after hepatic lineage (hepatic lineage for including more Species origins) to can be used for new drug development, artificial liver device kind careful
Multiple scientific research fields such as born of the same parents, Vitro hepatic inflammatory cell model.
Furthermore, it is necessary to explanation, although obtaining traditional hepatic lineage using direct transdifferentiation technology in embodiment,
And on this basis into line activating and further Fiber differentiation, still, those skilled in the art can according to above disclosure
Other tradition obtained in the prior art using other methods so that Vitro Culture Techniques described herein is expected to be equally applicable to
Hepatic lineage, such as the hepatic lineage that ES, iPS source obtain.
Although the application is using the in vitro culture of l cell and detects as exemplary embodiment, the application institute
The method stated be not limited to mouse cell generation hepatic lineage, apply also for optimization by the non-liver cell of multiple kinds (for example,
Fibroblast) hepatic lineage that generates, such as the non-liver cell of the non-liver cell in people source or rat source.The application also provides
By the hepatic lineage that people source fibroblast generates good medicine is generated after application optimization culture method described herein
Object choleresis index（BEI）With gene expression dose (close to parameters such as primary human liver cells) as evidence.
Specification Chinese and English abbreviation：
IHep, induced hepatocyte-like cell induce hepatic lineage；
HiHep, human induced hepatocyte-like cell, people source induction hepatic lineage；
Hnf4 α, hepatocyte nuclear factor4 α, Hepatocyte nuclear factor 4 α；
CDF, 5- (and-6)-carboxy-2 ', 7 '-dichloro-fluorescein, fluorescein substrate;
CLF, cholyl-lysyl-fluorescein, fluorescein substrate;
DPDPE,[D-Pen2,5] Enkephalin hydrate, enkephalins;
D8-TCA, d8-sodium taurocholate acid, isotope marks taurocholate;
TCA, taurocholic acid, taurocholate;
PB, phenobarbital, phenobarbital;
MTX, methotrexate, methotrexate (MTX);
CAMP, N6,2 '-O-Dibutyryladenosine3 ', 5 '-cyclic monophosphate sodium
Salt refers exclusively to dibutyryl cyclic adenosine monophosphate sodium salt here, referred to as cyclic adenosine monophosphate；
3-MC, 3-methylcholanthrene, 3-MECA;
VPA, valproic acid, valproic acid;
CLbile,int, intrinsic biliary clearance, inherent bile clearance rate;
BEI, biliary excretion index, choleresis index;
BA, bile acid, cholic acid;
SCMH, sandwich-culture mouse hepatocyte, sandwich mouse liver cell culture；
TTF, tail-tip fibroblasts, rat-tail fibrocyte;
PriHep, primary hepatocyte, primary hepatocyte；
FBS, fetal bovine serum, hyclone；
Mrp2, multidrug resistance-associated protein2, multidrug resistance associated protein 2;
Bsep, bile salt efflux protein, cholic acid efflux protein;
Ntcp, sodium taurocholate cotransporting polypeptide, sodium ion-taurocholate are common
Transport protein；
P-gp, p-glycoprotein, p- glycoprotein；
Cyp7a1, cholesterol7-alpha hydroxylase, cholesterol 7- hydroxylases；
PXR, pregnane X receptor, PgR；
FXR, farnesoid X receptor, method Buddhist nun's ester X receptors；
AhR, aryl hydrocarbon receptor, aryl hydrocarbon receptor；
CAR, constitutive androstane receptor, composing type androstane receptor；
PPARγ：Peroxisome proliferator-activated receptor alpha, peroxisome
Proliferator activated receptor；
Nrf2：NF-E2related factor-2, nuclear factor E2-related factor 2；
ES cell, embryonic stem cell, embryonic stem cell；
IPS cell, induced pluripotent stem cell induce versatile stem cell.
Claims (9)
1. it is a kind of optimized after hepatic lineage extracorporeal culturing method, including：
Step 1, using sandwich cell culture method culture tradition hepatic lineage；
Step 2 removes original culture medium, uses inducing culture instead and continues culture 3-5 days, hepatic lineage after being optimized；
Wherein, the inducing culture be serum free medium, and the inducing culture contain selected from cAMP, 3MC, PB,
A kind of nuclear receptors agonists in TCA and VPA either either containing cAMP, 3MC and PB or contain containing cAMP and PB
CAMP, PB and TCA or containing cAMP, 3MC, PB and TCA, above-mentioned each nuclear receptors agonists are in the inducing culture
Concentration range is respectively cAMP 1-10 μM, 3MC 1-10 μM, PB 10-100 μM, TCA 5-50 μM and VPA 0.2-4 μM；
Wherein, traditional hepatic lineage is direct transdifferentiation technology and embryonic stem cell or versatile stem cell induction differentiation skill
The hepatic lineage of art induction；
Wherein, traditional hepatic lineage has transfected hepatocyte nuclear factor, and the hepatocyte nuclear factor is selected from Hepatocyte nuclear factor 4
α (Hnf4 α) and Hepatocyte nuclear factor 4 A (HNF4A).
2. extracorporeal culturing method according to claim 1, wherein,
The inducing culture contains the cAMP that concentration is 2 μM and the PB that concentration is 50 μM or is 2 μM containing concentration
CAMP, concentration are 2 μM of 3MC and concentration is 50 μM PB or containing the cAMP that concentration is 2 μM, the PB that concentration is 50 μM and
Concentration is 20 μM of TCA or containing the cAMP that concentration is 2 μM, the 3MC that concentration is 2 μM, the PB that concentration is 50 μM and concentration
20 μM of TCA.
3. extracorporeal culturing method according to claim 1, wherein,
The tradition hepatic lineage is mouse source, rat source or people source tradition hepatic lineage.
4. extracorporeal culturing method according to claim 3, wherein,
The mouse source tradition hepatic lineage is to utilize direct transdifferentiation technology, is obtained by the non-liver cell in mouse source；The people source
Traditional hepatic lineage is to utilize direct transdifferentiation technology, is obtained by the non-liver cell in people source.
5. extracorporeal culturing method according to claim 4, wherein, the non-liver cell in mouse source is rat-tail fibroblast
Or rat embryo fibroblast cell；The non-liver cell in people source is mankind's embryo fibroblast.
6. according to the extracorporeal culturing method any one of claim 3-5, wherein,
The mouse source tradition hepatic lineage has transfected Hepatocyte nuclear factor 4 α (Hnf4 α)；The people source tradition hepatic lineage transfection
Hepatocyte nuclear factor 4 A (HNF4A).
7. hepatic lineage after a kind of optimization, the cell is obtained by extracorporeal culturing method according to any one of claims 1 to 6
.
8. hepatic lineage after optimization according to claim 7, the expression of drug transporters is traditional hepatic lineage
One times or more；And/or the expression of its bile acid transporter is significantly higher than traditional hepatic lineage；And/or its cholic acid synzyme
Expression be significantly higher than traditional hepatic lineage；And/or its drug choleresis index (BEI) and inherent bile clearance rate
(CLb,int) it is significantly higher than traditional hepatic lineage；And/or there is the polarization expression of drug transporters in it.
9. hepatic lineage after optimization according to claim 8, wherein, the bile acid transporter for Bsep, Mrp2 and/or
Ntcp；The drug transporters are P-gp, Bcrp, Oatp1b2, Oatp1a1 and/or Oatp1a4；The cholic acid synzyme is
Cyp7a1。
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