CN103045536A - Establishment and application for embryonic stem cell in vitro differentiation traditional Chinese medicine active substance pharmacodynamic screening model - Google Patents
Establishment and application for embryonic stem cell in vitro differentiation traditional Chinese medicine active substance pharmacodynamic screening model Download PDFInfo
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- CN103045536A CN103045536A CN2011103104935A CN201110310493A CN103045536A CN 103045536 A CN103045536 A CN 103045536A CN 2011103104935 A CN2011103104935 A CN 2011103104935A CN 201110310493 A CN201110310493 A CN 201110310493A CN 103045536 A CN103045536 A CN 103045536A
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Abstract
The invention discloses a pharmacodynamic screening model established by using an embryonic stem cell, which is used for preliminary screening and evaluation of pharmacological functions. Under drug induction, the embryonic stem cell is directionally differentiated into special-type cells so as to establish an efficient drug screening model which is used for preliminary screening and evaluation of pharmacological functions. The pharmacological functions of the drug can be evaluated preliminarily as long as the drug can take effect in one or a few stages. The model has an efficient drug screening characteristic, has direct and accurate evaluation index, and can evaluate the pharmacological functions of a plurality of natural drugs rapidly. Furthermore, the pharmacodynamic screening model takes less reagent, time and cost, has a wealth of cell differentiation information, has high efficiency and stimulation capability, and has both the advantages of pharmacological functions and mechanism research.
Description
Technical field
The invention belongs to the regenerative medicine field, relate to a kind of foundation and application of medicine efficacy screening model, mainly be that Curing Effect of Chinese medicine carries out rapid screening and assessment expeditiously about foundation and the application of drug-induced vitro differentiation of embryonic stem cells active ingredient of Chinese herbs medicine efficacy screening model.
Background technology
Classical medicine efficacy screening model comprises the medicine efficacy screening model of (1) whole animal level: its advantage is from integral level, intuitively to reflect the therapeutic action of medicine; Shortcoming is to need a large amount of animals, and experimental period is long, needs a large amount of trial-products, is not suitable for the preliminary Large-scale Screening of drug effect; (2) medicine efficacy screening model of histoorgan (such as isolated heart) level: main drawback is can not reflect neural and body fluid to the regulating effect of organ pharmacological effect, and its screen small scale, efficient is low, the sample demand is larger.(3) medicine efficacy screening model of cell levels: preferably representativeness is arranged, and the trial-product consumption is few, can realize Large-scale Screening.Its limitation is to reflect that medicine, can be less for the index of evaluating drug effect to the effect information of a certain cell type in continuing growth course.(4) medicine efficacy screening model of gene level can be on a large scale, high-throughput, quick screening drugs activeconstituents, evaluate efficacy.But this model still can not embody medicine makes the character (such as gene regulating, protein post-translational modification, signal transduction cascade etc.) that important vital process of time spent changes to the influence process of life entity activity and medicine and macromole.
Research embryonic stem cell (ES cell) vitro directed differentiation becomes single type cell (myocardial cell, neurocyte, endotheliocyte etc.) to originate in the nineties, is mainly used in (1) and transforms medical research; (2) regenerative medicine research; (3) developmental biology research.The ES cell has the external permanent multiplication capacity that does not break up, when being under the suitable culture condition orientable some single type cell that is differentiated to form.Abroad namely begin one's study in the beginning of this century vitro directed differentiation of ES cell, existing ES cell directional is divided into the report of myocardial cell, neurocyte, endotheliocyte etc., but the ES cell be divided into power far below the drug-induced power that is divided into.Stem cell is used for being operated in abroad of drug screening and just begins, and is domestic still in the starting stage.
Summary of the invention
The objective of the invention is to utilize the ES cell under given conditions, can be divided into the character of single type cell, drug-induced lower, make the ES cell become specific cells in vitro differentiation, make up the high-level efficiency medicine efficacy screening model, and utilize this model to carry out medicine effect preliminary screening and evaluation.The present invention mainly divides following step:
1. utilize the support of existing ES cell to cultivate;
2. drug-induced ES cell directional is divided into the cultivation of single type cell;
3. utilize this model to carry out medicine efficacy screening and assessment.
The present invention utilizes the support of existing ES cell to cultivate, and it is characterized in that: select mouse embryo stem cell D3 cell strain (ES-D3 cell strain), be inoculated on the trophocyte, the cultivation of going down to posterity according to a conventional method.
The drug-induced ES cell directional that the present invention carries out is divided into the cultivation of single type cell, it is characterized in that: when induced orientation is divided into the single type cell, this class cell comprises the myocardial cell, with substratum the ES-D3 cell is made single cell suspension, hanging drop is cultivated into embryoid body again, and embryoid body carries out adherent culture, adds for reagent thing co-cultivation, when special sign appears in the embryoid body cell mass, then with this index as the directed differentiation success.When inducing the ES cell directional to be divided into the myocardial cell, the special sign that the embryoid body cell mass occurs is the rhythm and pace of moving things is arranged to beat.The specific indexes that occurs according to drug-induced embryoid body cell mass, and time-effect relationship and the dose-effect relationship of drug-induced differentiation carry out statistical analysis, with this as the evaluating drug effect standard.Testing drug can be the active Chinese drug component materials such as the Radix Astragali, icarin, puerarin, ginkgolic flavone glycoside, the red sage root.
The used substratum of the present invention is DMEM substratum, foetal calf serum.
The multiple single type cell models such as myocardial cell's directed differentiation model, neurone directed differentiation model, blood vessel generation directed differentiation model be can make up with the inventive method, drug effect preliminary screening and the evaluation of related drugs are used for.
Utilize the inventive method to induce the ES cell directional to be divided into the myocardial cell, also can carry out cardiac muscle and grow research, the research of myocardial structural protein expression and the ionic channel formation research that the dependency special gene is expressed.The present invention has the following advantages:
1. application surface is very wide: the ES cell has the external unlimited multiplication capacity that does not break up, make up the medicine efficacy screening model that drug-induced ES cells in vitro directed differentiation forms single type cell (such as myocardial cell, neurocyte, endotheliocyte etc.), but the drug effect of the various new medicine of preliminary assessment take heart, nerve, blood vessel etc. as action target spot.Medicine is in Induction Process, may make the ES cell directional be divided into certain single type cell by intervening a plurality of links, therefore in the application of this model, as long as medicine has the wherein effect of one or several key link of intervention, but the drug effect of preliminary assessment medicine just.
2. large, the high specificity of bioinformation amount: ES cells in vitro directed differentiation system be owing to can substantially simulate and even reappear growth and histoorgan generative process complicated in the body, and possessing can Artificial Control, the characteristics of intervention; And can pass through in specific manner the ES cell in the directed differentiation process, in the effect of the aspect altitude simulation medicines such as cell signalling, gene expression regulation in whole animal, reappear medicine to the biological phenomena essence of biomacromolecule regulation and control, for new drug development provides simple and effective external drug effect evaluation model.
3. to the original new drug of inducing the ES cell directional to be divided into certain single type cell is arranged, this screening model also can provide the research of drug effect specificity target spot, and might find the gene that some have the treatment function owing to can present the whole process of differentiation.
4. the observation and appraisal index is intuitive, clear, can reflect intuitively time-effect relationship and the dose-effect relationship of medicine, is used for the objective evaluation drug effect.After cultivating altogether such as ES cell and the original new drug with treatment heart disease drug effect, whether has spontaneous rhythmic pulsation as assessment endpoint take the plastidogenetic embryoid body of ES, with control group significant difference is arranged such as time-effect relationship and the dose-effect relationship data that obtains, can clear and definite its ES cell directional being broken up has inducing action.
5. this medicine efficacy screening model still keeps the general characteristic of the cell aspect screening of medicaments activity of routine,, when joint few such as required trial-product consumption save money, efficient is high, but grow the dependency bioinformation owing in the directed differentiation process, be rich in, can provide more target spot for evaluating drug effect, therefore be that a high-level efficiency, high emulation take into account, drug effect and Mechanism Study and the medicine efficacy screening model of depositing are in newtype drug development, that the active Chinese drug component material pesticide effect is estimated the field application prospect is very wide.
Description of drawings
Fig. 1 is that the traditional Chinese medicine monomer ginkgolic flavone glycoside induces the ES-D3 cell directional to be divided into myocardial cell's time-effect relationship curve and amount effect relation curve figure.
Embodiment
Embodiment one: the traditional Chinese medicine monomer ginkgolic flavone glycoside induces the ES cell directional to be divided into myocardial cell's research
1.ES the support of cell is cultivated:
The ES-D3 cell is pressed 2x10 in conditioned medium (containing DMEM substratum, foetal calf serum)
7～5x10
7The density of individual/ml is inoculated on trophocyte's (l cell or STO cell strain), the cultivation of going down to posterity according to a conventional method.
2. drug-induced ES cell directional is divided into myocardial cell's cultivation:
1. the ES-D3 cell is made single cell suspension with trysinization, is inoculated in by 20～30uL on the cover inner surface of culture dish, adds 10mL ES-Hanks solution in the culture dish, and the lid upset is formed hanging drop, puts in the incubator hanging drop and cultivates 2 days.After this hanging drop is changed in the culture dish that fills 10mL ES cytodifferentiation substratum (DMEM substratum, foetal calf serum), carried out ES cell colony suspension culture 3 days, form ES cell embryoid body.2. the differentiation culture that adheres to the ES cell: embryoid body is transferred in 24 orifice plates with suction pipe at microscopically, hatched 4～5 hours, after making its absorption, slowly add 1ml ES division culture medium in every hole, add traditional Chinese medicine monomer ginkgolic flavone glycoside and ES cell embryoid body and hatch altogether.Begin with inverted microscope observation of cell every day the initial time that begins to beat to catch the myocardial cell this moment.
2. carry out evaluating drug effect with this model:
If the embryoid body cell mass rhythmicity can be arranged is beaten, then with this index as the directed differentiation cardioblast, make the medicine ginkgolic flavone glycoside to time-effect relationship curve and the amount effect relation curve of the differentiation of ES-D3 cell induction, the results are shown in accompanying drawing 1, with this index as evaluating drug effect.In the accompanying drawing 1, the ginkgolic flavone glycoside final concentration is respectively: serial 1:0, serial 2:10
-8Mol/L, serial 3:10
-7Mol/L, each concentration n=400 embryoid body, identical experiment repeats to have done 6 times.
Respectively 5 kinds of clear and definite medicines (such as ginkgolic flavone glycoside, the red sage root, the Radix Astragali, icarin, puerarin etc.) of heart disease curative effect are induced the differentiation test.The result wherein have 3 kinds of medicines respectively with ES-D3 co-culture of cells in the time of 5～7 days, rhythmic pulsation occurs with regard to the cell clone more than 90% is arranged, and heart plumule scleroproein up-regulated arranged.Other had 2 kinds of medicines and ES-D3 co-culture of cells in the time of 9～11 days, all had the cell clone more than 80% rhythmic pulsation to occur, and visible cardiac muscle is grown the dependent gene rise simultaneously.Wherein the inducing action with the traditional Chinese medicine monomer ginkgolic flavone glycoside is the most obvious, and has good dose-effect relationship.
Embodiment two: cardiac muscle is grown the research that the dependency special gene is expressed
If medicine can induce the ES cell directional to be divided into the myocardial cell, then respectively before medicine is cultivated altogether, when medicine is cultivated altogether rear embryoid body and formed and embryoid body when beating, carry out following research:
(1) extracts phage DNA after DNA library clone → cultivation and make template, the order-checking of PCR reaction amplification → amplified production;
(2) Semiquantitative reverse transcription polymerase chain reaction evaluation → some genes are carried out function supposition research;
(3) difference expression gene is carried out RT → PCR sxemiquantitative to identify → some gene is carried out function supposition research.
Embodiment three: the research of myocardial structural protein expression
(1) Electronic Speculum shows myocardial structural protein characteristic collection of illustrative plates, and whether to occur and maturity shows drug effect.
(2) protein electrophoresis, western blot, whether estimate has differential expression, and modify after expressing etc.Beat in case of necessity embryoid body electrical signal collection and analysis.
With medicine efficacy screening model of the present invention can be efficiently rapidly preliminary assessment have the myocardial function of improvement, anti-arrhythmia, improve blood supply of cardiac muscle, anti-ageing year dementia, anti-parkinson, antitumor and metastases, the original new drug of the drug effects such as treatment cardiovascular and cerebrovascular diseases, treatment diabetes, anti-hepatopathy.
Claims (6)
1. the construction and application of an embryonic stem cell vitro directed differentiation medicaments sifting model, it is characterized in that: drug-induced lower, make embryonic stem cell (ES) directed differentiation become the single type cell, make up the high-level efficiency medicine efficacy screening model, and utilize this model to carry out active ingredient of Chinese herbs medicine efficacy screening and assessment, the present invention mainly divides following step:
(1) utilize the support of existing ES cell to cultivate;
(2) drug-induced ES cell directional is divided into the cultivation of single type cell;
(3) utilize this model to carry out medicine efficacy screening and assessment.
2. the support cultivation that utilizes existing ES cell as claimed in claim 1 is characterized in that: select mouse embryo stem cell D3 cell strain (ES-D3 cell strain), be inoculated on the trophocyte, the cultivation of going down to posterity according to a conventional method.
3. drug-induced ES cell directional as claimed in claim 1 is divided into the cultivation of single type cell, it is characterized in that: when induced orientation is divided into the single type cell, this class cell comprises the myocardial cell, with substratum the ES-D3 cell is made single cell suspension, hanging drop is cultivated into embryoid body again, and embryoid body carries out adherent culture, adds for reagent thing co-cultivation, with the embryoid body cell mass special sign appears, as the index of directed differentiation success.
4. drug-induced ES cell directional as claimed in claim 3 is divided into the cultivation of single type cell, it is characterized in that: when induced orientation was divided into the myocardial cell, the special sign that the embryoid body cell mass occurs was the rhythm and pace of moving things is arranged to beat.
5. drug-induced ES cell directional as claimed in claim 3 is divided into the cultivation of single type cell, it is characterized in that: testing drug can be the active Chinese drug component materials such as ginkgolic flavone glycoside, the Radix Astragali, icarin, puerarin, the red sage root.
6. the construction and application of embryonic stem cell vitro directed differentiation medicaments sifting model as claimed in claim 1, it is characterized in that: utilize the method, myocardial cell's directed differentiation model, neurone directed differentiation model, blood vessel generation directed differentiation model and other multiple single type cell models be can make up, drug effect preliminary screening and assessment are used for.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109294975A (en) * | 2018-10-23 | 2019-02-01 | 山东华思生物科技有限公司 | A kind of stem cell drugs screening technique |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403586A (en) * | 2002-09-27 | 2003-03-19 | 浙江大学 | Construction and application of in vitro directional embryonic stem cell-differentiating medicine efficacy screening model |
| CN1425763A (en) * | 2002-12-30 | 2003-06-25 | 浙江大学 | Use of icariin in inducing external oriented differentiation of embryo stem cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1403586A (en) * | 2002-09-27 | 2003-03-19 | 浙江大学 | Construction and application of in vitro directional embryonic stem cell-differentiating medicine efficacy screening model |
| CN1425763A (en) * | 2002-12-30 | 2003-06-25 | 浙江大学 | Use of icariin in inducing external oriented differentiation of embryo stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 朱丹雁, 楼宜嘉: "淫羊藿苷诱导小鼠胚胎干细胞体外定向分化为心肌细胞方法学研究", 《中国药学杂志》, vol. 42, no. 9, 31 May 2007 (2007-05-31), pages 667 - 671 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109294975A (en) * | 2018-10-23 | 2019-02-01 | 山东华思生物科技有限公司 | A kind of stem cell drugs screening technique |
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Addressee: Beijing QMLC Stem Cell Technology Co., Ltd. Document name: Notification of Publication and of Entering the Substantive Examination Stage of the Application for Invention |
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Address after: 100084, C building, Tsinghua Science and technology building, No. 1 Zhongguancun East Road, Beijing, Haidian District, 301 Applicant after: Beijing QMLC Stem Cell Technology Co., Ltd. Address before: 100097 Beijing city Haidian District minzhuang Road No. 3, Tsinghua Science Park building 204 Yuquan Huigu 7 Applicant before: Beijing QMLC Stem Cell Technology Co., Ltd. |
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Application publication date: 20130417 |