CN101775431A

CN101775431A - Method for high content screening of therapeutic drugs for diabetes

Info

Publication number: CN101775431A
Application number: CN200910155755A
Authority: CN
Inventors: 徐铭恩; 葛亚坤
Original assignee: Hangzhou Electronic Science and Technology University
Current assignee: Hangzhou Dianzi University; Hangzhou Electronic Science and Technology University
Priority date: 2009-12-25
Filing date: 2009-12-25
Publication date: 2010-07-14
Anticipated expiration: 2029-12-25
Also published as: CN101775431B

Abstract

The invention relates to a diabetes pathological model based on cell assembly technology and application thereof in high content drug screening. Adipose-derived stem cells and simulated extracellular matrix material are assembled to form a cell-containing three-dimensional structure with certain porosity; the inducing differentiation condition is controlled to enable the adipose-derived stem cells on the surface of the inner passage of the three-dimensional structure to differentiate into endothelial cells and to enable the adipose-derived stem cells inside the matrix material to differentiate into fat cells; and pancreas islet separated out from the body of an animal is directly assembled in the gaps of the three-dimensional structure. The system model is excited for a long time by high-concentration dextrose, fatty acid insulin, TNF or cytokine secretion of fat cells and the like so as to induce the system model to produce pathology and symptom of diabetes. The drug to be tested is put into a culture system, and by detecting the level of the relevant markers of the model under normal condition and pathological condition, the pharmacological activity of the drug to be tested can be analyzed and thereby high content screening can be carried out to the drug.

Description

A kind of high intension screening method of Remedies for diabetes

Technical field

The invention belongs to biological technical field, specifically, relate to a kind of high intension drug screening method of diabetes medicament

Background of invention

Life science, information science combine with manufacturing science, are the important trend of 21st century development in science and technology.The controlled assembling of cell three-dimensional of making theoretical and organizational project theory based on the advanced person is for research fields such as organizational project, drug screening have been expanded new theory and technology space.In the medicament research and development field, high flux screening (the High-Throughput Screening of global medicament research and development mechanism widespread use since the nineties, drug screening evaluation technique such as HTS) faces predicament (Bleicher KH, the et al.Nat Rev Drug Discov.2003 of low success rate; 2 (5): 369-78).Its reason is because there are greatest differences in existing external drug screening system and real internal milieu, and this species diversity causes the low success rate that screens.Therefore making up the in-vitro evaluation screening system consistent with internal milieu, is the gordian technique that present medicament research and development field must solve.And be used for drug screening with the 3D many cells system that the groups of cells packing technique can make up the emulation internal milieu, such technology is that bridge has been set up in organizational project and drug research field, all has important scientific meaning and using value for groups of cells packing technique and drug screening technology.

Be based upon computer 3D modeling and the advanced cell three-dimensional package technique of making on the discrete accumulation theoretical basis, be under the anatomy digital model of histoorgan directly drives, location assembling viable cell/material cell, the technology of the many cells precursor of manufacturing tissue or organ.As the 3D bio-printer technology of people such as the Lee W of Harvard University exploitation, can spray cell and material, successively print fibroblast and keratinocyte and form multilayer skin texture (Lee W, et al.Biomaterials.2009; 30:1587-95).The 3D-cell printer technology of people such as Mironovf F of Univ. of South Carolina and the Boland R of Clemson university exploitation, spray cell and special substrate material by the repacking printer head, print activated three-dimensional cell system (Zhang C, et al.Biomaterials, 2008; 29 (28): 3781-91).The 3D bioassembly of the industry Li Sangna Smith CM of university exploitation, based on direct writing technology assembling cell, and systematic study various environmental factorss to the influence of assembling cell activity (Smith CM, et al.TissueEng.2007; 13 (2): 373-83).The Vacanti JP of MIT and De Leipo laboratory Borenstein J etc. also are fabricated material with the cell, in conjunction with chip manufacturing process, produce capillary blood vessel system (Fidkowski C, et al.TissueEng.2005; 11 (1-2): 302-9).Developed the cell three-dimensional controlled group packing technique based on rapid shaping (RP) principle domestic Tsing-Hua University face Yongnian etc., assembled tissue (Yan YN, et al.Biomaterials.2005 such as can keeping 3 months active artificial livers, artificial fat; 26 (29): 5864-71).

Yet because the meticulous and complicacy of organ structure, the groups of cells packing technique also has a lot of problems to need to be resolved hurrily in the face of making complex organs such as liver, kidney, heart.Meanwhile, the groups of cells packing technique has shown important theory and using value owing to can make up complicated 3D cell culture model in other field of life science.The investigator utilizes the three-dimensional multicell of tissue engineering technique manufacturing to conduct a research in recent years, obtains remarkable achievement.People such as Basu S such as the Princeton University utilize artificial three-dimensional multicell, have studied the pattern of developmental biology and have built up problem (Basu S, et al.Nature.2005,28; 434 (7037): 1130-4).Hotary KB etc. utilizes three-dimensional multicell, finds that MT1-MMP by changing the cell spaces geometrical shape, influences propagation (Hotary KB, the et al.Cell.2003 of tumour cell in 3D matrix; 114 (1): 33-45).Basu S points out, constructs and study three-dimensional multicell, can improve the understanding to biological development and function, and can be applicable to biomaterial, in the biological sensing research.The three-dimensional multisystem of the simple technique construction of these research and utilizations has obtained effective achievement in fields such as biology and drug screenings, proves that the groups of cells packing technique has important research and using value in the drug screening field.

In the medicament research and development field, since the nineties, (High-Throughput Screening HTS), becomes the main means of new drug development to the high flux screening technology that detects based on molecule and cell levels information.But since integral body be by various kinds of cell at the three-dimensional space ordered arrangement, different iuntercellulars carry out the complex system of signal conduction, regulation and control; Basis with the HTS technology: the single drug targets of in vitro study, the cultivation of cell two dimension, cellularstructure and function are imperfect to be diverse.This species diversity pair cell 26S Proteasome Structure and Function causes remarkably influenced, finds to compare two dimension as Kang X and cultivates, and adipocyte is in the dimensional culture environment, its form, propagation, differentiation, genetic expression, physiological functions such as metabolism exist notable difference (KangX, et al.Tissue Eng.2005; 11 (3-4): 458-68).This species diversity just, caused present puzzlement whole world medicament research and development mechanism problem---the HTS rapid screening goes out medicine guide thing, but wherein mostly in integral experiment, do not have a pharmacologically active.HTS presses for the high intension screening that develops into high success rate, and (High Content Screening HCS) has become common recognition (Bleicher KH, the et al.Nat Rev Drug Discov.2003 in drug research field; 2 (5): 369-78).At home, because limited fund, medicament research and development influence expensive and that excessive risk is brought is even more serious.Simultaneously, large multinational companies such as Luo Shi, Novartis, Merck enter the medicament research and development field of China in succession over 04 year, grab the valuable natural resources of Chinese medicinal materials of China, and China presses for the high efficiency medicament research and development technology of developing low-cost.

The prerequisite of HCS is to keep cellularstructure, functional integrity, this require for cell provide to body in similar growing environment, the investigator begins to make up the three-dimensional cell system and carries out drug research for this reason.The Fischbach C of Harvard etc. utilize the three-dimensional rack culturing cell to set up tumor model, show that features such as deterioration, susceptibility near true tumour, are good tumor drug screening instrument (Fischbach C, et al.Nat Methods.2007; 4 (10): 855-60).Liana Adam carries out tumour medicine research [18] with the so three-dimensional multicell of cytocyst.Because liver cell can be used for studying drug metabolism and toxicity, Griffith LG (Sivaraman A, et al.Curr Drug Metab.2005; 6 (6): 569-91), made up the three-dimensional many cells of liver cell system; Sun W has made up three-dimensional little hepatic tissue with Multi-nozzle depositio technology assembling cell, is used for drug metabolism study (Chang R, et al.Tissue Engineering.2008; 14 (2): 157-66).Yamada KM points out that the three-dimensional cell model can fill up the breach between two-dimentional cell cultures and experimentation on animals, to drug screening significant (Yamada KM, et al.Cell.2007; 130 (4): if 601-10). make up and to have more similar three dimension system in complicated functional organization's structure and the body, will further promote the development of drug screening technology.

By above analysis as seen, the modern technique of organizational engineering can be applicable to other research field and bears fruit.In the medicament research and development field, develop into high intension screening because defective, high flux screening that the high flux screening technology has a low success rate press for, and present high intension screening is keeping on cellularstructure and the functional completeness not bigger progress.The present situation in these fields and problem guide us to propose to utilize the outer pathological model of theory and technology construct of cell assembling, are applied to high intension drug screening, and this technology has important theory to be worth and application prospects.

Summary of the invention

The high intension drug screening method that the purpose of this invention is to provide a kind of Remedies for diabetes

Physiological system is combined together by the organ or tissue of a plurality of enforcement difference in functionalitys to be formed.In each physiological system of people, both just like circulation, urinary system is made up of a plurality of histoorgans like this, space structure is required accurately baroque system, simple relatively system on the structure of also having living space, the system that energy metabolism system comes to this.Energy metabolism system is mainly by the cell of several difference in functionalitys: adipocyte, and islet cells, formations such as neurocyte in certain space structure, by cell communication means such as internal secretions, are formed the metabolic complex networks system of regulation and control human body energy.Studies confirm that in a large number that in recent years β cell and adipocyte constitute adipocyte-Regular Insulin axle and regulate energy metabolism, endotheliocyte then is the regulation of secretion biological barrier that constitutes the selective permeation endocrine factor.Insulin secretion disorder, insulin resistant, complicated influencing each other and acting between adipocyte and the inner skin cell function damage is the unusual main mechanism of various pathologic, physiologics of contact metabolic syndrome.

The basic structure of animal energy metabolic system and parameter (is example with the human body):

(1) normal human's lipid content accounts for 10% of human body body weight, and the adipocyte sum is about 26.9 * 10 ⁹± 1.8 * 10 ⁹, and the pancreas islet sum has 1,000,000 approximately in normal people's pancreas.Obtain thus, in normal people's adipocyte-Regular Insulin axle, the ratio of adipocyte and pancreas islet is about 3 * 10 ⁴Adipocyte (individual)/pancreas islet (individual).

(2) body fat cell tissue is widely distributed, under normal circumstances, around the most subcutaneous layers, meso-omentum, kidney and marrow etc. locate a large amount of fatty depositss, constitute the architecture basics of energy metabolism system.

(3) pancreas islet is concentrated the cell mass that is dispersed in the pancreas, constitutes the point that energy metabolism is regulated.

(4) between adipocyte and pancreas islet, the excretory regulatory factor need pass through the blood vessel barrier separately, is diffused into active position by blood transport then, so endotheliocyte constitutes the biological barrier of selective permeation capacity control endocrine factor.

And with groups of cells packing technique assembling with induce various kinds of cell and control their exact position, the structure that makes up various kinds of cell theoretical and technical all be feasible.By to adipocyte, beta Cell of islet and the endotheliocyte spatial relation in vivo and the research of quantitative proportion, we have designed a kind of diabetes pathological model, and utilizing this model to be used for the high intension drug screening of Remedies for diabetes, this drug screening method comprises the steps:

(1) fat stem cell and substrate material assembling are formed the three-dimensional structure that contains cell with certain porosity;

(2) induce differentiation condition by control, make the fat stem cell of three-dimensional structure internal surface be divided into endotheliocyte, and be self-assembled into the endothelium spline structure at channel surface, the fat stem cell of substrate material inside then is divided into adipocyte;

(3) will in animal body, directly be assembled in the space of three-dimensional structure by isolating islet cells;

(4) above-mentioned three-dimensional structure is given the stimulator such as glucose, lipid acid, Regular Insulin, TNF or adipocyte excreted factor of high density, induced lipolysis cell, islet cells and endotheliocyte produce the diabetes pathological symptom;

(5) in above-mentioned culture systems, add medicine to be measured, the mark of correlation thing level of detection model under normal and pathological conditions, the pharmacologically active of analyzing medicine to be measured carries out drug screening.

In the above-mentioned steps, the quantity of fat stem cell is 10 ⁶-10 ⁸Individual/the 1ml substrate material, preferred 1-5 * 10 ⁷Individual/the 1ml substrate material.Islet cells quantity is 5-50, is preferably 10-20.Fat stem cell and islet cells derive from animal, and for example rat, mouse, rabbit, pig, ox or people etc. preferably derive from rat and people.

Extracellular matrix constitutes the external environment of cells survival; they be in the growth course from the cell internal secretion to extracellular various macromole; constitute the gel or the fibrous network of high degree of hydration then at cell peripheral; play support, protection, vegetative cell, the differentiation of pair cell, motion, communication have material impact.The artificial substratum material that is applied to cell assembling should help the formation of three-dimensional group assembling structure and stable, helps cell to form cell mass in suitable three-dimensional open environment, and transmission chemistry and mechanical signal are to cell.

The complicated component of extracellular matrix comprises collagen protein, protein-polysaccharide, glycosaminoglycan, laminin, fibronectin and elastin etc., and the different molecule that has of these compositions constitutes molecular length, and structural performance.The present invention considers that from the bionics angle cell epimatrix material of assembling process should be made of multiple material, and single-material also is to be difficult to satisfy mechanically shape, to make up the demand of multilevel hierarchy and controlled degradation etc.Take all factors into consideration above requirement to material, gelatin (Gelatine), sodium alginate (Sodium alginate) and the Fibrinogen of having selected to have good biocompatibility, controlled degradation and cellular affinity mix as substrate material.

Substrate material is the composition that comprises gelatin, sodium alginate, three kinds of materials of Fibrinogen, and gelatin, sodium alginate and fibrinogenic proportioning are 1: 0.2-1: 0.2-1.Gelatin and sodium alginate can be dissolved in respectively in the PBS damping fluid, with fibrinogenolysis in the DMEM nutrient solution, face the time spent with three kinds of materials with 1: the even blend of 0.2-1: 0.2-1, preferably with blend in 1: 1: 1.Because substrate material is a blend, therefore, its gelation mode is more than the gelation mode of single-material, both can pass through controlled temperature (gelatin) and realize gelation, also can be by introducing linking agent and enzyme gelation.What have more advantage is to use the degraded of enzyme and inducible factor control texture.

The fat stem cell of intermingling material and cultivation is mixed, obtain concentration and be about 10 ⁶-10 ⁸The fat stem cell of individual fat stem cell/mL-matrix material intermingling material is preferably preferred 1-5 * 10 ⁷Individual fat stem cell/1ml intermingling material.

In the above-mentioned steps, can will be dissolved in fat stem cell assembled formation in the substrate material by the groups of cells packing technique.The special-purpose experimental system that is used for the controlled assembling of cell three-dimensional mainly comprises the exclusive data process software, Solidworks for example, Cark delamination software etc., based on four-axis movement control card and stepper motor driven three-dimensional platform, material transports subsystem, and the working chamber of independent temperature and the shaping base plate of split-type design.Coordination control by total system has realized the accurate control to assembling process, the three-dimensional structure that contains cell that obtains having certain porosity after shaping is finished, and porosity is preferably 65%-85%.

Gelatin-based intermingling material/cell assembled structural body was cultivated 3-5 days, electron micrograph after the lyophilize shows, gelatin/sodium alginate/Fibrinogen intermingling material, structure porosity height, because the molecular chain behind the Fibrinogen solidification aggregation is longer than sodium alginate, easier formation has the structure of 10 ~ 30um diameter hole in a large number.Cell freely stretches at three-dimensional space, forms reticulated structure and a large amount of space and can supply nutrient solution go deep into inside configuration.Above-mentioned experimental result has fully confirmed employing sodium alginate, Fibrinogen and gelatin-based intermingling material, can construct the micro unit structure that is fit to the cell growth.

In the above-mentioned steps, Fibrinogen/gelatin/sodium alginate/cell is by the low temperature stack shaping, again through Ca2+ ion and zymoplasm double cross connection, Stability Analysis of Structures, both had certain rigidity, also have certain tensile properties, but structure stable maintenance more than 1 month, and the passage of design remains clear debating.Its reason is, the molecular chain of sodium alginate molecular chain relatively short and the fibrin polymerization body is longer, thus form two kinds of structures that mechanical characteristic is different, when two kinds of material-mix are used together, two kinds of molecular structure actings in conjunction have improved the bulk strength and the stability of packaging assembly.

In the above-mentioned steps, induce differentiation condition can select to induce with endothelial cell growth factor (ECGF) (EGF) earlier, preferred endothelial cell growth factor (ECGF)-1 (EGF-1), make the fat stem cell of three-dimensional structure internal surface be divided into endotheliocyte earlier, these last eventually cells that break up will lose the ability that is divided into adipocyte; Then with dexamethasone, (isobutyl-methylxanthine, IBMX), inducing culture makes the fat stem cell of substrate material inside can be divided into sophisticated adipocyte to isobutyl methylxanthine.The differentiation of different positions fat stem cell in the accurate control texture body of this method energy, inductive step and fat stem cell residing position in structure is depended in the differentiation of cell.

After treating that induction is finished, will be assembled in the three-dimensional structure from isolated islet cells location in the animal body.The islet cells that for example available syringe is drawn the structural integrity of cultivating is injected in the space of three-dimensional structure, and islet cells quantity is 5-50, is preferably 10-20.

The stimulator such as glucose, lipid acid, Regular Insulin, TNF or adipocyte excreted factor that give high density after the three-dimensional structure of many cells system assembles stimulate for a long time, and induced lipolysis cell, islet cells and endotheliocyte produce the diabetes pathological symptom.

As a kind of optimized technical scheme, can adopt 10-25mM, preferred 15mM glucose culture solution was cultivated after 5-10 days, induced to produce the diabetes pathological model.

Another optimized technical scheme can adopt 6-20 μ M, and preferred 10 μ M insulin nutrient solutions were cultivated after 5-10 days, induces to produce the diabetes pathological model.

Another optimized technical scheme can adopt 30-80ng/mL, and preferred 40ng/mL TNF-a nutrient solution was cultivated after 5-10 days, induces to produce the diabetes pathological model.

Above-mentioned diabetes pathological model can reflect diabetes to a certain extent, comprises the physiological and pathological situation of type i diabetes and type ii diabetes.Diabetes are a kind of common metabolism endocrinopathys, are because the absolute or relative shortage of the interior Regular Insulin of human body institute extremely, is a principal character with the hyperglycemia; The morbidity of diabetes not only has peripheral tissues to resistance of insulin and reducing, also has β emiocytosis function undesired, be that the excreting insulin ability lowers, discharge slowly, the delay at secretion peak can not be kept normal running balance between pancreatic beta cell secreting function and the insulin action.The Western medicine kind of treatment diabetes mainly contains Regular Insulin and analogue antidiabetic drug thereof at present, (competitive inhibition is positioned at the various alpha-glucosidases of small intestine to the alpha-glucosidase inhibitor antidiabetic drug, delay the enteron aisle carbohydrate absorption), berculon B class antidiabetic drug (directly stimulates the beta Cell of islet uelralante, the intensifier target histocyte is to the susceptibility of Regular Insulin), the biguanides antidiabetic drug (promotes peripheral tissues's ingestion of glucose such as muscle, quicken the anaerobic carbohydrate metabolism), thiazolidinediones antidiabetic drug (the enhancing body tissue improves the beta Cell of islet function to the susceptibility of Regular Insulin) and Drugs Promoting Insulin Secretion antidiabetic drug (simulation physiological insulin secretion) etc.

Before mention that the diabetes pathological model that we make up can be in the pathogeny of in-vitro simulated diabetes.Diabetes are that energy metabolism system gets muddled, and are closely related with glycolipid metabolism, thereby glucose and metabolism of fat can be used as the detection sign of glycolipid metabolism.The main regulatory factor of fat-pancreas islet endocrine axis comprises in addition: islet cells excretory Regular Insulin, and the C peptide, hyperglycemic-glycogenolytic factor, Somatostatin or the like, the wherein most important regulation of energy factor is a beta Cell of islet excretory Regular Insulin; Hormones such as adipocyte excretory leptin, phylaxin, adiponectin, Angiotensin, tumour necrosis factor-a (TNF-a), sexual hormoue, prostaglandin E2, wherein important is leptin, phylaxin and adiponectin, and these all can be used as diabetes certification mark thing.Because the diabetes pathological model can be in the pathogeny of in-vitro simulated diabetes, in culture systems, add medicine to be measured, the mark of correlation thing level of detection model under normal and pathological conditions, analyze the pharmacologically active of medicine to be measured, can efficiently filter out target compound with potential treatment diabetes disease.

(1) glucose

Sugar is the energy supply material that body weight for humans is wanted, and is again one of moiety of wanting of body weight for humans.Metabolism obstacles of blood glucose in the diabetes at first causes human body energy to supply with obstacle, can produce a series of metabotic change thus, finally causes multi-faceted metabolism disorder.Glucose is equivalent to the intravital blood sugar of machine in the nutrient solution, by observing adipocyte in the model to the conversion consumption of glucose, can reflect the synthetic fat vigor of adipocyte to a certain extent and to the susceptibility of Regular Insulin.Therefore the consumption of glucose in the nutrient solution can be used as glycometabolic detection sign.

(2) fat

Lipid mobilization is the significant process of energy metabolism, under pathology or starvation conditions, is stored in the fat in the adipocyte, progressively is hydrolyzed to free fatty acids (FFA) and glycerine and is released into blood for other tissue oxidizing utilizations by lipase.Lipid mobilization is subjected to the adjusting of hormone, and FFA had both provided the important substance of energy, and free fatty acids too high in the blood can damage tissue again, comprises bringing out insulin resistant, atherosclerosis and damage islet cells.So, mobilize by observing lipid acid, can reflect the physiological and pathological situation of energy metabolism system to a certain extent.The release synthetic and FFA of fat can be used as lipometabolic detection sign.

(3) lipogenesis

Adipocyte is as the important storage cell of energy, when blood glucose concentration is high, be that lipid acid stores with conversion of glucose is the critical function of adipocyte.Lipogenesis under the normal physiological situation can be stored fat, lowering blood glucose, and the lipogenesis dysfunction under the pathologic condition is then causeed fat, deleterious fatty hormone secretion, pathologic reactions such as hyperglycemia.So the lipogenesis of fat stem cell can reflect to a certain extent that fat stem cell is divided into the ability of adipocyte and synthetic fat.Adopt fat in the Oil red O solution-dyed adipocyte, the Oil red O dyestuff in the isopropanol extraction adipocyte, the method that microplate reader detects the absorbancy at 510nm place detects lipogenesis.Fat stem cell in the display model because culture environment approaches situation in the true body, breaks up sensitivity as a result, has the vigor of very strong synthetic fat.

For the long-time lipogenesis that brings out of cultivating of high concentration glucose, demonstrate significantly long-time promoter action on this model of rosiglitazone.

(4) the metabolic regulation quality testing is surveyed---insulin secretion

Regular Insulin is the most important regulatory factor of islet cells excretory, and Regular Insulin is the major hormone of regulating sugar, protein, fatty three big substance metabolisms and storage in the body by beta Cell of islet secretion, and influences many other important physiological function.The morbidity of one of the important diseases of metabolic syndrome type ii diabetes not only has peripheral tissues to resistance of insulin and reducing, also has β emiocytosis function undesired, and promptly the excreting insulin ability lowers, and discharges slowly the delay at secretion peak.

By the influence of detection of drugs to the kinetic parameter of insulin secretion, can screening of medicaments.For the secretion of the islet cells in the detected energy metabolic system model, set up filling system and carry out time dependent glucose stimulation Regular Insulin release experiment.Being presented at 15M glucose stimulates down, and the islet cells insulin secretion of assembling has very high activity.After the glucose induction of 15mM was cultivated for a long time, the insulin secretion under islet cells stimulates 15mM glucose in the model acutely descended, and rosiglitazone (belonging to the thiazolidinediones antidiabetic drug) can increase the insulin secretion that islet cells is suppressed by high sugar.Islet cells postpones 2min to the insulin secretion peak of glucose induction under normal circumstances, nateglinide (belonging to the Drugs Promoting Insulin Secretion antidiabetic drug) can be revised this inductive insulin secretion peak and postpone.Our model has not only reproduced the disorder of insulin secretion under the pathological conditions, has also detected the pharmacological action of nateglinide to the insulin secretion disorder, has proved that this model is to screening the validity of such medicine.Model also detects the disorderly pharmacological action to insulin secretion of rosiglitazone simultaneously, has proved that further this model is to screening the effective of thiazolidinediones and insulin secretion accelerating class medicine

(5) adipocyte excretory regulatory factor

A. leptin

Leptin is by ob gene (ob gene) coding, by adipocyte excretory peptide hormone.Leptin has biological effect widely, and leptin both can act on hypothalamic feeding center, reduces appetite, increases energy expenditure.Can be used as the part of fat-pancreas islet endocrine axis again, participate in the endocrine adjusting of Regular Insulin.Also can influence adjusting of blood pressure etc. by various mechanism.Regular Insulin can stimulate leptin secretion, increases leptin mRNA and expresses, and leptin can directly suppress insulin secretion again simultaneously.Leptin can prevent the overexpression of the β cell proinsulin gene of incretin and glucose induction as the inhibitory control signal that fatty tissue provides, and restrains the excessive generation of Regular Insulin and the formation of hyperinsulinemia.Leptin promotes steatolysis, suppresses lipogenesis.Leptin also can make IRS-1 (IRS-1) tyrosine phosphorylation weaken, and directly influences the insulin signaling transduction.At skeletal muscle, leptin energy stimulated muscle increases muscle to the picked-up of glucose and the susceptibility of Regular Insulin to the picked-up of glucose under Regular Insulin and the normal situation of hyperglycemic-glycogenolytic factor concentration.

Except the influence to the Regular Insulin effect, leptin also can weaken the function of beta Cell of islet excreting insulin by number of mechanisms, thereby obesity and β cell function defective are connected.Under the pathologic condition, leptin receptor susceptibility weakens, and leptin causes the beta Cell of islet hyperpolarization, and the ability that suppresses Regular Insulin release weakens, and causes hyperinsulinemia.Suppress the secretion of the high sugared plain peptide-1 of enteron aisle hormones (GLP-1) in addition by activating phosphatase diesterase 3B (PDE3B), thereby its usefulness that stimulates insulin secretion is weakened.

In a word, leptin has the important physical effect, and its metabolic effect is mutually short of money with the effect of Regular Insulin.Form two-way feedback regulation by leptin and Regular Insulin between fatty tissue and the β cell, leptin is closely related with insulin resistant, plays an important role in energy metabolism.

B. phylaxin

Phylaxin is that its gene specific is expressed in the white adipose tissue by a kind of peptide hormone of adipocyte excretory.Be divided in the sophisticated adipocyte process at preceding adipocyte, the level of phylaxin mRNA significantly increases, and nutrition simultaneously and hormone can be regulated and control the expression of phylaxin, and behind hello the Hi CHO, its expression level can significantly increase by 25 times.Phylaxin is considered to get in touch fat and insulin resistant and the important point of connection of diabetes.Phylaxin can make the effect of sugar tolerance and Regular Insulin impaired.Phylaxin can make insulin-induced 3T3 one L1 adipocyte that the picked-up of glucose is reduced, and the picked-up of sealing back glucose with phylaxin antibody increases again.At the obesity mice of heredity obesity and diet induced, serum phylaxin level significantly increases.Phylaxin antibody can make the obesity mice blood sugar of diet induced descend, and the susceptibility of Regular Insulin is recovered.These researchs show that all the secreted phylaxin of adipocyte can cause the insulin resistant of the many organs of whole body to the effect of synalbumin.The antidiabetic medicine rosiglitazone can significantly be reduced the expression of phylaxin mRNA, and the expression of downward modulation phylaxin is the important mechanisms of TZD class medicine performance anti-diabetic effect.Phylaxin also can reduce the intrinsic activity that grape is warded off transporter, and the rising plasma glucose concentration.In a word, the research of phylaxin just launches, and many mechanism are unclear fully, but present research conclusion can be determined phylaxin and participated in the formation of type ii diabetes insulin resistant state.

C. adiponectin

Adiponectin (adiponectin) is a kind of neurophysin of adipocyte excretory.Adiponectin can specificity combine with I, III and the collagen type v of tunica intima, and when function of vascular endothelium was impaired, the adiponectin in the circulation can be deposited on the vessel wall.TZD class medicine can promote the expression and the proteic secretion of adipocyte apm 1 gene.Discover that in the pathogenic process of type ii diabetes low adiponectin mass formed by blood stasis and insulin resistant and hyperinsulinemia have significant dependency.The fat monkey that the blood plasma adiponectin concentration is low, the picked-up of the periphery glucose under the insulin stimulating obviously weakens.The apm 1 gene knock-out mice proves that because of the shortage of adiponectin, fatty acid transport protein mRNA level reduces in the muscle, thereby causes severe insulin resistance.Adiponectin can promote the expression of fatty acid oxidase, uncoupling protein, thereby improves insulin resistant.The adiponectin that studies show that physiological dose of Yamauchi etc. can promote the burning and the energy consumption of liver and myocyte's lipid acid, thereby reduces TG level in the cell, improves fat mouse and type ii diabetes patient's hyperlipidaemia and insulin resistant.

Adiponectin has atherosclerosis and antiphlogistic effect, and it can suppress the expression of vascular endothelial cell adhesion factor and reduce scavenger cell excretory cytokine quantity.Adiponectin can be by suppressing endothelial cell adhesion molecule VCAM-1, and the expression of ICAM-1 suppresses inflammatory factor and activates.Adiponectin can combine with endotheliocyte, suppresses the expression of the monocyte adhesion molecule of TNF-a mediation.In addition, in the metabolic syndrome patient, the blood plasma level of adiponectin is obviously low, shows that the dyssecretosis of adiponectin and the generation of these diseases all have confidential relation, shows that adiponectin is having its important function aspect the metabolism of fat and sugar.

In a word, adiponectin has the adjusting lipid metabolism, improves insulin sensitivity, thus effect vascular endothelial cell atherosclerosis and antiphlogistic effect.

The present invention sets up medicaments sifting model with groups of cells packing technique and stem cells technology, has following major advantage:

Based on physical crosslinking, chemically crosslinked and biology crosslinked the substep crosslinking process, compare physics and chemically crosslinked technology, biological crosslinking technological is reliable and stable, not only can control it and solidify but also can control degradation.Finish the selection of the cell-shaped material that is fit to external physiological system model construction and proportioning, material aftertreatment technology, system assembles flow scheme design according to this technology.

Set up with adipocyte, endotheliocyte and pancreas islet are main external energy metabolism system modeling framework.Designed the three-dimensional hierarchical structure model that makes up the energy metabolism system model in order to instruct.The application cell package technique has been finished the assembling of project organization.Studies show that adipocyte, islet cells, endotheliocyte that model is cultivated than the plane can better the intravital physiological and pathological situations of emulation.

Based on the basic theories of fat stem cell origin and differentiation, experimental design the method for directed differentiation fat stem cell.Studies show that, by adopting substep control and accurate inductive method, can be with in the assembly: the cytodifferentiation of structured material inside be an adipocyte, and the cytodifferentiation on structure channel surface is an endotheliocyte, and the endotheliocyte that differentiates is self-assembled into the structure of endodermis sample at channel surface.

From the physiological significance of glycolipid metabolism and the importance energy metabolism, selected glucose consumption, lipid acid mobilize and lipogenesis as the glycolipid metabolism mark, and set up corresponding detecting method.The energy metabolism system model of assembling is than 2D culture systems and lack the 3D system of pancreas islet: glycolipid metabolism is more near truth in the body, more be easy to generate the pathology of Diabetes Mellitus state under the long time high concentration glucose induction, the pharmacological action of rosiglitazone is more remarkable.

According to fat-pancreas islet endocrine axis theory, selected the Regular Insulin Physiology and biochemistry sign of islet secretion.The main regulatory factor secretion of fat-pancreas islet endocrine axis changes very near intravital truth.The diabetes pathological state that the long time high concentration glucose induction produces down is also more near truth in the body, and the adipocyte in the model structure can influence the insulin secretion kinetics of β cell.The pharmacological action of medicine rosiglitazone and nateglinide is more remarkable and meet body innerlich anwenden situation.

This model and the technology of setting up model, have wide practical use in the drug research field, to make external drug study more system, accurately near testing in the body, can remedy the defective of high flux screening technology low success rate, reduce the drug development cost and risk, the pathogenesis and the study medication of one of principal disease of promotion 21 century threat human health diabetes.

Description of drawings

Glucose uptake experiment under Fig. 1 different condition.2D:2D culture systems wherein; 3D:3D trains system; 3D+ β-cell: the 3D culture systems of having assembled pancreas islet.Normal: the normal cultivation; HG: high concentration glucose (15mM) is cultivated 7d; HG+ROS: added rosiglitazone (3 μ g/mL) on the 11st day.Data?are?mean±s.d.，n＝6.

Free fatty acids release experiment under Fig. 2 different condition.2D:2D culture systems wherein; 3D:3D trains system; 3D+ β-cell: the 3D culture systems of having assembled pancreas islet.Normal: the normal cultivation; HG: high concentration glucose (15mM) is cultivated 7d; HG+ROS: added rosiglitazone (3 μ g/mL) on the 11st day.Data?are?mean±s.d.，n＝6.

The insulin secretion kinetics of the free pancreas islet of Fig. 3.Normal wherein; Normal cultivation group; HG: high concentration glucose (15mM) is cultivated 5d; HG+ROS: high sugar is cultivated and was added rosiglitazone (3 μ g/mL) on the 11st day; HG+NATE: high sugar is cultivated and was added nateglinide (5ug/ml) on the 11st day.Data?are?mean±s.d.，n＝3.

Fig. 4 assembles the insulin secretion kinetics of pancreas islet.Normal wherein; Normal cultivation group; HG: high concentration glucose (15mM) is cultivated 5d; HG+ROS: high sugar is cultivated and was added rosiglitazone (3 μ g/mL) on the 11st day; HG+NATE: high sugar is cultivated and was added nateglinide (5ug/ml) on the 11st day.Data?are?mean±s.d.，n＝3.

Fig. 5 is an adipocyte leptin secretion under the diabetes pathological model.2D:2D culture systems wherein; 3D:3D trains system; 3D+ β-cell: the 3D culture systems of having assembled pancreas islet.Normal: the normal cultivation; HG: high concentration glucose (15mM) is cultivated 5d; HG+ROS: added rosiglitazone (3 μ g/mL) on the 12nd day.Data?are?mean±s.d.，n＝6.

Fig. 6 is adipocyte phylaxin secretion under the diabetes pathological model.2D:2D culture systems wherein; 3D:3D trains system; 3D+ β-cell: the 3D culture systems of having assembled pancreas islet.Normal: the normal cultivation; HG: high concentration glucose (15mM) is cultivated 5d; HG+ROS: added rosiglitazone (3 μ g/mL) on the 12nd day.Data?are?mean±s.d.，n＝6.

Fig. 7 is adipocyte adiponectin secretion under the diabetes pathological model.2D:2D culture systems wherein; 3D:3D trains system; 3D+ β-cell: the 3D culture systems of having assembled pancreas islet.Normal: the normal cultivation; HG: high concentration glucose (15mM) is cultivated 5d; HG+ROS: added rosiglitazone (3 μ g/mL) on the 12nd day.Data?are?mean±s.d.，n＝6.

Embodiment

The invention will be further described below in conjunction with embodiment, and protection scope of the present invention is not limited to embodiment, every according in disclosed by the invention perhaps principle any this area of implementing be equal to replacement, all belong to protection scope of the present invention.

Embodiment 1 seed cell is cultivated

Selection derives from the seed cell of the fat stem cell of rat as the construction system basis, and fat stem cell is a multipotential stem cell, can be to directed differentiation such as adipocyte, scleroblast, muscle cells.

(1) after the anesthesia of rat (100-150g) abdominal injection vetanarcol, with 70% alcohol immersion 10min, the aseptic abdominal subcutaneous adipose tissues of getting is put into aseptic beaker rapidly, is transferred to super clean bench;

(2) after aforesaid operations is finished, in super clean bench,, use D-Hank ' s solution ((Ca with reticular tissue and the clot on the mosquito forceps rejecting fatty tissue ²⁺, Mg ²⁺-free Hank ' s, pH7.4): 8.175g NaCl, 0.403g KCl, 0.114g Na ₂HPO4, be dissolved in the 950mL redistilled water, add HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid, the 4-hydroxyethyl piperazine ethanesulfonic acid) adjust pH is settled to 1L) clean three times, change in the new beaker, fatty tissue is cut into the little fast of diameter 2 * 2mm, add D-Hank ' s solution, transfer in the centrifuge tube centrifugal 10 minutes of 1500rpm;

(3) gentle aspiration upper strata buoyant fatty tissue is in beaker, 0.1% collagenase (II type) solution that adds 2mL then, 37 ℃ of digestion 45min are with the nylon leaching net filtration Digestive system of 250-mm, liquid after the filtration is transferred in the centrifuge tube, centrifugal 10 minutes of 1000rpm;

(4) inhale and to remove the sophisticated adipocyte in upper strata, sedimentary cell D-Hank ' s solution is made into cell suspension once more, filters centrifugal 10 minutes of filtrate 1000rpm once more with the nylon leaching net of 25-mm;

(5) remove supernatant, add DMEM (Sigma) substratum that contains 15%FCS (foetal calf serum), blow and beat all cells group of scattering, cell concn to 1.5 ~ 3 * 10 are adjusted in numeration ⁵Cells/mL is inoculated in 25cm ²(cross with 0.2% gelatin immersion treatment in advance) in the culturing bottle; Be incubated in 37 ℃ of carbonic acid gas constant incubators;

(6) hatch 12 hours in the incubator after, rock culturing bottle gently, not adherent material (tissue and cell debris) is suspended, topple over and fall nutrient solution, PBS washs once gently, adds the new DMEM substratum that contains 15%FCS and continues cultivation.

(7) general about three days, cell reached individual layer and converges, and after cell converges, carried out the routine cultivation of going down to posterity;

(8) go down to posterity: inhale and remove nutrient solution, the pancreatin solution 1mL of adding 0.2%, inhale behind the washing bottle wall rapidly and go, add 0.2% pancreatin solution 1mL again, hatch about 5min for 37 ℃, microscopic, treat 80% cell rounding after, inhale gently and remove pancreatin solution, add the DMEM substratum that contains 15%FCS rapidly and stop digestion; Piping and druming culturing bottle wall is made into cell suspension once more; Adjust cell concn to 1.5 ~ 3 * 10 ⁵Cells/mL is inoculated in the new culturing bottle.

(9) the cell cultivation of can normally going down to posterity was gone down to posterity once in per three days.

The separation and the cultivation of embodiment 2 rat Langerhans islets

(1) SD rat, carbrital (30mg/kg) intramuscular anesthesia.Sterilization is cut open the belly, and appears pancreas and common bile duct, respectively wears a suture through the back at nearly duodenal common bile duct far-end, the nearly hepatic portal of common bile duct place, wouldn't ligation.

(2) in the common bile duct stage casing distad direct motion insert No. 4.5 syringe needles of people (connected the syringe that pre-temperature digests parting liquid is housed), the nearly hepatic portal of ligation common bile duct place suture, in common bile duct, inject a small amount of digestion parting liquid, make the bile row people duodenal lumen in the common bile duct, ligation common bile duct far-end suture afterwards, in the slowly retrograde notes people of the 5% collagenase solution 10-15ml ductus pancreaticus with pre-temperature, pancreas is fully expanded.

(3) the rapid complete pancreas of winning moves in the people 50ml Erlenmeyer flask and (the above-mentioned digestion parting liquid 6-10ml of pre-temperature is housed) in it, and places 38 ℃ of water-baths to shake (25-30) min gently, 20 order stainless (steel) wires sieve, adding ice-cold Hanks liquid (contains 10% new-born calf serum, NCS), stops digestion.

(4) 4 ℃, the centrifugal 3min of 1000r/min abandons supernatant, washes 2 times with method.The pancreas digested tissue does not continue above-mentioned steps, until organizing complete digestion.Throw out separates with Ficoll 400 density gradient centrifugations, and the concentration that adds the preparation of people Hank liquid successively is respectively 25%, 23%, and 20% and 11%Ficoll liquid.

(5) 4 ℃, the centrifugal 20min of 1000r/min, (23-20) % and (20%-11%) interface collect pancreas islet, wash 3 times through Hanks liquid, it is standby to add the new DMEM culture medium culturing that contains 10%FCS.

Embodiment 3 many cells system model building processs

(1) gelatin, sodium alginate, Fibrinogen and the preparation of cell intermingling material: gelatin is dissolved in the PBS damping fluid, form 20% solution, sodium alginate is dissolved in the PBS damping fluid, forms 5% solution, use among the NaOH respectively and remaining acetic acid, regulate pH value to 7.1,70 ℃ of baking oven discontinuous formula sterilizations.Under the aseptic condition with fibrinogenolysis in the DMEM nutrient solution, form 5% solution.Face the time spent with three kinds of materials with even blend in 1: 1: 1.The fat stem cell of material and cultivation is mixed, obtain concentration and be about 1 * 10 ⁷/ mL fat stem cell-matrix material intermingling material.

(2) assembling of energy metabolism system was made of system-based assembling and two steps of pancreas islet assembling.Disposable syringe with fat stem cell/matrix material intermingling material injection 1mL places 4 ℃ of refrigerator precoolings more than 30 minutes;

(3) select the Cell Assembly II equipment of Yin Hua company for use,, open shaping room temperature control switch and make working chamber's cooling at the sterilized shaping base plate of working chamber's internal fixing, monitor temperature in real time, when treating that temperature is reduced to 6 ℃, mounting of syringe and stationary nozzle;

(4) 3D model and the selected forming parameter by the Solidworks design directly drives the assembling shaping, the cad model of system's tertiary structure is designed to 10mm * 10mm * 3mm cube structure, the secondary structure characteristic dimension is 300mm, and transmission feature is of a size of 350mm, and ranks are counted n=15.According to secondary structure characteristic dimension and the corresponding different size shower nozzle of material rate of expansion, select the shower nozzle of model 6# for use.Sweep velocity 30mm/s extrudes flow 1.2mm ³/ s.

(5) three-dimensional structure that contains cell that obtains having 82% porosity after shaping is finished.Structure is stored in the aseptic 4 ℃ environment, forms aftertreatment in 1 hour.

(6) structure that assembles was immersed in 5% sterilization calcium chloride solution crosslinked 1 minute,, uses the crosslinked 10min of 100U/mL Taka-proteinase then with DMEM washing three times;

(7) from shaping base plate cutting structure body, transfer in the culture dish, add and contain 10%FCS, 1 μ mol/L insulin, the DMEM nutrient solution of 50ng/mL EGF-1and 50U/mL aprotinin, 37 ℃, 5%CO ₂Environment is cultivated 3d down.

(8) cultivate after 3 days, nutrient solution is converted to and contains 10%FCS, 1 μ M insulin, 1 μ M dexamethasone, 0.5mmol/L, isobutyl-methylxanth _InE (IBMX; Sigma), the DMEM nutrient solution of 50u/mL aprotinin, 37 ℃, 5%CO ₂Environment is cultivated 3d down.

(9) at the 6th day, use syringe (4# syringe needle) to draw 10 islet cellss of the structural integrity of cultivating, the location is assembled in the structure.The next day change liquid, collect culture supernatant 0.5mL and put into test tube and seal up for safekeeping ,-20 ℃ of refrigerators are frozen to be measured.

Control experiment: after assembling finished, the many cells system in the culture dish added earlier and contains 10%FCS, 1 μ M insulin, 1 μ M dexamethasone, 0.5mmol/L, isobutyl-methylxanthine (IBMX; Sigma), the DMEM nutrient solution of 50u/mLaprotinin, 37 ℃, the 5%CO2 environment is cultivated 3d down.Cultivate after 3 days, nutrient solution is converted to and contains 10%FCS DMEM, 10%FCS, and 1 μ mol/L insulin, the DMEM nutrient solution of EGF and 50U/mL aprotinin, 37 ℃, the 5%CO2 environment is cultivated 3d down.At the 6th day, 10 islet cellss of the structural integrity of draw cultivating with syringe (4# syringe needle), the location is assembled in the structure, the next day change liquid.

Observe under fluorescent microscope through different treatment step cultured cells assembly, CD31 dyeing and oil red O are two to be dyed.For cultivating the cell assembly of inducing with EGF earlier, the fat stem cell of material internal is divided into adipocyte (redness), and channel surface has only a few cell to be divided into fat stem cell.For the cell assembly of not using the EGF inducing culture earlier, the cell on viewability channel surface also is divided into adipocyte in a large number.This result shows, induce under the control in our differentiation condition of design, earlier induce the fat stem cell that can cause channel surface to be divided into endotheliocyte earlier with EGF, these last eventually cells that break up will lose the ability that is divided into adipocyte, cause then using dexamethasone, IBMX inducing culture, have only the fat stem cell of material internal can be divided into sophisticated adipocyte.Opposite, if induce with dexamethasone, IBMX earlier, will cause the fat stem cell of channel surface to be divided into sophisticated adipocyte earlier, lose the ability that is divided into endotheliocyte.This result proves, can induce the method for differentiation condition by control, the accurately differentiation of different positions fat stem cell in the control texture body, the fat stem cell of channel surface is divided into endotheliocyte, and be self-assembled into the endothelium spline structure at channel surface, constitute the biological barrier of selective permeation endocrine factor, the fat stem cell of substrate material inside then is divided into adipocyte, forms the physiological system realistic model that is similar to body fat cell position structure.

Glucose consumption under the embodiment 4 diabetes pathological models detects

(1) the many cells system after assembling is finished is with containing 10%FCS, the DMEM nutrient solution of normal glucose (5mM), 37 ℃, 5%CO ₂Environment is cultivated down 7d, the next day change liquid;

(2) adopt high concentration glucose (15mM) to cultivate simultaneously, induce the pathology of Diabetes Mellitus state that produces;

(3) changed liquid at the 12nd day, 37 ℃, 5%CO ₂Environment was cultivated down after 24 hours, and collection culture supernatant 0.5mL puts into test tube and seals up for safekeeping, and-20 ℃ of refrigerators are frozen to be measured;

(4), behind the nutrient solution before collecting, nutrient solution changed into contain 10%FCS, the DMEM nutrient solution of 25mM glucose, 37 ℃, 5%CO at the 13rd day ₂Environment was cultivated 24 hours down;

(5) at the 14th day, collection culture supernatant 0.5mL puts into test tube and seals up for safekeeping, and-20 ℃ of refrigerators are frozen to be measured;

(6) control group (fat stem cell 5 * 10 is cultivated on no pancreas islet many cells system and plane ⁴Be inoculated in 24 orifice plates, adopt and induce differentiation in a like fashion, as the contrast of 2D culture systems), carry out identical processing;

(7) glucose concn in the supernatant liquid of Shou Jiing is monitored with the glucose detection test kit;

(8) glucose consumption with glucose concn (M) among the DMEM that does not cross with co-culture of cells, deducts concentration (N) expression of glucose in the culture supernatant;

(9) glucose consumption rate C=[M (13d)-N (13d)]/[M (12d)-N (12d)]

(10) the many cells system of unassembled pancreas islet and 2D culture systems adopt identical method to detect glucose consumption;

(11) positive control medicine rosiglitazone (3 μ g/mL) is in the DMEM nutrient solution of cultivating that joined high glucose (15mM) on the 11st day.

By experimental result (Fig. 1) as seen, under the glucose of 15mM stimulates, than the plane culture system, the glucose consumption of adipocyte obviously increases in the 3D packaging assembly, this result shows the adipocyte in the 3D packaging assembly, because culture environment more approaches situation in the true body, has more vigor.Be both under the 3D culture environment, the glucose consumption of 3D system of assembling pancreas islet is 3 times of system of not assembling pancreas islet.This is because the pancreas islet of assembling can secrete a large amount of Regular Insulin, and Regular Insulin can stimulate adipocyte ingestion of glucose synthetic fat to the sugared stimulation responses of height.

Using 15mM glucose culture solution inducing culture after 7 days, the adipocyte glucose consumption of three culture systems all has decline, but the 3D system glucose consumption of assembling pancreas islet descends the most remarkable.This is because long high concentration glucose is cultivated and can be induced simultaneously, adipocyte produces insulin resistant, beta Cell of islet is replied susceptibility to glucose and descended, the stack of two kinds of effects, the glucose consumption that causes high concentration glucose to stimulate obviously descends, and this point meets truth in the body.Therefore easier the inducing of the 3D system of proof assembling pancreas islet produces significant pathological change, is fit to the pathology modeling.Under the pathologic condition that the glucose consumption that long-time cultivation is brought out for high concentration glucose descends, rosiglitazone (thiazolidinediones antidiabetic drug) is for the therapeutic action of the pathological change that brings out, and the energy metabolism system of assembling is more more remarkable than the 3D package system of 2D culture systems and shortage pancreas islet.This result has proved that fully the energy metabolism system of assembling is in the potentiality of screening treatment diabetes medicament.These experiments simultaneously also point out this pathological model can be used for the screening active ingredients of other unknown medicines for the treatment of diabetes.In culture systems, add medicine to be measured, detect the glucose consumption level of pathological model under normal and pathological conditions, can analyze the therapeutic action of medicine to be measured, have the diabetes of treatment disease potential drug thereby can filter out to the pathological change that brings out.

Lipid mobilization under the embodiment 5 diabetes pathological models detects

(3) at the 15th day, elder generation changes nutrient solution into and contains 10%FCS, the DMEM nutrient solution of 2% fatty acid free BSA, 37 ℃, 5%CO ₂Environment was cultivated 4 hours down

(4) collect culture supernatant 0.5mL and put into test tube and seal up for safekeeping ,-20 ℃ of refrigerators are frozen to be measured

(5) nutrient solution is changed into contain 10%FCS then, 2% fatty acid free BSA and 10 μ Misoproterenol DMEM nutrient solutions, 37 ℃, the 5%CO2 environment was cultivated 4 hours down

(6) collect culture supernatant 0.5mL and put into test tube and seal up for safekeeping ,-20 ℃ of refrigerators are frozen to be measured

(7) control group is cultivated on no pancreas islet many cells system and plane, carries out identical processing;

(8) the FFA concentration in the supernatant liquid detects with the lipid acid detection kit;

(9) FFA release rate is recently represented with the FFA concentration that does not add the isoproterenol stimulation with the FFA that adds under the isoproterenol stimulation situation.

(10) the many cells system of unassembled pancreas islet and 2D culture systems adopt identical method to detect FFA and discharge consumption;

(11) positive control medicine rosiglitazone (3 μ g/mL) added in the DMEM nutrient solution at the 11st day that cultivates.

By experimental result (Fig. 2) as seen, under norepinephrine stimulates, than the plane culture systems, the FFA of adipocyte discharges obviously in the 3D culture systems increases, this result shows that the adipocyte in the 3D culture system is more responsive to the lipid mobilization that norepinephrine stimulates, and has more vigor.And be both under the 3D culture environment, the FFA of the 3D culture systems of assembling pancreas islet discharges significantly and raises.This is because Regular Insulin can suppress adipocyte FFA release, the pancreas islet of assembling can be to the norepinephrine stimulation responses, suppress insulin secretion, the retarding effect that causes adipocyte FFA is discharged weakens, the effect stack that this effect and norepinephrine directly stimulate adipocyte FFA to discharge, thus cause the FFA secretion to increase.

Using the 15mM glucose culture solution, inducing culture is after 7 days, and the adipocyte FFA of three culture systems discharges all increase, and the FFA of 3D system adipocyte discharges will be higher than the 2D system, and the FFA release of the 3D system of assembling pancreas islet increases the most remarkable.This is owing to high concentration glucose cultivation for a long time, and energy induced lipolysis stem cell simultaneously is divided into adipocyte in a large number, and adipocyte can synthesize the significant quantities of fat storage, causes free FFA release increase under the norepinephrine stimulation situation.And differentiation of the fat stem cell of 3D system and lipogenesis are stronger than 2D system, and the more fat of storage is so free FFA discharges also high.This result proves that easier the inducing of the adipocyte of 3D system produces significant pathological change, is fit to the pathology modeling.The long-time free FFA that brings out that cultivates discharges increase for high concentration glucose, and rosiglitazone only in the 3D system of assembling pancreas islet, demonstrates tangible pharmacological action.This model can detect the pharmacological action of the inhibition FFA release of rosiglitazone, and this is consistent with the result who measures the outlook on human body, has proved that this model is to screening the validity of such medicine.

Insulin secretion kinetic measurement under the embodiment 6 diabetes pathological models

(1) the many cells system of assembling after finishing, with containing 10%FCS, the DMEM nutrient solution of normal glucose (5mM), 37 ℃, the cultivation 5 days down of 5%CO2 environment, the next day change liquid;

(3) ((10/group) are inoculated in gelatin-based culture dish kind, fat stem cell 5 * 10 with pancreas islet to be seeded in pancreas islet on the plating medium ⁴Be inoculated in 24 orifice plates, adopt and induce differentiation in a like fashion, as 2D culture systems contrast) with identical step process;

After (4) 5 days, the many cells system is transferred in the flow cell of 1mL, contain the DMEM nutrient solution of 2mM glucose with peristaltic pump with the flow perfusion of 0.2mL/min;

(5) at 37 ℃, the 5%CO2 environment is cultivated perfusion one hour down, collects effusive perfusion liquid, puts into test tube to seal up for safekeeping, and-20 ℃ of refrigerators are frozen to be measured, as the basal level of insulin secretion;

(6) perfusion was changed to perfusion liquid the DMEM nutrient solution that contains 15mM glucose after one hour;

(7) preceding 20min every the 2min sampling once puts into test tube and seals up for safekeeping, and-20 ℃ of refrigerators are frozen to be measured;

(8) behind the 20min, perfusion liquid is changed to the DMEM nutrient solution that contains 2mM glucose;

(9) take a sample once every 5min in the 15min, put into test tube and seal up for safekeeping ,-20 ℃ of refrigerators are frozen to be measured;

(10) insulin concentration is monitored with immunofluorescence Regular Insulin detection kit in the perfusate of Shou Jiing;

(11) unassembled pancreas islet adopts identical method to detect insulin secretion;

(12) positive control medicine rosiglitazone (3 μ g/mL) and nateglinide (nateglinide) are (5ug/ml) in the DMEM nutrient solution that joined high sugar (15mM) on the 11st day of cultivation.

The dynamic process of insulin secretion is significant to the energy metabolism regulation and control, and the deficiency of insulin secretion, the delay at insulin secretion peak or the like dynamic variation are the important pathomechanisms of diabetes.For being contained in islet cells in the energy metabolism system model, test group whether can imitate in vivo secreting function, we have done time dependent glucose with filling system stimulates Regular Insulin release experiment (Fig. 3, Fig. 4), the pancreas islet secretion of insulin pattern under glucose stimulates that has shown free or assembling.Under 15M glucose stimulated, the islet cells that the islet cells insulin secretion of assembling is cultivated than the plane obviously increased.The islet cells that the islet cells of proof assembling is cultivated than the plane maintains higher activity.

Can to cause the islet cells pathological change in order testing to be exposed in the high concentration glucose whether for a long time, and then to influence insulin secretion that we cultivate the plane and the islet cells of assembling is cultivated in the glucose of 15mM.After 5 days, result of study shows the insulin secretion all violent decline of islet cells under 15mM glucose stimulates of two kinds of training methods, but the height that the islet cells insulin secretion rate of descent of assembling is cultivated than the plane.The high sugar inhibition insulin secretion that is subjected to that nateglinide can increase by two kinds of training method islet cellss descends, but rosiglitazone has only increased the insulin secretion that the islet cells of assembling is suppressed by high sugar.

It should be noted that, after the long-time cultivation of high sugar with 15mM, the insulin secretion peak of the islet cells glucose induction of assembling postpones 2min under normal circumstances, nateglinide (insulin secretion accelerating class antidiabetic drug) can be revised this inductive insulin secretion peak and postpone, and this is consistent with experiment at human body.Yet this phenomenon does not observe on the islet cells of plane cultivation.The dynamic (dynamical) disorder of insulin secretion is the main features of diabetes.These results suggest, the adipocyte in the 3D structure help islet cells to obtain more diabetes pathological characters.The mechanism that further will show this phenomenon subsequently to the research of fatty hormone secretion.The mechanism of action of nateglinide be with Regular Insulin β cell on the sulfonylurea receptors bind, it is open to suppress the ATP sensitive potassium channel, causes the cytolemma depolarize, impels voltage-dependent ca channel open, causes that calcium concn raises in the endochylema, thereby promotes Regular Insulin to discharge.Nateglinide can stimulate diabetic subject's Regular Insulin to discharge rapidly and momently, improves the beta Cell of islet reactivity, promotes shifting to an earlier date of insulin secretion peak.Our model has not only reproduced the disorder of insulin secretion under the pathological conditions, has also detected the pharmacological action of nateglinide to the insulin secretion disorder, has proved that this model is to screening the validity of such medicine.Model also detects the disorderly pharmacological action to insulin secretion of rosiglitazone simultaneously, has further proved the validity of this model to screening thiazolidinediones and insulin secretion accelerating class medicine.

Adipocyte hormone under the embodiment 7 diabetes pathological models detects

(1) the many cells system of assembling after finishing, with containing 10%FCS, the DMEM nutrient solution of 5mM glucose, 37 ℃, the 5%CO2 environment is cultivation down, the next day change liquid;

(3) be seeded in adipocyte on the plating medium with identical step process;

(4) changed liquid at the 18th day, 37 ℃, the 5%CO2 environment was cultivated down after 3 days, and collection culture supernatant 1mL puts into test tube and seals up for safekeeping, and-20 ℃ of refrigerators are frozen to be measured;

(5) Leptin in the sample, the concentration of resistin and adiponectin is used leptin ELISA detection kit (RB respectively, USA), rat resistin ELISA detection kit (TPI, USA) and ratadiponectin ELISA detection kit detects, according to the specification sheets operation of detection kit;

(6) at the 21st day, collected culture supernatant after, collect assembled structural body and the acellular assembled structural body stay, disperse and digested 20 hours for 60 ℃ with the 0.14mg papoid;

(7) behind Hoechst 33258 (Sigma) dyeing, fluoroscopic examination, with calf thymus 1 type DNA as the examination criteria product;

(8) value that records of not celliferous structure deducts from the value that pattern detection obtains;

(9) value of fatty hormone secretion is represented with the ratio that ELLSA detects the dna content that obtains hormone concentration and record;

(10) the many cells system of unassembled pancreas islet and 2D culture systems adopt identical method to detect Leptin, the secretion of resistin and adiponectin;

(11) positive control medicine rosiglitazone (3 μ g/mL) added in the DMEM nutrient solution at the 12nd day that cultivates.

Experimental result shows (Fig. 5), and than the plane culture system, the leptin secretion of adipocyte obviously increases in the 3D culture system, proves the adipocyte in the 3D culture system because culture environment more approaches situation in the true body, and the vigor of secretion leptin is higher.Under same 3D culture environment, the leptin secretion of 3D culture systems of having assembled pancreas islet is lower than the system of not assembling pancreas islet.Using the 15mM glucose culture solution, inducing culture is after 12 days, and the leptin of three culture systems is expressed all significantly to be increased.This is because long high concentration glucose is cultivated, and can induce the significant quantities of fat differentiation of stem cells simultaneously is adipocyte, and adipocyte gathers a large amount of fat, thereby causes the secretion of leptin to increase.Increase for the long-time leptin secretion that brings out of cultivating of high concentration glucose, rosiglitazone is in the 3D system of assembling pancreas islet and unassembled pancreas islet, all demonstrate more significant pharmacological action, wherein to the pharmacological action maximum of 3D system of assembling pancreas islet, the situation of this and real human body is consistent.This experimental result proves that this model can detect the pharmacological action of rosiglitazone, proves that this model can screen such medicine from the angle of improving leptin secretion.

Phylaxin is considered to get in touch fat and insulin resistant and the important point of connection of diabetes.Fat stem cell is divided in the process of sophisticated adipocyte and adipocyte hypertrophy, and the level of phylaxin mRNA significantly increases, and nutrition and hormone can be regulated and control the expression of phylaxin.Phylaxin can make the effect of sugar tolerance and Regular Insulin impaired, and the antidiabetic medicine rosiglitazone can significantly be reduced the expression of phylaxin mRNA, and the expression of downward modulation phylaxin is the important mechanisms of TZD class medicine performance anti-diabetic effect.Experimental result shows (Fig. 6), and than the plane culture system, the leptin secretion of adipocyte slightly increases in the 3D culture system, shows the adipocyte in the 3D culture system.Under same 3D culture environment, assembled the phylaxin secretion of the 3D culture systems of pancreas islet and do not had significant difference with the system of not assembling pancreas islet.

Using the 15mM glucose culture solution, inducing culture is after 12 days, and the phylaxin of three culture systems is expressed all significantly to be increased.This is that inducing the significant quantities of fat differentiation of stem cells is adipocyte because high concentration glucose is cultivated for a long time, and adipocyte gathers a large amount of fat and hypertrophy, thereby causes the secretion of phylaxin to increase.In three culture systems, the inducing of high sugar down, the phylaxin secretion of having assembled the 3D culture systems of pancreas islet increases minimum, and the existence of pancreas islet makes system present a kind of stable status more, and this more approaches situation about observing in the body.Secrete increase for the long-time phylaxin that brings out of cultivating of high concentration glucose, rosiglitazone is in the 3D system of plane culture systems, assembling pancreas islet and unassembled pancreas islet, all demonstrate more significant pharmacological action, wherein the most obvious to the pharmacological action of 3D system of assembling pancreas islet.This experimental result proves that this model can screen such medicine from phylaxin excretory angle under the inhibition pathologic condition.

Adiponectin has the adjusting lipid metabolism; improve insulin sensitivity; improve metabolic syndrome patient hyperlipidaemia and insulin resistant; suppress the expression of vascular endothelial cell adhesion factor and reduce scavenger cell excretory cytokine quantity; protection endothelial function, atherosclerosis and antiphlogistic effect.Experimental result shows (Fig. 7), than the plane culture system, the adiponectin secretion level of adipocyte significantly increases in the 3D culture system, shows the adipocyte in the 3D culture system, culture environment more approaches situation in the true body, has more the vigor of secretion adiponectin.Under same 3D culture environment, assembled the adiponectin secretion of the 3D culture systems of pancreas islet and do not had significant difference with the system of not assembling pancreas islet.

Using the 15mM glucose culture solution, inducing culture is after 12 days, and the adiponectin expression of three culture systems all significantly lowers.In three culture systems, under the inducing of high sugar, assembled the phylaxin secretion reduction minimum of the 3D culture systems of pancreas islet, the existence of pancreas islet makes the adiponectin secretion level of system present a kind of stable status more, and this is the situation that approaches to observe in the body.Secrete reduction for the long-time adiponectin that brings out of cultivating of high concentration glucose, rosiglitazone is in the 3D of plane culture systems and unassembled pancreas islet system, all do not demonstrate tangible pharmacological action, but the pharmacological action of 3D system to the assembling pancreas islet is remarkable, the secretion level of adiponectin return to normal circumstances under secretion level do not have significant difference, this result uses rosiglitazone to observe situation about obtaining with true diabetic subject to be consistent, to prove that this model can screen such medicine from adiponectin excretory angle.

Claims

1. the screening method of a Remedies for diabetes may further comprise the steps:

(3) will in animal body, directly be assembled in the space of three-dimensional structure by isolating pancreas islet;

2. drug screening method according to claim 1 is characterized in that: the quantity of fat stem cell is 10 in the described method ⁶-10 ⁸Individual/the 1ml substrate material, islet cells quantity is 5-50.

3. drug screening method according to claim 1, it is characterized in that: in the step of described method (1), the special-purpose experimental system that is used for the three-dimensional controlled assembling of stroma cell mainly comprises the exclusive data process software, based on four-axis movement control card and stepper motor driven three-dimensional platform, material transports subsystem, the shaping base plate of the working chamber of independent temperature and split-type design.

4. drug screening method according to claim 1, it is characterized in that: in the step of described method (1), described substrate material is gelatin, sodium alginate and fibrinogenic composition, and wherein gelatin, sodium alginate and fibrinogenic proportioning are 1: 0.2-1: 0.2-1.

5. drug screening method according to claim 1, it is characterized in that: in the step of described method (2), it is to induce with endothelial cell growth factor (ECGF) earlier that differentiation condition is induced in described control, make the fat stem cell of three-dimensional structure channel surface be divided into endotheliocyte earlier, these last eventually cells that break up will lose the ability that is divided into adipocyte; Then, make the fat stem cell of substrate material inside be divided into sophisticated adipocyte with dexamethasone, isobutyl methylxanthine inducing culture.

6. drug screening method according to claim 1 is characterized in that: described three-dimensional structure has the microvoid structure of 10-30um diameter hole.

7. drug screening method according to claim 1 is characterized in that: described method produces the diabetes pathological model for adopting the cultivation of 10-25mM glucose culture solution after 5-10 days, inducing.

8. pharmaceutical methods according to claim 1 is characterized in that: described method induces to produce the diabetes pathological model for adopting the cultivation of 30-80ng/mL TNF-a nutrient solution after 5-10 days.

9. pharmaceutical methods according to claim 1 is characterized in that: described method induces to produce the diabetes pathological model for adopting the cultivation of 6-20 μ Minsulin nutrient solution after 5-10 days.

10. pharmaceutical methods according to claim 1 is characterized in that: described certification mark thing is glucose, free fatty acids, Regular Insulin, leptin, phylaxin, adiponectin etc.