CN105238745A - 3T3-L1 preadipocyte differentiation inducing reagent composition and application thereof - Google Patents
3T3-L1 preadipocyte differentiation inducing reagent composition and application thereof Download PDFInfo
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- CN105238745A CN105238745A CN201510565191.0A CN201510565191A CN105238745A CN 105238745 A CN105238745 A CN 105238745A CN 201510565191 A CN201510565191 A CN 201510565191A CN 105238745 A CN105238745 A CN 105238745A
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Abstract
The invention provides a composition capable of promoting 3T3-L1 preadipocyte differentiation. The composition comprises a differentiation inducing reagent containing ATP (adenosine-triphosphate), and the differentiation inducing reagent is mainly composed of adenosine-triphosphate, insulin, dexamethasone and IBMX (3-isobutyl-1-methylxanthine). The composition is made by adding the adenosine-triphosphate on the basis of a traditional 'cocktail' inducing reagent, and after adoption of the inducing composition for differentiation inducement for 8 days, 90% of 3T3-L1 preadipocytes can be differentiated into typical mature adipocytes, cell volume is increased, cell bodies are rounded, lipid droplets in cytoplasm are evident, and bigger lipid droplets are formed as compared with those formed according to a typical 'cocktail' inducing method. The composition is excellent in differentiation effect and high in differentiation efficiency.
Description
Technical field
The present invention relates to biomedical sector, specifically a kind ofly promote the composition that 3T3-L1 PECTORAL LIMB SKELETON breaks up and the application in PECTORAL LIMB SKELETON is differentiation-inducing thereof.
Background technology
PECTORAL LIMB SKELETON is the specific precursor cell that a class has to Adipocyte Differentiation potentiality, is generated by multipotential stem cell or ES cell differentiation.Adipocyte is the main component of white adipose tissue (WAT), and itself can not be bred, and induces form mainly through PECTORAL LIMB SKELETON under various hormonal action.In beginning before Adipocyte Differentiation, similar inoblast in PECTORAL LIMB SKELETON form, in spindle shape.After starting differentiation, PECTORAL LIMB SKELETON becomes round gradually, and the content of intracellular three fat rises gradually simultaneously, and the content of cytoskeletal protein etc. declines.Start in cell to occur that fat drips, PECTORAL LIMB SKELETON transforms to mature fat cell gradually.
The PECTORAL LIMB SKELETON of animal, as the cell model of fat disease and diabetes conditions research, is usually used in the medicine of the above-mentioned two kinds of diseases of in-vitro screening treatment.3T3-L1 PECTORAL LIMB SKELETON be Green and Kehinde in isolated a kind of cell strain with differentiation potential from the Swiss3T3 mouse embryo of 19 days in 1974, ripe adipocyte can be divided under suitable inductive condition.At present, this cell strain as one of the most frequently used cell model of in vitro study metabolism of fat regulation and control, is widely used in the scientific research in the fields such as metabolism of fat disease and new drug development.
Classical " hormone cocktail " applied in a large number PECTORAL LIMB SKELETON differentiation-inducing in, its main component is only containing IBMX, dexamethasone and insulin.Under the stimulation of Regular Insulin, dexamethasone and IBMX, by increasing the concentration of second messenger-cAMP in cell, promote that PECTORAL LIMB SKELETON is divided into ripe adipocyte.
The significant energy material of Triphosaden except transmitting as intracellular energy.As signaling molecule, transmembrane signal can also be participated in P2 (adenine nucleotide) receptors bind and conducts, play its different physiological roles in extracellular.It has wide prospect in cancer therapy, inflammation on controlling.
Summary of the invention
Adipocyte 3T3-L1 breaks up to the object of the present invention is to provide a kind of agent combination significantly to promote, is specifically to promote that the differentiation of autologous PECTORAL LIMB SKELETON and fat drip composition and the application thereof of formation.
For realizing above-mentioned purpose of the present invention, the invention provides following technical scheme:
System component:
The differentiation-inducing liquid of A:
(1) Triphosaden final concentration 0 μM, 50 μMs, 100 μMs, 150 μMs and 200 μMs.
(2) dexamethasone final concentration 1 μM.
(3) Regular Insulin final concentration 850nM.
(4) IBMX final concentration 0.25nM.
B maintain liquid:
(1) Triphosaden final concentration 0 μM, 50 μMs, 100 μMs, 150 μMs and 200 μMs.
(2) Regular Insulin final concentration 850nM.
Triphosaden is ubiquitous a kind of high-energy phosphate compound in various viable cell.In vivo in histocyte as the direct sources of all life movable institute energy requirement, store and transmit chemical energy, when body absorption, secretion, Muscle contraction and carry out biochemical building-up reactions need energy time, namely Triphosaden resolves into adenosine diphosphate (ADP) and phosphate, releases energy simultaneously.Triphosaden also as a kind of coenzyme, can strengthen the metabolic activity of cell, participates in synthesis and the metabolism of protein, fat, sugar and Nucleotide.Be combined with P2 receptor-specific in extracellular, regulate cell physiological function.
In vitro in experiment, murine preadipocyte cell 3T3-L1 is as common experimental material, and " hormone cocktail " usually through classics is induced to differentiate into ripe adipocyte.Ripe adipocyte is compared with PECTORAL LIMB SKELETON, and form becomes and becomes greatly round, can see that obvious fat drips by dyeing.In atomization, add the present invention, find the differentiation that significantly can promote 3T3-L1 cell in finite concentration situation.Existing about the report of Triphosaden in have no its report on PECTORAL LIMB SKELETON differentiation remarkably influenced.The present invention can effectively promote that PECTORAL LIMB SKELETON breaks up, and has good utilization prospect to quickening the differentiation-inducing of PECTORAL LIMB SKELETON.
Accompanying drawing explanation
Fig. 1 is artificial induction 3T3-L1 PECTORAL LIMB SKELETON atomization schematic diagram.
Fig. 2 is the impact (oil red O stain) that different concns Triphosaden breaks up 3T3-L1 PECTORAL LIMB SKELETON.
Fig. 3 is the impact (Nile red dyeing) of Triphosaden on content of triglyceride in 3T3-L1 adipocyte.
Fig. 4 is that Image-proPlus6.0 image processing software drips the statistics several, fat drips the total area, fat drips average area, fat drips the index such as diameter, quantity to 3T3-L1 adipocyte lactones.
Fig. 5 is that 3T3-L1 adipocyte lactones drips mean diameter statistics.
Fig. 6 is that 3T3-L1 adipocyte lactones drips average area statistics.
Embodiment
The various laboratory apparatus laboratory apparatuss that the present embodiment is used:
Bechtop (Thermo, the U.S.), microplate reader (Thermo, the U.S.), refrigerated centrifuge (Eppendorf, Germany), high-pressure sterilizing pot: (Tomy, Japan), cell CO
2incubator (Thermo, the U.S.), inverted microscope (OlympusIX71, Japan), liquid-transfering gun (Eppendorf, Germany), electronic balance (Sai Duolisi company, the U.S.), deionized water system (Millpore, the U.S.).
Experiment reagent:
Foetal calf serum (Gibco, the U.S.), DMEM/HighGlucose substratum (Hyclone, the U.S.), IBMX(Sigma, the U.S.), dexamethasone (Sigma, the U.S.), Regular Insulin (Sigma, the U.S.), Triphosaden (sigma, the U.S.), NaOH solution, oil red O dyestuff (), PBS (Hyclone, the U.S.), Nile red dye (Lonza, the U.S.), 10% paraformaldehyde, 75% alcohol, ultrapure water, filter paper, 48 orifice plate (Coring, the U.S.), 0.25% pancreatin (Hyclone, the U.S.), anhydrous isopropyl alcohol, Tissue Culture Flask (Coring, the U.S.), Hepes buffer reagent (Sigma, the U.S.).
Embodiment 1: the preparation of required all ingredients:
(1) insulin solutions preparation:
10mg dry powder insulin is dissolved in 0.02MHCl solution, obtains 1.7mM insulin solutions; Get 0.1ml1.7mM solution and add one-tenth 170 μMs of insulin solutions in 0.9ml basic culture solution.
(2) dexamethasone solution preparation:
Get dexamethasone dry powder 3.925mg to be dissolved in 1ml dehydrated alcohol, obtain 10mM dexamethasone; The dexamethasone getting 0.1ml10mM adds in 0.9mlPBS the dexamethasone solution becoming 1mM.
(3) IBMX solution preparation:
5.56mgIBMX dry powder is dissolved in the KOH(0.5M of 0.5ml) in solution, obtain 50mMIBMX solution.
(4) complete culture solution preparation:
Get DMEM (HighGlucose) nutrient solution one bottle of 500ml, add foetal calf serum (FBS) 55ml and 1.38gHepes buffer reagent, adjust pH=to 7 with 1mol/LNaOH solution.Degerming with the millipore filter of 0.22 μm, load 500ml culture jar.
(5) differentiation-inducing nutrient solution:
Get (1) described complete culture solution 50ml and add the IBMX solution 250 μ l that (3) described concentration is 50mM, (2) described concentration is the dexamethasone solution 50 μ l of 1mM is 170 μMs of insulin solutions 250 μ l with (1) described concentration, the obtained differentiation-inducing liquid containing 0.25mMIBMX, 1 μM of dexamethasone and 850nM Regular Insulin.
(6) maintain liquid:
Getting (1) described complete culture solution 50ml, to add (1) described concentration be 170uM insulin solutions 250 μ l, the obtained maintain liquid containing 850nM Regular Insulin.
(7) containing the preparation of the differentiation-inducing liquid of Triphosaden:
Take 24.2mg Triphosaden and be dissolved in 40ml(5) described in differentiation-inducing liquid in, obtained concentration is 1mM Triphosaden solution, removes with the millipore filter of 0.22 μm.Differentiation-inducing liquid containing 1.0mM Triphosaden is diluted 20,10,6.7 and 5 times by differentiation-inducing liquid respectively that add respectively again described in (5), makes its concentration be respectively 50 μMs, 100 μMs, 150 μMs and 200 μMs.
(8) containing the preparation of Triphosaden maintain liquid:
Take 24.2mg Triphosaden and be dissolved in 40ml(6) described in maintain liquid in, make its concentration be 1mM, 0.22 μM of membrane filtration is degerming.Maintain liquid containing 1.0mM Triphosaden is diluted 20,10,6.7 and 5 times by maintain liquid respectively that add respectively again described in (6), makes its concentration be respectively 50 μMs, 100 μMs, 150 μMs and 200 μMs.
(9) oil red O dye liquor:
Getting 0.7g oil red O adds in 200mL Virahol, room temperature hold over night, filters by double-layer filter paper, and collecting filtrate becomes stoste, room temperature for storage; Get 6 parts of stostes before use and add 4 parts of ddH
20 mixing, now with the current.
(10) Baker ' s formalin calcium liquid:
40% formaldehyde 10ml, 1.1g calcium chloride is added 90mlddH
2in O, regulate PH to 7.0, obtained 4%Baker ' s formalin calcium liquid.
(11) 3T3-L1 Preadipocyte:
Purchased from Chinese Academy of Sciences's Shanghai cell bank.
The recovery of embodiment 2:3T3-L1 PECTORAL LIMB SKELETON and cell cultures:
(1) freeze-stored cell is taken out from liquid nitrogen, be positioned over rapidly in the water-bath of 37 DEG C, after frozen storing liquid rapid solution, cell is poured in 15ml sterile centrifugation tube, with the centrifugal 3min of 1000rpm/min in refrigerated centrifuge.
(2) supernatant liquor is abandoned, resuspended again with the centrifugal 3min of 1000rpm/min after adding PBS.
(3) abandon supernatant liquor and add fresh complete culture solution, make single cell suspension, with 1 × 10
5individual/ml is seeded in culturing bottle, and liquid is changed once in 2 ~ 3 days in interval.
(4) treat bottom cell proliferation to confluent culture bottle, in bottle, add the pancreatin of about 1ml 0.25%, and observe under being placed in inverted microscope, when cell retraction becomes circle, add equivalent complete culture solution, stop digestion, at the bottom of repeatedly blowing and beating bottle with suction pipe, collecting cell suspension is to sterile centrifugation tube, with the centrifugal 3min of 1000rpm/min, abandon supernatant liquor, make cell Eddy diffusion with 2-3ml complete culture solution, then by 1 × 10
5individual/ml density reaches new culturing bottle and continues to cultivate.
Embodiment 3:3T3-L1 PECTORAL LIMB SKELETON differentiation-inducing:
(1) by 3T3-l1 PECTORAL LIMB SKELETON according to 2 × 10
4the density in individual/hole is seeded to 48 orifice plates, and every hole adds 300 μ l complete culture solutions.
(2) treat that cell attachment grows to fusion, inhale and abandon nutrient solution, be changed to the differentiation-inducing nutrient solution containing different concns Triphosaden (0,50 μMs, 100 μMs, 150 μMs and 200 μMs).
Be changed to the maintain liquid containing above-mentioned concentration Triphosaden after (3) 2 days, within every 2 days afterwards, change a not good liquor, till the 8th day.
Embodiment 4: Nile red fluorescent dye detects content of triglyceride in adipocyte:
(1) the differentiation-inducing culture plate to 8d is taken out, discard nutrient solution, wash 2 times with PBS.
(2) cover 48 holes with 200 μ lPBS, every hole adds 12 μ l Nile red dye.
(3) hatch 10 minutes at 37 DEG C of incubators, avoid illumination.
(4) fluorescent value intensity (excitation wavelength 485nm, emission wavelength 572nm) is detected by all wave band microplate reader, record fluorescent value intensity.
Embodiment 5: oil red O stain:
(1) the differentiation-inducing culture plate to 8 days is taken out, discard nutrient solution, wash 2 times with PBS, at 4 DEG C, with 10%Baker ' s formalin calcium liquid fixed cell 30min.
(2) formalin solution in culture dish is discarded, and with liquid-transfering gun remaining liquid blotted and clean 3 times with PBS again.
(3) PBS is abandoned in suction, and every hole adds fresh oil red O dye liquor 200 μ l and covers cell surface, and dye under room temperature 10min, sucking-off dye liquor, and rinse 2 times with PBS.
(4) be placed on observations under inverted microscope, take pictures.
Embodiment 6: fat drips diameter, quantity and area statistics:
Use Image-ProPlus6.0 image processing software statistics fat after oil red O stain to drip fat to drip diameter, quantity and fat and drip area.
Claims (6)
1. a formula for the differentiation-inducing reagent of 3T3-L1 PECTORAL LIMB SKELETON, is characterized in that: be made up of Triphosaden, Regular Insulin, IBMX and dexamethasone.
2. according to claim 1, it is characterized in that: each component proportions is followed successively by Triphosaden 50-200 μM, Regular Insulin is 0.17-1.5mM, IBMX0.25-1mM, dexamethasone 0.1-2 μM.
3. according to claim 1, it is characterized in that: each composition optimal concentration is followed successively by Triphosaden 100 μMs, Regular Insulin is 850nM, IBMX0.25mM, dexamethasone 1 μM.
4. according to claim 1, it is characterized in that: each composition optimal concentration is followed successively by Triphosaden 150 μMs, Regular Insulin is 850nM, IBMX0.25mM, dexamethasone 1 μM.
5. according to claim 1,2,3,4, claim this be combined in 3T3-L1 PECTORAL LIMB SKELETON cultivate and differentiation-inducing in application.
6., according to claim 1,2,3,4, this In vitro cell model being combined in fat disease of claim is about this Combination application.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109097323A (en) * | 2018-08-27 | 2018-12-28 | 华南理工大学 | A method of fat cell is divided into the external evoked mouse embryonic fibroblasts of oleic acid |
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| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
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| 吕桂芝: "三磷酸腺苷对人横纹肌肉瘤细胞系诱导分化作用的研究", 《解剖学报》 * |
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| CN109097323A (en) * | 2018-08-27 | 2018-12-28 | 华南理工大学 | A method of fat cell is divided into the external evoked mouse embryonic fibroblasts of oleic acid |
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Application publication date: 20160113 |