CN103060267A - An induced differentiation method for 3T3-L1 preadipocytes - Google Patents
An induced differentiation method for 3T3-L1 preadipocytes Download PDFInfo
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- CN103060267A CN103060267A CN2012105764309A CN201210576430A CN103060267A CN 103060267 A CN103060267 A CN 103060267A CN 2012105764309 A CN2012105764309 A CN 2012105764309A CN 201210576430 A CN201210576430 A CN 201210576430A CN 103060267 A CN103060267 A CN 103060267A
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Abstract
The invention discloses an induced differentiation method for 3T3-L1 preadipocytes, and the method comprises: the recovery of a 3T3-L1 preadipocyte cell line, the culture of the 3T3-L1 preadipocyte cell line, the passage of the 3T3-L1 preadipocyte cell line and the induced differentiation of the 3T3 -L1 preadipocyte cell line. Eight days after the induced differentiation of the 3T3-L1 preadipocytes by using the above method of the invention, about 90% of 3T3-L1 preadipocytes in vision filed are differentiated into typical mature fat cells, wherein the cell volume increases, the cell body turns round, cytoplasmic lipid droplets are obvious, and part of the lipid droplets integrate into larger lipid droplets. By using the present invention, relatively good differentiation efficiency of and differentiation results are obtained.
Description
Technical field
The present invention relates to biomedical sector, specifically a kind of 3T3-L1 Preadipocyte method of inducing differentiation.
Background technology
Preadipocyte is that a class can be bred and to the precursor cell of specialization of Adipocyte Differentiation.It is the precursor of mature fat cell.Before Adipocyte Differentiation, similar inoblast is spindle shape on the Preadipocyte form in beginning.After beginning differentiation, Preadipocyte is lost the form of spindle shape gradually, changes the contents increased of triglyceride in the while cell, the content decrease of cytoskeletal protein etc. to spheroidal.Occur fat in the cell and drip, Preadipocyte transforms to mature fat cell gradually.Because Preadipocyte does not contain fat and drips, volume is little, and the Angiogensis characteristic is arranged, and more can tolerate ischemic and wound than ripe adipocyte in the collection of cell and migration process, especially the ischemic stage after fat transfer, individual cells is survived by cytogamy than block fat is easier.
Studies show that suppressing RAS can reduce the morbidity of diabetes and the generation of complication, but feritin is not found in diabetic subject's blood in multinomial research or Angiotensin II increases to some extent, along with many tissues comprise the discovery of expressing each composition of RAS in pancreatic tissue, fatty tissue, the heart tissue, whether the activation that people more and more pay close attention to local RAS plays an important role in the genesis of diabetes and complication thereof.
Studies confirm that the various compositions that human adipocyte can produce RAS comprise proangiotensin, feritin or feritin sample active substance, Zinc metallopeptidase Zace1, angiotensinⅡ 1 type and angiotensin II type 2 receptor.Research in recent years finds that more human adipose tissue also can be expressed feritin acceptor and angiotensin-converting enzyme 2.The proangiotensin of its release of fatty tissue is not only the important sources of local RAS, the important composition composition of the RAS that circulates especially.There is the related component of nearly all RAS in result of study prompting fatty tissue part, and it not only acts on local organization and acts on each tissue of whole body as the important composition of circulation RAS especially.Therefore material impact occurs also to have in its morbidity and complication to diabetes.
Because fatty tissue is widely distributed at body, comprise around fat around subcutaneous lipids, the internal organ, the blood vessel fat etc., therefore its sphere of action of local RAS is extensive.But renin-angiotensin system not only retroactive effect affects its hyperplasia differentiation and metabolism of fat in adipocyte.The angiotensinⅡ of adipocyte secretion can also promote various inflammatory factors such as interleukin-6, TNF-α in addition, I chemotactic molecule-1 (ICAM-1), expression and the release of vascular cell chemotactic molecule-1 (VCAM-1) etc., thereby the pathophysiological process of participation onset diabetes and complication.
It is various that yet the expression of RAS changes the adjusting factor in the fatty tissue, and mechanism is still not clear.And owing to the proangiotensin great expression in fatty tissue as the Angiotensin precursor, many researchs mainly concentrate on generation, adjusting and the release aspect of proangiotensin in the fatty tissue, and very not clear and definite to the variation of other each compositions of RAS.
In order to set up the Preadipocyte culture system, the activation of RAS plays a role in the genesis of diabetes and complication thereof and has realistic meaning in the research regional adipose tissue.
Summary of the invention
The object of the present invention is to provide a kind of Preadipocyte method of inducing differentiation, the activation that is used for research regional adipose tissue RAS plays a role in the genesis of diabetes and complication thereof.
A kind of 3T3-L1 Preadipocyte method of inducing differentiation may further comprise the steps:
(1) recovery of adipocyte strain before the 3T3-L1: take out cell strain from the liquid nitrogen kind, place immediately 37 ℃ of water baths, take out when dissolving fully to enchylema, the cell of recovery is added in the centrifugal 5min of room temperature 1000rpm, and supernatant discarded adds nutrient solution again, blow and beat resuspended, resuspended cell and nutrient solution are sucked in the culture dish, and microscopically observation of cell form and quantity place 5%CO with culture dish
237 ℃ of constant incubators in cultivate;
(2) cultivation of adipocyte strain before the 3T3-L1: microscopically is seen cell attachment, and it is capable to be shuttle, and is bright, and cell was changed 1 nutrient solution in per 2 days, and the inclination culture dish is removed nutrient solution with transfer pipet, add nutrient solution along wall;
(3) going down to posterity of the front adipocyte strain of 3T3-L1: microscopically is seen cell attachment, be fusiformis or polygon, cell density is about 80%, the inclination culture dish, nutrient solution is removed with pipettor, add warm PBS solution 6ml along wall, rock culture dish after, remove with pipettor, repeat once, add Trypsin and digest, microscopically sees that cell is shrunk to circle by irregular polygon or fusiformis, about 30 seconds of process, add nutrient solution and stop digestion, with pipettor piping and druming, make at the bottom of the cell detachment culture dish, suck in the centrifuge tube, the centrifugal 4min of 1000rpm, the supernatant that inclines adds nutrient solution, and piping and druming disperses cell; Get the nutrient solution that contains cell and add orifice plate, place 5%CO
237 ℃ of constant incubators in cultivate;
(4) the front adipocyte strain of 3T3-L1 induce differentiation: above-mentioned passage cell is cultured to cover with fully and began to induce differentiation in rear 3 days; Add the DMEM nutrient solution that contains inductor (1.0uM dexamethasone, 1.0mM IBMX, 1.7uM Regular Insulin) and calf serum (10%), cultivated 2 days; Microscopically sees that a few cell is rounded, and there have fat to ooze to be existing; Adding the DMEM nutrient solution that 1.7uM Regular Insulin (1.7uM), calf serum (10%) are only arranged continues to cultivate; After continuing to induce 6 days, microscopically sees that cell is substantially rounded, and cell contains a plurality of larger fat and drips, and namely is divided into ripe adipocyte.
Use aforesaid method of the present invention to advance to induce differentiation to inducing rear 8 days to the 3T3-L1 Preadipocyte, about 90% cytodifferentiation is typical mature fat cell in the visual field, and cell volume increases, and cell space becomes circle, and the endochylema lactones drips obviously, and meromixis becomes larger fat to drip.As seen the present invention has preferably differentiation effect and differentiation efficiency.
Description of drawings
Fig. 1 is ripe 3T3-L1 adipocyte oil red O stain result (20 *).
Embodiment
The all ingredients that present embodiment is used:
(1) each PCR primer: the handsome Bioisystech Co., Ltd in Shanghai is synthetic, is made into 10mmol/L concentration with DEPC water ,-20 ℃ of preservations.
(2) oil red O dye liquor: 0.25g oil red O adds the 100ml Virahol, and 56 ℃ dissolved 1 hour, and cooled and filtered is as storage liquid.During use, get above-mentioned storage liquid and ddH2O with 6:4 ratio mixing, leave standstill 10min and be use liquid.
(3) 10mM dexamethasone storage liquid (10000 *): behind anhydrous alcohol solution ,-20 ℃ of storages.
(4) 0.5IBMX storage liquid (1000 *): after the DMSO dissolving ,-20 ℃ of storages.
(5) phosphoric acid buffer (PBS): with 10 * PBS storage liquid, with the 1:9 proportional arrangement, the pH value is transferred to 7.2, and is for subsequent use behind the high-temperature sterilization with ultrapure water.
(6) formaldehyde stationary liquid: 40% formaldehyde 10ml, 1.1g calcium chloride are added among the 90mlddH2O, regulate PH to 7.0.
(7) 3T3-L1 Preadipocyte is available from U.S. ATCC(American Type Culture Collection).
Induce differentiation specifically to comprise:
(1) recovery of adipocyte strain before the 3T3-L1:
(1) takes out cell strain from the liquid nitrogen kind, place immediately 37 ℃ of water baths, take out when dissolving fully to enchylema;
(2) the centrifugal 5min of room temperature 1000rpm is equipped with in the 15ml centrifuge tube of 10ml nutrient solution in the cell adding of recovery;
(3) supernatant discarded adds the 6ml nutrient solution again, blows and beats resuspended;
(4) resuspended cell and nutrient solution are sucked in the 35mm culture dish;
(5) microscopically observation of cell form and quantity;
(6) culture dish is placed 37 ℃ of constant incubators of 5%CO2 cultivate;
(2) cultivation of adipocyte strain before the 3T3-L1:
(1) microscopically is seen cell attachment, and it is capable to be shuttle, and is bright, and cell was changed 1 nutrient solution in per 2 days;
(2) the inclination culture dish is removed nutrient solution with transfer pipet, adds nutrient solution along wall;
(3) going down to posterity of the front adipocyte strain of 3T3-L1:
(1) microscopically is seen cell attachment, is fusiformis or polygon, cell density about 80%;
(2) the inclination culture dish is removed nutrient solution with pipettor;
(3) add warm PBS solution 6ml along wall, rock culture dish after, remove with pipettor, repeat once;
(4) add 1mlTrypsin and digest, microscopically sees that cell is shrunk to circle by irregular polygon or fusiformis, about 30 seconds of process;
(5) add the 2m nutrient solution and stop digestion;
(6) with pipettor piping and druming, make at the bottom of the cell detachment culture dish, suck in the centrifuge tube the centrifugal 4min of 1000rpm;
(7) supernatant that inclines adds the 40ml nutrient solution, and piping and druming disperses cell;
(8) get the nutrient solution that contains cell and add 12 orifice plates, every hole adds 1ml, places 37 ℃ of constant incubators of 5%CO2 to cultivate;
(4) before the 3T3-L1 adipocyte strain induce differentiation:
(1) above-mentioned passage cell is cultured to cover with fully and began to induce differentiation in rear 3 days;
(2) add the DMEM nutrient solution that contains inductor (1.0uM dexamethasone, 1.0mM IBMX, 1.7uM Regular Insulin) and calf serum (10%), cultivated 2 days;
(3) microscopically sees that a few cell is rounded, and there have fat to ooze to be existing;
(4) adding the DMEM nutrient solution that 1.7uM Regular Insulin (1.7uM), calf serum (10%) are only arranged continues to cultivate;
(5) continue to induce 6 days after, microscopically sees that cell is substantially rounded, cell contains a plurality of larger fat and drips, and namely is divided into ripe adipocyte.
Oil red dyeing:
(1) removes nutrient solution, slowly add PBS1ml along wall, remove after the flushing, repeat 2 times.
(2) with 10% formaldehyde stationary liquid fixed cell 10min.
(3) the PBS1ml flushing is 3 times.Remove PBS.
(4) fixed cell is dried 20min.
(5) add oil red O dye liquor, make dyestuff all cover cell surface, place 10min.
(6) suck oil red O dye liquor, with 60% washed with isopropyl alcohol cell 2 times.
(7) add ddH2O and clean, repeat 3-4 time.
(8) observations under the inverted microscope is taken pictures.
The differentiation and identification of 3T3-L1 adipocyte:
Induce the metamorphosis that breaks up in the whole process with the inverted phase contrast microscope observation of cell in the test.Began to induce differentiation rear the 2nd day, the 3T3-L1 cell gradually becomes circular by initial shuttle is capable, occurs tiny fat in the part endochylema and drips, and after this contains the cytosis that fat drips, fat drips and increases, increases, to inducing rear 8 days, about 90% cytodifferentiation is typical mature fat cell in the visual field, and cell volume increases, cell space becomes circle, the endochylema lactones drips obviously, and meromixis becomes larger fat to drip, and the oil red coloration result is seen Fig. 1.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the disclosed content of this embodiment.So everyly do not break away from the equivalence of finishing under the spirit disclosed in this invention or revise, all fall into the scope of protection of the invention.
Claims (1)
1. a 3T3-L1 Preadipocyte method of inducing differentiation is characterized in that, may further comprise the steps:
(1) recovery of adipocyte strain before the 3T3-L1: take out cell strain from the liquid nitrogen kind, place immediately 37 ℃ of water baths, take out when dissolving fully to enchylema, the cell of recovery is added in the centrifugal 5min of room temperature 1000rpm, and supernatant discarded adds nutrient solution again, blow and beat resuspended, resuspended cell and nutrient solution are sucked in the culture dish, and microscopically observation of cell form and quantity place 5%CO with culture dish
237 ℃ of constant incubators in cultivate;
(2) cultivation of adipocyte strain before the 3T3-L1: microscopically is seen cell attachment, and it is capable to be shuttle, and is bright, and cell was changed 1 nutrient solution in per 2 days, and the inclination culture dish is removed nutrient solution with transfer pipet, add nutrient solution along wall;
(3) going down to posterity of the front adipocyte strain of 3T3-L1: microscopically is seen cell attachment, be fusiformis or polygon, cell density is about 80%, the inclination culture dish, nutrient solution is removed with pipettor, add warm PBS solution 6ml along wall, rock culture dish after, remove with pipettor, repeat once, add Trypsin and digest, microscopically sees that cell is shrunk to circle by irregular polygon or fusiformis, about 30 seconds of process, add nutrient solution and stop digestion, with pipettor piping and druming, make at the bottom of the cell detachment culture dish, suck in the centrifuge tube, the centrifugal 4min of 1000rpm, the supernatant that inclines adds nutrient solution, and piping and druming disperses cell; Get the nutrient solution that contains cell and add orifice plate, place 5%CO
237 ℃ of constant incubators in cultivate;
(4) the front adipocyte strain of 3T3-L1 induce differentiation: above-mentioned passage cell is cultured to cover with fully and began to induce differentiation in rear 3 days; Add the DMEM nutrient solution that contains inductor (1.0uM dexamethasone, 1.0mM IBMX, 1.7uM Regular Insulin) and calf serum (10%), cultivated 2 days; Microscopically sees that a few cell is rounded, and there have fat to ooze to be existing; Adding the DMEM nutrient solution that 1.7uM Regular Insulin (1.7uM), calf serum (10%) are only arranged continues to cultivate; After continuing to induce 6 days, microscopically sees that cell is substantially rounded, and cell contains a plurality of larger fat and drips, and namely is divided into ripe adipocyte.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361307A (en) * | 2013-06-24 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | Induced differentiation method of C3H10T1/2 mesodermal multipotential embryonic stem cell stain |
| CN105087476A (en) * | 2015-08-31 | 2015-11-25 | 中国医学科学院北京协和医院 | Induced differentiation method of 3T3-L1 preadipocytes line |
| CN105238745A (en) * | 2015-09-08 | 2016-01-13 | 四川农业大学 | 3T3-L1 preadipocyte differentiation inducing reagent composition and application thereof |
| CN115044541A (en) * | 2022-05-24 | 2022-09-13 | 南京农业大学 | High-differentiation-efficiency immortalized pig fat precursor cell line and construction method and application thereof |
| WO2023225995A1 (en) * | 2022-05-27 | 2023-11-30 | 汕头得宝投资有限公司 | Use of sodium alginate-gelatin 3d scaffold in supporting differentiation of preadipocytes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101214338A (en) * | 2007-01-05 | 2008-07-09 | 陆华 | New use of Chinese medicine compound pharmaceutical composition |
| CN101311175A (en) * | 2007-05-23 | 2008-11-26 | 华北制药集团新药研究开发有限责任公司 | New xanthones compounds, preparation method and use thereof |
| WO2012004310A1 (en) * | 2010-07-06 | 2012-01-12 | Basf Beauty Care Solutions France S.A.S. | Adipose tissue model and preparation process |
-
2012
- 2012-12-26 CN CN2012105764309A patent/CN103060267A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101214338A (en) * | 2007-01-05 | 2008-07-09 | 陆华 | New use of Chinese medicine compound pharmaceutical composition |
| CN101311175A (en) * | 2007-05-23 | 2008-11-26 | 华北制药集团新药研究开发有限责任公司 | New xanthones compounds, preparation method and use thereof |
| WO2012004310A1 (en) * | 2010-07-06 | 2012-01-12 | Basf Beauty Care Solutions France S.A.S. | Adipose tissue model and preparation process |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361307A (en) * | 2013-06-24 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | Induced differentiation method of C3H10T1/2 mesodermal multipotential embryonic stem cell stain |
| CN103361307B (en) * | 2013-06-24 | 2015-07-29 | 上海交通大学医学院附属瑞金医院 | The method of inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain |
| CN105087476A (en) * | 2015-08-31 | 2015-11-25 | 中国医学科学院北京协和医院 | Induced differentiation method of 3T3-L1 preadipocytes line |
| CN105087476B (en) * | 2015-08-31 | 2018-12-07 | 中国医学科学院北京协和医院 | The method of inducing differentiation of 3T3-L1 pre-adipose cell lines |
| CN105238745A (en) * | 2015-09-08 | 2016-01-13 | 四川农业大学 | 3T3-L1 preadipocyte differentiation inducing reagent composition and application thereof |
| CN115044541A (en) * | 2022-05-24 | 2022-09-13 | 南京农业大学 | High-differentiation-efficiency immortalized pig fat precursor cell line and construction method and application thereof |
| CN115044541B (en) * | 2022-05-24 | 2023-08-15 | 南京农业大学 | Immortalized pig fat precursor cell line with high differentiation efficiency, and construction method and application thereof |
| WO2023225995A1 (en) * | 2022-05-27 | 2023-11-30 | 汕头得宝投资有限公司 | Use of sodium alginate-gelatin 3d scaffold in supporting differentiation of preadipocytes |
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Application publication date: 20130424 |