WO2015010417A1 - 利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞 - Google Patents

利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞 Download PDF

Info

Publication number
WO2015010417A1
WO2015010417A1 PCT/CN2013/088992 CN2013088992W WO2015010417A1 WO 2015010417 A1 WO2015010417 A1 WO 2015010417A1 CN 2013088992 W CN2013088992 W CN 2013088992W WO 2015010417 A1 WO2015010417 A1 WO 2015010417A1
Authority
WO
WIPO (PCT)
Prior art keywords
final concentration
cells
adipocytes
fibroblasts
culture
Prior art date
Application number
PCT/CN2013/088992
Other languages
English (en)
French (fr)
Inventor
吴东海
聂涛
徐爱民
李鹏
Original Assignee
中国科学院广州生物医药与健康研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院广州生物医药与健康研究院 filed Critical 中国科学院广州生物医药与健康研究院
Publication of WO2015010417A1 publication Critical patent/WO2015010417A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present invention relates to a cell differentiation-inducing cytokine composition and cell transdifferentiation culture method, particularly to promote fibroblast transdifferentiation into an adipocyte-determined cytokine composition and use the composition to promote fibroblast transdifferentiation into fat Cell culture method.
  • the reason for obesity is that when the energy in the body is out of balance and the energy consumed is greater than the energy consumed, the excess energy is mainly stored in the form of fat, and the weight of the fat increases to cause obesity.
  • Fat is mainly stored in the body's adipose tissue.
  • the increase in adipose tissue is mainly manifested by the increase in the volume of fat cells and the increase in the number of fat cells.
  • the volume of fat cells is limited, and it is impossible to grow infinitely. Therefore, excessive energy storage mainly relies on more newborn fat cells. Therefore, the differentiation of fat cells has become a major concern for the inhibition of obesity.
  • the mouse 3T3-L1 cell line is the most widely used fat cell differentiation research system, a recognized fat precursor cell line. Under the stimulation of the lipid-forming factors IBMX, DEX and Insulin, most cells can be induced to differentiate into adipocytes. According to years of research, peroxisome proliferator-activated receptor gamma (PPAR ⁇ ) is the most critical adipocyte differentiation transcription factor, and PPAR ⁇ knockout mice are embryonic lethal, caused by defects in adipose tissue. In addition, the addition of PPAR ⁇ ligands to adipose precursor cells also promotes their differentiation into adipocytes. The CCAAT/enhancer binding protein family (C/EBPs) also plays an important role in adipocyte differentiation.
  • C/EBPs CCAAT/enhancer binding protein family
  • C/EBP ⁇ and C/EBP ⁇ can promote the expression of PPAR ⁇ in the early stage of differentiation, while C/EBP ⁇ can maintain the expression of PPAR ⁇ .
  • C/EBP ⁇ and PPAR ⁇ together directly activate many related genes after adipocyte differentiation.
  • many other transcriptional activators that promote adipocyte differentiation have been discovered, such as CREB, SREBP, and the like.
  • fat cells are differentiated from adipose precursor cells, which are also thought to be mainly present in the tissue stem cell microenvironment of adipose tissue. When new fat cells are required, these fat precursor cells will differentiate. For fat cells, like muscle precursor cells, liver precursor cells and the like are present in muscles and liver. However, the marker genes of adipose precursor cells have not been determined so far, so it has not been possible to completely isolate true fat precursor cells from the body for research. In addition, in normal animals and human bodies, adipose tissue is mainly present in the subcutaneous cavity and subcutaneously in the abdomen.
  • adipocytes may not only have fat precursor cells, but under certain conditions, some non-fat precursor cell fibroblasts can also differentiate into adipocytes.
  • adipocytes By transducing non-fatty precursor cells, such as fibroblasts, into adipocytes, the mechanism of transdifferentiation of adipocytes can be more clearly understood, which in turn guides the development of drugs that inhibit obesity.
  • An object of the present invention is to provide a cytokine composition which can transduce fibroblasts into adipocytes, and a transdifferentiation culture solution and a culture method based on the cytokines.
  • a composition for promoting cell transdifferentiation the onset factors being A, B, C, D and E, the final concentrations in the culture solution are:
  • B hepatocyte growth factor, the final concentration is 10-30 ng/ml
  • E PPAR gamma agonist, final concentration 0.5 to 2 [mu]M.
  • the final concentrations of A, B, C, D, and E in the culture solution are respectively:
  • B hepatocyte growth factor, the final concentration is 20-30 ng/ml
  • E PPAR gamma agonist, final concentration of 1-2 ⁇ M.
  • the PPAR gamma agonist is selected from the group consisting of thiazolidinediones, and in particular, the thiazolidinedione is selected from the group consisting of ciglitazone, englitazone and troglitazone, pioglitazone, rosiglitazone, faglitazone, Daglitazone.
  • a method for culturing fibroblasts into adipocytes comprises the following steps:
  • the present invention explores a combination of cytokines for inducing transdifferentiation of fibroblasts into non-fatty precursor cells into adipocytes, and provides a new platform for studying adipocyte differentiation, indicating that the source of fat cells in the body may not only have fat precursors. cell.
  • the method of the invention has a simple induction process and can effectively induce fibroblasts into adipocytes.
  • Figure 1 is a diagram of fat cells after transdifferentiation
  • Figure 2 is a test result of transdifferentiation of mouse rat tail fibroblasts into adipocytes
  • Figure 3 is a graph showing the results of cell induction of the lowest and highest cytokine concentrations
  • Figure 4 is a graph showing the results of induction of NIH-3T3 cells.
  • a composition for promoting cell transdifferentiation the onset factors being A, B, C, D and E, the final concentrations in the culture solution are:
  • hepatocyte growth factor which is used to activate the STAT5 signaling pathway of cells, with a final concentration of 10-30 ng/ml;
  • C dexamethasone, used to activate the expression of C/EBP ⁇ , the final concentration is 100-200 nM;
  • E A PPAR gamma agonist that potentiates the activity of PPAR gamma with a final concentration of 0.5 to 2 ⁇ M.
  • the NIH-3T3 cell line selected for use in the present invention was purchased from ATCC, USA.
  • the above growth medium is a conventional cell culture medium, and those skilled in the art can make certain adjustments as needed.
  • Oil red working solution The oil red storage solution and water were mixed at a ratio of 6:4, and then allowed to stand at room temperature for 20 minutes and then filtered through a 0.22 ⁇ m filter.
  • RNA extraction Extracted by Trizol method, the specific operation is as follows:
  • RNA concentration OD260 ⁇ dilution factor ⁇ 0.04 Gg/ ⁇ L, OD260nm/OD 280nm
  • RNA concentration OD260 ⁇ dilution factor ⁇ 0.04 Gg/ ⁇ L
  • OD260nm/OD 280nm The purity of RNA extracted at 1.8 to 2.0 is considered to be high.
  • RNA reverse transcription synthesis of cDNA using SuperScriptTM IIRT reverse transcription kit
  • Fluorescence real-time quantitative PCR using Takara SYBR? RT-PCR Kit (Perfect Real Time) Quantitative PCR kit, according to the instructions for quantitative PCR reactions.
  • Pre-denaturation 95 ° C for 20 seconds
  • the reaction was repeated for 38 to 45 cycles, and the dissolution curve was performed at 65 to 95 ° C.
  • the plate was read every 0.5 ° C for 1 second.
  • the resuscitated NIH-3T3 cells were subcultured twice with cell growth medium, and then induced to differentiate. Take a well of 24-well plate culture as an example. After cell growth and aggregation, the medium was changed to cell induction medium (0.5 ml). In the induction medium, the final concentration of cytokines is as follows:
  • A epidermal growth factor, final concentration 20 ng / ml;
  • the induction medium was changed every two days and cultured for two weeks.
  • the transdifferentiation culture conditions of NIH-3T3 cells were different from those of the ABCDE group except that they did not use a cell-inducing medium and only cell growth medium was used.
  • the transdifferentiation culture conditions of NIH-3T3 cells were different from those of the ABCDE group, in that the cytokines added in the induced culture were as follows:
  • the culture medium was such that the induction medium for the first two days after the cell was full was a growth medium to which MDIR was added, and the subsequent induction medium was changed to a growth medium to which IR was added, and the solution was changed every two days.
  • the specific concentrations are as follows:
  • IBMX isobutylmethylxanthine, M: final concentration of 0.5 mM
  • Insulin (I) final concentration of 5 ⁇ g / ml
  • Rosiglitazone (R) The final concentration is 1 ⁇ M.
  • Isolation and culture of rat tail cells 3 cm of mouse tail cells were taken from the outside, soaked in 75% alcohol for 30 s, then transferred to medium supplemented with 5 times double antibody, and the rat tail was transferred to the culture plate in the cell console. Then, use a surgical scissors to cut the tail of the rat; add 37 ° C preheated medium, after 5 days, you can see the cells adherent, after 12 days, the cells are digested, plated into a 12-well culture plate, waiting for cell aggregation .
  • the induction differentiation step was identical to the NIH-3T3 cells of Example 1, cultured for 5 weeks and observed.
  • MDIR-induced group The induction differentiation step was the same as the MDIR induction test of Example 1.
  • Photo of transdifferentiated cells is shown in Figure 2.
  • the conventional adipogenic factor MDIR does not promote the differentiation of fibroblastic tail cells of primary non-fat precursor cells into adipocytes, and the cytokine combination of the present invention can promote differentiation into adipocytes.
  • the resuscitated NIH-3T3 cells were subcultured twice with cell growth medium, and then induced to differentiate. Take a well of 24-well plate culture as an example. After cell growth and aggregation, the medium was changed to cell induction medium (0.5 ml). In the induction medium, the final concentration of cytokines is as follows:
  • A epidermal growth factor, final concentration 10 ng/ml
  • the induction medium was changed every two days and cultured for four weeks.
  • the resuscitated NIH-3T3 cells were subcultured twice with cell growth medium, and then induced to differentiate. Take a well of 24-well plate culture as an example. After cell growth and aggregation, the medium was changed to cell induction medium (0.5 ml). In the induction medium, the final concentration of cytokines is as follows:
  • A epidermal growth factor, final concentration 30 ng/ml
  • the induction medium was changed every two days and cultured for two weeks.
  • the resuscitated NIH-3T3 cells were subcultured twice with cell growth medium, and then induced to differentiate. Take a well of 24-well plate culture as an example. After cell growth and aggregation, the medium was changed to cell induction medium (0.5 ml). In the induction medium, the final concentration of cytokines is as follows:
  • A epidermal growth factor, final concentration 20 ng/ml
  • the induction medium was changed every two days and cultured for 18 days.
  • FIG. 4 A photograph of oil red staining after induction culture in Example 5 is shown in Fig. 4.
  • pPAR ⁇ agonist pioglitazone can also induce differentiation of NIH-3T3 into adipocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Rheumatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供了能促进成纤维细胞转分化为脂肪细胞的组合物，其起效因子为肝细胞生长因子，表皮细胞生长因子，地塞米松和胰岛素。本发明也提供诱导了成纤维细胞转分化为脂肪细胞的培养液和方法。

Description

利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞
技术领域
本发明涉及一种细胞转分化诱导细胞因子组合物及细胞转分化的培养方法，特别促进成纤维细胞转分化为脂肪细胞确定的细胞因子组合物以及使用该组合物促进成纤维细胞转分化为脂肪细胞培养方法。
背景技术
肥胖发生的原因是当体内能量失衡，摄入的能量大于消耗的能量时，过多的能量主要以脂肪的形式储存起来，脂肪的重量增加从而导致肥胖的发生。脂肪主要储存在机体的脂肪组织内，脂肪组织的增多主要表现为脂肪细胞的体积变大和脂肪细胞数目的增加。但是脂肪细胞的体积是有一定限度的，不可能无限的变大，所以过多的能量储存主要是依靠更多的新生的脂肪细胞。因此，脂肪细胞的分化便成为抑制肥胖的主要关注热点。
小鼠的3T3-L1细胞系是目前运用最广泛的脂肪细胞分化研究体系，公认的脂肪前体细胞系。在成脂因子IBMX，DEX和Insulin刺激下，绝大多数细胞都能够被诱导分化为脂肪细胞。根据多年的研究，过氧化物酶体增生物激活受体γ (PPARγ)是最关键的脂肪细胞分化转录因子，PPARγ的敲除小鼠是胚胎致死的，由脂肪组织的缺陷造成。此外，脂肪前体细胞只需添加PPARγ的配体也可促进其分化为脂肪细胞。CCAAT/增强子结合蛋白家族(C/EBPs)也在脂肪细胞分化中起着重要作用。C/EBPβ和C/EBPδ在分化早期能够促进PPARγ的表达，而C/EBPα能够维持PPARγ的表达。C/EBPα和PPARγ一起可以直接激活许多脂肪细胞分化后的相关基因。近些年，除了C/EBPα和PPARγ外，还有很多其他的促进脂肪细胞分化的转录激活因子被发现，像CREB，SREBP等等。
通常认为脂肪细胞是由脂肪前体细胞分化而来，脂肪前体细胞也被认为主要存在于脂肪组织的组织干细胞微环境中，当需要新的脂肪细胞生成时，这些脂肪前体细胞便会分化为脂肪细胞，如同肌肉前体细胞，肝脏前体细胞等存在于肌肉和肝脏中。但是脂肪前体细胞的标志基因到目前为止尚未有定论，因此也一直无法完美的从体内分离出真正的脂肪前体细胞进行研究。此外，在正常动物和人类的机体内，脂肪组织主要存在于腹腔内和腹部的皮下内。但是肥胖发生后，机体各个部位都会出现脂肪组织，肾脏周围，肠系膜周围，各个部位的皮下内等等，同时腹腔内的脂肪组织也会急剧增重。这些证据说明可能分化为脂肪细胞的细胞可能不仅有脂肪前体细胞，在一些特定条件下，一些非脂肪前体细胞的成纤维细胞也能够分化为脂肪细胞。
通过将非脂肪前体细胞，如成纤维细胞转分化为脂肪细胞，可以更为清楚地了解脂肪细胞的转分化机制，进而指导抑制肥胖药物的开发。
发明内容
本发明的目的在于提供一种可以将成纤维细胞转分化为脂肪细胞的细胞因子组合物，及基于该细胞因子的转分化培养液和培养方法。
本发明所采取的技术方案是：
一种促进细胞转分化的组合物，其起效因子为A、B、C、D和E，其在培养液中的终浓度分别为：
A：表皮生长因子，终浓度10～30ng/ml；
B：肝细胞生长因子，终浓度10～30ng/ml；
C：地塞米松，终浓度100～200nM；
D：胰岛素，1～10μg/ml；
E：PPARγ激动剂，终浓度为0.5～2μM。
作为本发明的进一步改进，A、B、C、D、E在培养液中的终浓度分别为：
A：表皮生长因子，终浓度20～30ng/ml；
B：肝细胞生长因子，终浓度20～30ng/ml；
C：地塞米松，终浓度150～200nM；
D：胰岛素，5～10μg/ml；
E：PPARγ激动剂，终浓度为1～2μM。
PPARγ激动剂选自噻唑烷二酮类药物，特别的，噻唑烷二酮类药物选自环格列酮、恩格列酮和曲格列酮、吡格列酮、罗格列酮、法格列酮、达格列酮。
一种成纤维细胞转分化诱导培养液，培养液中添加有上述的组合物。
一种将成纤维细胞转分化为脂肪细胞的培养方法，包括如下步骤：
1) 分离成纤维细胞并培养在培养液中；
2) 在培养液中加入上述的组合物，
3) 继续培养，得到脂肪细胞。
本发明的有益效果是：
本发明探索出了用于诱导非脂肪前体细胞的成纤维细胞转分化为脂肪细胞的细胞因子组合，为了研究脂肪细胞分化提供了新的平台，表明体内的脂肪细胞来源可能不仅有脂肪前体细胞。
本发明方法诱导过程简单，可以有效地将成纤维细胞诱导为脂肪细胞。
附图说明
图1是转分化后的脂肪细胞图；
图2是小鼠鼠尾成纤维细胞转分化为脂肪细胞的试验结果；
图3是最低和最高细胞因子浓度的细胞诱导结果图；
图4是NIH-3T3细胞的诱导结果图。
具体实施方式
一种促进细胞转分化的组合物，其起效因子为A、B、C、D和E，其在培养液中的终浓度分别为：
A：表皮生长因子，用于激活细胞的STAT5信号通路，终浓度10～30ng/ml；
B：肝细胞生长因子，用于激活细胞的STAT5信号通路，终浓度10～30ng/ml；
C：地塞米松，用于激活C/EBPδ的表达，终浓度100～200nM；
D：胰岛素，加速脂滴的形成，1～10μg/ml；
E：PPARγ激动剂，增强PPARγ的活性，终浓度为0.5～2μM。
下面结实施例及实验数据，进一步说明本发明。
本发明选用的NIH-3T3细胞系购自美国ATCC公司。
以下实施例中所用培养基配方如下：
1）细胞生长培养基
高糖DMEM培养基（Hyclone）添加胎牛血清（终浓度为10 % v/v）和双抗（青霉素和链霉素（Gibco）），双抗浓度为(1% v/v)。
2）诱导培养基
添加了细胞转分化促进因子的细胞生长培养基；
3）油红储存液
0.7g油红染料，200mL异丙醇，震荡过夜，使用0.2μm过滤器过滤，4℃保存备用。
上述的生长培养基为常规的细胞培养基，本领域技术人员可以根据需要对其进行一定的调整。
脂肪细胞的形态鉴定-油红染色
油红工作液：油红储存液和水以6:4的比例混合，然后再室温下静置20分钟后用0.22μm滤膜过滤。
1) 将分化后的细胞从培养箱中取出，移除全部的细胞培养液；
2) 加入500μl的10%的福尔马林，在室温下放置5分钟；
3) 移除10%的福尔马林，加入500μl新鲜的福尔马林，在室温下至少孵育1个小时；
4) 移除福尔马林，然后加入500μl的60%异丙醇，移除60%异丙醇，让培养板晾干；
5) 加入500μl油红工作液，孵育10分钟；
6) 移除油红工作液，立即用水洗涤，用水洗涤4次
7) 移除水，完全晾干，加入100%异丙醇萃取油红，孵育10分钟或者更长；
8) 将200μl萃取后的异丙醇转移到ELISA板中，500nm读数，用于定量分析甘油三酯（TG）含量。
脂肪细胞的基因表达鉴定
RNA提取：利用Trizol法提取，具体操作如下：
1) 将分化后的细胞取出，移除培养基，用PBS洗涤一次，然后加入1ml Trizol，裂解30分钟，然后转移到EP管中；
2) 加入200 μl氯仿/ml Trizol,用力振荡混匀15秒，室温放置3分钟，4 ℃，12,000 g离心15分钟；
3) 溶液分成三层，品分为三层：无色的上清水相、中间的白色层及粉红色的下层有机相，小心吸取上清至新的离心管；
4) 加入500 μl异丙醇，轻轻混匀，室温放置10分钟，4 ℃，12,000 g离心10分钟；
5) 小心去除上清，加入1ml 75% 乙醇(DEPC水配制)，轻轻混匀，悬浮沉淀，4 ℃，7500 g离心5分钟；
6) 去除上清，自然烘干5分钟左右，加入40 μl DEPC水溶解RNA沉淀；
7) 取5 μl进行电泳检测提取RNA的质量，28S条带亮度是18S条带两倍的RNA是质量较好的RNA，凝胶与电泳缓冲液需新鲜配制；
8) 取2 μl测定RNA浓度，RNA的浓度＝OD260×稀释倍数×0.04 μg/μL，OD260nm/OD 280nm 在1.8～2.0视为抽提的RNA的纯度很高。
RNA逆转录：利用SuperScriptTM ⅡRT 逆转录试剂盒合成cDNA；
荧光实时定量PCR：采用Takara公司SYBR? RT-PCR Kit (Perfect Real Time) 定量PCR试剂盒，根据说明进行定量PCR反应。
反应条件：
预变性：95 ℃ 20秒；
变性：95 ℃，10秒；
退火：60 ℃，20秒；
孵育：70 ℃ 1秒；
根据基因的表达量重复38～45个循环，65～95 ℃做溶解曲线，每0.5 ℃读一次板，时间为1秒。
实施例1 NIH-3T3细胞的转分化
ABCDE组：
将复苏的NIH-3T3细胞用细胞生长培养基传代培养两次，之后进行诱导分化，以24孔板培养的一孔为例，细胞生长汇聚后，培养基更换为细胞诱导培养基（0.5ml），诱导培养基中，细胞因子的终浓度如下：
A：表皮生长因子，终浓度20ng/ml；
B：肝细胞生长因子，终浓度20ng/ml；
C：地塞米松，终浓度100nM；
D：胰岛素，5μg/ml；
E：罗格列酮，终浓度为1μM；
每两天更换一次诱导培养基，培养两周。
空白对照组：
NIH-3T3细胞的转分化培养条件同ABCDE组，不同之处在于其未使用细胞诱导培养基，仅使用细胞生长培养基。
MDIR诱导组：
NIH-3T3细胞的转分化培养条件同ABCDE组，不同之处在于其诱导培养中的中添加的细胞因子如下：
培养条件为在细胞长满后的开始两天的诱导培养基是添加MDIR的生长培养基，在后面的诱导培养基更换为添加IR的生长培养基，每两天换液一次。具体浓度如下：
IBMX (isobutylmethylxanthine, M)：终浓度为0.5mM；
地塞米松Dexamethasone (D)：终浓度为1μm；
胰岛素Insulin (I)：终浓度为5μg/ml；
罗格列酮Rosiglitazone (R)：终浓度为1μM。
实验结果：
培养两周后，转分化的细胞照片如图1 A所示，从图中可以看出，传统的成脂因子MDIR不能促进非脂肪前体细胞的成纤维细胞分化为脂肪细胞（MIDR组），而本发明的新的细胞因子组合可以促进非脂肪前体细胞的成纤维细胞NIH-3T3分化为脂肪细胞（ABCDE组）。如图所示，在添加诱导因子14天约有50%的细胞分化。随着诱导时间的增长，会有更多的细胞分化为脂肪细胞直至100%；
分别检测不同培养组中细胞中PPARγ和aP2 mRNA的相对表达量，结果如图1 B所示，空白对照组和MIDR诱导组中，没有检测到有PPARγ和aP2表达，在ABCDE组中，PPARγ和aP2的表达量较大，说明该组中已经形成了较大量的脂肪细胞；
分别检测不同培养组中细胞中的TG含量，结果如图1 C所示，从图中可以清楚地看出，空白对照组和MIDR诱导组中几乎检测不到TG，仅有ABCDE组中的TG量较多，说明该组中已经形成了较大量的脂肪细胞。
实施例2 小鼠鼠尾成纤维细胞的转分化
鼠尾细胞的分离和培养：室外取小鼠的鼠尾细胞3cm，用75%酒精浸泡30s，然后转移到添加5倍双抗的培养基中，在细胞操作台中，将鼠尾转移到培养板中，然后用手术剪把鼠尾剪碎；加入37摄氏度预热的培养基，5天后，可以看到有细胞贴壁，12天后，将细胞消化，铺板到12孔培养板中，等待细胞汇聚。
ABCDE组：
鼠尾成纤维细胞的分化：诱导分化步骤和实施例1的NIH-3T3细胞完全一致，培养5周并观察。
空白对照组：培养步骤同实施例1的空白对照试验。
MDIR诱导组：诱导分化步骤同实施例1的MDIR诱导试验。
转分化的细胞照片如图2 A所示，从图中可以看出传统成脂因子MDIR不能促进原代非脂肪前体细胞的成纤维鼠尾细胞分化为脂肪细胞，而本发明的细胞因子组合可以促进其分化为脂肪细胞。在添加诱导因子35天约有30%的细胞分化为脂肪细胞。
分别检测不同培养组中细胞中PPARγ mRNA的相对表达量，结果如图2 B所示，空白对照组和MIDR诱导组中，没有检测到有PPARγ表达，在ABCDE组中，PPARγ的表达量较大，说明该组中已经形成了较大量的脂肪细胞；
分别检测不同培养组中细胞中的TG含量，结果如图2 C所示，从图中可以清楚地看出，空白对照组和MIDR诱导组中几乎检测不到TG，仅有ABCDE组中的TG量较多，说明该组中已经形成了较大量的脂肪细胞。
实施例3
将复苏的NIH-3T3细胞用细胞生长培养基传代培养两次，之后进行诱导分化，以24孔板培养的一孔为例，细胞生长汇聚后，培养基更换为细胞诱导培养基（0.5ml），诱导培养基中，细胞因子的终浓度如下：
A：表皮生长因子，终浓度10 ng/ml；
B：肝细胞生长因子，终浓度10 ng/ml；
C：地塞米松，终浓度100 nM；
D：胰岛素，1 μg/ml；
E：罗格列酮，终浓度为0.5 μM；
每两天更换一次诱导培养基，培养四周。
实施例4
将复苏的NIH-3T3细胞用细胞生长培养基传代培养两次，之后进行诱导分化，以24孔板培养的一孔为例，细胞生长汇聚后，培养基更换为细胞诱导培养基（0.5ml），诱导培养基中，细胞因子的终浓度如下：
A：表皮生长因子，终浓度30 ng/ml；
B：肝细胞生长因子，终浓度30 ng/ml；
C：地塞米松，终浓度200 nM；
D：胰岛素，10 μg/ml；
E：罗格列酮，终浓度为2 μM；
每两天更换一次诱导培养基，培养两周。
实施例3和4诱导培养后的细胞照片如图3所示，从图中可以看出，两实施例中均可以诱导出脂肪细胞。高浓度的细胞因子下，诱导的速率更快；低浓度下，也可以成功地诱导出脂肪细胞。
实施例5
将复苏的NIH-3T3细胞用细胞生长培养基传代培养两次，之后进行诱导分化，以24孔板培养的一孔为例，细胞生长汇聚后，培养基更换为细胞诱导培养基（0.5ml），诱导培养基中，细胞因子的终浓度如下：
A：表皮生长因子，终浓度20 ng/ml；
B：肝细胞生长因子，终浓度20 ng/ml；
C：地塞米松，终浓度100 nM；
D：胰岛素，5 μg/ml；
F：吡格列酮，终浓度为1 μM；
每两天更换一次诱导培养基，培养18天。
实施例5诱导培养后的油红染色的照片见图4，从图中可以看出，PPARγ的激动剂吡格列酮也可以诱导NIH-3T3分化为脂肪细胞。

Claims (6)

  1. 一种促进细胞转分化的组合物，其起效因子为A、B、C、D和E，其在培养液中的终浓度分别为：
    A：表皮生长因子，终浓度10～30ng/ml；
    B：肝细胞生长因子，终浓度10～30ng/ml；
    C：地塞米松，终浓度100～200nM；
    D：胰岛素，1～10μg/ml；
    E：PPARγ激动剂，终浓度为0.5～2μM。
  2. 根据权利要求1所述的组合物，其特征在于：A、B、C、D、E在培养液中的终浓度分别为：
    A：表皮生长因子，终浓度20～30ng/ml；
    B：肝细胞生长因子，终浓度20～30ng/ml；
    C：地塞米松，终浓度150～200nM；
    D：胰岛素，5～10μg/ml；
    E：PPARγ激动剂，终浓度为1～2μM。
  3. 根据权利要求1或2所述的组合物，其特征在于：PPARγ激动剂选自噻唑烷二酮类药物。
  4. 根据权利要求3所述的组合物，其特征在于：噻唑烷二酮类药物选自环格列酮、恩格列酮和曲格列酮、吡格列酮、罗格列酮、法格列酮、达格列酮。
  5. 一种成纤维细胞转分化诱导培养液，其特征在于：所述培养液中添加有权利要求1～4任意一项所述的组合物。
  6. 一种将成纤维细胞转分化为脂肪细胞的培养方法，包括如下步骤：
    1) 分离成纤维细胞并培养在培养液中；
    2) 在培养液中加入权利要求1～4任意一项所述的组合物，
    3) 继续培养，得到脂肪细胞。
PCT/CN2013/088992 2013-07-25 2013-12-10 利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞 WO2015010417A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310317410.4 2013-07-25
CN201310317410.4A CN104342401B (zh) 2013-07-25 2013-07-25 利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞

Publications (1)

Publication Number Publication Date
WO2015010417A1 true WO2015010417A1 (zh) 2015-01-29

Family

ID=52392661

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/088992 WO2015010417A1 (zh) 2013-07-25 2013-12-10 利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞

Country Status (2)

Country Link
CN (1) CN104342401B (zh)
WO (1) WO2015010417A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754935A (zh) * 2016-04-07 2016-07-13 浙江大学 一种诱导成纤维细胞转分化为脂肪细胞的诱导培养基及其应用
WO2019073055A1 (en) 2017-10-13 2019-04-18 Imba - Institut Für Molekulare Biotechnologie Gmbh ENHANCED REPROGRAMMING OF SOMATIC CELLS

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110093309B (zh) * 2018-01-29 2021-07-02 中国科学院动物研究所 一种诱导成纤维细胞转分化为脂肪细胞的方法
WO2019144968A1 (zh) 2018-01-29 2019-08-01 中国科学院动物研究所 一种细胞诱导的方法
CN110093310B (zh) * 2018-01-29 2021-07-02 中国科学院动物研究所 一种将成纤维细胞转化为永生化细胞的方法及其应用
CN110592003B (zh) * 2019-09-17 2021-06-18 北京大学 体外诱导人皮肤成纤维细胞分化为脂肪细胞的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168065A (zh) * 2011-02-17 2011-08-31 暨南大学 一种体外诱导人脐带间充质干细胞成为肝细胞的方法与应用
CN102250829A (zh) * 2011-06-29 2011-11-23 天津和泽干细胞科技有限公司 人脐带间充质干细胞向肝细胞定向分化的诱导方法
WO2012022725A2 (en) * 2010-08-19 2012-02-23 F. Hoffmann-La Roche Ag Conversion of somatic cells to induced reprogrammed neural stem cells (irnscs)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102382800B (zh) * 2011-12-08 2013-01-23 吉林大学 一种细胞信号通路抑制剂诱导猪脂肪细胞形成的方法
CN103160460B (zh) * 2013-04-02 2014-12-24 中国农业科学院北京畜牧兽医研究所 一种诱导人成纤维细胞重编程成脂肪细胞的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012022725A2 (en) * 2010-08-19 2012-02-23 F. Hoffmann-La Roche Ag Conversion of somatic cells to induced reprogrammed neural stem cells (irnscs)
CN102168065A (zh) * 2011-02-17 2011-08-31 暨南大学 一种体外诱导人脐带间充质干细胞成为肝细胞的方法与应用
CN102250829A (zh) * 2011-06-29 2011-11-23 天津和泽干细胞科技有限公司 人脐带间充质干细胞向肝细胞定向分化的诱导方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, FANGQION G ET AL.: "Effects of Rosiglitazone and Serum on Gene Expressions of PPARa and PPARy in the Induced Differentiation Process of Pig Preadipocyte", JOURNAL OF ANHUI AGRICULTURAL SCIENCES, vol. 39, no. 15, 31 December 2011 (2011-12-31) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754935A (zh) * 2016-04-07 2016-07-13 浙江大学 一种诱导成纤维细胞转分化为脂肪细胞的诱导培养基及其应用
CN105754935B (zh) * 2016-04-07 2019-01-04 浙江大学 一种诱导成纤维细胞转分化为脂肪细胞的诱导培养基及其应用
WO2019073055A1 (en) 2017-10-13 2019-04-18 Imba - Institut Für Molekulare Biotechnologie Gmbh ENHANCED REPROGRAMMING OF SOMATIC CELLS

Also Published As

Publication number Publication date
CN104342401A (zh) 2015-02-11
CN104342401B (zh) 2017-04-05

Similar Documents

Publication Publication Date Title
WO2015010417A1 (zh) 利用确定的细胞因子组合促进成纤维细胞转分化为脂肪细胞
Wu et al. Role of kruppel-like transcription factors in adipogenesis
WO2012026712A2 (ko) Ｎｏｄ２의 아고니스트를 처리한 줄기세포 또는 그 배양물을 포함하는 면역질환 또는 염증질환의 예방 또는 치료용 약학조성물
WO2016048107A1 (ko) 인터페론-감마 또는 인터류킨-1베타를 처리한 줄기세포 또는 그 배양물을 포함하는 면역질환 또는 염증질환의 예방 또는 치료용 약학조성물
WO2013009100A2 (ko) 탯줄 추출물의 제조방법 및 그의 용도
WO2018124642A9 (ko) 치수줄기세포의 상아 모전구세포로의 분화용 조성물 및 상아모전구세포 표면에 특이적으로 결합하는 IgG 또는 IgM 타입 단일 클론 항체
WO2021045373A1 (ko) 증식 가능한 간 오가노이드
WO2021033990A1 (ko) 유도만능줄기세포-유래 중간엽 줄기세포 전구세포로부터 유래된 엑소좀을 포함하는 비알콜성 지방간염의 예방 또는 치료용 조성물
WO2019022482A2 (ko) 황칠나무 추출물을 유효성분으로 포함하는 섬유화 질환의 예방 또는 치료용 조성물
WO2009151304A2 (ko) 에이케이에이피일이를 함유하는 조성물 및 동물 모델로서 에이케이에이피일이 돌연변이 제브라피쉬의 용도
WO2018026212A2 (ko) 섬유증 질환 모델의 제조방법 및 섬유증 질환 모델의 용도
WO2020122498A1 (ko) 클로날 줄기세포를 포함하는 췌장염 치료용 약학적 조성물
WO2016111523A2 (ko) HNF4-α 길항제 및 이의 용도
WO2017030395A1 (ko) 비만 치료 또는 예방용 약학 조성물
WO2019088311A1 (ko) Ｓｔａｔ３ 저해활성을 갖는 화합물 및 이의 용도
WO2020130431A1 (ko) 클로날 줄기세포를 포함하는 이식편대숙주질환 치료용 약학적 조성물
WO2020149538A1 (ko) 클로날 줄기세포를 포함하는 아토피 피부염 예방 또는 치료용 약학적 조성물
WO2011155643A1 (ko) 포그 ２ 다섯 번째 징크 핑거 도메인을 포함하는 포스파티딜이노시톨３키나아제 활성 조절제
WO2013162330A1 (ko) 키메라 중간엽 줄기세포군, 그의 제조방법 및 편도줄기세포를 이용하여 부갑상선 호르몬을 생산하는 방법
WO2020091471A1 (ko) 근육 특이적 pck1 과발현 형질전환 개 생산
WO2020004698A1 (ko) 세포배양용 배지 조성물 제조방법, 세포배양용 배지 조성물 제조방법과 줄기세포 유효성분 3저 추출법을 이용한 줄기세포파쇄추출물(쉘드줄기세포) 제조방법, 이를 이용한 항관절염 치료용 조성물, 이를 이용한 항염증 치료용 조성물 및 세포재생 치료용 조성물
WO2009145488A2 (ko) 지방세포로의 분화 조절 기능을 갖는 지방세포 분화 표지자로서의 xbp1(s) 단백질 및 그 용도
WO2023096111A1 (ko) 신규 퀴니자린 유도체 및 이의 제조 방법
WO2011068260A1 (ko) 인슐린신호경로를 조절하는 마이크로알엔에이 및 그 표적의 작용을 제어하는 물질의 스크리닝 방법
WO2020189990A1 (ko) 세포 조성물, 이의 제조 방법 및 이를 포함하는 아토피 예방 또는 치료용 약학 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13889870

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13889870

Country of ref document: EP

Kind code of ref document: A1