天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (8): 993-995.doi: 10.11958/20160214

• 实验研究 • 上一篇    下一篇

小鼠前脂肪细胞3T3-L1培养与四联诱导分化方法的探讨

孙慧誌1 ， 田德润1△， 孟洁2 ， 赵楠2 ， 韩洁1 ， 甘椿椿3 ， 王勇2   

  1. 1天津医科大学人体解剖与组织胚胎学系 （邮编300070）， 2病理教研室， 3药学院
  • 收稿日期:2016-03-28 修回日期:2016-05-28 出版日期:2016-08-15 发布日期:2016-08-22
  • 通讯作者: 田德润 E-mail： tianderun@aliyun.com E-mail:tianderun@aliyun.com
  • 作者简介: 孙慧誌 （1998）， 女， 临床七年制在读， 主要从事肥胖发生机制的研究
  • 基金资助:
    1国家自然科学基金项目 （81270927）； 2天津医科大学基础医学院大学生科研基金

Discussion on cultivation and methodology of four-drug combination-induced differentiation in mouse preadipocytes 3T3-L1 cells

SUN Huizhi 1 ,TIAN Derun1△,MENG Jie2 ,ZHAO Nan2 ,HAN Jie1 , GAN Chunchun3 ,WANG Yong2   

  1. 1 Department of Anatomy and Histology and Embryology, 2 Department of Pathology, 3 Department of Pharmacy, Tianjin Medical University, Tianjin 300070, China
  • Received:2016-03-28 Revised:2016-05-28 Published:2016-08-15 Online:2016-08-22
  • Contact: TIAN Derun E-mail： tianderun@aliyun.com E-mail:tianderun@aliyun.com

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摘要： 摘要： 目的 改进小鼠前脂肪细胞 3T3-L1 的培养并诱导分化为成熟脂肪细胞的方法。方法 使用含有 10％胎牛血清 （FBS） 的高糖型 DMEM 液体培养基常规培养小鼠前脂肪细胞， 2~3 d 换液 1 次。细胞按诱导分化方式的不同分为三联诱导组和四联诱导组。三联诱导组诱导分化培养基Ⅰ成分为常规培养基基础上加用人胰岛素 10 mg/L， 1- 甲基-3-异丁基黄嘌呤 （IBMX）0.5 mmol/L， 地塞米松 （DEX） 1.0 μmol/L； 四联诱导组诱导分化培养基Ⅰ的成分为在三联诱导组基础上加入吲哚美辛 0.1 mmol/L。2 组均诱导分化培养基Ⅰ培养 2 d， 连续诱导 2 次后换用诱导分化培养基Ⅱ， 成分为： 高糖型 DMEM 培养基含 10 %胎牛血清、 100 U/mL 青霉素和 100 mg/L 链霉素混合液， 人胰岛素 10 mg/ L。培养 2 d， 连续诱导 2 次。倒置显微镜和油红 O 染色观察诱导前及诱导后 2 组细胞的形态变化。结果 小鼠前脂肪细胞 3T3-L1 状态良好， 呈现铺路石状生长， 均匀布满培养瓶底， 2 d 传代 1 次。四联诱导组诱导分化结果优于三联诱导组。三联诱导剂诱导前脂肪细胞后， 细胞形态并未发生明显变化。四联诱导剂诱导前脂肪细胞后， 可达到 90％以上的成熟脂肪细胞。成熟的脂肪细胞呈圆形， 有大量脂滴聚集， 油红 O 染色显现橘红色。结论 在三联诱导基础上加入吲哚美辛的四联诱导法可以更好地促进脂肪前体细胞分化。

关键词: 脂细胞, 体外研究, 细胞培养技术, 3T3-L1 细胞

Abstract: Abstract： Objective To optimize and establish the methodology for culturing and inducing differentiation of mouse preadipocytes 3T3-L1. Methods The mouse cells 3T3-L1 were incubated in DMEM medium contained with 10% FBS, during which the incubation medium was refreshed every 2 to 3 days. Two methods were used to introduce differentiation, including three- drug combination group and four- drug combination group. The protocol of medium Ⅰin three- drug combination group including insulin 10 mg/L, IBMX 0.5 mmol/L and DEX 1.0 μmol/L. The protocol of mediumⅠin four- drug combination group including indometacin 0.1 mmol/L based on those of three-drug combination group. Both of them were incubated for 2 days and continuous for 2 times. And medium Ⅱ included insulin 10 mg/L for 2-day culturing and continuous for 2 times. Oil red O staining was used to observe the morphological changes of two groups of cells before and after treatment under inverted microscope. Results Mouse preadipocytes 3T3-L1 appeared in good conditions and grew in a paving stone fashion. These cells covered homogeneously the bottom of incubators, the culture medium refreshed every 2 days. The results of four-drug combination group were better than those of three-drug combination group. After three-drug combination induced differentiation, there was no significant change in cell morphology. Comparing with three- drug combination induced differentiation, four- drug combination was successfully achieved in over 90% of the cell inducing, which were round-shaped, with jacinth ester droplets by oil-red O staining. Conclusion We have optimized the method for culturing and inducing differentiation of mouse preadipocytes 3T3-L1 by adding indometacin on the basis of the three-drug combination induced differentiation.

Key words: adipocytes, in vitro, cell culture techniques, 3T3-L1 cells