[甘精胰岛素对人皮下及网膜脂肪组织前脂肪细胞的原代培养及增殖、分化的影响]

[Primary culturing and effects of insulin glargine on proliferation, differentiation of human preadipocytes from subcutaneous and omental adipose tissue].

作者信息

Lu Hong-yun, Li Xiao-feng, Mu Pan-wei, Jiang Wei, Zeng Long-yi

机构信息

Department of Endocrinology, Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, China.

Department of Endocrinology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China, Email:

出版信息

Zhonghua Yi Xue Za Zhi. 2013 Sep 24;93(36):2861-6.

PMID:24373396

Abstract

OBJECTIVE

To compare the morphological and functional differences of human primary preadipocytes from different fat depots and explore the effects of insulin glargine on their proliferation and differentiation.

METHODS

Primary preadipocytes isolated from human subcutaneous and omental adipose tissue by collagenase I were passaged in vitro.Inverted phase contrast microscope was used to observe the morphological differences of two kinds of preadipocytes. Then two kinds of preadipocytes were cultured or induced to differentiation with different doses of insulin glargine. The methyl thiazolyl tetrazolium (MTT) assay was used to detect their proliferative differences.Reverse transcription-polymerase chain reaction (RT-PCR) was used to observe the effects of insulin on adipogenic gene expression.

RESULTS

(1) Both preadipocytes could be successfully cultured from adipose tissue and amplified in vitro.Subcutaneous preadipocytes were more slender and proliferated more quickly while omental preadipocytes were polygonal and aged easily.(2) MTT results showed that insulin glargine could inhibit the proliferation of omental preadipocytes in a dose-dependent fashion. After 72 h incubation, compared with negative control, the absorbance (A) value of 1000 nmol/L insulin glargine group decreased greatly (0.144 ± 0.021 vs 0.267 ± 0.040, P < 0.01). But it had no effect on subcutaneous preadipocytes (0.305 ± 0.045 vs 0.350 ± 0.037, P > 0.05). (3) Insulin at 500 nmol/L was a suitable concentration for inducing differentiation.RT-PCR analysis showed that, for subcutaneous adipocytes, adipogenic genes such as peroxisome proliferator-activated receptor gamma (PPARγ) (F = 31.31, P < 0.01) and CCAAT enhancer binding protein α (C/EBPα) (F = 9.86, P < 0.05) had the highest mRNA expression while preadipocytes gene Pref-1 had the lowest expression at this concentration. But insulin dose had no obvious effect on PPARγ or C/EBPα mRNA (P > 0.05) for omental adipocytes.

CONCLUSION

Insulin glargine could inhibit the proliferation of omental preadipocytes, and enhance the differentiation of subcutaneous and omental preadipocytes.

摘要

目的

比较不同脂肪储存部位的人原代前脂肪细胞的形态和功能差异，并探讨甘精胰岛素对其增殖和分化的影响。

方法

采用Ⅰ型胶原酶从人皮下和网膜脂肪组织中分离原代前脂肪细胞并进行体外传代培养。利用倒置相差显微镜观察两种前脂肪细胞的形态差异。然后用不同剂量的甘精胰岛素对两种前脂肪细胞进行培养或诱导分化。采用甲基噻唑基四氮唑（MTT）法检测其增殖差异。运用逆转录-聚合酶链反应（RT-PCR）观察胰岛素对脂肪生成基因表达的影响。

结果

（1）两种前脂肪细胞均能从脂肪组织中成功培养并在体外扩增。皮下前脂肪细胞更细长，增殖更快，而网膜前脂肪细胞呈多边形，易老化。（2）MTT结果显示，甘精胰岛素能以剂量依赖方式抑制网膜前脂肪细胞的增殖。孵育72小时后，与阴性对照相比，1000 nmol/L甘精胰岛素组的吸光度（A）值显著降低（0.144±0.021比0.267±0.040，P<0.01）。但对皮下前脂肪细胞无影响（0.305±0.045比0.350±0.037，P>0.05）。（3）500 nmol/L胰岛素是诱导分化的合适浓度。RT-PCR分析表明，对于皮下脂肪细胞，在该浓度下，过氧化物酶体增殖物激活受体γ（PPARγ）（F=31.31，P<0.01）和CCAAT增强子结合蛋白α（C/EBPα）（F=9.86，P<0.05）等脂肪生成基因的mRNA表达最高，而前脂肪细胞基因Pref-1的表达最低。但对于网膜脂肪细胞，胰岛素剂量对PPARγ或C/EBPα mRNA无明显影响（P>0.05）。