中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (16): 2577-2582.doi: 10.3969/j.issn.2095-4344.0278

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

沉默DEPTOR表达促进胰岛细胞释放胰岛素

邱  鸿,赖舒畅,潘道延,王  肖,沈  洁   

  1. 南方医科大学第三附属医院,广东省广州市  510630
  • 收稿日期:2018-02-15 出版日期:2018-06-08 发布日期:2018-06-08
  • 通讯作者: 沈洁,主任医师,教授,博士生导师,南方医科大学第三附属医院,广东省广州市 510630
  • 作者简介:邱鸿,女,1992年生,汉族,南方医科大学在读硕士,主要从事糖尿病发病机制研究。
  • 基金资助:

    国家自然科学基金资助项目(81770878);广州市科技创新委员会重大专项课题(201604020007)

DEPTOR gene silencing promotes β-cell insulin secretion

Qiu Hong, Lai Shu-chang, Pan Dao-yan, Wang Xiao, Shen Jie   

  1. the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • Received:2018-02-15 Online:2018-06-08 Published:2018-06-08
  • Contact: Shen Jie, Chief physician, Professor, Doctoral supervisor, the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • About author:Qiu Hong, Master candidate, the Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81770878; the Major Project of Guangzhou Science Technology and Innovation Commission, No. 201604020007

摘要:

文章快速阅读:


文题释义:
RNAi(RNA interference):通过导入高度保守的双链RNA(double-stranded,dsRNA)分子至细胞内后,促进与其同源的mRNA发生特异性的降解,从而高效并特异地阻断或抑制相应基因表达活性。RNA干扰是近年科研工作者们调控基因的表达、阐明细胞的信号通路和研究功能基因组学的重要技术。
siRNA:生物体内源性转录生成或外来导入的高度保守的双链RNA,被一种核酸酶Dicer切割后形成的长度为21-23个核苷酸的小片段RNA,即为siRNA。
摘要
背景:
mTOR复合物是调控胰岛β细胞质量与功能的关键蛋白,Deptor是复合物中的共有组分,Deptor的丢失是否会影响胰岛β细胞的胰岛素分泌功能,目前尚缺乏相关研究。
目的:利用siRNA干扰技术沉默小鼠胰岛素瘤NIT-1细胞DEPTOR基因的表达,探讨其对胰岛素瘤细胞分泌功能的影响及背后的机制。
方法:设计合成3对针对DEPTOR 基因的siRNA序列,利用脂质体法将siRNA转染至NIT-1细胞。实验组设为6组:①空白转染组(NIT-1细胞,转染复合液仅加Lipofectamin);②阴性对照转染组(NC-FAM);③阳性对照转染组(GAPDH);④siRNA deptor 1组(脂质体介导的转染复合+siRNA deptor385);⑤siRNA deptor 2组(脂质体介导的转染复合+siRNA deptor766);⑥siRNA deptor 3组(脂质体介导的转染复      合+siRNA deptor1275)。荧光显微镜下观察转染效率;QPCR检测DEPTOR mRNA的相对表达量;ELISA胰岛素试剂盒检测NIT-1细胞胰岛素分泌情况;Western blot检测DEPTOR下游关键蛋白的表达。
结果与结论:①在荧光显微镜下观察,siRNA能有效转入到NIT-1细胞内,呈绿色点状荧光;②PCR检测干扰后DEPTOR mRNA的相对表达量,发现设计合成3对siRNA序列中有两对siRNA序列(siDEPTOR385 and siDEPTOR766)对DEPTOR基因有干扰效果,干扰效果与阴性对照差异有显著性意义(P < 0.05);③ELISA胰岛素测定结果显示,在NIT-1细胞中沉默DEPTOR后,有效转染组细胞的胰岛素分泌显著升高  (P < 0.05);④Western-blot检测结果显示,Deptor下游关键蛋白S6、4EBP-1磷酸化明显增加,AKT磷酸化略有减少;⑤结果说明,利用RNAi技术在NIT-1细胞上可以有效地沉默DEPTOR 基因的表达,并促进细胞胰岛素的分泌,该现象可能与mTORC1通路的激活相关。

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程
ORCID: 0000-0002-9034-4725(邱鸿)

关键词: DEPTOR, 胰岛细胞, RNA干扰, 基因沉默, 胰岛素分泌

Abstract:

BACKGROUND: Mammalian target of rapamycin (mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein (DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet β cells affects insulin-secreting function has 
never been identified.
OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism.
METHODS: Three siRNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group (NIT-1 cells plus Lipofectamin), negative control group (NC-FAM), positive control group (GAPDH), siRNA deptor 1 group (siRNA deptor385), siRNA deptor 2 group (siRNA deptor766), and siRNA deptor 3 group (siRNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR mRNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay.
RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two (siDEPTOR385 and siDEPTOR766) of the three siRNA sequences could significantly disrupt the expression of DEPTOR mRNA, which had significant difference with negative control group (P < 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased (P < 0.05). Western blot assay results showed the grey levels of p-s6 and p-4EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that siRNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: Insulin-Secreting Cells, Insulinoma, Gene Silencing

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