中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (28): 4487-4492.doi: 10.3969/j.issn.2095-4344.0839

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

在INS-1细胞中构建胰腺组织硫氧还蛋白相互作用蛋白过表达模型:两种腺病毒感染方法的差异

曹竹杰,冯艳金,李  丹,王  瑾,焦向英   

  1. 山西医科大学生理学系,细胞生理学省部共建教育部重点实验室,山西省太原市  030001
  • 收稿日期:2017-12-26 出版日期:2018-10-08 发布日期:2018-10-08
  • 通讯作者: 焦向英,教授,博士生导师,山西医科大学生理学系,细胞生理学省部共建教育部重点实验室,山西省太原市 030001
  • 作者简介:曹竹杰,男,汉族,1992年生,山西省晋中市人,山西医科大学在读硕士,主要从事糖尿病及其发病机制方面的研究。
  • 基金资助:

    国家自然科学基金项目(30800399);山西省重点实验室建设项目(2014011049-12)

Differences in the construction of thioredoxin-interacting protein overexpression model in INS-1 cells by two adenovirus infection methods

Cao Zhu-jie, Feng Yan-jin, Li Dan, Wang Jin, Jiao Xiang-ying   

  1. Department of Physiology, Key Laboratory for Cellular Physiology of Ministry of Education, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Received:2017-12-26 Online:2018-10-08 Published:2018-10-08
  • Contact: Jiao Xiang-ying, Professor, Doctoral supervisor, Department of Physiology, Key Laboratory for Cellular Physiology of Ministry of Education, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Cao Zhu-jie, Master candidate, Department of Physiology, Key Laboratory for Cellular Physiology of Ministry of Education, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 30800399; the Shanxi Provincial Key Laboratory Construction Project, No. 2014011049-12

摘要:

文章快速阅读:
文题释义:
腺病毒载体:腺病毒是无包膜的线性双链DNA病毒,在自然界分布广泛,至少存在100种以上的血清型。它通过受体介导的内吞作用进入细胞内,然后腺病毒基因组转移至细胞核内,保持在染色体外,不整合进入宿主细胞基因组中。腺病毒基因组长约36 kb,两端各有一个反向末端重复区,反向末端重复区内侧为病毒包装信号。基因组上分布着4个早期转录元(E1、E2、E3、E4)承担调节功能,以及一个晚期转录元负责结构蛋白的编码。
硫氧还蛋白相互作用蛋白:属于硫氧还蛋白结合蛋白的一种,通过抑制硫氧还蛋白系统的功能而发挥介导氧化应激、抑制细胞增殖及诱导细胞凋亡等作用。硫氧还蛋白相互作用蛋白是胰岛素中炎症过程的核心分子,这种炎症会导致人体胰脏中胰岛细胞的死亡。
摘要
背景:
目前,通过腺病毒感染方法在细胞中构建蛋白质过表达模型的方法主要有2种,一是在腺病毒感染细胞    4 h后补充1640完全培养基,24 h后换为完全培养基,再继续培养到48 h(方法Ⅰ);另一种是在腺病毒感染细胞4 h后用PBS洗去含有腺病毒的1640培养基,更换4 mL完全培养基培养到48 h,中间不做任何处理(方法Ⅱ)。前者病毒的作用时间较长,感染效果较好,但病毒本身对细胞的损害程度较大,对实验模型的稳定性产生干扰。而后者病毒的作用时间短,对细胞的毒性低,但有时病毒感染效率较低,目的蛋白的表达效果欠佳。
目的:在大鼠INS-1胰岛β细胞中,通过比较相同腺病毒载体介导的不同感染细胞的方法而寻找一种更加稳定而高效的硫氧还蛋白相互作用蛋白(Thioredoxin-interacting protein,TXNIP)过表达模型。
方法:构建含有绿色荧光蛋白的腺病毒载体(Ad-GFP)和TXNIP过表达腺病毒载体(Ad-TXNIP-GFP)。分别设立正常组、空病毒组和TXNIP过表达组,按照上述2种不同的方法进行病毒转染。
结果与结论:①方法Ⅱ中的绿色荧光蛋白荧光强度和阳性效率高于方法Ⅰ;②方法Ⅱ中的细胞形态更好且细胞存活率更高;③方法Ⅱ中,目的基因TXNIP在mRNA水平和蛋白质水平的表达情况明显高于方法Ⅰ。由以上结果得出结论,腺病毒感染INS-1细胞4 h后,使用PBS将旧培养基洗去并更换新培养基,继续培养到48 h的方法更有利于在INS-1细胞中构建TXNIP过表达模型。

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: 0000-0003-1182-9052(焦向英)

关键词: 腺病毒载体, 绿色荧光蛋白, INS-1细胞, 硫氧还蛋白相互作用蛋白, TXNIP, 组织构建, 糖尿病, 存活率, 感染效率, 流式细胞术, 实时PCR, Western blot

Abstract:

BACKGROUND: There are two methods for constructing the protein overexpression model in cells by adenovirus infection. In Method I, 3 mL of 1640 medium was supplemented 4 hours after cell infection by adenovirus, and 24 hours later, culture medium was refreshed with complete medium for further 48 hour culture, In Method II, 4 hours after cell infection by adenovirus, PBS was used to wash 1640 culture medium containing adenovirus, and then 4 mL of complete culture was added for further culture till 48 hours. The main difference between the two methods is that the adenovirus in Method I has a longer action time and a better infection effect, but the damage of the adenovirus itself to cells is more severe, and it disturbs the stability of the experimental model. The effect of adenovirus in Method II is short lasting, and the toxicity of adenovirus to cells is lower, but the efficiency of adenovirus infection is weaker, and the expression of target protein is weaker.
OBJECTIVE: To find a more stable and efficient way to establish the thioredoxin-interacting protein (TXNIP) overexpression model by adenovirus infection in INS-1 islet β-cells.
METHODS: The adenovirus vector (Ad-GFP) with green fluorescent protein (GFP) and TXNIP overexpressing adenovirus vector (Ad-TXNIP-GFP) with green fluorescent protein (GFP) were constructed. Three groups (normal, Ad-GFP, Ad-TXNIP-GFP) were transfected with methods I and II, respectively.
RESULTS AND CONCLUSION: The GFP fluorescence intensity and the GFP positive percent in Method II were greater than those in the Method I. The cell morphology and survival rate in Method II were better than those in Method I. The expression of TXNIP at mRNA and protein levels in Method II was significantly higher than that in Method I. These findings show that the method II is more conducive to INS-1 cells in the construction of TXNIP overexpression model. 

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: Adenoviridae Infections, Gene Expression Regulation, Genes, Regulator, Insulin-Secreting Cells, Tissue Engineering

中图分类号: