中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (18): 3310-3313.doi: 10.3969/j.issn.1673-8225.2012.18.018

• 移植与基因 transplantation and gene • 上一篇    下一篇

真核过表达载体共转染3个胰岛发育关键基因在小鼠胎肝细胞内的表达*★

于静雯，张  振，徐艳花，陈容平，杨  锐，陈  宏   

  1. 南方医科大学附属珠江医院内分泌科，广东省广州市  510282
  • 收稿日期:2011-12-05 修回日期:2012-01-16 出版日期:2012-04-29 发布日期:2012-04-29
  • 作者简介:于静雯★，女，1986年生，天津市人，汉族，南方医科大学在读硕士，主要从事胰岛细胞新生研究。172581440@qq.com
  • 基金资助:

    广东省自然科学基金(10151051501000095)，课题名称：多顺反子核转染肝细胞重编程为胰岛素样细胞自体移植研究

     

Expression of three key pancreatic islet development genes in mice fetal hepatocytes following co-transfection of eukaryotic expression vectors

Yu Jing-wen, Zhang Zhen, Xu Yan-hua, Chen Rong-ping, Yang Rui, Chen Hong   

  1. Department of Endocrinology, Affiliated Zhujiang Hospital of Southern Medical University, Guangzhou  510282, Guangdong Province, China
  • Received:2011-12-05 Revised:2012-01-16 Online:2012-04-29 Published:2012-04-29
  • About author:Yu Jing-wen★, Studying for master’s degree, Department of Endocrinology, Affiliated Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China 172581440@qq.com
  • Supported by:

    Guangdong Natural Science Foundation No. 10151051501000095*

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被引次数

1

摘要：

背景：大量文献表明，向肝脏细胞引入一个多个胰岛发育的关键因子(如Pdx1、MafA及Ngn3)可以使肝细胞向胰岛细胞的方向进行转化。
目的：构建以pcDNA3.1(+)为骨架的质粒载体并共同转染胰十二指肠同源框蛋白(Pdx1)、神经源素3(Ngn3)及V型肌腱膜纤维肉瘤癌基因同源基因A(MafA)至小鼠胎肝细胞并检测其表达情况。
方法：以基因组或小鼠胰腺cDNA为模板扩增Pdx1、MafA及Ngn3三个目的基因，应用分子生物学技术，将3个目的片段克隆至真核表达载体pcDNA3.1(+)的多克隆位点中，构建小鼠系列真核过表达载体pcDNA3.1(+)-MafA ORF，pcDNA3.1(+)-Pdx-1 ORF，pcDNA3.1(+)-Ngn3 ORF通过Lipofectamine2000介导的脂质体转染小鼠胎肝细胞系BNL CL.2，用反转录PCR以及间接免疫荧光法检测3个目的基因的表达情况。
结果与结论：目的基因克隆正确,反转录PCR，间接免疫荧光法证实了脂质体转染后小鼠胎肝细胞中有Pdx1、Ngn3以及MafA的表达。结果表明，真核过表达载体pcDNA3.1(+)-MafA ORF，pcDNA3.1(+)-Pdx-1 ORF，pcDNA3.1(+)-Ngn3 ORF可成功构建，并能够将Pdx1、Ngn3以及MafA三个基因转至小鼠胎肝细胞进行表达。
关键词：胰十二指肠同源框蛋白；神经源素3；V型肌腱膜纤维肉瘤癌基因同源基因A；脂质体转染；BNL CL.2细胞株；胰岛细胞；基因表达
doi:10.3969/j.issn.1673-8225.2012.18.018

关键词: 胰十二指肠同源框蛋白, 神经源素3, V型肌腱膜纤维肉瘤癌基因同源基因A, 脂质体转染, BNL CL.2细胞株, 胰岛细胞, 基因表达

Abstract:

BACKGROUND: We have been studied that hepatocytes can be induced to differentiate into pancreatic B-cells through the introduction of a few factors of key islet development genes, such as pancreatic and duodenal homeobox-1 (Pdx-1), neurogenin 3 (Ngn3), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA).
OBJECTIVE: To construct the plasmids using the pcDNA3.1(+) frame to introduce the Pdx-1, MafA and Ngn3 into the mice fetal hepatocytes and to detect the expressions of these genes.
METHODS: We amplified the target genes of Pdx-1, MafA and Ngn3 using templates of the cDNAs or the genome and then cloned to the eukaryotic expression vector pcDNA3.1 (+) multiple cloning sites. The eukaryotic overexpression vectors of pcDNA3.1(+)-MafA ORF, pcDNA3.1(+) -Pdx-1 ORF and pcDNA3.1(+) -Ngn3 ORF were established by Lipofectamine2000 mediated liposome transfected with mouse fetal hepatocytes of BNL CL.2. The expressions of three target genes were tested through reverse-transcriptional polymerase chain reaction and indirect immunofluorescence.
RESULTS AND CONCLUSION: The target genes were cloned correctly. The expressions of Pdx-1, Ngn3 and MafA in the fetal hepatocytes were validated by the detection of reverse-transcriptional polymerase chain and immunofluorescence test. We successfully constructed the eukaryotic overexpressed plasmids of pcDNA3.1(+)-MafA ORF, pcDNA3.1(+) -Pdx-1 ORF and pcDNA 3.1 (+) -Ngn3 ORF which could express the genes of Pdx-1, Ngn3 and MafA in the fetal hepatocytes.

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