中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (7): 1169-1173.doi: 10.3969/j.issn.1673-8225.2011.07.007

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

重组人白细胞介素1受体拮抗蛋白荧光质粒在软骨细胞的表达

张  平1，刘  斌2，蔡道章2，钟志宏1，潘永谦1，张振山1   

  1. 1广州医学院第三附属医院骨外科，广东省广州市510150
    2中山大学附属第三医院骨外科，广东省广州市  510630
  • 收稿日期:2010-09-20 修回日期:2010-11-05 出版日期:2011-02-12 发布日期:2011-02-12
  • 作者简介:张平☆，男，1970年生，广东省广州市人，汉族，2008年中山大学毕业，博士，副主任医师，主要从事关节外科的研究。 644459349@qq.com
  • 基金资助:

    广东省科技计划项目(粤科规划字(2009)198号)，广州医学院博士启动项目(2008C20)。

Expression of recombinant human interleukin-1 receptor antagonist protein fluorescent plasmid in chondrocytes

Zhang Ping1, Liu Bin2, Cai Dao-zhang2, Zhong Zhi-hong1, Pan Yong-qian1, Zhang Zhen-shan1   

  1. 1Department of Orthopaedics, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou  510150, Guangdong Province, China
    2Department of Orthopaedics, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou  510630, Guangdong Province, China
  • Received:2010-09-20 Revised:2010-11-05 Online:2011-02-12 Published:2011-02-12
  • About author:Zhang Ping☆, Doctor, Associate chief physician, Department of Orthopaedics, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China 644459349@qq.com
  • Supported by:

    the Scientific and Technological Project of Guangdong Province, No.  (2009)198*, the Doctor Fund of Guangzhou Medical University, No.2008C20*

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摘要：

背景：白细胞介素1受体拮抗蛋白能延缓骨性关节炎进程，通过转基因方法可以使白细胞介素1受体拮抗蛋白表达的增加。
目的：观察重组人重组人白细胞介素1受体拮抗蛋白荧光质粒的构建及经脂质体转染软骨细胞的表达情况。
方法：双酶切法切取重组人白细胞介素1受体拮抗蛋白的c-DNA片段，通过T4DNA连接酶连接到pEGFP-C1载体上。体外分离培养兔关节软骨细胞，然后用构建的重组人白细胞介素1受体拮抗蛋白质粒经脂质体转染软骨细胞，通过荧光显微镜观察转基因的表达和荧光定量PCR检测其表达。
结果与结论：获得重组人pEGFP-C1-IL-1Ra真核表达载体质粒，酶切及测序结果证明表达质粒的DNA序列完全正确。荧光显微镜观察有绿色荧光蛋白表达，荧光定量PCR鉴定证实转染的软骨细胞基因得到表达。

关键词: 白细胞介素1受体拮抗蛋白, 基因转染, 荧光, 质粒, 软骨细胞, 表达

Abstract:

BACKGROUND: Interleukin-1 receptor antagonist (IL-1Ra) can delay osteoarthritis progress. The expression of IL-1Ra can be increased by gene transfection.
OBJECTIVE: To construct recombinant human IL-1Ra protein fluorescent plasmid and to observe its expression in chondrocytes by liposome gene transfection. 
METHODS: pGEM-T-IL-1Ra was digested with double enzymes restriction, and then it was linked with T4 DNA ligase and cloned into the pEGFP-C1 vector. Rabbit articular chondrocytes were isolated and cultured in vitro. The plasmids carrying the IL-1Ra gene were transfected into chondrocytes. Then the expression of the transgene was observed under a fluorescence microscope and detected by using real-time PCR assay.
RESULTS AND CONCLUSION: The sequence of obtained pEGFP-C1-IL-1Ra was identical to IL-1Ra and sequence in the Genbank. The expression of enhanced green fluorescent protein was observed. Real-time PCR analysis showed that the genes were expressed in chondrocytes.

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