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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

25卷

期数:

2019年05期

页码:

429-435

栏目:

基础医学

出版日期:

2019-09-20

文章信息/Info

Title:

Establishment and significance of SUZ12 overexpression and knockdown of stable malignant peripheral nerve sheath tumor cell lines

文章编号:

1006-8147(2019)05-0429-07

作者:

张 静¹; 高 雅²; 李新宇²; 朱香熹³; 孙硕遥¹; 肖 可⁴; 王 静²; 刘嘉岳²; 赵玉龙¹; 李光明¹; 朱 泽¹

（1.天津医科大学病原生物学系，天津300070；2.天津医科大学基础医学院，天津300070；3. 遵义医科大学珠海校区临床医学系，珠海 519090；4.多伦多大学圣乔治校区，多伦多 ONM5S）

Author(s):

ZHANG Jing¹; GAO Ya²; LI Xin-yu²; ZHU Xiang-xi³; SUN Shuo-yao¹; XIAO Ke⁴; WANG Jing²; LIU Jia-yue²; ZHAO Yu-long¹;  LI Guang-ming¹; ZHU Ze¹

（1. Department of Pathogen Biology，Tianjin Medical University，Tianjin 300070，China；2. School of Basic Medical Sciences, Tianjin Medical University，Tianjin 300070，China；3. Department of Clinical Medicine, Zhuhai Campus of Zunyi Medical University, Zhuhai 519090，China；4. University of Toronro-St.George Campus, Toronro ONM5S, Canada）

关键词:

SUZ12 ; 恶性外周神经鞘瘤; CRISPR/Cas9; 慢病毒

Keywords:

SUZ12;  MPNST;  CRISPR/Cas9;  lentivirus

分类号:

R739.43

DOI:

-

文献标志码:

A

摘要:

目的：构建SUZ12基因过表达和敲减的恶性外周神经鞘瘤(MPNST)稳定细胞株并探讨其意义。方法：PCR合成SUZ12基因全长并设计合成SUZ12基因的3对特异性sgRNA干扰靶点序列（sgRNA-1、sgRNA-2、sgRNA-3），构建重组表达质粒，转染293T细胞，包装慢病毒颗粒并用荧光法测定其滴度。确定慢病毒感染的最适MOI以及嘌呤霉素筛选的最适剂量，转染ST88-14细胞，构建过表达及敲减的MPNST稳定细胞株。荧光显微镜观察稳定株绿色荧光蛋白的表达情况，RT-qPCR在mRNA水平进行验证，MTT法检测SUZ12过表达和敲减组增殖活性的改变。结果：LV-SUZ12和LV-SUZ12-sgRNA转导入ST88-14细胞并稳定表达绿色荧光蛋白,RT-qPCR结果显示过表达稳定株SUZ12表达量明显升高（P<0.05），CRISPR/Cas9敲减稳定株SUZ12表达量明显下降（P<0.05）。MTT结果显示过表达SUZ12基因抑制MPNST细胞的增殖。结论：SUZ12过表达和敲减的稳定细胞株构建成功，初步验证结果表明过表达SUZ12基因抑制MPNST细胞的增殖，为针对SUZ12在MPNST中的生物学功能、作用机制和疾病诊断治疗的进一步研究提供了实验基础。

Abstract:

Objective: To establish the malignant peripheral nerve sheath tumor（MPNST） stable cell lines for over-expressed SUZ12 and knockdown SUZ12 and explore its significance. Methods: The full length SUZ12 gene was synthesized by PCR method,and three specific sgRNA interfere target sequences（sgRNA-1, sgRNA-2 and sgRNA-3）were designed and synthesized. Then to construct recombinant expression plasmids, 293T cells were transfected with these recombinant plasmids to package lentivirus, of which the titer were measured by fluorescence method. The optimal MOI for lentiviral infection and the optimal dose for purinomycin screening were determined, and ST8814 cells were transfected to construct the MPNST stable cell lines for overexpression and knockdown. The expression of green fluorescent protein in stably transfected strains was observed by fluorescence microscopy, and RT-qPCR was verified at the gene level. MTT assay was used to verify the proliferative activity of SUZ12 overexpression and knockdown groups. Results: LV-SUZ12 and LV-SUZ12-sgRNA were transduced into ST8814 cells and stably expressed green fluorescent protein. The results of RT-qPCR showed that the expression of SUZ12 overexpression stably increased(P<0.05), and the expression of CRISPR/Cas9 knockdown strain SUZ12 decreased significantly(P<0.05). MTT results show that overexpression of SUZ12 gene inhibits proliferation of MPNST cells. Conclusion: The stably transfected cell lines with overexpression and knockdown of SUZ12 were successfully constructed, and preliminary validation results indicate that overexpression of SUZ12 gene may indicate the growth of MPNST cells, providing experimental basis for further study on the biological function, mechanism and diagnosis and treatment of SUZ12 in MPNST.

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相似文献/References:

备注/Memo

备注/Memo:

基金项目 国家自然科学基金资助项目（81672650）
作者简介 张静（1994-），女，硕士在读，研究方向：恶性外周神经鞘瘤发病与发展的分子机制；通信作者：朱泽，E-mail: zhuze@tijmu.edu.cn，李光明，E-mail：li-guangming@hotmail.com。

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更新日期/Last Update: 2019-10-11