上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (4): 387-.doi: 10.3969/j.issn.1674-8115.2011.04.001

• 论著（基础研究） •    下一篇

抗PNAS-2单链抗体对白血病U937细胞凋亡的影响

肖珎予1, 陈芳源1, 王海嵘1, 吴英理2, 钟济华1, 钟 华1   

  1. 1.上海交通大学 医学院附属仁济医院血液科, 上海 200127； 2.上海交通大学 医学院细胞分化与凋亡教育部重点实验室, 上海 200025
  • 出版日期:2011-04-28 发布日期:2011-04-28
  • 通讯作者: 陈芳源, 电子信箱: chenfy04@yahoo.com.cn。
  • 作者简介:肖珎予（1984—）, 女, 硕士生；电子信箱: fedex2004@126.com。
  • 基金资助:

    仁济医院-交通大学基础医学院合作研究基金(ZD0704)和国家自然科学基金(81000212)

Effects of anti-PNAS-2 single chain antibody on apoptosis of human leukemic U937 cells

XIAO Zhen-yu1, CHEN Fang-yuan1, WANG Hai-rong1, WU Ying-li2, ZHONG Ji-hua1, |ZHONG Hua1   

  1. 1.Department of Hematology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China； 2.Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai |Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2011-04-28 Published:2011-04-28
  • Supported by:

    Research  Foundation  of  Renji  Hospital-Shanghai  Jiaotong  University  School  of  Medicine, ZD0704；National Natural Science  Foundation  of  China, 81000212

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摘要：

目的 观察抗PNAS-2单链抗体与PNAS-2抗原特异性结合的能力，并在此基础上探究其对急性单核细胞白血病U937细胞凋亡的影响。方法 运用反转录病毒转染法将抗PNAS-2单链抗体表达载体pMIG-PNAS-2ScFv转入U937细胞(pMIG-PNAS-2ScFv组)，同时设转染pMIG空载体(NC组)及未转染载体的U937细胞(U937组)为对照。流式细胞仪检测转染效率；激光共聚焦显微镜观察单链抗体表达情况及与靶抗原特异性结合的能力；流式细胞仪和激光共聚焦显微镜检测细胞凋亡；Western blotting观察PNAS-2表达及促凋亡因子caspase-3激活情况。结果 成功将抗PNAS-2单链抗体编码序列转入U937细胞，所表达的单链抗体能与PNAS-2抗原特异性结合。与NC组和U937组相比，pMIG-PNAS-2ScFv组细胞凋亡率明显上升（P<0.05），活化型caspase-3表达增加，但PNAS-2含量并无变化。结论 抗PNAS-2单链抗体能有效结合U937细胞内的PNAS-2抗原并有促进U937细胞凋亡的作用，其促凋亡机制可能与PNAS-2功能位点被封闭后功能失活而无法发挥抑凋亡作用有关。

关键词: PNAS-2, 单链抗体, U937细胞, 凋亡

Abstract:

Objective To observe specific binding ability of anti-PNAS-2 single chain antibody with PNAS-2 antigen and its effects on apoptosis of human leukemic U937 cells. Methods The pMIG-PNAS-2ScFv, anti-PNAS-2 single chain antibody expression vector, was transfected into U937 cell line by retroviral transfection method. Untransfected U937 cells and pMIG transfected with empty vector were used as controls. Transfection rate was measured by flow cytometry. Single chain antibody expression and ScFv-antigen specific binding were measured by laser confocal microscopy. Cell apoptosis was detected by flow cytometry and laser confocal microscopy. Expression of PNAS-2 and active caspase-3 was detected by Western blotting. Results Anti-PNAS-2 single chain antibody coding sequence was successfully transfected into U937 cells and its expressed single chain antibody could bind to PNAS-2 antigen with a high specificity. Compared with the control cells, pMIG-PNAS-2ScFv cells had a higher apoptosis rate (P<0.05) and increase in active caspase-3 expression，but no difference was found in PNAS-2 expression. Conclusion Anti-PNAS-2 single chain antibody can specifically bind PNAS-2 antigen and induce apoptosis of U937 cells, which may be related to the inactivation of PNAS-2 after closure of the functional site.

Key words: PNAS-2, single chain antibody, U937 cell, apoptosis