[靶向ICL基因的10-23脱氧核酶对巨噬细胞中异柠檬酸裂解酶表达及结核分枝杆菌感染的抑制作用]

[Inhibitory effects of 10-23 deoxyribozyme targeting ICL gene on the expression of isocitrate lyase and the infection of Mycobacterium tuberculosis in macrophages].

作者信息

Li Jun-ming, Li Na, Zhu Dao-yin, Yi Zheng-jun, Liu Ye-hua, Yang Chun, He Yong-lin

机构信息

Department of Microbiology and Immunology, Chongqing University of Medical Sciences, 400016 Chongqing, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2006 Aug;29(8):531-5.

PMID:17074266

Abstract

OBJECTIVE

To investigate the inhibitory effects of the 10-23 deoxyribozyme (DRZ) targeting ICL gene on the expression of isocitrate lyase (ICL) and the survival of Mycobacterium tuberculosis in macrophages.

METHODS

Five 10-23 DRZ targeting ICL genes (DZ1-DZ5) were designed according to the predicted secondary structure of Mycobacterium tuberculosis ICL mRNA. Their cleavage activity and specificity were identified in cell free conditions. Then Mycobacterium tuberculosis pretreated with DZ4 with or without subinhibitory concentration of isoniazid (INH) were used to infect THP-1 cells. The bacterial burden of the infected THP-1 cells was monitored at indicated times after infection. The effect of 10-23 DRZ on the growth of Mycobacterium tuberculosis in vitro was also assayed by plating Mycobacterium tuberculosis treated with INH alone or DZ4 plus INH on M7H10 agar directly.

RESULTS

Four of the five designed 10-23 DRZ, DZ1, DZ3, DZ4 and DZ5 could cleavage ICL mRNA efficiently and specifically. Treating Mycobacterium tuberculosis with 5 micromol/L DZ4 plus subinhibitory concentration of INH decreased the expression of ICL dramatically, by 34.9% - 46.7% (10 microg/L INH, P < 0.01) or 21.9% - 36.9% (5 microg/L INH, P < 0.01) when compared with corresponding concentration of INH alone. The survival of Mycobacterium tuberculosis in THP-1 cells was decreased significantly when Mycobacterium tuberculosis was pretreated with 5 micromol/L DZ4 plus subinhibitory concentration of INH. 4 or 7 days after infection, the bacteria burden in macrophages was decreased from 126.5 x 10(4) CFU, 307.5 x 10(4) CFU to 54.6 x 10(4) CFU, 114.3 x 10(4) CFU (when 10 microg/L INH used) or from 133.0 x 10(4) CFU, 325.4 x 10(4) CFU to 71.7 x 10(4) CFU, 174.4 x 10(4) CFU (when 5 microg/L INH used). 10-23 DRZ showed no obvious effect on the growth of Mycobacterium tuberculosis in M7H10 agar.

CONCLUSIONS

INH at the subinhibitory concentration can improve the entry of 10-23 DRZ in Mycobacterium tuberculosis. In the presence of subinhibitory concentration of INH, 10-23 DRZ targeting ICL gene can strongly inhibit the expression of ICL and decrease the survival of Mycobacterium tuberculosis in macrophages.

摘要

目的

研究靶向异柠檬酸裂解酶（ICL）基因的10-23脱氧核酶（DRZ）对巨噬细胞中异柠檬酸裂解酶（ICL）表达及结核分枝杆菌存活的抑制作用。

方法

根据结核分枝杆菌ICL mRNA的预测二级结构设计了5条靶向ICL基因的10-23 DRZ（DZ1-DZ5）。在无细胞条件下鉴定它们的切割活性和特异性。然后用DZ4预处理过的结核分枝杆菌，无论有无亚抑菌浓度的异烟肼（INH），感染THP-1细胞。在感染后的指定时间监测被感染的THP-1细胞中的细菌载量。通过将单独用INH或DZ4加INH处理过的结核分枝杆菌直接接种在M7H10琼脂平板上，也检测了10-23 DRZ对结核分枝杆菌体外生长的影响。

结果

设计的5条10-23 DRZ中的4条，即DZ1、DZ3、DZ4和DZ5能够高效、特异地切割ICL mRNA。用5 μmol/L DZ4加亚抑菌浓度的INH处理结核分枝杆菌，与相应浓度的单独INH相比，ICL的表达显著降低，当使用10 μg/L INH时降低34.9% - 46.7%（P < 0.01），当使用5 μg/L INH时降低21.9% - 36.9%（P < 0.01）。当用5 μmol/L DZ4加亚抑菌浓度的INH预处理结核分枝杆菌时，其在THP-1细胞中的存活显著降低。感染后4天或7天，巨噬细胞中的细菌载量从126.5×10⁴CFU、307.5×10⁴CFU降至54.6×10⁴CFU、114.3×10⁴CFU（当使用10 μg/L INH时）或从？133.0×10⁴CFU、325.4×10⁴CFU降至71.7×10⁴CFU、174.4×10⁴CFU（当使用5 μg/L INH时）。10-23 DRZ对结核分枝杆菌在M7H10琼脂平板上的生长无明显影响。