彭建华; 庞金伟; 吴越; 谢雨珂; 郭科成; 涂天琦; 母前程; 廖玉嫣; 曹芳; 刘亮; 陈礼刚; 孙晓川; 江涌

doi:10.3760/cma.j.issn.1001-8050.2019.03.015

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• 基础研究 •

长链非编码RNA F19对小鼠创伤性脑损伤后继发性脑损伤的影响

中华创伤杂志, 2019,35(3) : 267-273. DOI: 10.3760/cma.j.issn.1001-8050.2019.03.015

摘要

目的

探讨长链非编码RNA F19(lncRNA F19)对小鼠创伤性脑损伤(TBI)后继发性脑损伤的影响。

方法

(1)96只野生型C57BL/6J小鼠按随机数字表法分为假手术组、假手术＋空载病毒组、假手术＋lncRNA F19慢病毒组、TBI组、TBI＋空载病毒组及TBI＋lncRNA F19慢病毒组，每组下设TBI后1 d和3 d两个亚组，各亚组8只。检测lncRNA F19表达情况及沉默效率。(2)将96只野生型C57BL/6J小鼠按随机数字表法分为假手术组、TBI＋空载病毒组及TBI＋lncRNA F19慢病毒组，每组下设TBI后1 d和3 d两个亚组，各亚组16只。观察lncRNA F19对TBI后神经元凋亡的影响。采用控制性皮层损伤法(CCI)建立小鼠TBI模型。F19慢病毒及空载病毒为造模前5 d经侧脑室注射。伤后1 d，3 d，采用实时定量PCR(qRT－PCR)法检测lncRNA F19相对表达量(2^－ΔΔct)；Western blot法检测脑组织Toll样受体4(TLR4)、B淋巴细胞瘤－2(Bcl－2)及Bcl－2相关X蛋白(Bax)蛋白表达；TUNEL染色检测创伤灶周围神经元凋亡情况。

结果

从伤后第1天开始，无论是假手术还是TBI后，空载病毒对lncRNA F19的表达无影响(P>0.05)。伤后第1天，与假手术＋空载病毒组比较，假手术＋lncRNA F19慢病毒组脑组织lncRNA F19表达量明显下降(0.07±0.07 ∶ 0.93±0.17) ，与TBI＋空载病毒组比较，TBI＋lncRNA F19慢病毒组脑组织lncRNA F19表达量明显下降(2.91±1.18∶0.52±0.32)(P均<0.05)。与TBI＋空载病毒组比较，TBI＋lncRNA F19慢病毒组脑组织TLR4 (0.51±0.13∶0.66±0.15)、Bax (0.45±0.06∶0.67±0.16)蛋白相对表达量及TUNEL神经元凋亡比例[(23.55±6.85)%∶(31.58±7.52)%]均明显降低(P<0.05)，Bcl－2蛋白相对表达量(0.76±0.16∶0.47±0.12)明显升高(P<0.05)。伤后第3天，与假手术＋空载病毒组比较，假手术＋lncRNA F19慢病毒组脑组织lncRNA F19表达量明显下降(0.11±0.09∶0.96±0.09)，与TBI＋空载病毒组比较，TBI＋lncRNA F19慢病毒组脑组织lncRNA F19表达量明显下降(0.54±0.24∶3.39±0.90)(P均<0.05)。与TBI＋空载病毒组比较，TBI＋lncRNA F19慢病毒组脑组织TLR4 (0.60±0.20∶0.85±0.09)、Bax(0.60±0.12∶0.88±0.21)蛋白相对表达量及TUNEL神经元凋亡比例[(29.10±7.37)%∶(39.22±10.64)%]均明显降低(P<0.05)，Bcl－2蛋白相对表达量(0.66±0.12∶0.35±0.16)明显升高(P<0.05)。

结论

抑制lncRNA F19可有效减少小鼠TBI后TLR4介导的皮层神经元凋亡，从而减轻继发性脑损伤。

引用本文: 彭建华, 庞金伟, 吴越, 等. 长链非编码RNA F19对小鼠创伤性脑损伤后继发性脑损伤的影响 [J] . 中华创伤杂志, 2019, 35(3) : 267-273. DOI: 10.3760/cma.j.issn.1001-8050.2019.03.015.

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未经授权，不得转载、摘编本刊文章，不得使用本刊的版式设计。

除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。

创伤性脑损伤(traumatic brain injury, TBI)是神经系统常见的急性危重症之一，其死残率极高，严重威胁人类健康。TBI主要分为原发性损伤及继发性损伤两个过程，原发性损伤由于继发性损伤而恶化^[1]。神经细胞凋亡是TBI后继发性脑损伤主要病理过程之一，与TBI患者神经功能预后有着密切关系^[2]。近年来，国内外针对TBI后神经细胞凋亡相关研究已取得一定成果，然而目前临床上并没有可明确改善继发性脑损伤的有效靶点。本课题组前期的研究表明，TBI后脑组织大量长链非编码RNA(long non－coding RNAs, lncRNAs)表达发生改变，其中lncRAN F19与细胞凋亡、神经炎症等有着密切关系^[3]。在前期研究基础上，笔者拟通过建立小鼠TBI模型并结合慢病毒基因沉默技术，进一步探讨lncRNA F19与TBI后继发性脑损伤的关系，以期明确TBI诊治及预后判断的新靶点。

贡献者信息

彭建华

西南医科大学附属医院神经外科，泸州　646000

庞金伟

西南医科大学附属医院神经外科，泸州　646000

吴越

重庆医科大学附属第一医院神经外科　400010

谢雨珂

西南医科大学附属医院神经外科，泸州　646000

郭科成

西南医科大学附属医院神经外科，泸州　646000

涂天琦

西南医科大学附属医院神经外科，泸州　646000

母前程

西南医科大学附属医院神经外科，泸州　646000

廖玉嫣

西南医科大学附属医院神经外科，泸州　646000

曹芳

遵义医学院附属医院脑血管病科　563003

刘亮

西南医科大学附属医院神经外科，泸州　646000

陈礼刚

西南医科大学附属医院神经外科，泸州　646000

孙晓川

重庆医科大学附属第一医院神经外科　400010

江涌

西南医科大学附属医院神经外科，泸州　646000

通信作者

江涌

西南医科大学附属医院神经外科，泸州　646000

Email：jiangyong@swmu.edu.cn

关键词

脑损伤; 长链非编码RNA; Toll样受体4; 凋亡;

基金项目

国家自然科学基金（81771278, 81801176）

四川省科技厅苗子工程重点项目（2018RZ0090）

泸州市科技局项目（2017－R－74, 2016LZXNYD－Z02, 2016LZXNYD－J12）

作者声明

作者贡献声明　彭建华：实验操作、论文撰写；庞金伟、吴越、谢雨珂、郭科成、涂天琦：实验操作；母前程、廖玉嫣、曹芳、刘亮：数据整理、统计学分析；陈礼刚、孙晓川：研究指导、论文修改；江涌：研究指导、论文修改、经费支持

利益冲突

利益冲突　所有作者均声明不存在利益冲突

历史

出版日期：2019-03-15

收稿日期：2018-08-23

Basically Scientific Research

Effect of long non-coding RNA F19 on secondary brain injury after traumatic brain injury in mice

Jianhua Peng, Jinwei Pang, Yue Wu, Yuke Xie, Kecheng Guo, Tianqi Tu, Qiancheng Mu, Yuyan Liao, Fang Cao, Liang Liu, Ligang Chen, Xiaochuan Sun, Yong Jiang

Published 2019-03-15

Cite as Chin J Trauma, 2019, 35(3): 267-273. DOI: 10.3760/cma.j.issn.1001-8050.2019.03.015

Abstract

Objective

To investigate the effect of long non-coding RNA F19 (lncRNA F19) on secondary brain injury following traumatic brain injury (TBI) in mice.

Methods

(1) A total of 96 C57BL/6J male wild-type mice were divided into sham group, sham+ control lentivirus group, sham+ F19 lentivirus group, TBI group, TBI+ control lentivirus group and TBI+ F19 lentivirus group according to the random number table. Each group consisted of two subgroups of 1 day and 3 days after TBI, with eight mice per subgroup. The expression and silence efficiency of lncRNA F19 were detected. (2) A total of 96 C57BL/6J male wild-type mice were divided into sham group, TBI+ control lentivirus group and TBI+ F19 lentivirus group according to the random number table. Each group consisted of two subgroups of 1 day and 3 days after TBI, with 16 mice per subgroup. The effect of lncRNA F19 on neuronal apoptosis after TBI was recorded. The mice TBI model was established using the controlled cortical damage method (CCI). The lncRNA F19 lentivirus or control lentivirus were administrated by intracerebroventricular injection 5 days before injury. The expressions of lncRNA F19(2^-ΔΔct) were detected by real-time quantitative PCR (qRT-PCR) at 1 day and 3 days after injury. The Toll-like receptor 4 (TLR4), B lymphocyte tumor-2 (Bcl-2) and Bcl-2 related protein (Bax) expressions were detected by Western blot. The TUNEL was used to detect apoptosis around the traumatic lesions.

Results

From the first day after injury, both in the sham operation and TBI groups, the control lentivirus had no effect on the level of lncRAN F19 (P>0.05). One day after injury, compared with sham+ control lentivirus group, the levels of lncRNA F19 in sham+ F19 lentivirus group were significantly decreased (0.07±0.07∶0.93±0.17); compared with TBI+ control lentivirus group, levels of lncRNA F19 in TBI+ F19 lentivirus group were significantly decreased (2.91±1.18∶0.52±0.32) (P<0.05). There were significantly lower protein levels of TLR4 (0.51±0.13∶0.66±0.15), Bax (0.45±0.06∶0.67±0.16), lower TUNEL-positive neurons ratio [(23.55±6.85)%∶(31.58±7.52)%], but higher protein levels of Bcl-2 (0.76±0.16∶0.47±0.12) in TBI+ F19 lentivirus group compared with the TBI+ control lentivirus group (P<0.05). Three days after injury, compared with sham+ control lentivirus group, levels of lncRNA F19 in sham+ F19 lentivirus group were significantly decreased (0.11±0.09∶0.96±0.09); compared with TBI+ control lentivirus group, levels of lncRNA F19 in TBI+ F19 lentivirus group were significantly decreased (0.54±0.24∶3.39±0.90) (P<0.05). There were significantly lower protein levels of TLR4 (0.60±0.20)∶(0.85±0.09)], lower Bax (0.60 ± 0.12∶0.88±0.21), lower TUNEL-positive neurons ratio [(29.10±7.37)%∶(39.22±10.64)%], but higher protein levels of Bcl-2 (0.66±0.12∶0.35±0.16) in TBI+ F19 lentivirus group compared with the TBI+ control lentivirus group (P<0.05).

Conclusion

Inhibition of lncRNA F19 can significantly reduce the TLR4-induced neuronal apoptosis in cortex after TBI in mice and alleviate reduce the secondary brain injury.

Key words:

Brain injuries; Long non-coding RNAs; Toll-like receptor 4; Apoptosis

Contributor Information

Jianhua Peng