TLR4在大鼠坐骨神经损伤后Wallerian溃变过程中的体内作用研究*

张焕焕**,高楠楠,石玲玉,邵志伟,李雨婷,朱云,姚登兵

(南通大学江苏省神经再生重点实验室,生命科学学院,江苏226001

[]目的:研究TLR4Toll-like receptor 4)在大鼠坐骨神经损伤后,Wallerian溃变过程中的作用,为周围神经损伤修复与再生的研究提供实验依据。方法:制备大鼠损伤坐骨神经模型;利用修饰过的TLR4基因过表达质粒导入大鼠损伤坐骨神经远端组织;通过实时荧光定量PCRWestern Blot的方法分析损伤7d14d后基因和蛋白的表达,以及相关信号通路的变化。结果:在大鼠坐骨神经损伤7d14d后,实时荧光定量PCR显示,远端TLR4的过表达引起BaxBcl-2NF2NT3PKCα等基因的表达变化,Western Blot显示TLR4过表达后,p-AKT/AKTp-c-Jun/c-Junp-ERK/ERK等蛋白的表达发生显著性差异。结论:TLR4在大鼠坐骨神经损伤后,Wallerian溃变过程中可能发挥了重要的调控作用。

[关键词]Wallerian溃变;Toll-like receptor 4;神经再生;大鼠

[中图分类号]Q555.3 [文献标志码]A

The role of TLR 4 in Wallerian degeneration after rat sciatic nerve injury in vivo

ZHANG Huanhuan, GAO Nannan, SHI Lingyu, SHAO Zhiwei, LI Yuting, ZHU Yun, YAO Dengbing

( Jiangsu Key Laboratory of Neuroregeneration, School of Life Sciences , Nantong University , Nantong Jiangsu 226001)

[Abstract]Objective: To investigate the role of TLR4 (toll-like receptor 4TLR4) in Wallerian degeneration after the sciatic nerve injury in rats. Methods: All rats were anesthetized by using an injection of complex narcotics. The sciatic nerve was cut and a 1 cm segment was excised. The 0d animals received sham-operations. The modified pcDNA3.1(+)-TLR4 plasmid was used to overexpress TLR4 gene in injuried rat sciatic nerves. Real-time qPCR and Western Blot were used to analyze the mRNA and protein levels in sciatic nerves after 7 and 14 days. Results: The expressions of BaxBcl-2NF2NT3 and PKCα were changed in injuried sciatic nerves with TLR4 overexpression after 7 and 14 days. The overexpressed TLR4 also affected the expressions of p-AKT/AKT, p-c-Jun/c-Jun and p-ERK/ERK after sciatic nerve injury. Conclusion: TLR4 may play an important role in peripheral nerve degeneration and regeneration.

[Key words]Wallerian Degeneration(WD); TLR4; nerve regeneration; rat

纳米材料介孔硅承载阿霉素在C6胶质瘤中的实验研究*

张玮洵1**,侯仕强2,施金龙2

(南通大学1医学院;2附属医院神经外科,江苏226001

[]目的:探讨纳米材料介孔硅(MSN)承载阿霉素(DOX)在C6胶质瘤细胞和荷瘤大鼠的体内分布情况及其凋亡诱导作用。方法:将MSN按浓度梯度分别与C6胶质瘤细胞孵育,通过CCK-8细胞活力检测MSN细胞毒性;DOXMSN/DOX按时间梯度分别与C6胶质瘤细胞共培养,荧光显微镜观察药物吞噬;构建荷瘤大鼠模型,尾静脉分别注射DOXMSN/DOX后,通过免疫荧光及荧光分光光度计检测大鼠体内主要脏器的药物分布情况,Western Blot检测胶质瘤细胞凋亡。结果:MSN按浓度梯度1μg/mL10μg/mL50μg/mL加入C6胶质瘤细胞后,C6胶质瘤细胞增殖能力随介孔硅浓度递增差异无统计学意义(P>0.05);50μg/mL浓度梯度组1h6h12h24h酶标仪检测结果分析显示,C6胶质瘤细胞增殖能力差异无统计学意义(P>0.05)。荧光显微镜观察显示MSN/DOX组随时间增加在C6胶质瘤细胞中的DOX药物含量高于DOX组;MSN/DOX组在荷瘤大鼠胶质瘤组织中的DOX药物含量高于DOX组,且凋亡相关蛋白表达明显增加。结论:MSN细胞毒性低,可承载DOX更多地进入C6胶质瘤细胞,并促进C6胶质瘤细胞凋亡。

[关键词]纳米材料介孔硅;阿霉素;C6胶质瘤

[中图分类号]R730.264 [文献标志码]A

The application of Mesoporous silica nanoparticles sustained doxorubicin in C6 glioma

ZHANG Weixun, HOU Shiqiang, SHI Jinlong

( 1Medical School of Nantong University ; 2Department of Neurosurgery,

the Affiliated Hospital of Nantong University, Jiangsu 226001

[Abstract]Objective:To study the distribution of mesoporous silica nanoparticles sustaining doxorubicin in C6 glioma cells and glioma-bearing rats, and its inducing apoptosis of C6 glioma. Methods:C6 glioma cells were incubated with MSN by different concentrations, CCK-8 was used to detect the cytotoxicity of MSN. C6 glioma cells were randomly incubated with DOX and MSN/DOX. Fluorescence spectrophotometer was used to detect the change of DOX in the C6 glioma cells. The glioma-bearing rats were randomly injected with saline, DOX and MSN/DOX. The changes of proliferation and apoptosis were detected by Western Blot, and the distribution of DOX in the major organs was observed with Fluorescence spectrophotometer. Results:There was not statistic difference on MSN groups of different concentrations compared with the blank group by CCK-8 assay. The distribution of DOX in C6 glioma cells of MSN/DOX group was more than in DOX group, the same result was also observed in glioma-bearing rats, and MSN/DOX group induced more apoptosis compared with DOX group. Conclusion:The present studies prove that MSN has low cytotoxicity and remarkably increases the distribution of DOX in C6 glioma to induce apoptosis.

[Key words]mesoporous silica nanoparticles; doxorubic; C6 glioma