脊髓损伤早期Cathepsin基因表达和甲强龙干预后的变化

Expression of genes cathepsin family in acute spinal cord injury interfered with high-dose methylprednisolone

[目的]检测Cathepsin基因家族在脊髓损伤早期的表达和大剂量甲强龙干预后的变化,明确大剂量甲强龙是否通过溶酶体凋亡途径发挥神经保护作用。[方法]9只日本大耳兔随机分为3组:对照组(C组,n=3),模型组(M组,n=3)和药物组(D组,n=3)。C组仅进行椎板切除术,M组和D组进行椎板切除后采用Allen法建立急性脊髓损伤模型。D组在造模后2 h按人-兔等效剂量给予大剂量甲强龙冲击治疗。造模后8 h处死实验动物,分别获取损伤区为中心的长约8 mm的脊髓组织液氮保存,采用Trizol法提取总RNA,用9张Agilent兔子全基因4×44K芯片进行检测。采用GeneSpring 11.0软件,以P<0.05且倍数变化(FC)≥2筛选出差异表达基因,本文针对Ca-thepsin基因家族进行分析。[结果]成功建立脊髓损伤的动物模型并获得相应的组织标本。9组标本总RNA的质量均能满足基因芯片检测要求。基因芯片结果显示:在10个Cathepsin基因家族成员中,仅Cathepsin Z和proathepsin E在创伤后呈现差异性表达。Cathepsin C、D、F、K、L、S和W表达均无差异(M-C)。甲强龙冲击治疗后Cathepsin家族基因表达均无差异(D-M)。[结论]Cathepsin Z和E参与了脊髓损伤早期病理过程,但大剂量甲强龙抑制神经凋亡可能不通过溶酶体途径。

[Objective]To check expression and change of cathepsin genes family in acute spinal cord injury and to identify if high-dose methylprednisolone（MP） protect nerve cell by the lysosome apoptosis pathway.[Method]Nine rabbits were randomly divided into 3 groups： control group（group C,n=3）,model group（group M,n=3） and drug treatment group（group D,n=3）.Laminectomy at L4 was carried out on three groups.While acute spinal cord injury（SCI） model was established with Allen＇s Falling Strike Method in the groups M and D.High-dose methylprednisolone equivalent with human dose was adopted in group D at 2 h post-SCI.All rabbits were killed,and their damaged spinal cord tissues（about 8 mm in length） were obtained carefully and kept in liquid nitrogen.Total RNA was extracted with Trizol one-step method to examinate the gene expression profile by using Agilent Rabbit Oligo Microarray（4/44K）respectively.GeneSpring 11.0 analyzer software was used to filter potential candidate genes for statistical significance using Welchs t test,and only genes with P0.05 and fold change（FC） ≥2 were retained for further analysis.Especially,the expression of genes cathepsin family experienced close attention.[Result]The SCI model was successfully set up and these nine samples of damaged spinal cord tissues were acquired at 8 h post-SCI.The total RNAs of the 9 subsample of were qualified for microarray examination and data analysis was performed using Affymetrix GeneChip technology with standard operating procedures and quality control as recently described.Of 10 genes of cathepsin family,only cathepsin C and procathepsin E showed significant different expression（group D vs C）.All of cathepsin family genes didn＇t show significantly different expression under a circumstance of high-dose MP（group M vs D）.[Conclusion]Gene cathepsin C and procathepsin E take part in spinal cord injury at acute phase,but high-dose MP protecting nerve cells might not travel through the lysosome apoptosis pathway.