神经源性分化因子对小鼠放射性肠损伤的治疗作用

The therapeutic effect of NeuroD on radiation-induced intestinal injury in mice

目的 研究神经源性分化因子（NeuroD）对放射性肠损伤的治疗作用.方法 采用原核表达系统表达、纯化NeuroD与绿色荧光蛋白（EGFP）的融合蛋白（NeuroD-EGFP融合蛋白）,利用倒置荧光显微镜观察NeuroD-EGFP融合蛋白的体外进入细胞的效率.40只C57BL/6J小鼠采用随机数字表法分为正常对照组、磷酸盐缓冲液（PBS）组、EGFP组、NeuroD-EGFP融合蛋白组,每组10只.除正常对照组外,其余3组动物经9 Gy γ射线全身照射后,观察NeuroD-EGFP融合蛋白在小肠上皮细胞内的分布情况和NeuroD对放射性肠损伤的治疗作用.结果 纯化得到了NeuroD-EGFP融合蛋白.细胞上清中加入NeuroD-EGFP融合蛋白后,可见绿色荧光聚集在细胞内部,表明其可穿过细胞膜进入细胞内.小鼠腹腔注射NeuroD-EGFP融合蛋白5h后,小肠绒毛上皮细胞内部有绿色荧光聚集.小鼠照射后3.5d,NeuroD-EGFP组小鼠绒毛高度高于PBS及EGFP组（F =49.49,P＜0.01）,隐窝深度及隐窝数目大于PBS及EGFP组（F=16.72、10.32,P＜0.01）.结论 NeuroD蛋白可促进放射后肠道绒毛及隐窝的修复,对放射性肠损伤具有治疗作用.

Objective To evaluate the therapeutic effect of NeuroD protein on radiation-induced intestinal injuries.Methods The expression and purification of NeuroD-enhanced green fluorescent protein （EGFP） fusion protein was performed in prokaryotic expression system.The efficiency of the fusion protein transduction into cells was monitored under fluorescence microscope.C57BL/6J mice were randomly divided into four groups with 10 mice in each group：normal control group,PBS group,EGFP group,and NeuroD-EGFP group.Besides the normal control group,the other three groups of mice received 9 Gy γ-ray total body irradiation.Intestinal tissues were collected,frozen sections were prepared to monitor the distribution of NeuroD in mice intestinal tract under fluorescence microscope,and pathological sections were prepared for H＆amp;E staining to evaluate the therapeutic effect of NeuroD protein.Results The NeuroD-EGFP fusion protein was purified by Ni-NTA column and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）.Visible green fluorescence gathered within the cells after NeuroD-EGFP fusion protein was added in the culture medium,suggesting that NeuroD-EGFP could penetrate the cell membrane into the cells.Five hours after intraperitoneal injection of NeuroD-EGFP,visible green fluorescence gathered within the intestinal epithelial cells in villi.At 3.5 d after irradiation,NeuroD-EGFP treated mice showed significantly higher villus （F =49.49,P 〈 0.01） and crypt depth （F =16.72,P 〈 0.01） and more crypts per circumference （F =10.32,P 〈 0.01） compared with PBS and EGFP groups.Conclusion NeuroD protein can accelerate the post-irradiation recovery of injured villi and crypt of intestinal tract and could be used to treat radiation-induced intestinal injuries.