【作者】 张艳；

【导师】 朴丰源；

【作者基本信息】 大连医科大学 ， 劳动卫生与环境卫生学， 2016， 硕士

【摘要】 目的:研究间充质干细胞(mesenchymal stromal cells,MSCs)移植对正己烷的终毒物2,5-己二酮(2,5-Hexanedione,HD)暴露大鼠脊髓脱髓鞘损伤的保护作用,并探讨相关机制。方法:选用100只成年无特定病原菌(specified pathogen free,SPF),雄性SD大鼠,运用随机数字表将其随机分为:正常对照组(Control)、干细胞对照组(MSC)、染毒组(HD)、自身修复组(HD-NS)、干细胞修复组(HD-MSC),每组20只。以无菌生理盐水将HD稀释,通过腹腔注射方法染毒大鼠。染毒组的染毒剂量为400 mg/kg,注射量为0.3 ml/100g,对照组给予同等剂量的无菌生理盐水。每天白天染毒1次,染毒持续5周。HD模型成功后,干细胞修复组经尾静脉注射骨髓MSCs,自身修复组给予相同剂量无菌生理盐水。干细胞修复5周后,每组分别各取2只动物经4%多聚甲醛灌注固定脊髓,而每组其余的动物则提取脊髓,置于-80℃深冻冰箱中低温保存。运用卢卡斯快蓝(luxol fast blue,LFB)染色法和透射电镜法观察脊髓组织髓鞘形态的变化;采用实时定量聚合酶链反应(Quantitative real-time polymerase chain reaction,qRT-PCR)检测齿状蛋白 1(Jagged1)、Notch1、Notch胞内段(Notch intracellular domain,NICD)、无毛重组结合蛋白抑制剂(recombining binding protein suppressor of hairless,RBPJ)、发状分裂相关增强子 1(hairy and enhancer of split,Hes1)、肿瘤坏死因子(tumor necrosis factor,TNF)和核转录因子 RelB(avian reticuloendotheliosis viral oncogene-related B,RelB)基因水平的变化;应用Western Blot方法检测髓鞘碱性蛋白(myline basic protein,MBP)、Jagged1、NICD、RBPJ、Hes1、TNFα和RelB蛋白水平的变化。采用免疫荧光双染,观察 MBP 和 Hes1、RelB 和 2’,3’-环核苷酸-3’-磷酸二酯酶(2’,3’-cyclic-nucleo-tide-3’-phosphodiester-ase,CNPase)阳性表达关系。数据分析选用SPSS13.0统计软件,对实验结果进行统计学分析。结果:1.髓鞘形态变化LFB染色结果显示,HD暴露组大鼠脊髓轴索稀疏,排列不均匀,髓鞘脱失,轴突肿胀,空泡出现;HD-MSC组大鼠脊髓轴索纹理趋于正常。透射电镜结果显示,HD组髓鞘板层模糊,轴索变形,髓鞘变薄;HD-MSC组部分脱失的髓鞘修复,并有新生髓鞘。2.MBP蛋白表达Western Blot检测结果发现,HD组大鼠脊髓组织MBP蛋白的表达与Control组相比下调,差异有统计学意义(P<0.05),HD-MSC组MBP的表达与HD组相比上调,差异有统计学意义(P<0.05)。3.Hes1基因表达qRT-PCR检测结果显示,HD染毒组大鼠脊髓Hes1的mRNA表达与Control组相比上调,差异有统计学意义(P<0.01),HD-MSC组Hes1的mRNA表达与HD组相比下调,差异有统计学意义(P<0.01)。4.MBP和Hes1免疫荧光表达免疫荧光双染结果显示,HD组大鼠脊髓组织MBP 阳性表达面积低于Control组,差异有统计学意义(P<0.05),HD-MSCs组大鼠脊髓组织MBP 阳性表达面积高于HD组,差异有统计学意义(P<0.05)。HD组大鼠脊髓组织Hes1累积光密度值(IOD)值高于Control组,差异有统计学意义(P<0.01),与HD-MSCs组大鼠脊髓组织Hes1的IOD值低于HD组,差异有统计学意义(P<0.01)。5.Jagged1的mRNA和蛋白表达qRT-PCR检测结果显示,HD组大鼠脊髓组织Jagged1的mRNA表达与Control组相比上调,差异有统计学意义(P<0.05),HD-MSC组Jagged1的mRNA表达与HD组相比无统计学意义(P>0.05)。Jagged1蛋白的Western Blot检测结果与qRT-PCR检测结果一致。6.Notch1的mRNA表达qRT-PCR检测结果显示,HD组大鼠脊髓组织Notch1的mRNA表达水平与Control组相比上调,差异有统计学意义(P<0.05),HD-MSC组Notch1的mRNA表达与HD组相比,无统计学意义(P>0.05)。7.NICD的mRNA和蛋白表达qRT-PCR检测结果显示,HD组大鼠脊髓组织NICD的mRNA表达与Control组相比上调,差异有统计学意义(P<0.05),而HD-MSC组NICD的mRNA表达与HD组相比无显著差异(P>0.05)。NICD蛋白的Western Blot检测结果与qRT-PCR检测结果一致。8.RBPJ的mRNA和蛋白表达qRT-PCR和Western Blot检测结果均显示,各组之间无统计学差异(P>0.05)。9.TNF的mRNA表达qRT-PCR检测结果显示,HD染毒组大鼠脊髓TNF的mRNA表达与Control组相比上调,差异有统计学意义(P<0.01),HD-MSC组TNF的mRNA表达与HD组相比下调,差异有统计学意义(P<0.01)。10.TNFα蛋白表达Western Blot检测结果发现,HD染毒组大鼠脊髓组织TNFα的蛋白表达与Control组相比上调,差异有统计学意义(P<0.01),HD-MSC组TNFα的蛋白表达与HD组相比下调,差异有统计学意义(P<0.01)。11.RelB的mRNA和蛋白表达qRT-PCR和Western Blot检测结果显示,HD组大鼠脊髓组织RelB的mRNA和蛋白表达与Control组相比上调,差异有统计学意义(P<0.01),HD-MSC组RelB的mRNA和蛋白表达与HD组相比下调,差异有统计学意义(P<0.01)。12.CNPase和RelB免疫荧光表达免疫荧光双染结果显示,HD组大鼠脊髓组织CNPase 阳性表达细胞数量低于Control组,差异有统计学意义(P<0.01),HD-MSC组大鼠脊髓组织CNPase 阳性表达细胞数量高于HD组,差异有统计学意义(P<0.01)。HD组大鼠脊髓组织RelB阳性表达细胞数量高于Control组,差异有统计学意义(P<0.01),HD-MSC组大鼠脊髓组织中RelB阳性表达细胞数量低于HD组,差异有统计学意义(P<0.01)。结论:MSCs对HD脱髓鞘损伤具有保护作用;MSCs髓鞘保护作用与Notch通路相关蛋白表达无关,而与TNFα/RelB介导的Notch活性抑制有关。更多还原

【Abstract】 Objective:Study on the protective effect of mesenchymal stem cells(mesenchymal stromal cells,MSCs)transplantation on the final poison hexane 2,5-hexanedione(2,5-Hexanedione,HD)exposed rats with spinal cord demyelination injury,and explore related mechanism.Methods:100 adult SPF grade male SD rats were randomly divided into:control group(Control),stem cells in the control group(MSC),exposed group(HD),self-repair group(HD-NS),stem cells repair group(HD-MSC),20 in each group.0.9%sterile saline to HD diluted exposed rats by intraperitoneal injection.Exposure dose group was 400 mg/kg,injection volume 0.3 ml/100g,the control group received the same dose of saline.Exposed once a day for five weeks.After the HD model was successful,stem cells repair group via tail vein injection of bone marrow extract isolated MSCs,self-repair group was given the same dose of saline.Five weeks later,each group depicting two animals with 4%paraformaldehyde perfusion fixation spinal cord,while the rest of the animals in each group are extracted spinal cord,placed in a deep freeze-80℃ refrigerator cold storage.LFB staining and transmission electron microscopy was used to observe changes in the myelin sheath forms;qRT-PCR detection of Jaggedl,Notchl,NICD,RBPJ,Hesl,TNF and RelB gene level;Application Western Blot assay MBP,Jaggedl,NICD changes RBPJ,Hesl,TNFa and RelB protein levels.Immunofluorescence staining was observed MBP and Hesl,RelB and 2’,3’-cyclic nucleotide-3’-phosphodiester-ase(CNPase)expression relationships.With SPSS 13.0 statistical software for statistical analysis of test results.Results:1.Myelin morphological changes LFB staining showed morphological changes,HD-exposed rat spinal cord tissue sections were observed axonal light sparse,uneven arrangement,demyelination,axonal swelling and vacuoles.HD-MSC rat spinal cord tissue sections,axonal texture become normal.TEM results showed,HD group myelin lamellar blurred,deformable axonal myelin thinning.HD-MSC group demyelination myelin repair part,and a newborn myelin.2.MBP protein expression Western Blot test results found that rats exposed spinal cord HD group MBP protein expression down-regulated compared with the control group,the difference was statistically significant(P<0.05),HD-MSC group MBP expression and HD group compared raised,the difference was statistically significant(P<0.05).3.Hesl gene expression qRT-PCR test results showed,HD exposed rats spinal cord Hesl mRNA expression compared with the Control group increases,the difference was statistically significant(P<0.01),HD-MSC group of Hesl mRNA expression down-regulated compared with HD group,the difference was statistically significant(P<0.01).4.MBP and Hesl immunofluorescence expression immunofluorescence staining showed,HD exposed rats spinal cord MBP-stained area decreased compared with the Control group,the difference was statistically significant(P<0.05),the MBP-stained area of HD-MSCs group was increased compared with HD group,the difference was statistically significant(P<0.05).The value of Hesl integrated optical density(IOD)in HD exposed rats spinal cord was higher than the Control group,the difference was statistically significant(P<0.01),the HD-MSCs group was less than the HD group,the difference there was statistically significant(P<0.01).5.Jaggedl mRNA and protein expression qRT-PCR test results showed,HD exposed rats spinal cord Jagged1 mRNA expression compared with the Control group increases,the difference was statistically significant(P<0.05),HD-MSC group Jaggedl mRNA expression and HD group compared no significant difference(P>0.05).Western Blot test results of Jaggedl protein consistent with the qRT-PCR test results.6.mRNA expression of Notch1 qRT-PCR results showed that HD rat spinal cord tissue Notchl mRNA levels increase when compared with the Control group,the difference was statistically significant(P<0.05),HD-MSC group Notchl mRNA expression compared with the HD group was not statistically significant(P>0.05).7.NICD mRNA and protein expression qRT-PCR test results showed that HD group rat spinal cord tissue NICD mRNA expression compared with the Control group increases,the difference was statistically significant(P<0.05),but HD-MSC group NICD mRNA expression and HD group compared no significant difference(P>0.05).NICD protein Western Blot test results and qRT-PCR test results consistent.8.RBPJ mRNA and protein expression qRT-PCR and Western Blot test results showed the test results found no significant difference between the groups(P>0.05).9.TNF mRNA expression qRT-PCR test results showed,HD exposure group mRNA expression in rat spinal cord and the Control group compared increases of TNF,the difference was statistically significant(P<0.01),HD-MSC group TNFa protein was lower compared to HD group,the difference was statistically significant(P<0.01).10.TNFa protein expression Western Blot test results found,HD rats exposed spinal cord tissue TNFa protein expression compared with the Control group increases,the difference was statistically significant(P<0.01),HD-MSC group TNFa protein expression and HD group compared to lower,the difference was statistically significant(P<0.01).11.RelB mRNA and protein expression qRT-PCR and Western Blot test results showed,HD rats exposed spinal cord RelB mRNA and protein expression compared with the Control group increases,the difference was statistically significant(P<0.01),HD-MSC group RelB mRNA and protein expression and HD group compared to lower,the difference was statistically significant(P<0.01).12.CNPase and RelB immunofluorescence expression Immunofluorescence showed that HD exposure group spinal cord CNPase positive cells number was lower than the Control group,the difference was statistically significant(P<0.01),HD-MSC group’s number of CNPase positive cells was higher than the HD group,the difference was statistically significant(P<0.01).HD exposure group spinal cord tissue RelB expression cells number was higher than Control group,the difference was statistically significant(P<0.01),while HD-MSC spinal cord RelB positive cells number was lower than HD group,the difference was statistically significant(P<0.01).Conclusion:MSCs for HD demyelinating injury in rats,its protective effect of myelin has nothing to do with the Notch pathway-related protein expression,but with TNFa/RelB mediated inhibition of Notch activity.更多还原