【作者】 徐丹；

【导师】 王廷华；

【作者基本信息】 昆明医学院 ， 神经生物学， 2009， 硕士

【摘要】 [目的]调查大鼠脊髓全横断后miRNA的表达变化,筛查特异性变化miRNA。并在体外PC12、Hela细胞中查找特异性miRNA的靶蛋白。[方法]1、6只大鼠,随机分为假手术组（对照组）和脊髓全横断组（实验组）。术后3天取脊髓、肝脏提取总RNA,分离miRNA;2、用miRNA芯片miRCURY^TMArray筛查对照、实验组脊髓和肝脏中miRNA表达变化,查找特异性miRNA;3、TargetScan靶基因预测软件结合蛋白组学结果确定miR-138的5个靶蛋白;4、免疫组织化学检测胸段正常脊髓组织中5个靶蛋白表达情况;5、PC12、Hela细胞分为3组,分别为正常细胞组（正常组）,单纯脂质体转染组（脂质体组）和miR-138转染组（miR-138组）。分别于转染24h、48h和72h后计数PC12、Hela各组细胞数;6、分别于转染24h、48h和72h后对PC12、Hela细胞各组进行MTT检测,计算miR-138的细胞抑制率;7、免疫组织化学检测PC12、Hela细胞各组靶蛋白表达变化,每组5个样本,测定阳性染色的光密度值;8、WesternBlot检测PC12、Hela细胞各组靶蛋白表达变化,每组5个样本,测定条带光密度值;9、RT-PCR检测PC12、Hela细胞各组靶基因mRNA表达变化,每组设8个样本,检测条带灰度值;10、所有的数据用SPSS 13.0软件进行分析。【结果]1、miRNA芯片筛查出在损伤脊髓组织中miR-138特异性表达减少;2、TargetScan靶基因预测软件共预测出343个靶基因,结合蛋白组学结果共确定5个靶靶蛋白,分别是β-突轴核蛋白（synuclein,beta;Sncb）,波形蛋白（vimentin;Vim）,原肌球蛋白4（tropomyosin 4;Tpm4）,电压依从性阴离子通道2（voltage-dependent anion channel 2:VDAC2）,神经营养性酪氨酸激酶2型受体（neurotrophic tyrosine kinase,receptor,type 2;NTRK2,TrkB）;3、免疫组化检测到胸段正常脊髓组织中Sncb、Vim、Tpm4、TrkB和VDAC2都有表达;4、与PC12细胞正常组比较,脂质体组在转染后48h（P=0.01）细胞数目减少;与脂质体组比较,miR-138组在24h（P=0.001）、48h（P=0.008）和72h（P=0.000）细胞数目减少。在Hela细胞,与正常组比较,脂质体组在转染后72h（P=0.009）细胞数目减少;与脂质体组比较,miR-138组在24h（P=0.711）、48h（P=0.417）和72h（P=0.408）细胞数目没有变化;4、MTT检测结果为与正常组比较,PC12脂质体组在48h（P=0.011）和72h（P=0.000）细胞增殖活力受到明显抑制;与脂质体组比较,miR-138组在72h（P=0.045）细胞增殖活力受到明显抑制。与正常组比较.Hela脂质体组在24h（P=0.01）和72h（P=0.008）细胞增殖活力受到抑制;与脂质体组比较,miR-138组在24h（P=0.022）和48h（P=0.022）细胞增殖活力受到促进,但在72h细胞增殖活力受到明显抑制。PC12在24h细胞抑制率为-6.29275%.48h为22.62443%,72h为22.11101%;Hela在24h细胞抑制率为-15.6846%,48h为-17.299%,72h为7.813765%;5、免疫组化检测到Tpm4和TrkB在PC12和Hela细胞中都表达,Vim仅在Hela细胞中表达。Scnb和VDAC2在两种细胞中都未检测到表达。Tpm4在胞浆中着色,TrkB在胞浆和核仁中显色,Vim在胞浆和突起中显色。光密度值分析显示Hela细胞miR-138组TrkB表达减少,其它靶蛋白没有变化;6、Wetern Blot在PC12细胞中检测到Tpm4、Sncb和VDAC2表达,Sncb表达减少;在Hela细胞中检测到Tpm4和VDAC2表达,VDAC2表达减少;8、RT-PCR在两种细胞中都检测到Vim、Tpm4和VDAC2 mRNA,永检测到Sncb和TrkB表达,miR-138组与对照组比较都没有变化。[结论】1、miR-138可能在脊髓损伤的进程中发挥重要作用;2、miR-138抑制PC12、Hela细胞的增殖;3、miR-138的靶蛋白可能为Sncb、VDAC2和TrkB:4、miR-138的调控方式可能为抑制mRNA翻译,但并不降解靶基因。更多还原

【Abstract】 Objective To determined the specific miRNAs in the spinal cord after transsection and find its targets in PC12,Hela cells.Methods:1.6 adult rats were randomly devided into sham operated group(n=3) and operated group(n=3).At 3 days post operation,RNA were extracted from the spinal cord and the liver tissues for miRNA array analysis;2.total miRNAs were hybridized with miRNA array chip,then the results obtained from spinal cord and liver were compared between that of the control and the experiment group to find the specific miRNA in injuried spinal cord;3.according to primary proteomics results, TargetScan was employed to predict the possible targets of miR-138;4.To authenticate those results,target proteins obtained from thoracic segments of spinal cord in innocent rats,were investigated with immunohistochemistry;5.PC12,Hela cells were devided into 3 groups,including nomal group,lipofectamin group(control group) and miR-138 group.At 24h,48h and 72h after transfection,the numbers of PC12,Hela cell in all groups were counted;6.at 24h,48h and 72h post transfection, MTT assay were performed in all groups to evaluate the cell viability and to calculate the inhibition ratio;7.expressional changes and intracellular locations of the target proteins were detected by using immunocytochemistry and Western Blot;8.RT-PCR were used to investigate the mRNA levels of targets;9.data analysis was performed using SPSS13.0. Results.1.miR-138 was specially reduced in the spinal cord of cord transected rats;2. there were 343 targets predicted with TargetScan;assocaited with the primary proteomics results,5 targets were determined in this study;they were synuclein, beta(Sncb);vimentin(Vim);tropomyosin 4(Tpm4);voltage-dependent anion channel 2(VDAC2) and neurotrophic tyrosine kinase,receptor,type 2(NTRK2,TrkB).3. Sncb、Vim、Tpm4、TrkB and VDAC2 were detected in thoracic segments of nomal spinal cord;4.compared with that of nomal group,the numbers of PC12 cells of lipofectamin group decreased at 48h after transfection,while compared with that of the lipofectamin group,cells of miR-138 group were reduced after 24h,48h and 72h post operation.Howerver,similar phenomenon was not observed in Hela cell. lipofectamin group cells decreased at 72h after transfection.There were no significant differences between miR-138 group and lipofectamin group.5.compared with that of the nomal group,OD values of lipofectamin group decreased at 48h and 72h in PC12 cell,while at 24h,48h in Hela cell.At 72h post operation,compared with that of the control group,cell viabilities were inhibited in miR-138 group both in PC 12 and Hela cell.However,at 24h and 48h post operation,viabilities of Hela cell were promoted. The hibition ration of PC12 were -6.29275%at 24h,22.62443%at 48h and 22.11101%at 72h post cord transcection,for Hela cell they were -15.6846%, -17.299%and 7.813765%;6.Analyzed with immunocytochemistry,Tpm4 and TrkB were detected in both cell,while Vim were limited in Hela cell.Scnb and VDAC2 were not found in both cell.The immunoreactive(IR) of Tpm4 was located in cytoplasm,and immunostain of Vim were detected in cytoplasm and process,while TrkB IR could be found in cytoplasm and nucleolus.The ananlysis of grey scals value revealed that TrkB level decreased in Hela miR-138 group,compared with that of control group.7.The protein levels of Tpm4,Sncb and VDAC2 were detected in PC12 by using Western Blot,and Tpm4,VDAC2 in Hela cell.Protein expression of Sncb decreased in PC12 miR-138 group,while VDAC2 protein level decrease in Hela cell compared with that of control group.8.In both cell,mRNA expressions of Vim, Tpm4 and VDAC2 were amplified,but Sncb and TrkB could not detected.There were no significant differences of mRNA expression pattern between miR-138 group and control group.Conclusion.1.miR-138 might take an important part in spinal cord injury;2. miR-138 could inhibit the viability of PC12 and Hela cell;3.Sncb,VDAC2 and TrkB might be the potential targets of miR-138;4.The regular methods of miR-138 might be translation restrain,but not degradation of mRNA.更多还原