最新microRNA-9-1慢病毒载体构建及对小鼠骨髓间质干细胞诱-….doc

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1、Four short words sum up what has lifted most successful individuals above the crowd: a little bit more.-author-datemicroRNA-9-1慢病毒载体构建及对小鼠骨髓间质干细胞诱-microRNA-9-1慢病毒载体构建及对小鼠骨髓间质干细胞诱导分化为神经细胞的影响*MicroRNA-9-1慢病毒载体的构建及其对小鼠骨髓间质干细胞诱导分化为神经细胞的影响*景黎君，贾永林，鲁晶晶，韩 瑞，王舒阳，李尽义，彭 涛，贾延劼（郑州大学第一附属医院神经内科，河南 郑州 450052）摘 要 目

2、的：探讨microRNA-9-1在体外诱导小鼠骨髓间充质干细胞（MSCs）向神经细胞分化中的作用。方法: 构建小鼠microRNA-9-1慢病毒载体（microRNA-9-1-LV）并感染小鼠MSCs，筛选最适感染复数（MOI）；实验分为未感染组、感染组（感染microRNA-9-1-LV组）、阴性对照组（感染FU-RNAi-NC-LV）；采用-巯基乙醇诱导感染后小鼠MSCs分化为神经细胞。倒置荧光显微镜下观察MSCs感染后荧光表达情况；采用免疫细胞化学染色检测神经元烯醇化酶（NSE）、神经微管结合蛋白（MAP-2）和胶质纤维酸性蛋白（GFAP）的表达变化；PT-PCR检测MAP-2 mRNA

3、的表达变化；MTT方法检测细胞存活率。结果：(1) 阳性克隆PCR证明小鼠microRNA-9-1慢病毒载体构建成功，孔稀释法测定病毒滴度为11012 TU/L。(2) 倒置显微镜下观察小鼠microRNA-9-1慢病毒载体感染成功，MOI值为20，感染4 d时感染率最高，细胞存活率较高，感染率可达91.3% 4.2%。(3) -巯基乙醇可以诱导MSCs向神经细胞分化，其中以感染组诱导效果最佳，NSE和MAP-2的表达率显著高于其它各组（P0.05）。结论：(1) microRNA-9-1可高效感染小鼠MSCs。(2) 感染miRNA-9-1-LV后MSCs经-巯基乙醇诱导向神经细胞分化比率增

4、加。关键词 MicroRNA-9-1；骨髓间充质干细胞；神经元；慢病毒中图分类号 R329.21 文献标识码 A *基金项目 国家自然科学基金资助项目（No. 30770758）；河南省教育厅自然科学研究计划项目（No. 2008A320032）通讯作者 Tel: 0371-66295112; E-mail: jiayanjie1971Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neuronsJING Li-jun,

5、 JIA Yong-lin, LU Jing-jing, HAN Rui, WANG Shu-yang, LI Jin-yi, PENG Tao, JIA Yan-jie(Department of Neurology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China. E-mail: jiayanjie1971)ABSTRACT AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal s

6、tem cells (MSCs) to differentiate into neurons. METHODS: The lentiviral vector of microRNA-9-1 (microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group (transfected with microRNA-9-1-LV) and negative control group (transf

7、ected with FU-RNAi-NC-LV). MSCs were treated with -mercaptoethanol (-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtubulin-associated protein 2

8、 (MAP-2) was detected by RT-PCR. The expression of neural cell specific markers, neuron-specific enolase (NSE), MAP-2 and glial fibrillary acidic protein (GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed succ

9、essful construction of mouse microRNA-9-1-LV. The virus titer was 11012 TU/L (TU, transduction unit). The best transfection efficiency (up to 91.3% 4.2%) and survival rate appeared when multiply of infection (MOI) was 20 and on the 4th day. -ME induced MSCs to differentiate into neurons and the best

10、 efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other groups (P0.05）；感染24 h，感染组MTT值显著下降，与各对照组MTT值比较均有显著差异（P0.05），各对照组之间无显著差异。感染48 h、72 h、96 h后MTT值虽较24 h有所上升（P0.05），但与其他组MTT比较仍有显著差异（P0.0

11、5），见表3。4 -巯基乙醇体外诱导MSCs分化为神经细胞未感染组MSCs诱导5 h，部分细胞出现神经细胞改变，胞体收缩呈多角形或圆形，突起细长，有数个分支，部分在局部形成网络。感染micro-RNA-9-1-LV组诱导后，细胞形态变化更为迅速和显著，诱导5 h多数细胞胞体收缩呈圆形、锥形，细胞突起细长，交织成网，形成较典型的神经细胞结构，少量细胞脱落、死亡。阴性对照组和未感染组类似，见图3。5 免疫细胞化学染色法感染组诱导5 h，NSE和MAP-2的表达率分别为87.2% 2.0%和85.5% 0.8%，显著高于其它各组（P0.05）。各组GFAP表达率均小于1%，无显著性差异，见图4、表4

12、。6 RT-PCR结果3组分别诱导分化5 h均表达MAP-2 mRNA，未转染组和阴性对照组无显著差异，感染microRNA-9-1组表达MAP-2 mRNA较前2组明显增高，有显著差异，见图5、图6。讨 论1 慢病毒载体感染技术 慢病毒基因转导载体包容量大，能够感染任何类型神经细胞，其携带的目的基因可整合到宿主细胞基因组使外源基因获得长期稳定表达，且不编码病毒蛋白，在中枢神经系统中也不会引起免疫反应5。在人和小鼠脑组织中成熟的microRNA-9有3种不同形式，microRNA-9-1、-9-2和-9-3，其原始转录本分别位于3、13、7号染色体上。通过用维甲酸处理小鼠P19细胞发现，mic

13、roRNA-9-1在诱导后有明显变化，经历急剧上升至迅速下降过程，且处理后神经特异性标志物MAP-2逐渐上升，提示microRNA-9-1在神经分化中起着重要调控作用和生物指示作用6。据此本研究采用采用三质粒表达系统即pGC-LV载体、pHelper 1.0载体和pHelper 2.0载体来构建慢病毒载体，建立microRNA-9-1增强体外模型，并探讨其在MSCs诱导分化为神经细胞中的作用。2 MicroRNA-9在MSCs向神经细胞分化中的作用 MicroRNA可以作为发育开关以及微调发育程序来调控生物体内多种发育过程7。MicroRNA-9是脑组织特异表达的一种小RNA，它在小鼠胚胎干细

14、胞和肿瘤细胞的分化中其重要调控作用8。研究发现，microRNA-9在神经干细胞向神经元分化过程中起重要调控作用9，通过影响转录激活因子STAT3信号通路来促进神经分化5。通过Fgf信号通路调控中脑后脑交接区（midbrain-hindbrain boundary，MHB）的组织活性10。 Krichevsky等4研究发现在胚胎发育的第5阶段microRNA-9/9*表达较高，此阶段是神经元前体向神经元和胶质细胞分化的关键时期，抑制神经前体细胞中microRNA-9表达可使神经元数量减少29% - 31%。MicroRNA-9调控着由胚胎干细胞衍生来的神经祖细胞的增殖和迁移，由其直接调控的一个

15、靶基因可以编码一种19 kD的磷酸蛋白stathmin，该蛋白特异性在神经祖细胞上表达，可以增加微观的不稳定性，从而促进细胞定向迁移11,12。本研究发现感染microRNA-9-1慢病毒载体的MSCs向神经细胞分化效率增加，考虑可能和microRNA-9的上述作用有关。MSCs是一种成体干细胞，可以向骨、软骨、脂肪等多种组织分化，研究发现其可以向神经细胞分化13,14。与神经干细胞定向分化所不同，MSCs分化为神经细胞是一种横向分化方式，其调控因子目前了解较少。Greco等3发现在人MSCs分化为神经细胞过程中，microRNA在其中起着重要调控作用。但与神经干细胞分化机制是否相同，micr

16、oRNA-9是否参与分化过程尚未见报道。本研究首次采用感染microRNA9-1慢病毒载体，构建microRNA-9-1增强模型，观察其在MSCs诱导分化中的作用。本研究发现，microRNA-9-1慢病毒载体可高效感染小鼠MSCs，感染后报告基因GFP可稳定表达。本研究MTT结果发现，感染microRNA-9-1慢病毒后，MSCs存活率下降，但感染仅含有报告基因的慢病毒载体后MSCs存活率变化不大，表明microRNA-9在MSCs保持细胞自我更新、分裂、凋亡等生理功能中起到一定作用。本研究发现，与未感染和感染仅含有GFP的慢病毒的MSCs相比感染microRNA9-1慢病毒后，MSCs向神

17、经细胞分化效率显著提高，神经细胞早、中期标志物NSE和成熟神经细胞特异性标志物MAP-2表达均明显增高，且未见GFAP表达。这提示microRNA-9-1可能在MSCs横向分化神经细胞中起作用，提高microRNA-9-1的表达，可以促进MSCs向神经细胞分化。单独感染microRNA-9-1慢病毒并不能使MSCs向神经细胞分化，还必须在诱导剂（-巯基乙醇等）的作用下才能分化。这说明microRNA9-1在MSCs在横向分化为神经细胞过程中不是唯一的，可能是对一些信号途径调控，如Notch信号途径15，从而提高诱导效率。但是具体机制尚不明确。综上所述，本研究采用慢病毒感染技术，建立microR

18、NA-9-1过表达细胞模型，从而促进MSCs向神经细胞的分化效率，提示microRNA-9-1在MSCs诱导分化为神经细胞过程中可能起促进作用。参考文献1 Lagos-Quintana M, Rauhut R, Yalcin A, et al. Identification of tissue-specific microRNAs from mouseJ. Curr Biol, 2002, 12 (9):735-739.2 原清涛, 邓宇斌, 李树浓. 骨髓间质干细胞诱导分化为神经元及其应用的研究进展J. 中国病理生理杂志, 2004, 20(11):2139-2142.3 Greco SJ,

19、 Rameshwar P. MicroRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cellsJ. Proc Natl Acad Sci U S A, 2007, 104 (39):15484-15489.4 Krichevsky AM, Sonntaq KC, Isacson O, et al. Specific microRNAs modulate embryonic stem cell-derived neurogene

20、sisJ. Stem Cells, 2006, 24 (4):857-864.5 Simmons A, Whitehead RP, Kolokoltsov AA, et al. Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vi

21、troJ. Virol J, 2006, 3:8.6 Ko MH, Kim S, Hwang do W, et al. Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cellsJ. FEBS J, 2008, 275 (10):2605-2616.7 Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, fun

22、ction and decay J. Nat Rev Genet, 2010, 11 (9):597-610.8 Delaloy C, Gao FB. microRNA-9 multitasking near organizing centersJ. Nat Neurosci, 2008, 11 (6):625-626.9 Conaco C, Otto S, Han JJ, Mandel G. Reciprocal actions of REST and a microRNA promote neuronal identityJ. Proc Natl Acad Sci U S A, 2006,

23、 103 (7):24222427.10 Leucht C, Stiqloher C, Wizenmann A, et al. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundaryJ. Nat Neurosci, 2008, 11 (6):641-648. 11 Delaloy C, Liu L, Lee JA, et al. MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-deri

24、ved neural progenitorsJ. Cell Stem Cell, 2010, 6 (4):323-335.12 Nobuko U. microRNA-9 controls a migratory mechanism in human neural progenitor cellsJ. Cell Stem Cell, 2010, 6 (4):294-296.13 Cho KJ, Trzaska KA, Greco SJ, et al. Neurons derived from human mesenchymal stem cells show synaptic transmiss

25、ion and can be induced to produce the neurotransmitter substance P by interleukin-1J. Stem cells, 2005, 23 (3):383-391.14 施雪英, 陈先文, 胡慧敏, 等. 三七总皂苷和维甲酸联合诱导大鼠MSCs分化为神经元样细胞J. 中国病理生理杂志, 2007, 23(3):612-613.15 Wang Z, Li Y, Kong D, et al. Cross-talk between miRNA and Notch signaling pathways in tumor deve

26、lopment and progressionJ. Cancer Lett, 2010, 292 (2):141-148.Figure 1. PCR results for constructed microRNA-9-1-LV vector. A: enzyme-digested product. 1: marker; 2: the pFU-GW-RNAi plasmid without digestion; 3: the pFU-GW-RNAI plasmid digested by Xba I and Hpa I. B: PCR for cloned target gene. 1: th

27、e group with ddH2O; 2: the group with empty vector; 3: the group with GAPDH; 4: marker; 5-12: the groups with transformants of microRNA-9-1.图1 慢病毒载体构建PCR结果Figure 2. The cell infection rate (48 h after transfection, 200). A: transfected group; B: negative control group.图2 慢病毒感染24 h细胞感染率Figure 3. -ME

28、induced MSCs into neurons (200). A: transfected group; B: negative control group.图3 -巯基乙醇诱导各组MSCs向神经细胞分化Figure 4. Expression of MAP-2 in -ME-induced differentiation of MSCs into neurons (5 h after induction, 200). A: transfected group; B: negative control group.图4 -巯基乙醇诱导各组MSCs向神经细胞分化过程中MAP-2的表达Figu

29、re 5. The RT-PCR for MAP-2. A: transfected group；B: negative control group; C: non-transfected group.图5 MAP-2 RT-PCR结果Figure 6. MAP-2 mRNA expression. MeanSD. n = 15. * P0.05 vs other groups.图6 MAP-2 mRNA各组比值表1 引物序列Table 1. Sequences of the primersNamePrimer sequenceProduct (bp)MicroRNA-9-1Forward:

30、5-TCATAAAGCTAGATAACCGAAGAT-3405Reverse: 5-TCCAGAGGCGACCCCAGAGC-3MAP-2Forward: 5-CCCAAGAACCAACAAGATGAA-3710Reverse: 5-AATCAAGGCAAGACATAGCGA-3-actinForward: 5-TGACGGGGTCACCCAACTGTGCCCATCTA-3105Reverse: 5-CTAGAAGCATTTGCGGACGATGGAGGG-3表2 细胞感染率Table 2. The cell infection rates (%. MeanSD. n=12)GroupBefor

31、e transfection24 h after transfection48 h after transfection72 h after transfection96 h after transfectionTransfected43.58.364.53.184.52.174.02.674.33.4Negative control42.59.163.82.982.52.372.62.273.92.8表3 MSCs MTT结果Table 3. The MTT values of MSCs (%. MeanSD. n=6)GroupBefore transfection24 h after t

32、ransfection48 h after transfection72 h after transfection96 h after transfectionNon-transfected73.54.271.32.273.92.274.02.674.33.4Transfected73.14.850.73.9*53.11.6*64.83.7*59.22.3*Negative control72.83.569.53.171.02.672.62.273.92.8* P0.05 s other groups.表4 -巯基乙醇诱导5 h各组细胞免疫化学染色结果Table 4. The results of immunocytochemical staining in each group 5 h after induction by -ME (%. MeanSD. n = 6