Brn-4在大鼠海马神经干细胞向神经元分化中的作用
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摘要
目的:研究Brn-4在海马神经干细胞向神经元分化中的作用,探讨穹窿海马伞切割后海马神经再生过程中NSCs向神经元分化的生物学机制。方法:实验一免疫荧光双标:分别制备切割穹窿海马伞侧和正常侧海马提取液,将体外培养的鼠胚海马神经干细胞球分成切割组、正常组和对照组,其中前两组中分别加入切割穹窿海马伞侧海马提取液和正常侧海马提取液。各组培养细胞分别于第3、7、14和21d行Brn-4/MAP-2免疫荧光双标检测。用图像处理系统分别计数每视野下Brn-4阳性细胞和Brn-4/MAP-2双标神经元数,并测量双标神经元的胞体面积、细胞周长及Brn-4的免疫荧光强度。用Stata8.0软件对数据进行方差分析和组间比较。实验二RNA干扰:将加入切割穹窿海马伞海马提取液、体外培养的鼠胚海马神经干细胞球分别接种入6孔培养板和24孔培养板中。筛选有效序列、优化转染条件后将接种于6孔培养板和24孔培养板中的细胞均随机分成沉默组和未沉默组,沉默组中加入针对Brn-4的有效siRNA序列。转染后第3、7、14和21d时分别提取6孔培养板中的细胞总RNA,行半定量RT-PCR,以各泳道中Brn-4与GAPDH条带的光密度比值表示Brn-4 mRNA的相对表达量;24孔培养板中的细胞于第3、7、14和21d时分别行时分别行Brn-4/MAP-2免疫荧光双标检测,用图像处理系统计数Brn-4阳性细胞和Brn-4/MAP-2双标神经元数,测量双标神经元的胞体面积、细胞周长以及Brn-4的免疫荧光强度。用Stata8.0软件对数据进行方差分析和组间比较。
结果:实验一各组中Brn-4/MAP-2双标神经元数、胞体面积和细胞周长随时间推移均呈现由低到高再逐渐降低、Brn-4的免疫荧光强度也呈现由低到高再逐渐降低的趋势,且两者均于培养后第14d达到高峰的时相性特点;同一时间点的三组中,切割组双标神经元最多、胞体最大、突起最丰富,且Brn-4的免疫荧光也最强,正常组次之,而对照组最差。统计分析表明,除3d时各组Brn-4免疫荧光强度相比差异无统计学意义(P>0.05)外,其余数据组间比较均有统计学意义(P<0.01)。实验二本实验优化的转染条件合适,转染试剂无明显毒副作用,转染效率为95%。6孔培养板中第3、7、14d时同一时间点的沉默组和未沉默组相比,沉默组中Brn-4 mRNA的相对表达量明显低于未沉默组,组间比较P均<0.01,第21d时组间差异缩小,P<0.05。24孔培养板中同一时间点的两组比较,沉默组Brn-4/MAP-2双标细胞数少,胞体小,突起不丰富,且Brn-4的免疫荧光弱,与未沉默组比较,P均<0.01。结论:在切割穹窿海马伞海马提取液促进体外培养的海马神经干细胞向神经元分化过程中,Brn-4的表达明显增强;加入针对Brn-4的siRNA后Brn-4 mRNA和蛋白表达量明显降低,且神经干细胞向神经元的分化明显受到影响。提示Brn-4在大鼠海马神经干细胞向神经元分化过程中可能起着重要作用。
Objective: To study the role of Brn-4 on the neural stem cells from hippocampus differentiating into neurons, and to investigate the biology mechanism of neural regeneration and reparation after fimbria/fornix transection. Methods: Immunofluorescence double-labeling in experiment one: NSCs isolated and expanded from the hippocampus of embryonic rat were divided into 3 groups: the transection group, the normal group and the control group. Then the extracts from the fimbria/fornix transected and intact hippocampi of adult SD rats previously obtained were respectively added into the transection and the normal group. The neurons differentiated from NSCs were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after culturing. The number of Brn-4 positive cells, the intensity of immuno- fluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double- labeling positive neurons were analyzed by image processing software respectively. Stata8.0 statistical software was adopted to analyze the results. RNA interference in experiment two: NSCs isolated and expanded from the hippocampus of embryonic rat and cultured in vitro were planted into 6-well and 24-well cell culture plate respectively with the extract from the fimbria/fornix transected hippocampi. Then the cells were divided into the silenced group and the unsilenced group from two plates respectively after siRNA sequence filtered and transfection condition optimized, and the effective fragment was added into the silenced group. The total RNA was extracted from cells of 6-well cell culture plate on the 3rd, 7th, 14th and 21st day after transfection. The relative level of Brn-4 mRNA expression was indicated by the ratio of the optical density value of Brn-4 to that of GAPDH by RT-PCR. The cells of 24-well cell culture plate were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after transfection. The number of Brn-4 positive cells, the intensity of immunofluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double-labeling positive neurons were measured respectively by image processing system. The analysis of variance and group comparison were applied with Stata8.0 statistical software. Results: In experiment one, the number of Brn-4/MAP-2 double-labeling positive neurons and its body area, perimeter increased gradually after transfection. And the peak appeared on the 14th day, then the growing status degraded later. The change of the intensity of immunofluorescence of Brn-4 showed the same tendency. Among three groups, the number, body area and perimeter of double-labeling cells were the best in the transection group. Compared with the control group, the growing status was better in the normal group. The differences were significant between any two groups except the intensity of immunofluorescence of Brn-4 on the 3rd day among three groups (P<0.01). The results in the experiment two showed that the optimized transfection condition was suitable and toxico side effect of transfection reagent was indistinct. It also suggested that the efficiency of transfection was 95 percent. The relative level of Brn-4 mRNA expression of the silenced group was obviously lower than the unsilenced group on the 3rd, 7th, 14th day respectively(P<0.01), and the difference decreased on the 21st day between the two groups(P<0.05). The Brn-4/MAP-2 double-labeling positive neurons were fewer, the bodies were smaller, and the intensity of immunofluorescence of Brn-4 was thinner in the silenced group compared with the unsilenced group significantly(P<0.01). Conclusion: The expression level of Brn-4 was enhanced obviously when the neural stem cells differentiated into neurons facilitated by the extracts from the fimbria/fornix transected hippocampi. The expression of Brn-4 mRNA and protein were degraded after the siRNA aimed directly at the Brn-4 gene, and the differentiation of the neural stem cells into neurons was influenced obviously. The results suggested that Brn-4 may play an important role in the process of neural stem cells differentiating into neurons.
结果:实验一各组中Brn-4/MAP-2双标神经元数、胞体面积和细胞周长随时间推移均呈现由低到高再逐渐降低、Brn-4的免疫荧光强度也呈现由低到高再逐渐降低的趋势,且两者均于培养后第14d达到高峰的时相性特点;同一时间点的三组中,切割组双标神经元最多、胞体最大、突起最丰富,且Brn-4的免疫荧光也最强,正常组次之,而对照组最差。统计分析表明,除3d时各组Brn-4免疫荧光强度相比差异无统计学意义(P>0.05)外,其余数据组间比较均有统计学意义(P<0.01)。实验二本实验优化的转染条件合适,转染试剂无明显毒副作用,转染效率为95%。6孔培养板中第3、7、14d时同一时间点的沉默组和未沉默组相比,沉默组中Brn-4 mRNA的相对表达量明显低于未沉默组,组间比较P均<0.01,第21d时组间差异缩小,P<0.05。24孔培养板中同一时间点的两组比较,沉默组Brn-4/MAP-2双标细胞数少,胞体小,突起不丰富,且Brn-4的免疫荧光弱,与未沉默组比较,P均<0.01。结论:在切割穹窿海马伞海马提取液促进体外培养的海马神经干细胞向神经元分化过程中,Brn-4的表达明显增强;加入针对Brn-4的siRNA后Brn-4 mRNA和蛋白表达量明显降低,且神经干细胞向神经元的分化明显受到影响。提示Brn-4在大鼠海马神经干细胞向神经元分化过程中可能起着重要作用。
Objective: To study the role of Brn-4 on the neural stem cells from hippocampus differentiating into neurons, and to investigate the biology mechanism of neural regeneration and reparation after fimbria/fornix transection. Methods: Immunofluorescence double-labeling in experiment one: NSCs isolated and expanded from the hippocampus of embryonic rat were divided into 3 groups: the transection group, the normal group and the control group. Then the extracts from the fimbria/fornix transected and intact hippocampi of adult SD rats previously obtained were respectively added into the transection and the normal group. The neurons differentiated from NSCs were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after culturing. The number of Brn-4 positive cells, the intensity of immuno- fluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double- labeling positive neurons were analyzed by image processing software respectively. Stata8.0 statistical software was adopted to analyze the results. RNA interference in experiment two: NSCs isolated and expanded from the hippocampus of embryonic rat and cultured in vitro were planted into 6-well and 24-well cell culture plate respectively with the extract from the fimbria/fornix transected hippocampi. Then the cells were divided into the silenced group and the unsilenced group from two plates respectively after siRNA sequence filtered and transfection condition optimized, and the effective fragment was added into the silenced group. The total RNA was extracted from cells of 6-well cell culture plate on the 3rd, 7th, 14th and 21st day after transfection. The relative level of Brn-4 mRNA expression was indicated by the ratio of the optical density value of Brn-4 to that of GAPDH by RT-PCR. The cells of 24-well cell culture plate were detected by immunofluorescence double-labeling of Brn-4 and MAP-2 on the 3rd, 7th, 14th and 21st day after transfection. The number of Brn-4 positive cells, the intensity of immunofluorescence of Brn-4 and the number, area, perimeter of Brn-4/MAP-2 double-labeling positive neurons were measured respectively by image processing system. The analysis of variance and group comparison were applied with Stata8.0 statistical software. Results: In experiment one, the number of Brn-4/MAP-2 double-labeling positive neurons and its body area, perimeter increased gradually after transfection. And the peak appeared on the 14th day, then the growing status degraded later. The change of the intensity of immunofluorescence of Brn-4 showed the same tendency. Among three groups, the number, body area and perimeter of double-labeling cells were the best in the transection group. Compared with the control group, the growing status was better in the normal group. The differences were significant between any two groups except the intensity of immunofluorescence of Brn-4 on the 3rd day among three groups (P<0.01). The results in the experiment two showed that the optimized transfection condition was suitable and toxico side effect of transfection reagent was indistinct. It also suggested that the efficiency of transfection was 95 percent. The relative level of Brn-4 mRNA expression of the silenced group was obviously lower than the unsilenced group on the 3rd, 7th, 14th day respectively(P<0.01), and the difference decreased on the 21st day between the two groups(P<0.05). The Brn-4/MAP-2 double-labeling positive neurons were fewer, the bodies were smaller, and the intensity of immunofluorescence of Brn-4 was thinner in the silenced group compared with the unsilenced group significantly(P<0.01). Conclusion: The expression level of Brn-4 was enhanced obviously when the neural stem cells differentiated into neurons facilitated by the extracts from the fimbria/fornix transected hippocampi. The expression of Brn-4 mRNA and protein were degraded after the siRNA aimed directly at the Brn-4 gene, and the differentiation of the neural stem cells into neurons was influenced obviously. The results suggested that Brn-4 may play an important role in the process of neural stem cells differentiating into neurons.
引文
1 Reynolds BA and Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992; 255(5052): 1707-1710
2 Richards LJ, Kilpatrick TJ and Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci USA, 1992; 89(18): 8591-8595
3 Morshead CM, Reynolds BA, Craig CG, et al. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron, 1994; 13(5): 1071-1082
4 Mckay R. Stem cells in the central nervous system. Scinece, 1997; 276 (5309): 66-71
5 Gage FH. Mammalian neural setm cells. Scinece, 2000; 287(5457): 1433-1438
6 Gage FH. Stem cells of the central nervous system. Curr Opin Neuro- biol, 1998; 8(5): 671-676
7 Frisen J, Lothian C, Lendahl U. Central nervous system setm cells in the embryo and adult. Cell Mol Life Sci, 1998; 54(9): 925-945
8 Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell, 1997; 88(3): 287-298
9 Barinaga M. Fetal neuron grafts pave the way for stem cell therapies. Science, 2000; 287(5457): 1421-1422
10 Winkler C, Fricker RA, Gates MA, et al. Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain. Mol Cell Neurosci, 1998; 11(3): 99-116
11 Bush G, diSibio G, Miyamoto A, et al. Ligand-induced signaling in the absence of furin processing of Notch 1. Dev Biol, 2001: 229(2): 494- 502
12 Ling ZD, Potter ED, Lipton JW, et al. Differentiation of mesen-cephalic progenitor cells into dopaminergic neurons by cytokines. Exp Neurol, 1998; 149(2): 411-423
13 Vicario-Abejon C, Collin C, Tsoulfas P, et al. Hippocampal stem cells differentiate into excitatory and inhibitory neurons. Eur J Neurosci, 2000; 12(2): 677-688
14 Josephson R, Muller T, Pickel J, et al. POU transcription factors control expression of CNS stem cell-specific genes. Development, 1998; 125(16): 3087-3100
15王永刚,冯忠堂,王延华,等.神经干细胞分化调控的研究.中华神经外科杂志. 2002; 18(5): 339-341
16 Gradwohl G, Fode C, Guillemot F. Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors. Dev Biol, 1996; 180(1): 227-241
17 Gehring WJ. The homeobox in perspective. Trends Biochem Sci, 1992; 17(8): 277-280
18 Lewis EB. Clusters of master control genes regulate the development of higher organisms. JAMA, 1992; 267(11): 1524-1531
19 Mihailescu D, Kury P and Monard D. An octamer-binding site is crucial for the activity of an enhancer active at the embryonic met-/mesencephalic junction. Mech Dev, 1999; 84(1-2): 55-67
20金国华,张新化,田美玲,等.大鼠海马内移植神经干细胞的存活和迁移.神经解剖学杂志, 2003; 19(4): 378-382
21张新化,金国华,秦建兵,等.穹窿海马伞切割侧海马对植入神经干细胞分化为神经元的影响.神经解剖学杂志,2004;20(4):360-364
22金国华,张新化,田美玲,等.穹窿海马伞切割侧海马提取液对神经干细胞分化为神经元的促进作用.解剖学报, 2004;40(2):141-145
23 Shimazaki T, Arsenijevic Y, Ryan AK, et al. A role for the POU-Ⅲtranscription factor Brn-4 in the regulation of striatal neuron precursor differentiation. EMBO J, 1999; 18(2): 444-456
24王磊,金国华,秦建兵,等.穹窿海马伞切割大鼠海马内Brn-4mRNA的表达变化.解剖学报, 2006; 37(4): 387-390
25王磊,金国华,秦建兵,等.切割穹窿海马伞大鼠海马内Brn-4 mRNA的表达变化-原位杂交法.解剖学报, 2007; 38(4): 385-389
26 Zhang xinhua, Jin guohua, Wang lei, et al. Brn-4 is up-regulated in the deafferented hippocampus and promotes neuronal differentiation of neural progenitors in vitro. Hippocampus, in press
27田美玲,胡文忠,金国华,等. Brn-4抗体对大鼠海马神经干细胞分化为神经元的影响.神经解剖学杂志, 2008; 24(1): 85-88
28 Fire A, Xu S, Montogomery MK, et a1. Potent and specific genetic interference by double stranded RNA in caenorhabditis elegans. Nature, 1998; 391(6669): 806-811
29 Baulcombe DC. RNA as a target and an initiator of post- transcriptional gene silencing in transgenic plants. Plant Mol Biol, 1996; 32(1-2): 79-88
30 Hammond SM, Boettcher S, Caudy AA, et al. Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science, 2001; 293 (5532): 1146-1150
31 Miyagishi M, Taira K. RNAi expression vectors in mammalian cells. Methods Mol Biol, 2004; 252: 483-491
32 Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296
33 Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409(6818): 363-366
34 Guo S,Kemphues KJ. Par-l,a gene required of establishing Polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asym- metrically distributed. Cell, 1995; 81(4): 611-620
35黄镇,金国华,张新化,等.提高成年大鼠神经干细胞单克隆形成率的方法.解剖学报, 2002; 33(6): 594-598
36衣昕,金国华,田美玲,等.壳聚糖支架与神经干细胞生物相容性的研究.神经解剖学杂志, 2007; 23(5): 506-510
37董传明,金国华,秦建兵,等.神经生长因子对穹窿海马伞切割后海马自体神经干细胞增殖和向神经元分化的影响.解剖学报, 2007; 38(6): 642-646
38 Dutton R,Bartlett PF.Precursor cells in the subventricular zone of the adult mouse are actively inhibited from differentiating into neurons. Dev Neurosci, 2000; 22(1-2): 96-105
39纪家武,王玮.神经干细胞诱导分化的研究进展.解剖与临床, 2004; 9(4): 286-288
40 Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci, 1992; 12(11): 4565-4574
41 Bartlett PF, Brooker GJ, Faux CH, et al. Regulation of neural stem csll differentiation in the forebrain. Immunol Cell Biol, 1998; 76(5): 414- 418
42 Andersen B, Rosenfeld MG. POU Domain Factors in the Neuroendocrine System: lessons from developmental biology provide insights into human disease. Endocr Rev, 2001, 22(1): 2-35
43 Le Moine C ,Young WS 3rd. RHS2, A POU domain-containing gene, and its expression in developing and adult rat. Proc Natl Acad Sci USA, 1992, 89(8): 3285-3289
44陈蓉,金国华,田美玲,等.海马中56KD蛋白诱导人神经干细胞迁移的作用.神经解剖学杂志, 2005; 21(4): 372-376
45张蕾,金国华,田美玲,等.切割海马伞大鼠海马中65KD和83KD差异蛋白诱导神经干细胞迁移和向神经元分化的作用.解剖学杂志, 2006; 29(6): 677-681
46 Wu H, Friedman WJ, Dreyfus CF. Differential regulation of neuro- trophin expression in basal forebrain astrocytes by neuronal signals. J Neurosci Res, 2004; 76(1): 76-85
47 Weskamp G, Gasser UE, Dravid AR, et al. Fimbria-fornix lesion increases nerve growth factor content in adult rat septum and hippocampus. Neurosci Lett, 1986; 70(1): 121-126
48 Larkfors L, Stromberg I, Ebendal T, et al. Nerve growth factor protein level increases in the adult rat hippocampus after a specific cholinergic lesion. J Neurosci Res, 1987; 18(4): 525-531
49 Cellerino A. Expression of messenger RNA coding for the nerve growth factor receptor trkA in the hippocampus of the adult rat. Neuroscience, 1996; 70(3): 613-616
50 Murray B, Alessandrini A, Lu CL, et al. Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity. Proc Natl Acad Sci USA, 1998, 95(20): 11975- 11980
51 Cellerino A. Expression of messenger RNA coding for the nerve growth factor receptor trkA in the hippocampus of the adult rat. Neuroscience, 1996, 70(3): 613-616
52 Alvarez-Bolado G, Rosenfeld MG, Swanson LW. Model of forebrain regionalization based on spatiotemporal patterns of POU-Ⅲhomeobox gene expression,birthdates,and morphological features. J Comp Neurol, 1995; 355(2): 237-295
53 Malik KF, Kim J, Hartman AL, et al. Binding preferences of the POU domain protein Brain-4: implications for autoregulation. Brain Res Mol Brain Res, 1996; 38(2): 209-221
54 Mathis JM, Simmons DM, He X, et al. Brain-4:a novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression. EMBO J, 1992; 11(7): 2551-2561
1. Fire A, Xu S, Montogomery MK, et a1. Potent and specific genetic interference by double stranded RNA in caenorhabditis elegans. Nature, 1998; 391(6669):806-811
2. Baulcombe DC. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol, 1996; 32(1-2):79-88
3. Verma NK, Dey CS. RNA-mediated gene silencing: mechanisms and its therapntic applications. Journal of Clinical Phamacy & Therapeutics, 2004; 29(5):395-404
4. Sledz CA, Williams BR. RNA interference and double-strand-RNA-activated Pathways. Biochem Soc Trans, 2004; 32(6):952-956
5. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell, 1990; 2(4):279-289
6. Vander Krol A, Mur L, Beld M, et a1. Flavonoid gene in petunia: ddltion of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell, 1990;2(4):291-299
7. Jorgensen R, Cluster PD, Napoli C. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs.antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol Biol, 1996; 31(5): 957-973
8. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995;81(4):611-620
9. Waterhouse PM, Graham MW. Wang MB. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA, 1998; 95(23):13959-13964
10. Ngo H, Tschudi C. Gull K, et al. Double-stranded RNA induces mRNA degradation in Trypanosoma bruceil. Proc Natl Acad Sci USA, 1998;95(25):14687-14692
11. Cogoni C, Macino G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature, 1999; 399(6732): 166-169
12. Lohmann JU, Endl I, Bosch TC. Silencing of developmental genes in Hydra. Dev Biol, 1999;214(1):211-214
13. Sanchez Alvarado A, Newmark PA. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci USA, 1999;96(9):5049-5054
14. Pal-Bhadra M, Bhadra U, Birchler JA. Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences. Cell, 1999;99(1):35-46
15. Hammond SM, Bernstein E, Beach D, et a1. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000;404(6775):293-296
16. Yan L, Ming Y, Kui W, et al. Study on dsRNA mediated gene silencing in saccharomyces cerevisiae: suppressing the expression of GRE3 gene. Progress in Biochemistry and Biophysics, 2007; 34(3): 292-298
17. Tuschl T, Zamore PD, Lehmann R, et a1. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999;13(24):3191-3197
18.纪虹,孙开来.小RNA的研究现状.国际遗传学杂志, 2006;29(1):30-34
19. Provost P, Dishart D, Doucet J, et al. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J, 2002; 21(21): 5864- 5874.
20. Hammond SM, Boettcher S, Caudy AA, et al. Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science, 2001; 293 (5532): 1146-1150
21. Fire A. RNA-triggered gene silencing. Trends Genet, 1999,15(9):358-363
22. Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing.Cell, 2001;107(4):465-476
23. Brantl S. Antisense-RNA regulation and RNA interference. Biochemica et Biophysica Acta, 2002;1575(1-3):15-25
24. Lee Y, Jeon K, Lee JT, et al. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J, 2002;21(17):4663-4670
25.何晨,谭军,陈薇,等. MicroRNA研究进展.生物技术通讯, 2005;16(6): 674-676
26. Dostie J, Mourelatos Z, Yang M, et a1. Numerous microRNPs in neuronal cells containing novel microRNAs. RNA, 2003; 9(2):180-186
27. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002;3(10):737-747
28. Davenport RJ. Gene silencing: A faster way to shut down genes. Science, 2001; 292 (5521):1469-1471
29. McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature, 2002, 418(6893): 38-39
30. Svoboda P,Stein P,Hayashi H,et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development, 2000; 127(19): 4147-4156
31. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature, 2002; 418(6896):430-434
32. Matzke MA, Birchler JA. RNAi-mediated Pathways in the nucleus. Nature reviews Genetics, 2005;6(1):24-35
33. Bohula EA, Salisbury AJ, Sohail M, et al. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.J Biol Chem, 2003;278(18):15991-15997
34. Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.Nucleic Acids Res, 2002;30(10):46-55
35. Tuschl T. Expanding small RNA interference. Nat Biotechnol, 2002;20(5): 446-448
36. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002; 16(8):948-958
37. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002;296(5567):550-553
38. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001;411(6836):494-498
39. Lieberman J, Song E, Lee SK, et a1. Interfering with disease:opportunities and roadb locks to harnessing RNA interference. Trends Mol Med, 2003; 9(9):397-403
40. Jacque JM, Triques K, Srevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002; 418(13):435-438
41. Lee NS, Dohjima T, Bauer G, et a1. Expression of small interference RNAs targeted against HIV-1 rev transcriptions in human cells. Nat Biotechnol, 2002;20(5):500-505
42. Novina CD, Murray MF, Dykxhoorn DM, et al. siRNA-directed inhibition of HIV-1 infection. Nat Med, 2002;8(15):681-686
43. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003;21(6):639-644
44. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA, 2003;100(4):2014-2018
45. Lin JP, Yu YL, Wang LY. RNAi design to the Sprotein of SARS-CoV. Letters in Biotechnology, 2004;4(15):335-337
46. Brumelkamp TR, Bernards R, Afami R. Stable suppression of tummor origenicity by virus-mediated RNA interference. Cancer Cell, 2002;2(3): 243-247
47. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short—interferingRNKs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA, 2002; 99(9): 6047-6052
48. McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins. RNA, 2002;8(6):842-850
49. Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol, 2002;20(5):497-500
50. Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cels. Cancer Res, 2003;63(7):1515-1519
51. Caplen NJ, Taylor JP, Statham VS, et al. Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference. Hum Mol Genet, 2002;11(2):175-184
52. Takasugi N, Tomita T, Hayashi L, et a1. The role of presenilin cofactors in the gamma-secretase complex. Nature, 2003;422(6930):438-441
53. Carthew RW. RNA interference: the fragile X syndrome connection. Curr Biol, 2002;12(24):852-854
54. Ishizuka A, Siomi MC, Siomi H. A drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev, 2002; 16(19): 2497-2508
55. Downward J. RNA interference. BMJ, 2004;328(7450):1245-1248
2 Richards LJ, Kilpatrick TJ and Bartlett PF. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci USA, 1992; 89(18): 8591-8595
3 Morshead CM, Reynolds BA, Craig CG, et al. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron, 1994; 13(5): 1071-1082
4 Mckay R. Stem cells in the central nervous system. Scinece, 1997; 276 (5309): 66-71
5 Gage FH. Mammalian neural setm cells. Scinece, 2000; 287(5457): 1433-1438
6 Gage FH. Stem cells of the central nervous system. Curr Opin Neuro- biol, 1998; 8(5): 671-676
7 Frisen J, Lothian C, Lendahl U. Central nervous system setm cells in the embryo and adult. Cell Mol Life Sci, 1998; 54(9): 925-945
8 Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell, 1997; 88(3): 287-298
9 Barinaga M. Fetal neuron grafts pave the way for stem cell therapies. Science, 2000; 287(5457): 1421-1422
10 Winkler C, Fricker RA, Gates MA, et al. Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain. Mol Cell Neurosci, 1998; 11(3): 99-116
11 Bush G, diSibio G, Miyamoto A, et al. Ligand-induced signaling in the absence of furin processing of Notch 1. Dev Biol, 2001: 229(2): 494- 502
12 Ling ZD, Potter ED, Lipton JW, et al. Differentiation of mesen-cephalic progenitor cells into dopaminergic neurons by cytokines. Exp Neurol, 1998; 149(2): 411-423
13 Vicario-Abejon C, Collin C, Tsoulfas P, et al. Hippocampal stem cells differentiate into excitatory and inhibitory neurons. Eur J Neurosci, 2000; 12(2): 677-688
14 Josephson R, Muller T, Pickel J, et al. POU transcription factors control expression of CNS stem cell-specific genes. Development, 1998; 125(16): 3087-3100
15王永刚,冯忠堂,王延华,等.神经干细胞分化调控的研究.中华神经外科杂志. 2002; 18(5): 339-341
16 Gradwohl G, Fode C, Guillemot F. Restricted expression of a novel murine atonal-related bHLH protein in undifferentiated neural precursors. Dev Biol, 1996; 180(1): 227-241
17 Gehring WJ. The homeobox in perspective. Trends Biochem Sci, 1992; 17(8): 277-280
18 Lewis EB. Clusters of master control genes regulate the development of higher organisms. JAMA, 1992; 267(11): 1524-1531
19 Mihailescu D, Kury P and Monard D. An octamer-binding site is crucial for the activity of an enhancer active at the embryonic met-/mesencephalic junction. Mech Dev, 1999; 84(1-2): 55-67
20金国华,张新化,田美玲,等.大鼠海马内移植神经干细胞的存活和迁移.神经解剖学杂志, 2003; 19(4): 378-382
21张新化,金国华,秦建兵,等.穹窿海马伞切割侧海马对植入神经干细胞分化为神经元的影响.神经解剖学杂志,2004;20(4):360-364
22金国华,张新化,田美玲,等.穹窿海马伞切割侧海马提取液对神经干细胞分化为神经元的促进作用.解剖学报, 2004;40(2):141-145
23 Shimazaki T, Arsenijevic Y, Ryan AK, et al. A role for the POU-Ⅲtranscription factor Brn-4 in the regulation of striatal neuron precursor differentiation. EMBO J, 1999; 18(2): 444-456
24王磊,金国华,秦建兵,等.穹窿海马伞切割大鼠海马内Brn-4mRNA的表达变化.解剖学报, 2006; 37(4): 387-390
25王磊,金国华,秦建兵,等.切割穹窿海马伞大鼠海马内Brn-4 mRNA的表达变化-原位杂交法.解剖学报, 2007; 38(4): 385-389
26 Zhang xinhua, Jin guohua, Wang lei, et al. Brn-4 is up-regulated in the deafferented hippocampus and promotes neuronal differentiation of neural progenitors in vitro. Hippocampus, in press
27田美玲,胡文忠,金国华,等. Brn-4抗体对大鼠海马神经干细胞分化为神经元的影响.神经解剖学杂志, 2008; 24(1): 85-88
28 Fire A, Xu S, Montogomery MK, et a1. Potent and specific genetic interference by double stranded RNA in caenorhabditis elegans. Nature, 1998; 391(6669): 806-811
29 Baulcombe DC. RNA as a target and an initiator of post- transcriptional gene silencing in transgenic plants. Plant Mol Biol, 1996; 32(1-2): 79-88
30 Hammond SM, Boettcher S, Caudy AA, et al. Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science, 2001; 293 (5532): 1146-1150
31 Miyagishi M, Taira K. RNAi expression vectors in mammalian cells. Methods Mol Biol, 2004; 252: 483-491
32 Hammond SM, Bernstein E, Beach D, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296
33 Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409(6818): 363-366
34 Guo S,Kemphues KJ. Par-l,a gene required of establishing Polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asym- metrically distributed. Cell, 1995; 81(4): 611-620
35黄镇,金国华,张新化,等.提高成年大鼠神经干细胞单克隆形成率的方法.解剖学报, 2002; 33(6): 594-598
36衣昕,金国华,田美玲,等.壳聚糖支架与神经干细胞生物相容性的研究.神经解剖学杂志, 2007; 23(5): 506-510
37董传明,金国华,秦建兵,等.神经生长因子对穹窿海马伞切割后海马自体神经干细胞增殖和向神经元分化的影响.解剖学报, 2007; 38(6): 642-646
38 Dutton R,Bartlett PF.Precursor cells in the subventricular zone of the adult mouse are actively inhibited from differentiating into neurons. Dev Neurosci, 2000; 22(1-2): 96-105
39纪家武,王玮.神经干细胞诱导分化的研究进展.解剖与临床, 2004; 9(4): 286-288
40 Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci, 1992; 12(11): 4565-4574
41 Bartlett PF, Brooker GJ, Faux CH, et al. Regulation of neural stem csll differentiation in the forebrain. Immunol Cell Biol, 1998; 76(5): 414- 418
42 Andersen B, Rosenfeld MG. POU Domain Factors in the Neuroendocrine System: lessons from developmental biology provide insights into human disease. Endocr Rev, 2001, 22(1): 2-35
43 Le Moine C ,Young WS 3rd. RHS2, A POU domain-containing gene, and its expression in developing and adult rat. Proc Natl Acad Sci USA, 1992, 89(8): 3285-3289
44陈蓉,金国华,田美玲,等.海马中56KD蛋白诱导人神经干细胞迁移的作用.神经解剖学杂志, 2005; 21(4): 372-376
45张蕾,金国华,田美玲,等.切割海马伞大鼠海马中65KD和83KD差异蛋白诱导神经干细胞迁移和向神经元分化的作用.解剖学杂志, 2006; 29(6): 677-681
46 Wu H, Friedman WJ, Dreyfus CF. Differential regulation of neuro- trophin expression in basal forebrain astrocytes by neuronal signals. J Neurosci Res, 2004; 76(1): 76-85
47 Weskamp G, Gasser UE, Dravid AR, et al. Fimbria-fornix lesion increases nerve growth factor content in adult rat septum and hippocampus. Neurosci Lett, 1986; 70(1): 121-126
48 Larkfors L, Stromberg I, Ebendal T, et al. Nerve growth factor protein level increases in the adult rat hippocampus after a specific cholinergic lesion. J Neurosci Res, 1987; 18(4): 525-531
49 Cellerino A. Expression of messenger RNA coding for the nerve growth factor receptor trkA in the hippocampus of the adult rat. Neuroscience, 1996; 70(3): 613-616
50 Murray B, Alessandrini A, Lu CL, et al. Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity. Proc Natl Acad Sci USA, 1998, 95(20): 11975- 11980
51 Cellerino A. Expression of messenger RNA coding for the nerve growth factor receptor trkA in the hippocampus of the adult rat. Neuroscience, 1996, 70(3): 613-616
52 Alvarez-Bolado G, Rosenfeld MG, Swanson LW. Model of forebrain regionalization based on spatiotemporal patterns of POU-Ⅲhomeobox gene expression,birthdates,and morphological features. J Comp Neurol, 1995; 355(2): 237-295
53 Malik KF, Kim J, Hartman AL, et al. Binding preferences of the POU domain protein Brain-4: implications for autoregulation. Brain Res Mol Brain Res, 1996; 38(2): 209-221
54 Mathis JM, Simmons DM, He X, et al. Brain-4:a novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression. EMBO J, 1992; 11(7): 2551-2561
1. Fire A, Xu S, Montogomery MK, et a1. Potent and specific genetic interference by double stranded RNA in caenorhabditis elegans. Nature, 1998; 391(6669):806-811
2. Baulcombe DC. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol, 1996; 32(1-2):79-88
3. Verma NK, Dey CS. RNA-mediated gene silencing: mechanisms and its therapntic applications. Journal of Clinical Phamacy & Therapeutics, 2004; 29(5):395-404
4. Sledz CA, Williams BR. RNA interference and double-strand-RNA-activated Pathways. Biochem Soc Trans, 2004; 32(6):952-956
5. Napoli C, Lemieux C, Jorgensen R. Introduction of a Chimeric Chalcone Synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell, 1990; 2(4):279-289
6. Vander Krol A, Mur L, Beld M, et a1. Flavonoid gene in petunia: ddltion of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell, 1990;2(4):291-299
7. Jorgensen R, Cluster PD, Napoli C. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs.antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol Biol, 1996; 31(5): 957-973
8. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell, 1995;81(4):611-620
9. Waterhouse PM, Graham MW. Wang MB. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA, 1998; 95(23):13959-13964
10. Ngo H, Tschudi C. Gull K, et al. Double-stranded RNA induces mRNA degradation in Trypanosoma bruceil. Proc Natl Acad Sci USA, 1998;95(25):14687-14692
11. Cogoni C, Macino G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature, 1999; 399(6732): 166-169
12. Lohmann JU, Endl I, Bosch TC. Silencing of developmental genes in Hydra. Dev Biol, 1999;214(1):211-214
13. Sanchez Alvarado A, Newmark PA. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci USA, 1999;96(9):5049-5054
14. Pal-Bhadra M, Bhadra U, Birchler JA. Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences. Cell, 1999;99(1):35-46
15. Hammond SM, Bernstein E, Beach D, et a1. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000;404(6775):293-296
16. Yan L, Ming Y, Kui W, et al. Study on dsRNA mediated gene silencing in saccharomyces cerevisiae: suppressing the expression of GRE3 gene. Progress in Biochemistry and Biophysics, 2007; 34(3): 292-298
17. Tuschl T, Zamore PD, Lehmann R, et a1. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 1999;13(24):3191-3197
18.纪虹,孙开来.小RNA的研究现状.国际遗传学杂志, 2006;29(1):30-34
19. Provost P, Dishart D, Doucet J, et al. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J, 2002; 21(21): 5864- 5874.
20. Hammond SM, Boettcher S, Caudy AA, et al. Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science, 2001; 293 (5532): 1146-1150
21. Fire A. RNA-triggered gene silencing. Trends Genet, 1999,15(9):358-363
22. Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing.Cell, 2001;107(4):465-476
23. Brantl S. Antisense-RNA regulation and RNA interference. Biochemica et Biophysica Acta, 2002;1575(1-3):15-25
24. Lee Y, Jeon K, Lee JT, et al. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J, 2002;21(17):4663-4670
25.何晨,谭军,陈薇,等. MicroRNA研究进展.生物技术通讯, 2005;16(6): 674-676
26. Dostie J, Mourelatos Z, Yang M, et a1. Numerous microRNPs in neuronal cells containing novel microRNAs. RNA, 2003; 9(2):180-186
27. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002;3(10):737-747
28. Davenport RJ. Gene silencing: A faster way to shut down genes. Science, 2001; 292 (5521):1469-1471
29. McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature, 2002, 418(6893): 38-39
30. Svoboda P,Stein P,Hayashi H,et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development, 2000; 127(19): 4147-4156
31. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature, 2002; 418(6896):430-434
32. Matzke MA, Birchler JA. RNAi-mediated Pathways in the nucleus. Nature reviews Genetics, 2005;6(1):24-35
33. Bohula EA, Salisbury AJ, Sohail M, et al. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.J Biol Chem, 2003;278(18):15991-15997
34. Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.Nucleic Acids Res, 2002;30(10):46-55
35. Tuschl T. Expanding small RNA interference. Nat Biotechnol, 2002;20(5): 446-448
36. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 2002; 16(8):948-958
37. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002;296(5567):550-553
38. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001;411(6836):494-498
39. Lieberman J, Song E, Lee SK, et a1. Interfering with disease:opportunities and roadb locks to harnessing RNA interference. Trends Mol Med, 2003; 9(9):397-403
40. Jacque JM, Triques K, Srevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002; 418(13):435-438
41. Lee NS, Dohjima T, Bauer G, et a1. Expression of small interference RNAs targeted against HIV-1 rev transcriptions in human cells. Nat Biotechnol, 2002;20(5):500-505
42. Novina CD, Murray MF, Dykxhoorn DM, et al. siRNA-directed inhibition of HIV-1 infection. Nat Med, 2002;8(15):681-686
43. McCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003;21(6):639-644
44. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA, 2003;100(4):2014-2018
45. Lin JP, Yu YL, Wang LY. RNAi design to the Sprotein of SARS-CoV. Letters in Biotechnology, 2004;4(15):335-337
46. Brumelkamp TR, Bernards R, Afami R. Stable suppression of tummor origenicity by virus-mediated RNA interference. Cancer Cell, 2002;2(3): 243-247
47. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short—interferingRNKs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA, 2002; 99(9): 6047-6052
48. McManus MT, Petersen CP, Haines BB, et al. Gene silencing using micro-RNA designed hairpins. RNA, 2002;8(6):842-850
49. Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3’overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol, 2002;20(5):497-500
50. Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cels. Cancer Res, 2003;63(7):1515-1519
51. Caplen NJ, Taylor JP, Statham VS, et al. Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference. Hum Mol Genet, 2002;11(2):175-184
52. Takasugi N, Tomita T, Hayashi L, et a1. The role of presenilin cofactors in the gamma-secretase complex. Nature, 2003;422(6930):438-441
53. Carthew RW. RNA interference: the fragile X syndrome connection. Curr Biol, 2002;12(24):852-854
54. Ishizuka A, Siomi MC, Siomi H. A drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev, 2002; 16(19): 2497-2508
55. Downward J. RNA interference. BMJ, 2004;328(7450):1245-1248