乳杆菌代谢产物RNA组分序列分析及对其部分生物学功能的研究
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摘要
目的:
1.分析乳杆菌代谢产物RNA组分的序列,明确该核酸成分的性质
2.通过RNA组分在体外对Caco-2细胞TLR-9通路及对细胞增殖影响的研究,探讨其作用机制及对肿瘤细胞的作用
3.通过RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态恢复情况的研究,探讨其对肠道机械屏障及免疫细胞的作用
方法:
1.乳杆菌代谢产物RNA组分序列分析:应用Solexa上机前处理过程对样品RNA进行处理,经过加接头反转录后对连接产物进行PCR扩增,进而对PCR产物做TA克隆测序。(测序委托深圳华大基因科技有限公司进行)
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响
(1)实验分为四组,阴性对照组、阳性对照组、RNA100ug/ml组、RNA200ug/ml组。实验组中加入不同浓度RNA组分,阳性对照组中加入CPG-ODN
(2)RT-PCR法检测TLR9、NF-κBp65、IL-6 mRNA水平的变化;免疫组化法检测NF-κBp65蛋白水平的变化;ELISA法检测Caco-2细胞培养液中IL-6的变化
(3)MTT法检测RNA组分对Caco-2细胞增殖的影响
3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响
(1)SPF级Balb/c小鼠随机分为正常组,重度失衡组,自然恢复组,RNA恢复组。采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型后,以RNA组分10mg/kg/天的剂量,连续3天灌胃调节,并从灌服之日起第7天提取标本,检测指标
(2)HE染色及透射电镜观察菌群失衡小鼠通过RNA组分恢复后肠粘膜绒毛形态变化
(3)透射电镜观察菌群失衡小鼠通过RNA组分恢复后派氏结中细胞形态变化
结果:
1.乳杆菌代谢产物RNA组分序列分析:所得39条序列,经Blast比对其中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响
(1)与对照组相比RNA组分组TLR9及NF-κBp65、IL-6在mRNA及蛋白水平的表达量均高于阴性对照组,100ug/mlRNA组表达量低于阳性对照组,200ug/mlRNA组表达量高于阳性对照组
(2)RNA组分对Caco-2细胞增殖有抑制作用,显示出明显的时间剂量依赖性
3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响
(1)肠粘膜绒毛形态:应用回灌RNA进行恢复后,通过HE染色发现与自然恢复组相比,肠上皮细胞层细胞排列紧密,肠绒毛排列规整,肿胀、断裂消失,绒毛顶端未见坏死脱落的肠上皮细胞,且炎性细胞浸润减少。通过电镜观察发现与自然恢复组相比,微绒毛恢复整齐,细胞间连接更加紧密
(2)派氏结中细胞形态:经过回灌RNA进行恢复后,通过电镜观察发现肠壁派氏结重度失衡组与正常组相比较,淋巴细胞之间的间隙明显变大;树突状细胞伪足变短,通过回灌RNA进行恢复后,与自然恢复组相比,淋巴细胞间隙明显缩窄,树突状细胞伪足延伸
结论:
1.乳杆菌代谢产物RNA组分中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR通路具有活化作用
3.乳杆菌代谢产物RNA组分对人结肠腺癌Caco-2细胞增殖有抑制作用
4.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态具有恢复作用
Objective:
1.The sequence analysis of RNA components from Lactobacillus metabolites .
2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation influence of Caco - 2 cell .
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
Methods:
1.Sequence analysis: Processing samples RNA applies Solexa pre-treatment process.TA cloning sequencing PCR products after samples RNA reverse transcription add joints connecting. (Sequencing entrust shenzhen huada gene technology Co., LTD)
2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation.
(1)Experiments are divided into four groups and the negative control, positive control, RNA100ug/ml group, RNA200ug/ml group. To join different concentration into RNA Group, join CPG - ODN into positive control.
(2)Detecting the change of TLR9, NF-κBp65 ,IL-6 by RT-PCR and Detecting the change of NF-κBp65 protein by Immunohistochemistry, assaying the change of IL-6 in culture of Caco-2 by ELISA.
(3)Detecting the effect of RNA components on proliferation of Caco-2 cell line by MTT.
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
(1)Balb/c mice SPF were randomly divided into four groups, control,severe unbalance group, natural recovery group and RNA recovery group. We established mice model for dysbiosis of Intestinal flora by ceftriaxone, and then detect targets at the seventh day by the secretive RNA of DM9811 10mg/kg / day for 3 days.
(2)Observing the bowel structure change of flora imbalances mice after the restoration of RNA components by HE dyed and electron microscopic.
(3)Observing the change of Peyer’spatches in flora imbalances mice after the restoration of RNA components by electron microscopic.
Results:
1.Sequence analysis: Income 39 of the sequence. The samples relevant sequence is Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu.
2.The effect of RNA components from Lactobacillus metabolites on Caco-2cell line.
(1)Compared with control group TLR - 9 NF -κBp65 andIL - 6 in mRNA and protein level is higher than negative control, 100ug/mlRNA group expression is less than positive control group, 200ug/mlRNA group is higher than positive control group.
(2)The effect of RNA components on Caco - 2 cell proliferation inhibitory showed time dose dependent obviously.
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
(1)Intestinal mucosa villi: Through recharge with RNA,and compared with natural recovery, intestinal epithelium cells group cell layers closely-arranged villi with arrangement, neat, swelling, fracture disappear, fluffy top has not seen the necrosis of intestinal epithelium cells off, and inflammatory cells infiltrating decrease. Through recharge with RNA and compared with natural recovery group microvilli restore tidy, cell connections between closer.
(2)Peyer’spatches: Compared with severe imbalance flora group and the normal group the lymphocyte clearances between apparent greatens; Dendritic cells pseudopods become shorter, through recharging after recovery, compared with RNA natural recovery group , lymphocytes clearance obviously become narrower, dendritic cells pseudopods outspread.
Conclusion:
1.Some ingredients of Lactobacillus metabolites RNA component are most similar to Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu.
2.The Lactobacillus metabolites RNA components have activatory function on Caco - 2 cells TLR pathways.
3.The effect of Lactobacillus metabolites RNA components on human adenocarcinoma Caco - 2 cell line proliferation is inhibition.
4.The Lactobacillus metabolites RNA components have recovery function on intestinal flora imbalances mice intestine structural integrity and Peyer’spatches.
1.分析乳杆菌代谢产物RNA组分的序列,明确该核酸成分的性质
2.通过RNA组分在体外对Caco-2细胞TLR-9通路及对细胞增殖影响的研究,探讨其作用机制及对肿瘤细胞的作用
3.通过RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态恢复情况的研究,探讨其对肠道机械屏障及免疫细胞的作用
方法:
1.乳杆菌代谢产物RNA组分序列分析:应用Solexa上机前处理过程对样品RNA进行处理,经过加接头反转录后对连接产物进行PCR扩增,进而对PCR产物做TA克隆测序。(测序委托深圳华大基因科技有限公司进行)
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响
(1)实验分为四组,阴性对照组、阳性对照组、RNA100ug/ml组、RNA200ug/ml组。实验组中加入不同浓度RNA组分,阳性对照组中加入CPG-ODN
(2)RT-PCR法检测TLR9、NF-κBp65、IL-6 mRNA水平的变化;免疫组化法检测NF-κBp65蛋白水平的变化;ELISA法检测Caco-2细胞培养液中IL-6的变化
(3)MTT法检测RNA组分对Caco-2细胞增殖的影响
3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响
(1)SPF级Balb/c小鼠随机分为正常组,重度失衡组,自然恢复组,RNA恢复组。采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型后,以RNA组分10mg/kg/天的剂量,连续3天灌胃调节,并从灌服之日起第7天提取标本,检测指标
(2)HE染色及透射电镜观察菌群失衡小鼠通过RNA组分恢复后肠粘膜绒毛形态变化
(3)透射电镜观察菌群失衡小鼠通过RNA组分恢复后派氏结中细胞形态变化
结果:
1.乳杆菌代谢产物RNA组分序列分析:所得39条序列,经Blast比对其中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响
(1)与对照组相比RNA组分组TLR9及NF-κBp65、IL-6在mRNA及蛋白水平的表达量均高于阴性对照组,100ug/mlRNA组表达量低于阳性对照组,200ug/mlRNA组表达量高于阳性对照组
(2)RNA组分对Caco-2细胞增殖有抑制作用,显示出明显的时间剂量依赖性
3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响
(1)肠粘膜绒毛形态:应用回灌RNA进行恢复后,通过HE染色发现与自然恢复组相比,肠上皮细胞层细胞排列紧密,肠绒毛排列规整,肿胀、断裂消失,绒毛顶端未见坏死脱落的肠上皮细胞,且炎性细胞浸润减少。通过电镜观察发现与自然恢复组相比,微绒毛恢复整齐,细胞间连接更加紧密
(2)派氏结中细胞形态:经过回灌RNA进行恢复后,通过电镜观察发现肠壁派氏结重度失衡组与正常组相比较,淋巴细胞之间的间隙明显变大;树突状细胞伪足变短,通过回灌RNA进行恢复后,与自然恢复组相比,淋巴细胞间隙明显缩窄,树突状细胞伪足延伸
结论:
1.乳杆菌代谢产物RNA组分中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等
2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR通路具有活化作用
3.乳杆菌代谢产物RNA组分对人结肠腺癌Caco-2细胞增殖有抑制作用
4.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态具有恢复作用
Objective:
1.The sequence analysis of RNA components from Lactobacillus metabolites .
2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation influence of Caco - 2 cell .
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
Methods:
1.Sequence analysis: Processing samples RNA applies Solexa pre-treatment process.TA cloning sequencing PCR products after samples RNA reverse transcription add joints connecting. (Sequencing entrust shenzhen huada gene technology Co., LTD)
2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation.
(1)Experiments are divided into four groups and the negative control, positive control, RNA100ug/ml group, RNA200ug/ml group. To join different concentration into RNA Group, join CPG - ODN into positive control.
(2)Detecting the change of TLR9, NF-κBp65 ,IL-6 by RT-PCR and Detecting the change of NF-κBp65 protein by Immunohistochemistry, assaying the change of IL-6 in culture of Caco-2 by ELISA.
(3)Detecting the effect of RNA components on proliferation of Caco-2 cell line by MTT.
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
(1)Balb/c mice SPF were randomly divided into four groups, control,severe unbalance group, natural recovery group and RNA recovery group. We established mice model for dysbiosis of Intestinal flora by ceftriaxone, and then detect targets at the seventh day by the secretive RNA of DM9811 10mg/kg / day for 3 days.
(2)Observing the bowel structure change of flora imbalances mice after the restoration of RNA components by HE dyed and electron microscopic.
(3)Observing the change of Peyer’spatches in flora imbalances mice after the restoration of RNA components by electron microscopic.
Results:
1.Sequence analysis: Income 39 of the sequence. The samples relevant sequence is Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu.
2.The effect of RNA components from Lactobacillus metabolites on Caco-2cell line.
(1)Compared with control group TLR - 9 NF -κBp65 andIL - 6 in mRNA and protein level is higher than negative control, 100ug/mlRNA group expression is less than positive control group, 200ug/mlRNA group is higher than positive control group.
(2)The effect of RNA components on Caco - 2 cell proliferation inhibitory showed time dose dependent obviously.
3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.
(1)Intestinal mucosa villi: Through recharge with RNA,and compared with natural recovery, intestinal epithelium cells group cell layers closely-arranged villi with arrangement, neat, swelling, fracture disappear, fluffy top has not seen the necrosis of intestinal epithelium cells off, and inflammatory cells infiltrating decrease. Through recharge with RNA and compared with natural recovery group microvilli restore tidy, cell connections between closer.
(2)Peyer’spatches: Compared with severe imbalance flora group and the normal group the lymphocyte clearances between apparent greatens; Dendritic cells pseudopods become shorter, through recharging after recovery, compared with RNA natural recovery group , lymphocytes clearance obviously become narrower, dendritic cells pseudopods outspread.
Conclusion:
1.Some ingredients of Lactobacillus metabolites RNA component are most similar to Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu.
2.The Lactobacillus metabolites RNA components have activatory function on Caco - 2 cells TLR pathways.
3.The effect of Lactobacillus metabolites RNA components on human adenocarcinoma Caco - 2 cell line proliferation is inhibition.
4.The Lactobacillus metabolites RNA components have recovery function on intestinal flora imbalances mice intestine structural integrity and Peyer’spatches.
引文
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4.Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell, 2004,116 (2) :281–297.
5.Cullen, B. R.Transcription and processing of human microRNA precursors[J].Mol.Cell, 2004, 16(6):861–865.
6.Nair V, Zavolan M. Virus-encoded microRNAs: novel regulators of geneexpression [J]. Trends Microbiol,2006,14(4):169-175
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16.Victor Ambros. The evolution of our thinking about microRNAs[J]. Nature Medicine, 2008,14:1036-1040
17.Pfeffer, S. et al.Identification of microRNAs of the herpesvirus family[J]. Nat Methods, 2005,2:269–276
18.Gottwein E, Cullen BR. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity[J]. Cell Host Microbe ,2008,3:375–87
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20.Omoto S, Fujii Y R. Regulation of human immunodeficiency virus 1 transcription by nef microRNA[J]. J Gen Virol, 2005,86:751-755
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22.Shen B, Goodman H M. Uridine addition after microRNA-directed cleavage[J].Science, 2004,306(5698):997
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30.McCormick, C. and Ganem, D. The kaposin B protein of KSHV activates the p38/MK2 pathway and stabilizes cytokine mRNAs[J]. Science, 2005, 307:739–741
31.Sullivan, C.S, Ganem D. MicroRNAs and viral infection[J]. Mol Cell,2005,20(1):3-7
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33.Lecellier, C.H. et al. (2005) A cellular microRNA mediates antiviral defense in human cells[J]. Science 308, 557–560
34.Baskerville, S. and Bartel, D.P.Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes[J]. RNA , 2005,11: 241–247
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36.Miska, E.A. et al. Microarray analysis of microRNA expression in the developing mammalian brain[J]. Genome Biol, 2004, 5: R68
37.Sun, Y. et al. Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs[J]. Nucleic Acids Res, 2004, 32: e188
38.Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development[J]. Science, 2005, 309: 310–311
39.Monticelli, S. et al. MicroRNA profiling of the murine hematopoietic system[J]. Genome Biol, 2005, 6:R71
40.Nelson, P.T. et al. Microarray-based, high-throughput gene expression profiling of microRNAs [J]. Nat. Methods, 2004, 1:155–161
41.Thomson, J.M. et al. A custom microarray platform for analysis of microRNA gene expression[J]. Nat. Methods ,2004, 1:47–53
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44.徐娜,刘明,李宝玉,柳纪省. RNA干扰抑制口蹄疫病毒复制研究进展[J].动物医学进展,2010, 31(3):82-86
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1.Ambros, V, B. Bartel, D. P. Bartel, C. B. Burge, J. C. Carrington, G.Ruvkun, andT. Tuschl. A uniform system for microRNA annotation[J]. RNA,2003,9:277–279.
2.Carrington J C,Ambros V.Role of microRNAs in plant and animal development[J]. Science,2003,301(5631):336-338
3.Pfeffer S,Zavolan M,Grasser F A,et al.Identification of virus-encode microRNAs[J].Science, 2004,304(5671):734-736
4.Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell, 2004,116 (2) :281–297.
5.Cullen, B. R.Transcription and processing of human microRNA precursors[J].Mol.Cell, 2004, 16(6):861–865.
6.Nair V, Zavolan M. Virus-encoded microRNAs: novel regulators of geneexpression [J]. Trends Microbiol,2006,14(4):169-175
7.Sullivan C S, Grundhoff A T, Tevethia S, Pipas J M and Ganem D. SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxictcells[J]. Nature, 2005, 435 (7024):682–686.
8.Kim do N, Chae H S,et al. Expression of viral microRNAs in Epstein-Barrvirus-associated gastric carcinoma[J]. J Virol, 2007, 81(2):1033-1036
9.Reinhart B J, Slack F J, Basson M, Bettinger J C, Pasquinelli A E, Rougvie A.E, Horvitz H.R, and Ruvkun G. The 21 nucleotide let-7 RNA regulates development altiming in Caenorhab-ditis elegans[J]. Nature,2000,403:901–906
10.Slack F J, Basson M, Liu Z, Ambros V, Horvitz H. R, and Ruvkun G. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor[J].Mol.Cell,2000,5:659–669.
11.Lee R C. et al. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993, 75,843–854
12.Bartel D. P. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell, 2004,116: 281–297
13.Griffiths-Jones S. The microRNA Registry[J]. Nucleic Acids Res, 2004, 32: 109–111.
14.Griffiths-Jones S. et al. MiRBase: microRNA sequences, targets and gene nomenclature[J]. Nucleic Acids Res, 2006, 34:140–144
15.Carl D.Novina and Phillip A. Sharp. The RNAi revolution[J]. Nature, 2004,430:161-164
16.Victor Ambros. The evolution of our thinking about microRNAs[J]. Nature Medicine, 2008,14:1036-1040
17.Pfeffer, S. et al.Identification of microRNAs of the herpesvirus family[J]. Nat Methods, 2005,2:269–276
18.Gottwein E, Cullen BR. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity[J]. Cell Host Microbe ,2008,3:375–87
19.Obbard D J, Gordon K H, Buck A H, Jiggins F M. The evolution of RNAi as a defence against viruses and transposable elements[J]. Philos Trans R Soc Lond B Biol Sci, 2009, 364:99–115.
20.Omoto S, Fujii Y R. Regulation of human immunodeficiency virus 1 transcription by nef microRNA[J]. J Gen Virol, 2005,86:751-755
21.Omoto S. et al. HIV-1 nef suppression by virally encoded miRNA[J]. Rertovirology, 2004, 1:44
22.Shen B, Goodman H M. Uridine addition after microRNA-directed cleavage[J].Science, 2004,306(5698):997
23.Saenz-Robles, M.T. et al. Transforming functions of Simian Virus 40[J]. Oncogene, 2001, 20: 7899–7907
24.Umbach JL, Kramer MF, Jurak I, Karnowski HW, Coen DM, Cullen BR. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs[J].Nature, 2008, 454: 780–783
25.Barth S, Pfuhl T, Mamiani A et al. Epstein–Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5[J]. Nucleic Acids Res ,2008,36:666–675
26.Stern-Ginossar N, Elefant N, Zimmermann A et al. Host immune system gene targeting by a viral miRNA[J]. Science ,2007,317:376–81
27.Pfeffer S, Voinnet O. Viruses, microRNAs and cancer[J]. Oncogene, 2006,25(46):6211-6219
28.Young L S. and Rickinson A B. Epstein-Barr virus: 40 years on[J]. Nat. Rev.Cancer, 2004, 4: 757–768
29.Cai, X. et al. Kaposi’s sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells[J]. Proc. Nat. Acad. Sci. U. S. A[J]. 2005 , 102 (15) : 5570–5575
30.McCormick, C. and Ganem, D. The kaposin B protein of KSHV activates the p38/MK2 pathway and stabilizes cytokine mRNAs[J]. Science, 2005, 307:739–741
31.Sullivan, C.S, Ganem D. MicroRNAs and viral infection[J]. Mol Cell,2005,20(1):3-7
32.Jopling, C.L. et al. (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA[J]. Science 309, 1577–1581
33.Lecellier, C.H. et al. (2005) A cellular microRNA mediates antiviral defense in human cells[J]. Science 308, 557–560
34.Baskerville, S. and Bartel, D.P.Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes[J]. RNA , 2005,11: 241–247
35.Babak, T. et al. Probing microRNAs with microarrays: tissue specificity and functional inference [J]. RNA, 2004, 10: 1813–1819
36.Miska, E.A. et al. Microarray analysis of microRNA expression in the developing mammalian brain[J]. Genome Biol, 2004, 5: R68
37.Sun, Y. et al. Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs[J]. Nucleic Acids Res, 2004, 32: e188
38.Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development[J]. Science, 2005, 309: 310–311
39.Monticelli, S. et al. MicroRNA profiling of the murine hematopoietic system[J]. Genome Biol, 2005, 6:R71
40.Nelson, P.T. et al. Microarray-based, high-throughput gene expression profiling of microRNAs [J]. Nat. Methods, 2004, 1:155–161
41.Thomson, J.M. et al. A custom microarray platform for analysis of microRNA gene expression[J]. Nat. Methods ,2004, 1:47–53
42.Shingara, J. et al.An optimized isolation and labeling platform for accurate microRNA expression profiling [J]. RNA , 2005, 11: 1461–1470
43.Weiler J, Hunziker J, Hall J. Anti-mRNA oligonucleotides (AMOs) : ammunition to target miRNAs implicated in human disease [J]? Gene Therapy, 2006, 13:496-502
44.徐娜,刘明,李宝玉,柳纪省. RNA干扰抑制口蹄疫病毒复制研究进展[J].动物医学进展,2010, 31(3):82-86