经典H-2分子mRNA探针的制备及在C57小鼠中枢神经系统原位杂交中的应用-东南大学学报医学版

经典H-2分子mRNA探针的制备及在C57小鼠中枢神经系统原位杂交中的应用

单位：东南大学医学院病原生物学与免疫学系,发育与疾病相关基因教育部重点实验室,江苏南京 210009

分类号：Q786;R-33

出版年·卷·期（页码）：2013·32·第二期（135-140）

摘要：

目的:研究经典H-2基因在C57小鼠中枢神经系统中的表达谱,设计合成地高辛标记的经典H-2 mRNA探针。方法:利用PCR扩增出针对经典H-2分子的特异性保守片段,然后将PCR产物纯化后与pGEM-T Easy载体连接,转化到大肠杆菌DH5α中。测序后获得目的基因的单克隆,扩增并提取质粒,采用体外转录的方法合成地高辛标记的经典H-2 mRNA的正、反义RNA探针。运用原位杂交方法分析经典H-2基因在C57小鼠中枢神经系统中的表达情况。结果:成功构建了321 bp经典H-2 mRNA的正、反义探针,原位杂交方法检测到经典H-2 mRNA在出生后15 d小鼠中枢神经系统中反义探针有杂交信号,正义探针无杂交信号。结论:正义探针无杂交信号表明合成的321 bp经典H-2 mRNA反义探针具有特异性。

Objective: In order to find out the expression pattern of classical H-2 in the central nervous system of C57 mice thus synthesize RNA probe marked by Digoxigenin.Methods: The probes for PCR amplification was designed specifically to amplify the classical H-2 molecules.The PCR product was purified and later linked into pGEM-T Easy Vector.The plasmid was sequenced for confirmation the correct sequence of the gene.The sense and antisense RNA probe were synthesized by using the method of in vitro transcription of the vector.The expression of classical H-2 mRNA in the central nervous system of mice was analyzed by the method of in situ hybridization by using this probe.Results: 321 bp classical H-2 mRNA's sense and anti-sense probes were successfully conformed.With in situ hybridization,the anti-sense probe yielded hybridization signal while sense probe had no signal in the central nervous system of P15 mice.Conclusion: The sense probe is used as a negative control and it yields no signal,thus demonstrating the specificity of the anti-sense probe.

参考文献：

[1] LAMPSON L A.Interpreting MHC classⅠ expression and classⅠ/classⅡ reciprocity in the CNS:reconciling divergent findings[J].Microsc Res Techniq,1995,32:267-285.
[2] NEUMANN H,SCHMIDT H,CAVALIE A,et al.Major histocompatibility complex(MHC) classⅠ gene expression in single neurons of the central nervous system:differential regulation by interferon(IFN)-γ and tumor necrosis factor(TNF)-α[J].Exp Med,1997,185:305-316.
[3] CORRIVEAU R A,HUH G S,SHATZ C J,et al.Regulation of classⅠ MHC gene expression in the developing and mature CNS by neural activity[J].Neuron,1998,21:505-520.
[4] GENE S H,LISA M B,DU H P,et al.Functional requirement for classⅠ MHC in CNS development and plasticity[J].Science,2000,290:2155- 2159.
[5] GODDARD C A,BUTTS D A,SHATZ C J,et al.Regulation of CNS synapses by neuronal MHC classⅠ[J].PNAS,2007,104:6828-6833.
[6] MASATO O,HIDETOSHI I,JERZY K K,et al.Major histocompatibility complex(MHC) classⅠb gene duplications,organization and expression patterns in mouse strain C57BL/6[J].BMC Genomics,2008,9:178.
[7] 谢维.MHC基因多态性和肿瘤的发生[J].现代免疫学,2004,24(6):441-444.
[8] KLEIN J.Seeds of time:fifty years ago Peter A.Gorer discovered the H-2 complex[J].Immunogenetics,1986,24:331- 338.
[9] SNELL G D.Some recollections of Peter Gorer and his work on this fiftieth anniversary of his discovery of H-2[J].Immunogenetics,1986,24:339-340.
[10] RALL G F,MUCKE L,OLDSTONE M B,et al.Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class Ⅰ-expressing neurons in vivo[J].Exp Med,1995,182:1201-1212.
[11] 顾香芳,甘光明,王芳,等.应用原位杂交技术分析ZNF216基因在成年小鼠各主要组织中的表达模式[J].东南大学学报:医学版,2005,24(1):1-4.
[12] SHATZ C J.MHC classⅠ:an unexpected role in neuronal plasticity[J].Neuron,2009,64:40-45.
[13] MCCONNELL M J,HUANG Y H,DATWANI A,et al.H2-Kb and H2-Db regulate cerebellar long-term depression and limit motor learning[J].Science,2009,106:6784-6789.
[14] BOULANGER L M.Immune proteins in brain development and synaptic plasticity[J].Neuron,2009,64:93-109.
[15] ADELSON J D,BARRETO G E,XU L J,et al.Neuroprotection from stroke in the absence of MHC Ⅰ or Pir B[J].Neuron,2012,73:1100-1107.
[16] ZHONGQI P W,TINA B,NATHALIE E B,et al.Major histocompatibility complex class Ⅰ-mediated inhibition of neurite outgrowth from peripheral nerves[J].Immunology Letters,2011,135:118-123.
[17] ZHONGQI P W,LORRAINE W,TINA V B,et al.Enhanced neuronal expression of major histocompatibility complex classⅠ leads to aberrations in neurodevelopment and neurorepair[J].J Neuroimmunol,2011,232:8-16.
[18] DENG X H,AI W M,LEI D L,et al.Lipopolysaccharide induces paired immunoglobulin-like receptor B(PirB) expression,synaptic alteration,and learning-memory deficit in rats[J].Neuroscience,2012,209:161-170.

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