Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

doi:10.1016/S1001-9294(11)60020-5

Chinese Medical Sciences Journal ›› 2011, Vol. 26 ›› Issue (1): 54-59.doi: 10.1016/S1001-9294(11)60020-5

• Original Article • 上一篇下一篇

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Bo Bai¹, Hai-qing Liu², Jing Chen¹, Ya-lin Li², Hui Du², Hai Lu¹, and Peng-li Yu¹

1Department of Neurobiology, Jining Medical College , Jining 272067, China 2Department of Physiology, Taishan Medical College , Taian 271000, China

收稿日期:2009-09-23 修回日期:2011-04-18 出版日期:2011-04-18 发布日期:2011-04-18
通讯作者: Bo Bai E-mail:bbai@mail.jnmc.edu.cn

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Bo Bai¹, Hai-qing Liu², Jing Chen¹, Ya-lin Li², Hui Du², Hai Lu¹, and Peng-li Yu¹

1Department of Neurobiology, Jining Medical College , Jining 272067, China 2Department of Physiology, Taishan Medical College , Taian 271000, China

Received:2009-09-23 Revised:2011-04-18 Online:2011-04-18 Published:2011-04-18
Contact: Bo Bai E-mail:bbai@mail.jnmc.edu.cn
About author:Tel: 86-537-3616002, Fax: 86-537-3616777

摘要/Abstract

摘要： Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85?1.98) compared with normal striatal neurons (28.43?1.46, P<0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81?2.04 vs. 44.20?1.31, P<0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10?2.17 vs. 50.11?2.01, P<0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.

关键词: neuron, morphine tolerance, suppression subtractive hybridization

Abstract: Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH).Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85?1.98) compared with normal striatal neurons (28.43?1.46, P<0.01). Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 (Mtch1; 96.81?2.04 vs. 44.20?1.31, P<0.01) and thymoma viral proto-oncogene 1 (Akt1; 122.10?2.17 vs. 50.11?2.01, P<0.01), showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.

Key words: neuron, morphine tolerance, suppression subtractive hybridization

基金资助:

The National Natural Science Foundation of China (81070961,30770676, and 30870932 ) , the Natural Science Foundation of ShandongProvince (ZR2009DZ004), and the Science and Technology Bureau Foundation ofShandong Province (2006GG2202037).

Bo Bai, Hai-qing Liu, Jing Chen, Ya-lin Li, Hui Du, Hai Lu, and Peng-li Yu. Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine[J].Chinese Medical Sciences Journal, 2011, 26(1): 54-59.

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Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

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