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 Sbtra...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 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma 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. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.展开更多
Objective To investigate the changes of β-adrenergic receptor (βAR) in peripheral lymphocytes and β2AR mRNA levels at different stages of bronchial asthma. Methods β2AR density and β2AR mRNA level in peripheral l...Objective To investigate the changes of β-adrenergic receptor (βAR) in peripheral lymphocytes and β2AR mRNA levels at different stages of bronchial asthma. Methods β2AR density and β2AR mRNA level in peripheral lymphocytes, cAMP and cGMP levels in blood plasma were estimated by radioligand binding assay, radioimmunoassay and RT-PCR.Results (1) Maximum bound volume (Bmax) and equilibrium dissociation constant (Kd) of β2AR of lymphocyte in asthma patients at remission stage were markedly higher than that in normal subjects, while cAMP levels in blood plasma showed no difference. Bmax of β2AR and cAMP levels in asthma patients at acute exacerbation stage were significantly lower than that in normal subjects, and Kds between these two groups were not much different. (2) Expression of β2ARmRNA in peripheral lymphocytes of asthmatics at remission stage was not significantly different compared with that in normals. Conclusions Amount and function of βAR and β2ARmRNA levels are related to asthmatic conditions. Changes of βAR and β2ARmRNA in asthma might rather be a pathological change accompanied by the course of the disease than a primary defect.展开更多
基金Supported by the National Natural Science Foundation of China (81070961,30770676,and 30870932)the Natural Science Foundation of Shandong Province (ZR2009DZ004)the Science and Technology Bureau Foundation of Shandong Province (2006GG2202037)
文摘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 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma 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. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.
基金theNationalNatureScienceFoundationofChina (No 395 70370 )
文摘Objective To investigate the changes of β-adrenergic receptor (βAR) in peripheral lymphocytes and β2AR mRNA levels at different stages of bronchial asthma. Methods β2AR density and β2AR mRNA level in peripheral lymphocytes, cAMP and cGMP levels in blood plasma were estimated by radioligand binding assay, radioimmunoassay and RT-PCR.Results (1) Maximum bound volume (Bmax) and equilibrium dissociation constant (Kd) of β2AR of lymphocyte in asthma patients at remission stage were markedly higher than that in normal subjects, while cAMP levels in blood plasma showed no difference. Bmax of β2AR and cAMP levels in asthma patients at acute exacerbation stage were significantly lower than that in normal subjects, and Kds between these two groups were not much different. (2) Expression of β2ARmRNA in peripheral lymphocytes of asthmatics at remission stage was not significantly different compared with that in normals. Conclusions Amount and function of βAR and β2ARmRNA levels are related to asthmatic conditions. Changes of βAR and β2ARmRNA in asthma might rather be a pathological change accompanied by the course of the disease than a primary defect.
