Objective To establish and identify tetracycline controlled gene inducible system in immortalized rat astrocyte strains. Methods The PT67 cells were transfected with pRevTet-On vector. The transfected cells were selected in medium containing G418. RT-PCR assay was used to confirm that Rev Tet-On virus was packed correctly. The viral titer was assayed by infecting NIH3T3 cells. The immortalized astrocyte was infected by RevTet-On virus and individual clones were selected. All individual clones were transiently transfected with pRev TRE-Luc, which carried luciferase gene. The clone with low background and high induction expression was selected and its inducibility was tested. Results The RT-PCR assay showed that RevTet-On virus was packed successfully. The highest viral titer of RevTet-On was 7.4?05 CFU/ml. Rev Tet-On infected immortalized astrocyte and 48 individual clones were isolated. Clone 6 was selected for its highest induction of the luciferase activity in response to doxycycline and the lowest leakiness (activity in the absence of doxycycline) and its inducible fold was 20.6. The expression of luciferase was induced in a dose-dependent manner by doxycycline at the concentrations between 100 and 2 000 ng@ml-1. The expression of luciferase began 1h after doxycycline administration (1000 ng?ml-1) and reached the maximum level 48 h later. Conclusion The tetracycline controlled inducible system is established successfully in immortalized rat astrocyte strains, which is useful for the study of control exogenous gene expression.

Objective To construct a recombinant retroviral vector with human preproenkephalin gene regulated by tetracycline. Methods Human preproenkephalin gene was amplificated by polymerase chain reaction (PCR) and was cloned into retrovirus tetracycline responsive plasmid. Then this recombinant plasmid and regulatory plasmid pRev Tet-On were transferred into packaging cell PT67 respectively. The transfected PT67 / Tet-On and PT 67 / TREhPPE cells were selected by the corresponding antibiotics and identified by RT-PCR. The selected cells were enriched and virus liter was assayed using NIH3T3. Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant RevTRE/hPPE vector was constructed successfully. The virus liter of PT 67/TREhPPE was 3.2 ? 103 CFU?ml-1 and the virus titer of PT67/Tet-On was 2.6?105 CFU?ml-1.Conclusion A recombinant retroviral vector with human preproenkephalin gene regulated by trtracycline and stable virus producing lines have been successfully constructed, providing a good basis for further research on regulated cell therapy of chronic pain.

Objective To investigate the effect of transplantation of bone marrow mesenchymal stem cells (MSCs) in subarachnoid space on chronic neuropathic pain in rats. Methods One hundred and forty female SD rats weighing 180-200 g were used in this study. Chronic neuropathic pain (NP) was induced by ligation and separation of tibial and common fibular nerves (SNI). Two weeks after the surgery the animals were randomly divided into4 groups (n=35 each):group Ⅰ NP; group Ⅱ NP+ MSC (MSCs); group Ⅲ NP+ phosphate buffer saline (PBS) and group Ⅳ NP+ bone marrow monocyte (BNMCs). MSCs 10 μl, PBS 10 μl and BNMCs 10 μl were injected into subaraclmoid space at 2 weeks after surgery in group Ⅱ , Ⅲ and Ⅳ respectively. Paw withdrawal threshold to yon Frey filament stimulation (PWT) was measured before surgery (T0, baseline), at 2 weeks after surgery (T1) and 1, 2, 3, 4 and 5 weeks after subarachnoid injection (T2-6). Five animals were killed at T1-6 in each group and their lumbar enlargements were removed for determination of BDNF mRNA expression. Results PWT was significantly decreased by SNI at T1 in all 4 groups, and at T2-6 in group Ⅰ , Ⅲ and Ⅳ as compared with the baseline at T0 (P ＜ 0.05). Subarachnoid MSC transplantation significantly reduced mechanical hyperalgesia at T2-6 and up-regulated BDNF mRNA expression at T2-4 as compared with that at T1 (P ＜0.05). There was no significant change in PWT and BDNF mRNA expression after subarachnoid PBS and BNMCs injection in group Ⅲ and Ⅳ (P ＞ 0.05). Compared with group Ⅰ , PWT was significantly increased and BDNF mRNA expression was up-regulated in group Ⅱ (P ＜ 0.05), but no significant change in PBS and BNMCs was found in group Ⅲ (P ＞ 0.05) .Conclusion Up-regulation of BDNF mRNA expression in the spinal cord may be involved in the amelioration of chronic neuropathic pain by subarachnoid bone marrow mesenchymal stem cell transplantation.

Objective To evaluate the role of ATP-sensitive potassium (KATP) channel in spinal dorsal horn neurons in hyperalgesia after thoracotomy in rats.Methods Twenty-eight Sprague-Dawley rats,aged 7-9 weeks,weighing 250-350 g,in which intrathecal catheters were successfully implanted without complications on 14th day after chronic post-thoracotomy pain was induced,were randomly divided into 4 groups (n =7 each):control group,the solvent dimethyl sulfoxide (DMSO) group,KATP channel opener pinacidil group (group P) and KATP channel blocker glibenclamide group (group G).10％ DMSO 10 μl,pinacidil 10 μg/10 μl and glibenclamide 50μg/10μ1 were injected intrathecally in groups DMSO,P and G at 5 day after the intrathecal catheter was implanted,respectively.Paw withdrawal threshold to von Frey filament stimulation was measured before intrathecal administration and at 10,30 and 60 min after intrathecal administration and the acetone test was performed.Coldinduced pain threshold was measured.Results There was no significant difference in paw withdrawal threshold to yon Frey filament stimulation at each time point among the four groups (P ＞ 0.05).Compared with C and DMSO groups,cold-induced pain threshold was significantly increased in group P and decreased in group G (P ＜ 0.05).There was no significant difference in cold-induced pain threshold between C and DMSO groups (P ＞ 0.05).Conclusion KATP channel in spinal dorsal horn neurons is involved in the maintenance of hyperalgesia after thoracotomy in rats.

Objective To construct lentivirus vector expressing antigene RNA and ferritin gene.Methods Intermediate plasmid pGC-FU-HF was constructed by transfecting lentivirus vector pGC-FU with heavy chain ferritin subunit gene.The target plasmid pGC-agRNA-HF was subsequently constructed by transfecting the intermediate plasmid with β-arrestin 2 antigene RNA.The NG108-15 cells were transfected with the target plasmid.The titre of lentivirus vector was measured by RT-PCR.The expression of antigene RNA and ferritin gene was determined by Western blot and RT-PCR.Results Lentivirus vector was successfully transfected with antigene RNA and ferritin gene.The titre of lentivirus vector was 2.00 × 109 TU/ml.The expression of β-arrestin2 protein was down-regulated and the expression of ferritin protein up-regulated in the NG108-15 cells after being transfected with the lentivirus vector.Conclusion Lentivirus vector expressing antigene RNA and ferritin gene has been successfully constructed.

Descending nociceptive modulation from the supraspinal structures plays an important role in cancer-induced bone pain (CIBP). Rostral ventromedial medulla (RVM) is a critical component of descending nociceptive facilitation circuitry, but so far the mechanisms are poorly known. In this study, we investigated the role of RVM glial activation in the descending nociceptive facilitation circuitry in a CIBP rat model. CIBP rats showed significant activation of microglia and astrocytes, and also up-regulation of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and pro-inflammatory mediators released by glial cells (IL-1β, IL-6, TNF-α and brain-derived neurotrophic factor) in the RVM. Stereotaxic microinjection of the glial inhibitors (minocycline and fluorocitrate) into CIBP rats' RVM could reverse the glial activation and significantly attenuate mechanical allodynia in a time-dependent manner. RVM microinjection of p38 MAPK inhibitor (SB203580) abolished the activation of microglia, reversed the associated up-regulation of pro-inflammatory mediators and significantly attenuated mechanical allodynia. Taken together, these results suggest that RVM glial activation is involved in the pathogenesis of CIBP. RVM microglial p38 MAPK signaling pathway is activated and leads to the release of downstream pro-inflammatory mediators, which contribute to the descending facilitation of CIBP.

Descending nociceptive modulation from the supraspinal structures plays an important role in cancer-induced bone pain (CIBP).Rostral ventromedial medulla (RVM) is a critical component of descending nociceptive facilitation circuitry,but so far the mechanisms are poorly known.In this study,we investigated the role of RVM glial activation in the descending nociceptive facilitation circuitry in a CIBP rat model.CIBP rats showed significant activation of microglia and astrocytes,and also up-regulation of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and pro-inflammatory mediators released by glial cells (IL-1β,IL-6,TNF-a and brain-derived neurotrophic factor) in the RVM.Stereotaxic microinjection of the glial inhibitors (minocycline and fluorocitrate) into CIBP rats' RVM could reverse the glial activation and significantly attenuate mechanical allodynia in a time-dependent manner.RVM microinjection of p38 MAPK inhibitor (SB203580) abolished the activation of microglia,reversed the associated up-regulation of pro-inflammatory mediators and significantly attenuated mechanical allodynia.Taken together,these results suggest that RVM glial activation is involved in the pathogenesis of CIBP.RVM microglial p38 MAPK signaling pathway is activated and leads to the release of downstream pro-inflammatory mediators,which contribute to the descending facilitation of CIBP.

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