Objective:To construct prokaryotic expression vector carrying esat6 gene and express in E. coli. Methods: The MTb esat6 gene was amplified by PCR,then cloned into pQE30 plasmid, sequenced and then cloned into pET32a( + ) plasmid. Thus two kinds of prokaryotic expression vectors were constructed. Results: After being transformed into the E. coli and inducted with 1mM IPTG,no protein was expressed in the pQE30 - ESAT6 system, but a recombinant protein, about 19 kDa,was expressed in the pET32a( + ) - ESAT6 system. In the presence of 1mM IPTG for 4h,the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E. coli. It's antigenicity was confirmed by Westem blotting. The protein was purified through the Ni - NTA resin and the purity reached 92 % . Conclusion : The prokaryotic expression vector (pET32a( + ) - ESAT6) was constructed successfully,and the rESAT6 was obtained,providing an experimental basis for application of rESAT6.

Objective:To construct lhp-esat6 fusion gene and its prokaryotic expression vector and express it in E.coli.Methods:By Gene SOEing techniques,a fusion gene was constructed by splicing lhp gene and esat6 gene,then cloned into pQE30 plasmid and expressed in DH5a.Results:The fusion gene was identified by DNA sequencing.A fusion protein about 26kDa was expressed in the E.coli.In presence of 1mM IPTG for 4h,the fusion protein was expressed to the maximum.The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E.coli.Its antigenicity was confirmed by Western blotting.The fusion protein was purified through the Ni-NTA resin and the purity reached 98%.Conclusion:The lhp-esat6 fusion gene and the prokaryotic expression vector (pQE30-CFP10-ESAT6) were constructed successfully,and the fusion protein CFP10-ESAT6 was obtained,so that it can provide an experimental basis for application of the recombinant CFP10-ESAT6.

Objective:To reconstruct Mycobacterium shuttle expression plasmid with Mycobacterium tuberculosis HSP70 promoter based on shuttle plasmid pJEM11.Methods:The cDNA fragment of 229bp promoter and its correlative sequence was amplified by PCR.Multiple clone sites was added to the end of amplified fragment,and then it was cloned into the pJEM11 to construct the plasmid pJCH02.The pJCH02 was identified by digestion with restriction endonuclease and sequence analysis.The lhp-esat6 fusion gene was cloned into the pJCH02,then its expression in BCG was confirmed by SDS-PAGE.Results:The sequence of HSP70 promoter and its correlative sequence was in accordance with the predicted sequence.The lhp-esat6 fusion gene was expressed in BCG.Conclusion:The shuttle expression plasmid pJCH02 is constructed successfully.The study provides the basis for BCG polyvaccine.

BACKGROUND:For patients with lumbar spondylolisthesis combined with osteoporosis, appropriate fixation system for effective reset and good fixation stability is currently a hot issue of clinical concern. Pedicle screw screw-rod system after bone cement perfusion can achieve the effective fixation between pedicle screw system and the vertebral bone. OBJECTIVE:To observe the therapeutic effect of bone cement-augmented pedicle screw on patients with lumbar spondylolisthesis combined with osteoporosis. METHODS:17 cases of lumbar spondylolisthesis combined with osteoporosis were identified by bone density test. They received the posterior open reduction and internal fixation, and implanted with 68 bone cement-augmented pedicle screws. Their repair effects were observed by short-term fol ow-up. Patients were evaluated using low back pain Visual Analog Scale and lower limb Oswestry Disability Index before treatment, 1 week, 3 months and 1 year after treatment. Vertebral height, intervertebral height, screw loosening and bone cement leakage were observed using imaging.
RESULTS AND CONCLUSION:Compared with pre-treatment, low back pain Visual Analog Scale score and lower limb Oswestry Disability Index were significantly improved at 1 week, 3 months and 1 year after treatment (P<0.05). No significant difference was detected between post-treatment and fol ow-up (P>0.05), which indicated that clinical repair effect could be effectively maintained. At 3 months of fol ow-up, one screw loosening occurred in two patients. During fixation, mild bone cement leakage appeared in seven vertebral bodies with screw fixation, no symptoms or subsequent complications were observed. There were no significant differences in vertebral height and intervertebral height before and after treatment and during fol ow-up (P>0.05). These results suggest that bone cement-augmented pedicle screw for patients with lumbar spondylolisthesis combined with osteoporosis can effectively reset vertebral slippage, effectively provide good anti-pul-out force for a long term, and the effect was stable. Bone cement augmentation can perfectly strengthen fixation, shows good biocompatibility, and avoids osteoporosis around the screw-induced failure fixation.

Objective To determine whether or not capsazepine(CPZ),a transient receptor po-tential vanilloid-1(TRPV1)antagonist,attenuates lidocaine-induced cytotoxicity on rat dorsal root ganglion (DRG)neurons in vitro.Methods Adequate DRG neurons from 3-day neonatal Wistar rats were obtained,cultured and purified in vitro.To achieve higher cell viability,we reduced the concen-tration of trypsin to 0.125% compared with others.The purified DRG neurons were incubated with 0,2.5,5,10,20 and 40 mmol/L lidocaine for 10 min,respectively,their viabilities were examined using Cell Counting Kit(CCK-8)assay,and the lethal concentration 50(LC50 )of lidocaine on DRG neurons was calculated.Then,the variation of lidocaine-induced cell viability at LC50 ,when 0,1,10 and 100 μmol/L CPZ were respectively added to the incubations,was examined with CCK-8 assay. Results The purified percentage of DRG neurons was as high as 91% after digesting by 0.125%trypsin and purifying in vitro.Cell viability of DRG neurons in group L1,L2,L3,L4,L5 was signifi-cantly down regulated compared with the control group,to be specific,that of L3,L4,L5 being re-markably lower than that of L1,that of L4,L5 lower than that of L2 and that of L5 lower than that L3 and L4.After lidocaine induced DRG neurons for 10 min,LC50 was 30 mmol/L;10 μmol/L and 100 μmol/L CPZ significantly reduced LC50 DRG neuron toxicity induced by lidocaine (P <0.05). The effect of 10 μmol/L CPZ had reached the maximal effect,decreasing the cell viability decrease from 50% to 35%.Conclusion The novel method in this experiment is effective to obtain good DRG neurons,the LC50 of lidocaine on rat DRG neurons is 30 mmol/L,and CPZ attenuates the cytotoxicity induced by lidocaine on rat DRG neurons.

Renal fibrosis is the final common pathway for all kinds of chronic kidney diseases to end-stage renal diseases. Angiotensin Ⅱ plays an important role in the process of renal fibrosis. How to block angiotensin Ⅱ has been a focus of nephrology. This article reviewed the progress in traditional Chinese medicine against renal fibrosis with angiotensin Ⅱ as research target.

Dosage Forms ; Drug Carriers ; Drug Delivery Systems ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Nanotechnology

Hematologic Diseases ; diagnosis ; Hematologic Tests ; Humans

Objective To investigate the roles of reactive oxygen species (ROS) and phosphatidyl-inositol 3-kinase-Akt (PI3K-Akt) in neuroprotection against spinal cord ischemia-reperfusion (I/R) injury by ischemic postconditioning (IP) or controlled low perfusion pressure (LR). Methods One hundred and twenty-six adult male SD rats weighing 300-350 g were randomly divided into 7 groups (n = 18 each): group Ⅰ I/R; group ⅡIP; group Ⅲ LR; group Ⅳ IP + LY-294002 (IP + LY); group Ⅴ LR + LY-294002 (LR + LY); group Ⅵ IP +N-acetylcysteine (IP+ N) and group Ⅶ LR+ N-acetyl-cysteine (LR+ N). Spinal cord ischemia was induced by 9 min occlusion of the thoracic aorta combined with controlled hypotension (MAP = 40 mm Hg). In IP group the animals were subjected to 3 cycles of 30 s reperfusion interspersed with 30 s ischemia immediately after release of aortic occlusion. In LR group MAP was maintained at 40 mm Hg for another 5 min immediately after release of aortic occlusion, then increased to 100 mm Hg. In group Ⅳ-Ⅶ LY-294002 (PI3K inhibitor) 25 mg/kg or N-acetylcysteine (ROS scavenger) 100 mg/kg were administered at the onset of reperfusion. Twelve animals in each group were killed at 2 h of reperfusion. The lumbar segment of the spinal cord was removed for determination of the level of Akt phosphorylation and opening of mitochondrial permeability transition pore (mPTP). Neurological function was assessed and scored (15 = normal function, 0 = unable to move hind limb) at 4, 12, 24 and 48 h of reperfusion in another 6 animals in each group. The animals were then killed after last assessment and the lumbar segment of the spinal cord was removed for detection of apoptotic neurons. Results Compared with I/R group,both IP and LR significantly enhanced the level of Akt phosphorylation in the spinal cord, inhibited mPTP opening and neuronal apoptosis and increased neurological function scores. There was no significant difference in the protective effects exerted by IP and LR. The neuroprotective effects exerted by IP and LR were abolished by LY-294002 and N-acetylcysteine. Conclusion Ischemic postconditioning or controlled low perfusion pressure can protect the spinal cord from I/R injury by activating PI3K-Akt and inhibiting mitochondrial permeability.