BACKGROUND:An abroad study repoRed the distribution and expression of augmenter of liver regeneration(ALR)in the central nervous system.There are few literatures on how to prepare and evaluate ALR protein polyclonal antibody in recombinant rats,and how to construct prokaryotic expression vector.There are no repots concerning ALR in the central nervous system in China.OBJECTIVE:TO express ALR fusion protein in E coli BL21 and prepare and identify polyclonal antibody.METHODS:RNA was extracted from the hippocampus of Sprague Dawley rats.The prokaryotic expression plasmid pET28a-ALR was constructed and the positive recombinant plasmid was transformed into BL21.Protein ALR was expressed by inducing transformed BL21 with Isopropyl-β-D-thiogalactopyranoside(IPTG)and purified by Ni~(2+)affinity chromatography column after immune the rabbit for 4 times.the serum of rabbits was extracted from hear as polyclonal antibody.The titer and specificity of the rabbit's antiserum was respectively measured by ELISA and Western blotting The following parameters were measured:construction of prokaryotic expression plasmid pET26a-ALR;pET28a-ALR recombinant enzyme digestion evaluation;results of ELISA and Western-blotting.RESULTS AND CONCLUSION:Expecting bands were obtained by double enzyme digestion electrophoresis,respectively 5.3 kb and 0.4 kb.Nucleotide sequence analysis verified that prokaryotic expression vector pET28a-ALR was successfully constructed.The 19 ku fusion protein was successfuIly expressed.The titer of the antiserum measured by ELlSA could achieve 1:2 000 This indicated that antibody and purified recombinant ALR had a good reaction.and high titer.could meet the experimental require.Western blotting analysis proved that the antibody could identify the prokaryotic expression product of ALR.Prokaryotic expression system expressed ALR fusion protein,prepared and purified polyclonal antibody of ALR protein,and could meet the experimental require of ALR immunoblotting.

Objective To compare the effects on blood lipid level by immunosuppressive drugs in renal transplantation recipients. Methods Two hundred and eighty-three renal allograft recipients with tacrolimus(FK506), cyclosporine A(CsA) and rapamycin (SRL) immunosuppressive regimen were reviewed in this study. The variation of whose total cholesterol(TC) and triglyceride(TG) concentration in serum were compared before and after three immunosuppressive regimen. Results There was no significant difference in TC and TG before and after oral FK506 for 93 patients[(4.9± 1. 1) and (1. 4±0. 8)mmol/L vs (4. 9±1.1) and (1.4±1.0)mmol/L, respectively, P>0. 05]. The concentration of TC and TG from 106 patients with CsA[(4. 8±1. 0) and (1. 6±0. 8)mmol/L vs (6. 6±1. 7) and (3. 2±1. 0)mmol/L, respectively] and 29 patients with SRL was higher than those before taking drugs, P<0. 05. The concentration was increased after 12 to 24 weeks generally. The concentration of TC and TG of CsA from FK506 to tacrolimus for 51 patients[(6. 7±1. 1) and (2. 8± 1. 0)mmol/L vs (4. 7±1. 7) and (1. 5±1. l)mmol/L, respectively] were decreased after 12 weeks (P<0. 01). Conclusions Primary factor of dyslipidemia was that CsA and SRL were used for patients post-renal transplantation, which should be regarded. The FK506-based immunosuppressive regimen should be recomended in renal transplantation patients who have a hyperlipidmia.

Objective To assess the clinical value of three dimensional dynamic contrast enhanced MR angiography (3D DCE MRA) in the detection for intracranial aneurysm. Methods 3D DCE MRA was performed in 54 patients highly suspected with intracranial aneurysms. Then conventional digital subtraction angiography (DSA) and feasible endovascular treatment were performed simultaneously. A three dimensional fast imaging with steady state precession (3D FISP) was used for 3D DCE MRA(Gd DTPA dose, 0.2 mmol per kilogram for body weight; acquisition time, 10 seconds). The source images were subtracted from mask images and transferred to computer workstation. All images were subsequently post processed using three dimensional reconstruction. 3D DCE MRA images and DSA images were compared for demonstration of the aneurysm, its neck, and relationship with parent artery, and the usefulness for endovascular treatment was evaluated. Results There were 39 cases with 45 intracranial aneurysms. The sensitivity, specificity, and accuracy of 3D DCE MRA were 96%, 73%, and 90%, respectively. Aneurysm and its neck depiction at 3D DCE MRA was significantly better than that at DSA, especially for aneurysms adjacent to the cavernous sinus and near the PICA of vertebral artery. 3D DCE MRA could guide neurosurgeons to the desired DSA projection, and helped them make plan for interventional or surgical treatment in advance. But the diagnosis should be very carefully made for small aneurysms located in the periphery and the arterial bifurcation. Conclusion 3D DCE MRA is a fast, noninvasive and efficient technique for diagnosing intracranial aneurysms. Its three dimensional information is helpful for DSA demonstration and treatment planning. Any uncertain diagnosis requires DSA confirmation.

Objective To study the correlation between the post-transplant renal allograft function and the variation of serum cystatin C (CyC) concentration in renal allograft recipients. Methods One hundred and ninety-three renal allograft recipients accepted the same combination immunosuppressive regimen of tacrolimus, mycophenolate and prednisone were enrolled into the study. Patient's serum and urine samples were collected on day 5 post-transplant to detect serum cystatin C, serum and urine creatinine (SCr). Correlation analysis was used to analyze correlation between CyC concentration and SCr concentration or the calculated creatinine clearance rate (CkCCr) by using the Cockcroft-Gault equation and urine creatinine clearance rate (CCr). Specificity and sensitivity of using the CyC concentration to evaluate glomerular filtration rate (GFR) were calculated as well.Results The mean concentrations of serum CyC and SCr on day 5 post-transplant were (1.91±1.2)mg/L and (174.0±129.1)μmol/L, respectively. While the CCr and CkCCr were (67.9±27.3)ml/min and (68.1±27.8)ml/min, respectively. Forty-two patients had a CyC concentration below 1.25 mg/L, 102 patients'CyC concentrations were between 1.25 and 2.0 mg/L and 49 patients'CyC concentrations were above 2. 0 mg/L. As for SCr, 62 patients had a concentration below 125 μmol/L, 83 patients'concentrations were between 125 and 200 μmol/L and 48 patients'concentrations were above 200 μmol/L. For CkCCr, there were 52 cases with a concentration above 80 ml/min, 96 cases with a concentration between 80 and 60 ml/min and 45 cases with a concentration below 60 ml/min. Serum CyC concentration had a negative correlation with CkCCr (r=-0. 907, P＜0. 001) and had a significantly positive correlation with SCr concentration (r=0. 886, P＜0. 001). SCr had a significantly negative relationship with CkCCr (r=-0. 889 ,P＜0. 001). Serum CyC had higher correlation with CkCCr than the correlation between SCr and CkCCr. The ROC curves showed that areas under curve of CyC, SCr, CCr and CkCCr were 0. 877, 0. 771, 0. 832 and 0. 909, respectively. Specificity and sensitivity of CyC, SCr,CCr and CkCCr were 69.3%, 96.1%, 77.1%, 71.3%and 91.6%, 52.2%, 67.5%, 84.6%, respectively.Conclusions Serum CyC concentration elevates earlier than SCr concentration when there is slight renal function impairment. Serum CyC concentration might become a more sensitive marker to evaluate the post-transplant renal allograft function in renal transplant recipients.

Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from human peripheral blood and their total RNA was extracted.The gene fragment encoding GPI was amplified by RT-PCR using the obtained RNA as template and was inserted into the constructed recombinant plasmid pBudCE4.1-bcr/abl in order to anchor GPI and bcr/abl.The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI was transfected into COS-7 cells,and the expressions of objective fragment were detected by RT-PCR and Western blotting.Results The results of restriction analysis,PCR and DNA sequencing proved that GPI-anchored bcr/abl fusion fragment was correctly inserted into vector pBudCE4.1.The expression of bcr/abl fusion gene and fusion protein were identified in transfected COS-7 cells and on their membrane.Conclusion The recombinant plasmid pBudCE4.1-bcr/abl-GPI was successfully constructed and expressed on the membrane of COS-7 cells,which found a basis of cell immunity with GPI-anchored bcr/abl fusion gene.

Objective To explore the effectiveness of a respiratory function training instrument with stable chronic obstructive pulmonary disease (COPD) patients.Methods Sixty-seven COPD patients in the stable period were randomly divided into a treatment group of 36 and a control group of 31 using a random number table.Both groups were given conventional pulmonary rehabilitation,including half-closed lip respiration,abdominal respiration and upper limb training.The treatment group was additionally provided with 30 minutes of respiratory training using a respiration function training instrument 5 times per week for 6 months.Both groups were assessed for their mobility,life quality and pulmonary function using the 6-minute walk test (6 MWT),a COPD assessment test (CAT),the BODE index,forced vital capacity (FVC),forced expiratory volume in one second (FEV1) and surface electromyography (SEMG) of the respiratory muscles before and after the 6-month intervention.Results Before the treatment there were no significant differences between the two groups in terms of any of the measurements.After the treatment,significant improvement was observed in the average 6 MWT,CAT,BODE index and SEMG results in both groups,but with significantly greater improvement in the treatment group.The average FVC and FEV1 results did not improve significantly,so after the intervention there was still no significant difference between the groups.Conclusions Respiratory training using the pulmonary function training instrument can improve the mobility,life quality and the functioning of the respiratory muscles of COPD patients in the stable period.

Objective To investigate the anti-tumor activities of cell-penetrating peptide ( CPP) - mediated trichosanthin ( TCS) , which is a recombinant protein obtained from Radix Trichosanthis. Methods Cysteine residue was introduced to the C-terminus of TCS by protein recombinant technique, and then with the newly-formed terminal as the modification site, TCS was coupled with CPP. As a target protein, CPP-mediated TCS was isolated and purified by affinity chromatography. The expression of the target protein and its responsiveness to reducing substances were detected by using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cellular uptake rate of CPP-mediated TCS was determined by using cell uptake test, and its anti-tumor activity was measured by using methyl thiazolyl tetrazolium (MTT) assay. Results The TCS-CPP compound had been successfully developed in this study, and showed certain reducing responsiveness. After modified with CPP, TCS had higher cellular uptake rate and stronger anti-tumor effect on HeLa and MCF-7 cells. Conclusion TCS modified by CPP can enhance the anti-tumor activities of TCS.

Objectives To investigate the effects of ethanol on neural development and kainate receptor expression in young mice. Methods Fetal alcohol spectrum disorder model was established by administration of 20% ethanol solu?tion to 7-day-old Kunming mice and control animals received physiological saline (The number of treatment and control were 80 and 40, respectively ). Body weight and general biological features were observed every day. Morris water maze was used to test learning and memory ability. Fluoro-Jade B was used to examine neural cells 24 hours after treatment in additional thirty 7-day-old Kunming mice which were further divided into two groups:a treatment group receiving 20%ethanol solution (n=15) and a control group receiving physiological saline (n=15). The development of neural cells and expression levels of kainite receptors were examined by using immunofluorescence staining. Results The body weight was significantly lighter in treatment group than in control group(control:21.13 ± 1.72g,treatment:13.96 ± 2.98g,P<0.05). Morris test showed that model group had longer latency than control group to find hidden platform(control:21.05± 5.31s,treatment:34.15±3.26s,P<0.05). Spatial probe test revealed that the number of passing through the platform were significantly smaller in model group than in control group(control:2.70 ± 1.25 times,treatment:0.93 ± 0.80 times,P<0.05). Astrocyte development anomaly was evident after ethanol treatment for 7 days. The expression levels of kainite re?ceptor GluR-6 and KA2 were up-regulated in the CA region of the hippocampus after ethanol treatment for 7 days. Con?clusion Kainite receptor GluR-6 and KA2 in CA region of the hippocampus may contribute to ethanol-induced hippo?campal neural development anomaly.

Objective To explore the effects of action observation therapy on upper-extremity motor function after ischemic stroke and on the motor cortex using functional magnetic resonance imaging (fMRI).Methods Forty patients with ischemic stroke were randomly assigned to an observational group (n =20) or a control group (n =20).Both groups received conventional rehabilitation,while the observational group was additionally provided with action observation therapy for 8 weeks.Both groups were assessed using the Fugl-Meyer assessment (FMA) and the Barthel index (BI) before and after the 8 weeks of treatment and functional magnetic resonance imaging was performed before treatment.Two months after the treatment,nine patients of the experimental group and 8 of the control group who continued to receive their respective treatments after discharge were again assessed using functional magnetic resonance imaging.Results After the treatment the average FMA score and BI score of both the observational group and the control group had increased significantly.The increase in the average FMA score of the observational group was significantly greater than that of the control group.However,there was no significant difference between the two groups in the increases in BI score after 8 weeks of treatment.The fMRI results showed that there was a significantly greater rise in activity in the bilateral precentral gyrus,parietal lobe and the supplementary motor area of the patients in the observational group after the treatment compared with the control group.Conclusion Action observation therapy can improve upper extremity motor function and performance in the activities of daily living after ischemic stroke and induce changes in the excitability of the cerebral motor cortex.

Objective:To study the influence of protein transduction domain (PTD)-oligomerization domain (OD)-HA fusion proteins on apoptosis of bcr/abl-positive cell lines. Methods:bcr/abl-positive cells were treated with PTD-OD-HA protein. The apoptoses of the cells were detected by flow cytometry (FCM), DNA ladder and transmission electron microscopy (TEM), and the levels of apoptosis-related genes bax and bcl-2 were detected by RT-PCR and Western blotting. Results:FCM examination demonstrated that PTD-OD-HA protein induced the apoptosis of bcr/abl-positive cells; DNA ladder showed that the classic DNA ladders appeared in BaF3-P210 and K562 cells after 48 h treatment with PTD-OD-HA proteins; the apoptoses of BaF3-P210 cells were observed by TEM; the levels of bax in mRNA and protein increased in BaF3-P210 and K562 cells, and bcl-2 decreased. Conclusion:PTD-OD-HA proteins specifically induced the apoptosis of bcr/abl positive cells.