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 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.

Objective To investigate the rehabilitative effect of low frequency repetitive transcranial magnetic stimulation (rTMS) on convalescing patients with Broca's aphasia. MethodsTwenty-eight patients with Broca's aphasia recovering from cerebral infarction were randomly divided into a stimulation group and a control group with 14 subjects in each.Patients in the control group accepted conventional drugs,speech rehabilitation and sham stimulation,while patients in the stimulation group were in given low frequency rTMS in place of the sham stimulation.Their speech performance was evaluated using the China Rehabilitation Research Center's aphasia examination (CRRCAE) pre-stimulation,post-stimulation and 90 days later. ResultsCompared with before treatment and with the controls,the speaking scores of the stimulation group increased significantly after treatment and also 90 days later. ConclusionLow frequency rTMS can not only improve the speech performance of Broca's aphasia sufferers in the short term,but it also plays a lasting role.It may thus have clinical application for patients with Broca's aphasia.

Objective To assess the value of S index and FIB-4 for diagnosis of liver fibrosis in patients with chronic hepatitis B (CHB) by comparing with traditional indexes APRI and Forns.Methods A total of 361 patients with confirmed CHB from the First Hospital of Nanjing Medical University and Huashan Hospital Affiliated to Fudan University during January 2006 and December 2011 were enrolled in the study.The clinical,laboratory and pathological data of patients were collected.Four noninvasive score systems APRI,Forns,S index and FIB-4 were computed.With liver biopsy as the gold standard,the area under the ROC curve (AUROC) was used to assess the value of above 4 score systems in diagnosis of liver fibrosis,and Z test was performed to evaluate the effectiveness of above systems.Results The areas under ROC curve (AUCs) of APRI,Forns,S index and FIB-4 for significant fibrosis (≥S2) were (0.737 ±0.027),(0.716 ± 0.028),(0.745 ± 0.026) and (0.781 ± 0.025),respectively.When the cut off value of FIB-4 was set at 1.62,the sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) for diagnosis of significant fibrosis were 59.3％,85.8％,89.4％ and 51.2％,respectively,which were better than Forn index (Z =3.28,P =0.001).While for S4 (cirrhosis) the AUCs of APRI,Foms,S index and FIB-4 were (0.687 ± 0.035),(0.792 ± 0.028),(0.863 ± 0.024) and (0.832 ± 0.025),respectively.When the cut off value of S index was set at 1.06,the sensitivity,specificity,PPV and NPV for diagnosis of cirrhosis were 77.9％,85.5％,59.4％ and 93.5％,respectively,which were better than APRI and Forns (Z =6.74 and 3.21,P ＜ 0.01).Conclusions APRI,Forns,S index and FIB-4 are simple and accurate methods for assessing liver fibrosis.FIB-4 and S index are better than APRI and Forns in diagnosis of significant fibrosis and cirrhosis,which may replace liver biopsy in certain extend.

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.

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 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 investigate the changes of microRNA (miR)-146a ,miR-29b expression levels and the 3 kinds of meth-ylase DNMT1 ,DNMT3a and DNMT3b levels in K562 cell lines after BCR/ABL inhibitor Gleevec treatment .Methods The half maximal inhibitory concentration(IC50 ) of Gleevec on K562 cells was detected by the MTT method .The stem loop primers method and the fluorogenic quantitative PCR were adopted to detect miRNAs and the methylase gene level .Results IC50 of Gleevec acting on K562 cells was 40 .85μmol/L .After Gleevec action ,miR-29b showed the increasing trend ,but 3 kinds of methylase expression level were decreased to some extent .Gleevec could significantly increase the miR-146a level in K562 cells(P<0 .05) .Conclusion Gleevec can influence the expression of miR-146a ,miR-29b and DNMTs levels in K562 cells .