Objective To screen monoclonal antibodies against different binding sites of connexin 43(Cx43) and establish a double antibody sandwich ELISA method for quantitative detection of Cx43 protein in order to further study the structure,function and subcellular localization of full-length and truncated Cx43 protein.Methods The sequence of Cx43 protein was analyzed by bioinformatics method. The peptide fragments were designed and synthesized for different domains, and coupled with keyhole limpet hemocyanin(KLH) protein, which were used as immunogen to immunize 15 female BALB/c mice.Hybridoma cells were prepared by cell fusion technology, and cell lines stably secreting monoclonal antibodies against Cx43 protein were screened. The antibodies were purified by Protein G affinity chromatography, and its subtype, affinity and specificity were identified. Two high affinity monoclonal antibodies were used as capture antibody and detection antibody respectively(labeled with HRP) to establish a double antibody sandwich ELISA method. The sealing solution(5% milk-PBS, 3%BSA-PBS), washing solution [0. 01 mol/L PBS(pH 7. 2), 0. 01 mol/L PBS-0. 5% Tween 20(pH 7. 2)(i. e., PBST)] and sample diluent [3% BSA-PBS and 0. 01 mol/L PBS(pH 7. 2)]were optimized, and the method was verified for the linear range, sensitivity, specificity, precision and accuracy. The content of Cx43 protein in mouse brain tissue, intestine tissue, femur tissue and MC3T3-E1 mouse embryonic osteoblast lysate was detected by the established method.Results Three hybridoma cell lines secreting monoclonal antibodies against Cx43 were screened and named as 1-1E2, 2-1G11 and 3-2G12 respectively. The antibody subtypes were IgG1, IgG1 and IgG2b respectively, and the antibody sensitivity reached 0. 002 μg/mL, among which 2-1G11 and 3-2G12 had higher affinity. A double antibody sandwich ELISA was developed with 2-1G11 as capture antibody and HRP-labeled 3-2G12 as detection antibody. The optimum sealing solution was 3% BSA-PBS, washing solution was PBST, and sample diluent was 0. 01 mol/L PBS(pH 7. 2). Cx43 protein standard showed a good linear relationship with the mean value of A_(450)in the concentration range of 3. 12-200 ng/mL, R~2> 0. 98. Cx43 protein could be specifically detected by this method, and there was no cross reaction with KLH protein, bone morphogenetic protein-2(BMP-2) and human recombinant TGM2 protein. The CV of precision verification was 7. 72%. The recovery rates of high, medium and low concentrations of test samples were all in the range of 85%-115%. The content of Cx43 protein in MC3T3-E1 mouse embryonic osteoblasts was higher(58. 03 ng/mL), while the content in osteogenic tissue was lower(38. 4 ng/mL).Conclusion Specific monoclonal antibodies against different epitopes of human Cx43 were successfully prepared, and a double antibody sandwich ELISA method with high sensitivity and strong specificity for quantitative detection of Cx43 protein was established, which provided a reliable detection method for the study of structure and function of Cx43 protein.

Objective To evaluate the influence of laboratory critical value reporting on the efficacy of pediatric critical care.Methods A comparative analysis was conducted to evaluate the changes after the establishment of laboratory critical value reporting system.The parameters chosen for assessment included laboratory test turnaround time,medical intervention start time,survival rate,etc.Results Before the establishment of laboratory critical value reporting system,laboratory test turnaround time was (44.5±14.6)min,medical intervention start time was (40.7±5.3)min,and the success rate of the emergency treatment in ICU was (80.36±6.32)%[the rate in normal ward was(82.64±9.21)%].But after the establishment of laboratory critical value reporting system,laboratory test turnaround time,medical intervention start time,the success rate of the emergency treatment in ICU (normal ward) were (18.7±8.8)min,(23.9±6.7)min and (89.49±4.58)% [(90.04±6.45)%].Laboratory critical value reporting system shortened laboratory test turnaround time and medical intervention start time (P<0.05),and the successful rate of the emergency treatment improved evidently.Conclusion Laboratory critical value reporting system can improve successful rate of the emergency treatment significantly.

Cytokeratin 1 4 (CK1 4)has a different degree of expression in NSCLC,breast cancer,cer-vical cancer,esophageal cancer and other tumors,except in the normal basal cells.CK1 4 is mainly expressed in the peripheral part of the tumor,which is rarely expressed in the non-aggressive part.Usually the higher malignant of the tumor has the more expression of CK1 4.Given all that,CK1 4 gene plays an important role in the tumor progression and metastasis in a variety of tumors,which can be considered as an biomarker being used in the diagnosis,treatment and prognosis evaluation.

Objective To understand the rpoB gene mutation in M.tuberculosis isolates,and to evaluate their clinical value.Method 335 clinical isolates of mycobacterium tuberculosis(109 isolates drug susceptible,246 isolates rifampin-resistance or multidrug resistance including rifampin) were detected using polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP).Results SSCP pattern of reference mycobacterium tuberculosis H37Rv as control,no mutation was found in rifampin-suscepitible 109 strains.SSCP patterns of 225/246 rifampin resistant clinical isolates were different from the normal control.The sensitivity was 91 5%.31 resistant isolates,included 25 abnormal isolates and 6 normal isolates of SCCP were identified.Sequencing showed 29 isolates had rpoB gene mutations and 3 isolates were not found rpoB gene mutations.The 3 most frequent rpoB gene mutation situs were Leu-531(19 isolates.TCGTTG) and His-526(7 isolates,CACTAC) and Asp-516(3 isolates,GACGTC).Conclusions The results confirm that rpoB gene mutation is the most important mechanism in rifampin resistant tuberculosis mutation situs are 531 tryptophan and 526 histidine;respectively.It is feasible that using PCR-SSCP to detect drug resistance in mycobacterium tuberculosis.

BACKGROUND:At present, electrical stimulation technology has become a hot topic, but its effect on body’s electrical activity remains unclear. Moreover, previous studies on this aspect mainly focused on smal-sized animals. OBJECTIVE:Under the premise without prejudice to cardiac electrical activity, to find the maximum pulse voltage and frequency of electrostimulation that is taken on the liver area of pigs. METHODS:Three healthy male Rong-Chang pigs were obtained. Pulse electrical stimulator electrodes were fixed on right lobe of the surface projection of corresponding liver area. Fixed electrical stimulation parameters were set, including pulse width 10 ms, square wave, stimulation time 1 hour. They received voltage tolerance experiment between 5 V and 100 V and a frequency tolerance experiment between 1 Hz and 5 Hz. A detail record of pigs’ electrocardiogram, discomfort reaction and changes in life behavior of the pigs were obtained. RESULTS AND CONCLUSION:During voltage tolerance experiment, high- and low-voltage electrical stimulation pulse was positively associated with heart rate increment. When the voltage was greater than 35 V, mild arrhythmia such as sinus arrhythmia and ventricular premature beats appeared, without malignant arrhythmia or death. When the voltage was greater than 60 V, qRs formation induced by electrical stimulation wave occurred. In frequency tolerance experiment, when frequency was greater than 2 Hz, arrhythmia appeared, no malignant arrhythmia was found. Pulsed electrical stimulation can induce the formation of qRs wave. Above findings suggest that the appropriate frequency and voltage of pulse electrical stimulation are 2 Hz and 35 V. At > 35 V voltage or > 2 Hz frequency, pulsed electrical stimulation on the liver area can cause arrhythmia, mostly benign arrhythmia.

BACKGROUND:The mechanisms of mesenchymal stem cells directional y homing to infarcted myocardium post myocardial infarction are stil unclear.
OBJECTIVE:To investigate the role of stromal cellderived factor-1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4) axis on mesenchymal stem cellmigration promoted by mononuclear cells after myocardial infarction.
METHODS:Cardiomyocytes and mesenchymal stem cells were respectively isolated from suckling and adult Sprague-Dawley rats. Twelve healthy Sprague-Dawley rats were selected (six rats for myocardial infarction models and six for sham models), then circulating mononuclear cells were isolated. 4,6-Diamino-2-phenyl indole-labeled mesenchymal stem cells, cardiomyocytes and mononuclear cells were cultured into the upper, middle and lower layers of the tri-chamber coculture system, respectively. In this experiment, there were four groups:myocardial infarction group, AMD3100 (CXCR4 inhibitor) group, sham group and blank control group. After 48 hours, the number of migrating mesenchymal stem cells with blue-lighting nucleus was calculated under fluoroscope. Immunocytochemistry and immunofluorescent staining was used to detect SDF-1 expression in cardiomyocytes and CXCR4 expression in mesenchymal stem cells, respectively.
RESULTS AND CONCLUSION:Migrating mesenchymal stem cells with positive expression of CXCR4 were observed in each group other than the blank control group. The number of migrating mesenchymal stem cells was higher in the myocardial infarction group than in the other groups. Tumor necrosis factor-αneutralizing antibody and CXCR4 inhibitor AMD3100 could obviously reduce the number of migrating mesenchymal stem cells (P<0.05). Cardiomyocytes in each group expressed SDF-1 positively. The gray values of SDF-1 expression in the myocardial infarction and AMD3100 groups were significantly higher than those in the sham and blank control groups (P<0.05). SDF-1/CXCR4 axis plays a certain role in mesenchymal stem cells migration promoted by mononuclear cells after myocardial infarction.

Objective To study the feasibility and curative effect of laparoscopic radical resection of rectal carcinoma. Methods Twenty-eight cases undergoing laparoscopic radical resection (laparoscopic group) and 26 cases undergoing open radical resection (open group) were enrolled from January 2004 to December 2007. The following parameters: operation-related situations, postoperative recovery,result of radical resection, and postoperative outcome were compared between the two groups. Results The blood loss during operation in laparoscopic group was less than that in open group [(148.0±26.5) ml vs (396.0±79.6) ml, P<0.01]. The gastrointestinal tract and urination function in laparoseopic group recovered faster than those in open group[the time of diet in taking was (2.8±0.1) d vs (3.9±0.3) d,the time of detaining urethral catheter was(4.2±0.2) d vs (6.0±0.8) d] (P<0.05). The hospital stay was shorter in laparoscopic group than that in open group [(9.8±1.1) d vs(13.2±2.8) d, P<0.01]. The operation time, the number of cleared lymph nodes and complications of laparoscopic between the two groups were no significant difference (P>0.05). There was no significant difference in local recurrent and beyond metastases rates between the two groups. Conclusion Laparoscopic radical resection of rectal carcinoma parallels with open surgery is in safety and effectiveness, it has less traumatic and blood loss and faster in patients recovery.

Objective To evaluate the prospects of recombinant 38000 protein of Mycobacterium tuberculosis in tuberculosis epidemic investigation and subunit vaccine preparation.Methods Physicochemical characteristics of recombinant 38000 protein was detected by P I, peptide-mapping analysis and circular dichroism,guinea pig skin test,MTT stain,and peripheral blood macrophage phagocytosis were used to investigate the roles of recombinant 38000 protein in the cell immunity.Results Recombinant 38000 protein was acidic protein,its P I, was 4 67.The number of alkaline amino acid correspond with theoretic number;The secondary structure of recombinant 38000 protein was composed of ?-helix(32 6%),?-turn(31 6%) and random coil(35 8%) Recombinant 38000 protein could induce DTH in guinea pig sensitized by Mycobacterium tuberculosis Recombinant 38000 protein enhanced phagocytosis of macrophage in mice . PBMC from 30 8% healthy donors and 25% tuberculosis patients were stimulated by the recombinant 38000 protein.Conclusion Recombinant 38000 protein may be used as diagnostic reagent and as an candidate in development of subunit vaccine.

Objective To compare expression of extracellular proteins of virulent H37Rv and attenuated H37Ra in order to search differential proteins,to provide a train of thought for studing M.TB toxicity further.Methods Extracellular proteins were extracted from H37Rv and H37Ra which were inoculated and cultured on Suton's medium for three weeks.The first dimensional electrophoresis was performed on immobilized pH gradient rod gels(pH 3～10).Then the proteins in the rod gels were separated using SDS-PAGE gels.The silver-stained gels were dried and scanned with image scanner.The 2D image analysis was performed with image Master 2D Elite 3 10.Results The most protein spots deriving from extracellular proteins of H37Rv and H37Ra strains were in acidic range.In the basic range(pI more than 9 0),the number of protein spots belong to extracellular proteins of H37Rv and H37Ra was few.Three protein spots belong to low molecular range in H37Rv strain.However,absent in H37Ra strain.Conclusions Two-dimensional gel electrophoresis is useful to separate protein in Mycobacterium tuberculosis.

0. 05 ) , but the control group had(P