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.

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 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.

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.

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.

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.

The idea and methods of study on proteomics have many similarities to the whole view and the dialectical view of TCM,so it confirms with own characteristics of syndromes to introduce proteomics into studies of essence of syndromes.Appling the idea and methods of proteomics to study TCM differentiation of syndromes of hepatic disease and medicines for treatment and prevention of hepatic disease will comprehensively reveal the essence of generation,development and transformation of TCM hepatic disease,providing the most essential theoretical basis for TCM prevention and treatment of hepatic disease.To establish transform mode concept of correlation of proteomics with TCM hepatic disease will enrich greatly theory of TCM treatment of hepatic disease based on differentiation of syndromes,and also will provide a new thinking model for study on TCM syndrome types,and accelerate the process of TCM modernization.

Objective To investigate the clinical effect of using ambroxol hydrochloride and dexamethasone by nasal spray inhalation after endoscopic sinus surgery in treating chronic sinusitis.Methods One hundred and forty patients with chronic sinusitis receiving endoscopic sinus surgery in our hospital from January 2014 to December 2014 were selected as the research subjects.After surgery,the control group was given dexamethasone nasal spray inhalation,while the observation group was given ambroxol hydrochloride and dexamethasone by nasal spray inhalation.The curative efficacy,the score of visual analogue scale(VAS) was adopted to evaluate the subjective symptoms severity and the nasal endoscopy and CT examination results were evaluated by the Lund-Kennedy score.The serum levels of IL-5 and IL-12 were detected in the two groups.Results All cases were successful completed surgery and no severe adverse reactions appeared after surgery.The observation group had a total effective rate of 97.1 ％,which was signif icantly higher than 88.6 ％ in the control group,the difference was statistically significant(P＜0.05).In the aspects of sinusitis related symptoms after operation,compared with the control group,the VAS scores of nasal discharge,nasal obstruction,cheek pain,headache and hyposmia in the observation group were significantly decreased,the difference was statistically significant(P＜0.05).In the postoperative re-examination process,the score of assessment of nasal endoscopic score and paranasal sinuses CT score in the observation group were significantly decreased,the difference was statistically significant(P＜0.05).In the serological indicator determination,serum levels of IL-5 and IL-12 after treatment in the observation group were significantly lower than those in the control group,the difference was statistically significant(P＜0.05).Conclusion Using ambroxol hydrochloride combined with dexamethasone by nasal spray inhalation after endoscopic sinus surgery has good efficacy in treating chronic sinusitis,can decrease serum levels of IL-5 and IL-12.

BACKGROUND:In recent years, embryonic hepatic stem cel s have attracted more attention, but there are few reports on the potential of embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s as wel as the related differentiation conditions. OBJECTIVE:To investigate the moderate condition to induce mice embryonic hepatic stem cel s to differentiate into cardiomyocyte-like cel s in vitro with chemical reagents. METHODS:Dimethylsulfoxide in combination with 5-azacytidine with different concentrations and time were used to induce the embryonic hepatic stem cel s of 13.5 days mice and to observe the differentiation effect. RESULTS AND CONCLUSION:Under in vitro conditions, 0.8%dimethylsulfoxide+5μmol/L 5-azacytidine could induce the mouse embryonic hepatic stem cel s to express the specific markers of myocardial cel s, while increasing the concentration of the inducer and extending the induction time could not improve the induction efficacy.

10.3969/j.issn.2095-4344.2013.23.014