AIM: To prepare and purify the polyclonal antibodies against human myofibrillogenesis regulator 1 (hMR-1), then to characterize the purity, titer, specificity and the availability.METHODS: Two polypeptides named peptide 1 and 2 were synthesized based on the bioinformatics analysis of the sequence of hMR-1 by using software TMHMM and DNAStar, then coupled with keyhole limpet hemocyanin (KLH) for immunization. These peptides for immunization were mixed and injected into New Zealand rabbits to prepare antibodies specifically against hMR-1. ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, antibodies were identified by Western blotting and immunocytofluorescent assays. Applications of the antibodies on neonatal rat cardiomyocytes were also employed.RESULTS: (1)The titers of antibodies were 1:10~5. In WB assay, a specific 17kD band was detected, corresponding to the predicted molecular weight of hMR-1; the positive fluorescent signals were distinct. (2)On the neonatal rat cardiomyocytes model, we observed a peri-nucleus location. The fluorescent signal of hMR-1 overexpression group was much stronger than that in vector control and normal control groups.CONCLUSION: All these results indicate that the antibodies obtained from poly peptides mixture immunization have either human original or rat original antigens. The antibody is available for using in Western blotting or immunofluorescent assays.

10.3969/j.issn.1007-3969.2013.05.004

Objective To investigate the short-term efficacy and safety of gefitinib combined with dendritic cells-cytokine induced killer (DC-CIK) cells in the treatment of advanced lung adenocarcinoma.Methods Fifty patients with lung adenocarcinoma who in previous had underwent radiotherapy and first-line chemotherapy failure received DC-CIK in combination with gefitinib treatment,blood count changes,imaging data,carcinoembryonic antigen and changes in the quality of life before and after treatment were compared and evaluated.Results DC-CIK could improve effectively and relieve bone marrow suppression response after chemotherapy and significantly increase the WBC content of blood (P ＜ 0.01).After treatment,the tumor carcinoembryonic antigen (CEA) was significantly lower in patients (P ＜ 0.01).Fifty cases of patients in complete remission (CR) were 0 cases,partial remission (PR) were 24 cases,stable disease (SD) were 17 cases and progression (PD) were 9 cases,and the response rates (RR) were 48％;The quality of life of patients was significantly improved (P ＜ 0.05).Common adverse reactions were rash,diarrhea,chill and fever,but mild symptoms could be relieved after symptomatic treatment.Conclusions Gefitinib with autologous DC-CIK cell infusion second-line treatment of advanced lung cancer have a certain short-term efficacy,without significant adverse reactions.Gefitinib with autologous DC-CIK cell therapy can mitigate the response of bone marrow suppression in patients with advanced lung cancer and improve the quality of life of patients.Long-term effect remains to be investigated.

ObjectiveTo prepare paclitaxel-loaded nanoparticles (NPs),and to observe drug biodistribution after intravascular infusion of the NPs using a DispatchTM catheter into New Zealand rabbit abdominal aorta models.Methods Paclitaxel-loaded NPs were prepared by ultrasonication/emulsificcation/solvent evaporation technique using biodegradable poly (lactic-co-glycolic acid)(PLGA) as drug carrier.NP size and morphology was assessed by submicro-laser defractometer and scanning electron microscopy.In vitro release of paclitaxel from the NPs was performed by shaking in PBS at 37℃.The NPs was delivered into New Zealand rabbit abdominal aorta using a DispatchTM catheter.ResultsThe diameter of paclitaxel NPs was around 246 nm with very narrow size distribution.The NPs showed good spherical shape with smooth uniform surface.Paclitaxel loading in the NPs was about 19.06％ with encapsulation efficiency about 93.25％.The NPs maintained a sustained in vitro drug release for 30 days in PBS.After in vivo NP infusion,paclitaxel was detected in the vascular tissue around the infusion site and it retained in the site for 21 days.ConclusionPLGA nanoparticles as local drug delivery carrier showed great potential to maintain a high local drug concentration and prolonged drug resident time in animal model in vivo.

Objective: To study the expression and localization of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis. Methods: Using monoclonal antibody against TFAR19, the expression level and cell localization of TFAR19 were examined by fluorescence microscope, confocal laser scan microscope(CLSM) and flow cytometry. Simultaneously, we also analyzed the relationship of TFAR19 protein with phosphatidylserine (PS) externalization and cell nuclear DNA fragmentation. Results: The level of TFAR19 proteins expressed in TF-1 cells treated with GM-CSF withdrawal was significantly increased compared with normal TF-1 cells, then translocated rapidly from cytoplasm to the nucleus of cells. Appearance of TFAR19 in the nucleus of apoptotic cells preceded the detection of PS externalization and DNA fragmentation. Conclusion: Nuclear translocation of TFAR19 protein is one of the earliest events of cell apoptotic process. These data provided a new clue to further approach to the biological function of TFAR19 and study of cell apoptosis.

Objective To study the human lymphangiogenesis in the early stage of embryo and the expression of forkhead box C2(FOXC2) in human lymphangiogenesis.Methods Lymphatic vessel endothelial haluronic acid receptor-1(LYVE-1) was used as the special marker of lymphatic vessels.Immunohistochemical and double immunofluorescence staining were used to detect the expressions of FOXC2 and LYVE-1 in lymphatic vessels of 85 human embryos of 5 to 11 weeks pregnancy and analyze the features of lymphatic vessel genesis and development.Results LYVE-1 was initially detected in lymphatic vessels in 7-week human embryos.The lymphatic vessel endothelial cells presented brown staining for LYVE-1 in jugular and thoracic region.LYVE-1 was also found to express in human embryonic mesentery lymphatic vessels on the 70th day of pregnancy.The expression of FOXC2 in human embryo was detected prior to that of LYVE-1.At the beginning of the 6th week of pregnancy,FOXC2 was obviously seen in human embryonic mesoderm mesenchyme.FOXC2 expressed not only in human embryonic lymphatic vessels,but also in the vertebral body,cardiovascular wall and other tissues.The expressions of FOXC2 and LYVE-1 were still seen in human embryonic lymphatic vessel endothelial cells after 80 days of pregnancy.Conclusion Lymphangiogenesis of human embryo begins between the 7th and 8th weeks of pregnancy.FOXC2 and LYVE-1 could have some relation with the human embryonic lymphatic vessel genesis and development.

Objective To establish an inactivated viral particle-based ELISA for the detection of antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) in serum samples collected from a MERS-CoV associated case. Methods Serum samples were collected from 10 newborns and 40 healthy adults. A viral particle-based ELISA was established by using the inactivated MERS-CoV virions as antigen. The levels of IgM and IgG antibodies in the serum samples were detected by the established ELISA and the cut-off values for positive detection were determined. Then the inactivated MERS-CoV virion-based ELISA was used to detect the antibodies against MERS-CoV in 5 serum samples collected from the first im-ported MERS case in China. Results The cut-off values of IgM and IgG antibodies in serum samples for ELISA were determined to be A450 readings of 0. 32 and 0. 42, respectively. The titers of IgM and IgG anti-bodies in serum samples collected at early admission to hospital from the first imported MERS case in China were both 1 ︰ 40. Seroconversion occurred 2 weeks after his admission to hospital with the titers of IgM and IgG reaching to 1 ︰ 320. Conclusion The inactivated MERS-CoV virion-based ELISA was established successfully and could be used for the detection of serum antibodies (IgG and IgM) in MERS associated cases.

Objective To compare the sensitivity and specificity of venereal disease research laboratory (VDRL) test versus several other laboratory tests in the diagnosis of neurosyphilis. Methods Lumber puncture was conducted to obtain cerebrospinal fluid (CSF) from untreated outpatients with latent syphilis (LS) or serofast outpatients with LS. Then, VDRL test, rapid plasma regain (RPR) test, Treponema pallidum particle agglutination (TPPA) assay, fluorescent treponemal antibody-absorption (FTA-ABS) test and protein quantification were performed on these CSF samples. The sensitivity, specificity, positive predictive value and negative predictive value were compared between VDRL test and four other laboratory tests in the diagnosis of neurosyphilis. Results Totally, 61 cases of latent syphilis were included in this study. The sensitivity, specificity,positive predictive value and negative predictive value were 93.44% (57/61), 99.32%(293/295), 96.61%(57/59), 98.65% (293/297)for CSF-RPR, respectively, 91.80% (56/61), 82.71% (244/295), 52.34% (56/107),97.99 (244/249) for CSF-TPPA, respectively, 93.44% (57/61), 82.71% (244/295), 52.78%(57/108), 98.39%(244/248) for CSF-FTA-ABS, respectively, and 49.18%(30/61), 97.29% (287/295), 78.95% (30/38),90.25% (287/318) for CSF protein quantification, respectively. Conclusions CSF-VDRL cannot be replaced by CSF-RPR, -TPPA, -FTA-ABS, or CSF protein quantification in the diagnosis of neurosyphilis. CSF-RPR shows a high sensitivity and specificity in the diagnosis of neurosyphilis, with an increased diagnostic capability (area under the receiver operating characteristic curve) compared with CSF-TPPA, CSF-FTA-ABS or CSF protein quantification.

Objective To observe the ehanges in plasma OX40L level in patients with stable angina pectoris (SAP) after percutaneous coronary intervention (PCI) and to investigate the relationship between OX40L level and prognosis in patients with SAP.Methods We collected 200 cases of SAP after PCI.Plasma OX40L levels were examined at different time points,including before and after stent placement,in all patients withSAP.The patients with SAP were followed up for 18 months,and the end point was adverse cardiovascular events.Results The plasma OX40L level increased more apparently after PCI than before PCI (P ＜ 0.05) and peaked at 24 hours.We followed up 185 patients successfully,including 39 (21.08％) patients with and 146 (78.92％) without adverse cardiovascular events.The mean maximum OX40L level was higher in patients with than in those without adverse events (P ＜ 0.05).Multivariable logistic regression analysis revealed that the mean maximum OX40L level was related to adverse cardiovascular events (P ＜ 0.05).Conclusion PCI may cause upregulation of plasma OX40L expression.Mean maximum OX40L level showed good clinical predictive value of occurrence of cardiovascular events 18 months after PCI.

Near-infrared spectroscopy(NIRS) is a technique for continuous,noninvasive,bedside monitoring of inadequate tissue perfusion and oxygenation.Intestinal ischemia is an important pathophysiologic in mucosal injury and the development of necrotizing enterocolitis (NEC).So somebody thinks that NIRS might be a useful tool to diagnose the earliest stages of NEC and to predict its progression.This review will describe the feasibility,safety,sensor location and associated research result of NIRS in NEC.