10.3969/j.issn.1007-3969.2013.05.007

Objective To produce prokaryotic recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST?Bind~TM kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate (3-GAP). Results SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1-GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2 872 U min~-1ml~-1. Conclusion The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.

Using human acidic fibroblast growth factor Specific primers we tested the technique of screening cDNA library by a DNA polymerase chain reaction.With the method,we have suc-cessfully isolated encoding region cDNA of human interleukin 8 from both human dermal fibrob-last and PMA stimulated U937 cell cDNA libraries.

Objective To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. Methods A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamHⅠ and SalⅠ , the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E.coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni 2+ ) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. Results The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. Conclusion The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.

BACKGROUND: Previous results of our study show that oyster extract has some protective effects on apoptosis of the neuroepithelium in neural tube defects induced by hyperthermia in vivo.It remains unclear whether the extraet also protects in vitro cultured neural stem cells.OBJECTIVE: To investigate the protective effect of oyster extract on apoptosis of cerebral neural stem cells induced by hyperthermia.METHODS: The cerebral neural stem cells of embryonic mice of 13 days were cultured in vitro.Nestin expression was detected by immunofluorescence method to identify neural stem cells.The neural stem cells of passage 3 were divided randomly into 4groups: hyperthermia control group and oyster treated Ⅰ,Ⅱ,Ⅲ groups(mass concentration 2.5,5,10 g/L oyster extract solution).In addition,culture solution control group(no cells),and culture solution+oyster extract control group(no cells)were designed.All oyster extract groups and control groups were treated by hyperthermia over 39 ℃.The survival rate and the vitality of neural stem cells were detected by trypan blue staining and MTT assay.Western-blotting was employed to explore the expression of p53 in cerebral neural stem cells of each group.RESULTS AND CONCLUSION: The survival rate and the value of MTT assay in oyster treated groups Ⅱ and Ⅲ were significantly greater than hyperthermia control group(P < 0.05),but the expression of p53 in oyster treated groups Ⅱ and Ⅲ were weaker than hyperthermia control group.Oyster extract plays an important protective role in the apoptosis of neural stem cells induced by hyperthermia.

Objective To observe the effect of lipopolysaccharide (LPS)-mediated infection during pregnancy on the expression of mineralocorticoid receptor (MR) and density of dendritic spines in CA1 region of the dorsal hippocampus of rat offspring,so as to explore the mechanisms for learning and memory injury of rat offspring which were infected during prenatal period,then to provide scientific experimental evidence for the prevention of prenatal infection-induced delayed neuropsychiatric sequelae which contributed to learning and memory dysfunction.Methods Ten-week-old female Sprague-Dawley rats (n =30) were matched with male rats (1 ∶ 1).Pregnant rats were randomly divided into a control group (n =10) and an experimental group (n =20).The pregnant rats in experimental group were treated with LPS (66 μg/kg,intraperitoneally),and the pregnant rats in control group were intraperitoneally injected with same volume of saline on gestational day 10.On postnatal day 48,Morris water maze was used to estimate the ability of learning and memory;the brain tissues of offspring were taken and paraffin sections were stained with hematoxylin eosin (HE) for histological observation of CA1 region of the dorsal hippocampus;frozen sections were treated with indirect immunofluorescence to observe the expression of MR in CA1 region of the dorsal hippocampus;Golgi-Cox method was used to observe the density of dendritic spines of CA1 region.Results In Morris water maze test,from the third day the time of escape latency in experimental group [the 3rd day:(42.603 ± 9.837) s;the 4th day:(30.222 ± 9.789) s;the 5th day:(28.808 ± 12.526) s] was significantly higher than that of the control group [the 3rd day:(28.078±14.088) s;the 4th day:(20.692±13.099) s;the 5th day:(14.632 ±11.624) s] (the 3rd day:t =-3.274,P＜0.01;the 4th day:t =-2.257,P ＜0.05;the 5th day:t =-3.213,P＜0.01);the swimming time in the target quadrant [(14.660 ± 7.337) times] and the number of crossing platform [(0.933 ± 0.704) times] in experi mental group were significantly decreased compared with those of the control group [time:(23.820 ± 6.321) s;num bers:(2.000 ± 0.756) times] (t =3.663,4.000,all P ＜ 0.01).Hematoxylin eosin staining showed that the nerve cells of the hippocampus in the control group distributed in order,nucleuses were round or oval,nucleoli were obvious,and chromatins were homogeneous;but the cells in the experimental group distributed in disorder and pathological changes were detected,such as cellular swelling,necrosis and obvious nuclear pyknosis.By immunofluorescence staining,the average optical density (AOD) of MR in CA1 region decreased significantly in the experimental group (0.067 ± 0.017) compared with that of the control group (0.083 ± 0.009) (t =2.644,P ＜ 0.05).In Golgi-Cox method,the density of dendritic spines in experimental group [(7.705 ± 0.791)/10 μm] was below that of the control group [(9.655 ± 1.391)/10 μm] (t =3.852,P ＜ 0.01).Conclusions LPS-mediated infection during pregnancy might lead to hippocampus-dependent learning and memory dysfunction which might be associated with the reduced expression of MR and the low density of dendritic spines in CA1 region of the dorsal hippocampus.

To train the talents of clinical laboratory for the demand of clinical jobs,it is necessary to make the experimental teaching reform of clinical laboratory hematology.Comprehensive experimental teaching,which centers on case,has been explored in many ways like prefessional teachers' training,cases' choose,teaching method and experimental examination.And the practice has proved that the reform of comprehensive experimental teaching can give full play to the students' initiative and creativity,foster the students' ability to analyze and diagnose hematopathy.

Objective To investigate the influence of Hui medicine flatiron moxibustion therapy and nursing intervention on pain and serum IL-2,TNF-α in rheumatoid arthritis (RA) patients before and after treatment and care interventions.Methods 89 cases RA hospitalized patients from Traditional Chinese Orthopedics and Traumatology department of General Hospital of Ningxia Medical University were collected and were randomly divided into the control group (43 cases) and the treatment group (46 cases) from May 2011 to September 2013.Two groups used a combination method of overall and portion acupoints.The control group used warm acupuncture,30 min every time,1 time per day,routine care was given.The treatment group was given with Hui medicine flatiron moxibustion instrument by the professional Chinese medicine nurses,direct moxibustion acupoints site to portion skin slight degree redness,30 min every time,1 time per day,and with routine care,focused on the reasons things out the enlighten,emotion,suggested transferring,calm the nerves of the traditional Chinese medicine psychological care guidance.10 times as a course,to rest for 3～5 days on treatment interval,for efficacy evaluation after three courses.Meanwhile,before and after the application,to test the pain degree with NPRS; to assess the psychological and emotional status with SCL-90,serum IL-2 and TNF-α levels were tested with radioimmunoassay.Results The total effective rate was 89.13％ on the treatment group,the control group was 74.42％.After treatment,the NPRS,SCL-90 scores were significantly improved than those before treatment; the NPRS,SCL-90 scores of the treatment group were better than those of the control group.After treatment,serum IL-2 and TNF-α levels were significantly improved compared with those before treatment; after treatment,IL-2 and TNF-α levels of the treatment group were better than those of the control group.All the above comparison had statistical significance.Conclusions Hui medicine flatiron moxibustion therapy and nursing intervention may effectively improve pain in RA patients,ease the psychological anxiety,increase serum IL-2,TNF-α levels,enhance the quality of life for patients,and promote disease recovery.

Aim To observe the effects of low concentration ouabain(OUA)on intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes.Methods The guinea-pig ventricular myocytes were obtained by enzymatic dissociation technique, then were incubated with Fluo3-AM. The Fluo3-AM fluorescent signal was detected with confocal laser scanning microscopy. The change of [Ca2+]i was represented by the percentage of fluorescence intensity change [(FI-FI0)/FI0,%] (FI: fluorescent intensity after addimg OUA, FI0: control). Results In normal Tyrode,s solution and Ca2+-free Tyrode′s solution.OUA (1?10-9~1?10-6 mol?L-1)elevated [Ca2+]i in a concentration-dependent manner, in normal Tyrode′s solution by 16.7?6.8(P0.05).Genistein (GST) (1、10、50、100 ?mol?L-1)abolished the OUA-induced increases in[Ca2+]i in a concentration-dependent manner by 17.5?3.1、14.2?8.9、0.8?7.6(P

Aim By establishing mouse acute myocardial infarction model,to observe the protection of isoflavones on ischemic myocardium and research the mechanism.Methods Mouse acute myocardial infarction model was established by ligating the left anterior descending(LAD)coronary artery.Danshen was used as the positive control.The effect of isoflavones on myocardial infarct area,serum myocardium creatase and serum levels of SOD and MDA was observed.By Real Time PCR,it was found that isoflavones could affect the expression of β-adrenergic receptor kinase(β-ARK_1).Results Isflovones could obviously reduce the myocardial infarct area and lower the levels of serum myocardial creatase and MDA.It could downregulate the expression of β-ARK_1 as the doses are increased.Conclusions Isoflavones can protect the myocardium of acute myocardial infarction mouse.The mechanism is related to the reduction of the oxidative damage,and the downregulation of the expression of β-ARK_1.