Construction of recombinant eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes / 国际眼科杂志(Guoji Yanke Zazhi)

AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.

Impact of psychosocial stress on airway inflammation and its mechanism in a murine model of allergic asthma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has already been recognized that psychosocial stress evokes asthma exacerbation; however, the mechanism of how stress gets inside the body is not clear. This study aimed to observe the impact of psychosocial stress on airway inflammation and its mechanism in the ovalbumin-induced asthmatic mice combined with social disruption stress.</p><p><b>METHODS</b>Thirty-six male BALB/c mice were randomly divided into: control group, asthma group (ovalbumin-induced), asthma plus social disruption stress group (SDR), and SDR group. The open field video tracking system was used to assess animal behaviors. The invasive pulmonary resistance (RL) and dynamic lung compliance (cdyn) test system from Buxco was applied to detect pulmonary function. The enzyme-linked immunosorbent assay (ELISA) was utilized to determine OVA-IgE, T-helper type 2 (Th2) cytokines (IL-4, IL-5, IL-13) and corticosterone in mouse serum, the Th2 cytokines (IL-4, IL-5, IL-13, IL-6, TNF-α) in bronchoalveolar lavage fluid (BALF), and IL-6 and TNF-α levels in the supernatant of splenocytes cultured in vitro. Hematoxylin-eosin (H&E) staining was used to assess airway inflammation in lung histology. The cell count kit-8 assay (CCK-8) was applied to evaluate the inhibitory effect of corticosterone on splenocyte proliferation induced by lipopolysaccharide (LPS). Real time-PCR and Western blotting were utilized to determine glucocorticoid receptor (GR) mRNA and GR protein expression in lungs.</p><p><b>RESULTS</b>The open field test showed that combined allergen exposure and repeated stress significantly shortened the time the mice spent in the center of the open field (P < 0.01), increased ambulatory activity (P < 0.01) and the count of fecal boli (P < 0.01), but deceased vertical activity (P < 0.01). Results from pulmonary function demonstrated that airway hyperresponsiveness (AHR) was enhanced by psychosocial stress compared with allergy exposure alone. The ELISA results showed that cytokines in serum and BALF were significantly increased (P < 0.05). Moreover, the lung histology showed that infiltrated inflammatory cells were significantly increased in the asthma-SDR group compared with the asthma group (P < 0.05). Interestingly, serum corticosterone was remarkably raised by psychosocial stress (P < 0.05). In addition, the inhibitory effect of corticosterone on IL-6 and TNF-α in LPS-stimulated splenocyte cultures in vitro was diminished in the asthma-SDR group compared to the asthma group. The CCK-8 test revealed that the inhibition effect of corticosterone on splenocyte proliferation induced by LPS was significantly impaired in the SDR and asthma-SDR groups, while no significant effect was observed in the control and asthma groups. Furthermore, expression of GR mRNA and GR protein were significantly reduced in the lung tissues of the asthma-SDR group (P < 0.05).</p><p><b>CONCLUSIONS</b>Social disruption stress can promote anxiety behavior, activate the hypothalamic-pituitary-adrenal (HPA) axis, increase AHR and inflammation, and also impair glucocorticoid sensitivity and its function in a murine model of asthma. The down-regulation of GR expression induced by social disruption stress is in part associated with glucocorticoid insensitivity, which leads to asthma exacerbation.</p>

Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To prepare eukaryotic expression of rotavirus (RV) SA11 capsid protein VP7, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens.</p><p><b>METHODS</b>MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7.</p><p><b>RESULTS</b>The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg.</p><p><b>CONCLUSION</b>The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.</p>

Enhanced expression of the decoy receptor IL-13Ralpha2 in macrophages of Schistosoma japonicum-infected mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Type 2 cytokine interleukin (IL)-13 and its decoy receptor, IL-13 receptor (R) alpha2 appear to play a major role in tissue fibrosis of schistosomiasis and asthma. IL-13 is a key regulator of the extracellular matrix (ECM). It is known to signal to cells by binding to the IL-13Ralpha1, which then heterodimerizes with IL-4Ralpha. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 is known to down-regulate granulomatous inflammation and prolong host survival in Schistosoma mansoni (S. mansoni) infection, but little is known about the location and expression level of IL-13Ralpha2 in the context of S. japonicum infection.</p><p><b>METHODS</b>We established S. japonicum-infected mouse models. Kinetic serum levels of IL-13Ralpha2 were examined with ELISA. IL-13Ralpha2 mRNA and protein of liver tissues were determined by PCR and immunoblotting analysis, respectively. Detection of IL-13Ralpha2 expression and location in macrophages was performed by TaqMan PCR and fluorescent immunocytochemistry technique, respectively.</p><p><b>RESULTS</b>A marked elevation of mRNA and protein expression of IL-13Ralpha2 was observed in mice during S. japonicum infection. An enhanced expression of IL-13Ralpha2 was further demonstrated in primary macrophages of murine schistosomiasis.</p><p><b>CONCLUSIONS</b>IL-13Ralpha2 in macrophages may be a critical contributor to pathogenesis of schistosomiasis. The data highlight the potential importance of cell signaling and antifibrotic gene therapeutics in T helper 2 cell (Th2)-mediated diseases.</p>

Mesenchymal chondrosarcoma of the orbit: report of a case and review of the literature / 国际眼科杂志(Guoji Yanke Zazhi)

· AIM: To report a rare case of mesenchymalchondrosarcoma in the orbit and to explore its clinicmanifestations, pathologic characters, management andprognosis. · METHODS: We report a case of mesenchymalchondrosarcoma of the orbit. The clinical materials,including ophthalmological examination, computed tomo-graphy scan of the orbit, histopathology and immunohis-tochemistry of the biopsy specimen was reported, and itspertinent literatures were reviewed.· RESULTS: A 36-year-old female was seen with proptosisand decreased vision. Histopathology demonstrated anadmixture of undifferentiated mesenchymal cells andislands of mature hyaline cartilage. Immunohistochemicalstudies revealed positivity for vimentin and S-100, whichwas consistent with the diagnosis of mesenchymalchondrosarcoma.· CONCLUSION: Mesenchymal chondrosarcoma in theorbit is extremely rare malignant tumor. Multi-modalitytreatments (surgery, chemotherapy and radiotherapy)may lead to long-term survival.

Induction of heat shock protein in retinal ganglion cells and its role in a rat glaucoma model / 国际眼科杂志(Guoji Yanke Zazhi)

· AIM: To investigate whether heat shock protein 27 (HSP27) is induced in retinal ganglion cells (RGCs) in experimental rat glaucoma and whether the induction of HSP27 by intraocular pressure (IOP) elevation can increase serum autoantibody to HSP27 in the model.IOP elevation、Sham and normal groups by SPSS12.0. IOP was raised by electrocoagulating at least 3 episcleral veins and limbal veins on the right eye of each rat in IOP elevation group and its contralateral eye was used as controls. Immunohistochemical staining for HSP27 was performed in RGCs and retinal nerve fiber layer (RNFL) and serum immunoreactivity against HSP27 was detected by means of enzyme-linked immunosorbent assay (ELISA) in three groups.RNFL of the eyes with IOP elevation, while it was expressed weakly in untreated control eyes. Compared with sham and normal groups, serum autoantibody to HSP27 was slightly high at 1wk (P ＞0.05) and significantly increased at 2, 3, 4 and 8wk (P＜0.05) in IOP elevation group.enhanced expression of the endogenous HSP27 might play an important role in glaucomatous optic neuropathy.

A new method of duplication of the chronic intraocular hypertension model: underwater electrocoagulation / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the experimental method of inducing the chronic intraocular hypertension in rat eyes.METHODS: Twenty Wistar rats were randomly divided into ocular hypertension and Sham control groups. Intraocular pressure (IOP) was raised by electrocoagulating at least 3 episcleral and limbal veins on the right eye of each rat in ocular hypertension group and its contralateral eye was used as control. At 1, 2, 3, 4 and 8wk after the electrocoagulation of the veins, IOP were measured. RESULTS: The treatment of electrocoagulation caused a significant IOP increase of the right eyes over the baseline, over the contralateral eyes, and over the sham control eyes (repeated measures ANOVA, P＜0.001). At 1wk,IOP was (30.12± 5.L8 ) mmHg (1kPa=7.5mmHg), and maintained the high IOP up to 8wk.CONCLUSION: The chronic intraocular hypertension model could be successfully created by electrocoagulating three or more episcleral and limbal veins.

Effects of HBV infection on hepatic fibrosis and level of Th1/Th2 cytokines in the patients with Schistosomiasis japonica / 中华检验医学杂志

Objective The levels of Thl cytokines(IL-10 and IL-13)and Th2 cytokines(INF-? and TNF-?)were determined in the sera of patients with Schistosomiasis japonica in order to find the relationship between cytokines and severe hepatic fibrosis(HF)in schistosomiasis.Methods A total of 358 patients with advanced Schistosomiasis japonica were examined by ultrasound.68 HBsAg negative patients were chosen randomly as experimental control.Among them,39 patients were found to have mild HF and 29 were severe HF.The sera levels of Thl and Th2 cytokines were determined with ELISA.Results Among these 358 patients,83(23.2%)were HBsAg positive.Neither earlier nor severer hepatic fibrosis was noted in the patients who had been simultaneously infected with HBV than those only infected with schistosomiasis. There was a significant difference between mild[ 1.60(1.30-12.14)ng/L]and severe[ 4.20(1.43- 52.07)ng/L]HF patients in the level of IL-10(Z=-3.907,P0.05)was found in level of IFN-?,between severe[3.12(1.38-66.14)ng/L]and mild[5.87(1.33-216.33)ng/ L]HF subjects.Our observation did not reveal any obvious difference of TNF-? between severe[ 2.48(0.79 -19.86)ng/L]and mild[ 2.28(0.67-15.72)ng/L]HF groups.Conclusions Patients infected with advanced shistosomiasis may become more susceptible to HBV.The results of the present investigation showed that a high level production of IL-13 was associated with severe HF.