Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.

Objective To construct a recombinant expressing plasmid of the hy1 gene of Enterococcus faecium and to express the recombinant Hy1 protein in E.coil.To explore the immune response in mice fed orally with Hyl protein.Methods hy1 gene was amplified by polymerase chain reaction(PCR)and inserted into a prokaryotic expression vector pQE-30.The recomhinant plasmids were transfected into DH5_?to express Hy1 fusion proteins,which were purified by Ni~--column. Western blot was employed to confirm the immunogenicity of the purified protein.Mice were immu- nized by feeding with the fusion protein.The concentrations of antigen-specific antibody in the serum, mucosal fluid and faces were detected by enzyme-linked immunosorbent assay(ELISA).The role of these antibodies in the anti-infection response was evaluated after the mice were challenged with TX0016.Results hy1 gene was sequenced as 1662 bp,the fusion protein encoding polypeptides of 553 amino acid residues.The relative molecular weight was 60 000 when it was determined by sodium dodecylsulfatepo-lyacry-lamide gel electropboresis(SDS-PAGE).The dissolvable expression protein accounted for 38% of total cell protein.After processed by affinity chromatography,the purity of fusion protein was above 92%.Western blot analysis confirmed that fusion protein could be specifically recognized by the anti-TX0016 serum.The concentrations of serum IgA,serum IgG,faeces sIgA and intestmucosal fluid sIgA was 0.365?0.048,0.431?0.064,0.743?0.056 and 1.112?0.113 respectively in hy1 groups and 0.051?0.013,0.098?0.019,0.102?0.032 and 0.187?0.051 respectively in control group.The differences were statistically significant.The mice survival rate after TX0016 challenge was 70% in hyl group and 50% in control group.There was significant difference between these two groups.Conclusion The results indicate that oral immunization with hyl can induce effective mueosal immune response and produce high level sIgA.

Objective: To investigate the effect of protein ki nase C on signal transduction such as tyrosine phosphorylation, c-fos and c-ju n mRNA expression in antigen activated mast cells. Methods: RBL-2H3 cells either untreated or treated with phorbol 12-myristate 13 -acetate (PMA) were sensitized with anti-DNP IgE, and activated with DNP-BSA, histamine release and tyrosine phosphorylation were quantitatively measured by ELISA and flow cytometry, respectively. The effect of PKC on the ex pression of c-fos and c-jun in serum-deprived RBL-2H3 cells activated by DNP-BSA detected by ethidium staining of PCR-amplified cDNA, the amplified cDNA products were subjected to Southern blot hybridization using specific prob es to determine the veracity of amplification. Results: Tyr osine phosphorylation and histamine release were significantly reduced from (4.4 7±0.03)% to (2.79±0.07)% and (104.47±1.31) nmol/L to (60.75±1.38) nm ol/L, respectively, 45 min after DNP-BSA stimulation in sensitized cells pre treated with PMA for 48 h. Bands of the size predicted for the amplified cDNA we re obtained: 299 bp for c-fos, and 651 bp for c-jun, a decrease of 91% and 82% , respectively, for c-fos and c-jun mRNAs was observed in antigen stimulated c ells pretreated with PMA for 48 h. Conclusion: PKC plays an impo rtant role in modulating the tyrosine phosphorylation and histamine release resp onses and may upregulate the expression of c-fos and c-jun in antigen activate d mast cell.

Objective To investigate the effects of propofol on brain injury induced by intestinal ischemia-reperfusion (I/R) in rats.Methods Forty-eight adult male Sprague-Dawley rats,weighing 250-300 g,were randomly allocated to one of 3 groups (n =16 each) using a random number table:sham operation group (group Sham),I/R group,and propofol group (group P).Intestinal I/R was produced by occlusion of the superior mesenteric artery for 90 min followed by reperfusion.In group P,propofol 50 mg/kg was injected intraperitoneally at 30 min before reperfusion,and the equal volume of fat emulsion was given in the other two groups.Blood samples were collected at 24 h of reperfusion for determination of serum tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) concentrations.The cerebral cortex and hippocampus were isolated for measurement of TNF-α and IL-1β mRNA expression (by real-time reverse transcriptase-polymerase chain reaction) and myeloperoxidase (MPO) activity (using colorimetric method).Morris water maze test was carried out at 1,3 and 5 days of reperfusion.Results Compared with group Sham,the serum TNF-α and IL-1β concentrations were significantly increased,the expression of TNF-o and IL-1β mRNA in the cerebral cortex and hippocampus was up-regulated,the MPO activity was increased,and the escape latency was prolonged,and the frequency of crossing the original platform was decreased during reperfusion in group I/R (P＜0.05).In group I/R,the concentrations of serum TNF-αand IL-1β were significantly decreased,thc cxpression of TNF-α and IL-1β mRNA in the cerebral cortex and hippocampus was down-regulated,and the escape latency was shortened,and the frequency of crossing the original platform was increased during reperfusion (P＜0.05),and no significant change was found in MPO activity in group P (P＞0.05).Conclusion Propofol reduces brain injury induced by intestinal I/R through inhibiting systemic and local inflammatory responses in rats.

Objective To explore the effect of electroacupuncture on activation of microglia in peri-infarct cortex after cerebral isch-emia-reperfusion in rats. Methods 36 male Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and electroacupuncture group (n=12). The latter two groups were occluded the left middle cerebral arteries with modified Longa's method for 2 hours and reperfused. The electroacupuncture group received electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints for 3 days. The nerve cell damage in peri-infarct cortex was observed with HE staining, while the expression of ED1 was determined with immunohisto-chemical staining, and the expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and IL-6 were determined with Western blotting. Results The neurological deficits score improved significantly in the electroacupuncture group (P<0.05), with less nerve cell dam-age, less number of ED1 positive microglia (P<0.05) and less levels of TNF-α, IL-1βand IL-6 (P<0.05), compared with the model group. Conclusion The electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints can protect brain from ischemia-reperfusion injury, which might be associated with inhibiting the microglial activation and proinflammatory response in peri-infarct cortex.

OBJECTIVETo investigate the nephrotoxic effects of methyl cantharidimide tablets on urinary protein and enzymes in Beagle dogs.

METHODBeagle dogs were randomly divided into negative control group(blank tablet), methyl cantharidimide tablets group (6.11,12.21, 24.42 mg x kg(-1)), continuously 30 days of oral adminiStration, once a day. The drug and control group were collected and determined fresh urine in 1, 2, 3 and 4 weeks of the administration; Serum urea nitrogen (BUN), creatinine (Crea), total protein (TP) and albumin (ALB) as well as sodium, potassium, chloride electrolyte were determined on 15 and 30 days of the administration; Urine albumin (mAlb), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin( NGAL), N-acetyl-beta-D-glucosaminidase (NAG), clusterin, beta2-microglobulin (beta2-MG), alpha1-microglobulin (alpha1-MG), alanine aminopeptidase( AAP) and im- munoglobulins IgG were tested on 15 and 30 days of the administration.

RESULTCompared with the control group, urine protein and white blood cells was significantly increased in each dose group. On 15 days of the administration, mAlb were higher in each dose group, KIM-1, NGAL, clusterin, NAG and AAP were significantly higher in high-dose group, while the middle and low dose group had no significant difference, as well as blood SCr and BUN no obvious abnormalities. On 30 days, mAlb, KIM-1, clusterin, NAG, AAP were increased in each dose group, appearing dose-effect relationship, beta2-MG and NGAL levels were significantly increased in high-dose group. Contents above indicators were increased with significant dose and time relationship, and serum BUN, Scr were correlated, suggesting that urine mAlb, KIM-1, clusterin, NAG and AAP indicators that can sensitively respond the changes of proteins and enzymes in urine.

CONCLUSIONMethyl cantharidimide tablets has a renal toxicity, urine mAlb, KIM-1, clusterin, NAG and AAP can be used as the early nephrotoxic biomarkers of methyl cantharidimide tablets.

Animals ; Biomarkers ; urine ; Dogs ; Female ; Kidney ; drug effects ; Kidney Diseases ; chemically induced ; Male ; Proteins ; metabolism ; Tablets ; adverse effects ; Urine ; chemistry

Objective To compare the effects and complications between neuroendoscope-assisted and conventional shunt methods in patients with hydrocephalus,Methods The data of 299 patients with hydrocephalus admitted to our hospital from June 2002 and June 2009 were analyzed.Among these patients,98 adopted neuroendscope:78 were performed ventriculoperitoneal shunt(EVPS)combined with endoscopic third ventriculostomy(ETV)and the other 20 with obstructive hydrocephalus were performed ETV only.The other 201 patients adopt conventional operation:VPS was employed in 196 and ventriculoatrial shunt was performed in 5.We compared shunt effectiveness by calculating the pre-and post-operative ventricular indexes,shunt failure rates and complication rates during the follow-up between the 2 groups.Results No difference in etiology of hydrocephalus or clinical characteristics between the 2 groups was found.Compared with the conventional shunt group,neuroendscope-assisted group enjoyed a longer operative time,a higher surgery cost,a lower clogging incidence rote and a higher precision rote of putting the tube(P<0.05).Conclusion Applying of neuroendoscope is a very good option in treating patients with various kinds of hydrocephalus with good long-term follow-up results.

BACKGROUNDPlatelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma.

METHODSThe gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.

RESULTSPRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-beta1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P < 0.05), and the effects of fraction B and C showed no significant difference compared to the PRP group (P > 0.05). Fraction A and D showed no significant difference to the negative control group (P > 0.05).

CONCLUSIONSThe growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.

Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Chromatography, Gel ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Platelet Count ; Platelet-Derived Growth Factor ; isolation & purification ; pharmacology ; Platelet-Rich Plasma ; chemistry ; Transforming Growth Factor beta1 ; isolation & purification ; pharmacology

Accidents, Occupational ; statistics & numerical data ; China ; epidemiology ; Humans

Objective To explore the application value of retrobulbar nerve block combined with general anesthesia in the penetrating keratoplasty (PKP). Methods A total of 100 recipients undergoing PKP from January 2017 to January 2019 were recruited in this study. All recipients were divided into the observation group (n=50) and control group (n=50) by random number table method. In the control group, patients received laryngeal mask airway under general anesthesia, and in the observation group, patients received laryngeal mask airway under general anesthesia combined with retrobulbar nerve block. Hemodynamic changes of the PKP recipients before and after operation were observed in two groups. The dosage of analgesic drugs and the incidence of complications were observed in two groups. The degree of pain at postoperative 2-, 6- and 24-h was evaluated by visual analogue scale (VAS) in two groups. The awakening situation of the recipients in two groups was observed. The levels of inflammatory cytokines at 1 d before and after operation were statistically compared in two groups. Results The average arterial pressure and heart rate at intraoperative 15 min and after the surgery in the observation group were significantly higher than those in the control group (both P < 0.05). In the observation group, the dosage of remifentanil and propofol were (1.0±0.4) mg and (299±40) mg, significantly lower than (1.3±0.6) mg and (365±42) mg in the control group (both P < 0.05). The incidence of complications did not significantly differ between two groups (P > 0.05). In the observation group, the VAS scores at 2-, 6- and 12-h after operation were remarkably lower than those in the control group (all P < 0.01). The respiratory recovery time, eye opening time, directional force recovery time and extubation time of the recipients in the observation group were significantly shorter than those in the control group (all P < 0.05). The expression levels of including interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) at postoperative 1 d in the observation group were considerably lower than those in the control group (all P < 0.05). Conclusions Retrobulbar nerve block combined with general anesthesia can maintain hemodynamic stability during PKP, reduce the dosage of remifentanil and propofol and alleviate the degree of postoperative pain and inflammatory responses of the recipients.