Real-time dynamic recording of cerebral cortical vascular embolization and regeneration in rats / 生理学报

The purpose of this study was to establish a method to record the dynamic process of vascular regeneration and remodeling in rat cerebral ischemic regions. An animal brain window model was established to continuously observe the changes of rat cortical vascular ischemia in vivo, and the model of cerebral ischemia was established by photochemical embolization. Optical coherence tomography (OCT) was performed to record the formation of vascular blockage and the injury and regeneration of small vessels during cerebral ischemia recovery. The results showed that 30 min of laser irradiation could completely block the cortical vessels in rats. Within 24-48 h after ischemia, the degree of brain injury was the greatest, and the number of blood vessels in the ischemic region reached the minimum. Then the blocked blood vessels began to be dredged, and the small blood vessels around the ischemic area began to regenerate. Small blood vessels in the superficial/deep layers of the cortex disappeared significantly after laser irradiation. During 10 d after ischemia, the blocked blood vessels were gradually dredged and recovered. On the 10th day after laser irradiation, a large number of neovascularization appeared in the superficial layer of cortex, but the deep vessels did not recover. These results indicate that the method established in this study can observe the changes of blood vessel in cerebral ischemic region continuously, which lays a foundation for further quantitative study on the dynamics of embolized blood vessels and peripheral capillaries during the recovery of cerebral ischemia.

Effect of particulate matter 2.5 at urban centre of Hangzhou on lung impairment in rats / 中国应用生理学杂志

OBJECTIVE@#To explore the effect of ambient particle matter 2.5 (PM2.5) collected in the urban center of Hangzhou on the lung injury of rats and on the activating of endoplasmic reticulum pathway.@*METHODS@#PM2.5 samples were collected on quartz fiber filters using a PM2.5 high-volume air sampler in the urban area of Hangzhou. The collected PM2.5 particles were extracted in ultrapure water and concentrated by vacuum freeze-drying. Twenty-four male Sprague-Dawly (SD) rats were randomly divided into 3 groups:saline control group, low dose PM2.5 exposure group (5 mg/kg BW) and high dose PM2.5 exposure groups (25 mg/kg BW). Each group received intratracheal instillation of PM2.5, once a week for 4 weeks. Twenty-four hours after the last exposure, the rats were narcotized and sacrificed, left lung was isolated and fixed with 4% paraformaldehyde for histopathological detection. The bronchoalveolar lavage fluid (BALF) was collected from the right lung. The total antioxidant capacity (T-AOC) level, the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH) in BALF were detected by chemical colorimetry. The level of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) cytokines in BALF was measured by enzyme linked immunosorbent assay (ELISA). And the protein expressions of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase receptor-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor (p-eIF2α), transcription factors C/EBP homologue protein (CHOP), inositol-requiring enzyme 1α (IRE1α) and X-box binding protein 1 (XBP1) in lung tissue were determined by Western blotting.@*RESULTS@#Compared with control group, rats in both low dose (5 mg/kg) and high dose (25 mg/kg) PM2.5-treated groups showed obviously dose-dependent pulmonary toxicity including thickening of alveolar walls, narrowing of alveolar space, interstitial hyperplasia and inflammatory cell infiltration. Compared with control group, T-AOC level and the SOD activity in BALF in both PM2.5-treated groups were decreased dose-dependently (<0.05), whereas the LDH activity in BALF were increased in a dose-dependent manner (<0.05). Exposure to PM2.5 resulted in a increasing of the release of proinflammatory cytokines including TNF-α, IL-1β and IL-6 in rat lung in a dose-dependent manner (<0.05). The levels of GRP78, p-PERK, p-eIF2α, CHOP, IRE1α and spliced XBP1 (XBP1-S) were significantly up-regulated, whereas the level of unspliced XBP1 (XBP1-U) was down-regulated in the rat lung tissue of high-dose PM2.5 treated group.@*CONCLUSIONS@#The PM2.5 in the urban area of Hangzhou can significantly cause lung inflammatory injury in rats. Both oxidative stress and activation of ER stress pathways may be related to such PM2.5 inhalation-induced lung inflammatory injury.

Cleavage of in vitro transcripts of hepatitis B virus C gene by 10-23 DNA enzyme / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.</p><p><b>METHODS</b>10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.</p><p><b>RESULT</b>Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.</p><p><b>CONCLUSION</b>10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.</p>

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro.</p><p><b>METHODS</b>PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h.</p><p><b>RESULTS</b>After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity.</p><p><b>CONCLUSION</b>This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.</p>

Clinical features of chronic hepatitis B patients with YMDD mutation after lamivudine therapy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the clinical features of chronic hepatitis B (CHB) patients with tyrosine-methionine-aspartate-aspartate (YMDD) mutation after lamivudine therapy.</p><p><b>METHODS</b>This investigation was a retrospective study of 63 CHB patients with YMDD mutation during lamivudine therapy. Clinical data, including period and types of YMDD mutation; hepatitis B virus (HBV) DNA levels and alanine aminotransferase (ALT) levels before and after YMDD mutation were measured. YMDD mutation in the HBV DNA polymerase gene was determined using polymerase chain reaction (PCR) and direct sequencing. HBV DNA quantification was determined using real-time PCR. Relevant serum markers of HBV were measured. The follow-up period was 12 months after YMDD mutation.</p><p><b>RESULTS</b>YMDD mutation occurred 7-44 months (median, 21.5 months) after the start of lamivudine therapy. The majority of the cases (42/63, 66.6%) had YMDD mutants detected between 12 and 24 months. Four types of YMDD mutation were observed in this study, rtL180M/M204V mutation was the predominant type (26/63, 41.3%). A proportion of patients (16/63, 25.4%; 12/63, 19.1%) had higher HBV DNA levels and ALT levels (after mutation vs before mutation), respectively.</p><p><b>CONCLUSION</b>The majority of patients with YMDD mutants had similar or lower HBV DNA levels and ALT levels compared with baseline values. This subset of patients might have benefited from the continued lamivudine therapy. The patients with increased ALT and HBV DNA levels (breakthrough hepatitis) should benefit from the addition of a newer nucleotide analogue (e.g. adefovir).</p>

Character of HBV (hepatitis B virus) polymerase gene rtM204V/I and rtL180M mutation in patients with lamivudine resistance / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVES</b>To investigate the relationship between HBV (hepatitis B virus) polymerase gene 180 and 204 sites mutation and lamivudine resistance.</p><p><b>METHODS</b>One hundred forty-one patients with lamivudine resistance after lamivudine treatment and 60 chronic hepatitis B patients without lamivudine treatment were enrolled in this study. The serum HBV DNA mutation was analyzed by sequence detection via polymerase chain reaction (PCR). The sequences of the same patient were analyzed before and after lamivudine treatment.</p><p><b>RESULTS</b>One hundred and nine lamivudine resistance patients had HBV YMDD (tyrosine-methionine-aspartate-aspartate) mutation. Among them, 45 patients had rtL180M/M204V mutation (41.28%), 28 patients had rtL180M/M204I mutation (25.70%) and 36 patients had rtM204I mutation (33.02%). There were 6 patients with rtL180M mutation in 32 lamivudine resistance patients. Sixty chronic hepatitis patients without lamivudine treatment had no mutations.</p><p><b>CONCLUSIONS</b>HBV mutations, which play an important role in lamivudine resistance usually locate at polymerase gene 204 site; 180 site mutation was also observed in these patients. Evaluation of the anti-virus therapy by surveillance of the two sites mutations is of importance.</p>

Development of a system for quick screening of efficient HBx-siRNA / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a system for quick screening of efficient siRNA targeted HBx mRNA.</p><p><b>METHODS</b>Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis.</p><p><b>RESULT</b>(1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388.</p><p><b>CONCLUSION</b>Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.</p>

Detection and analysis of the new kind of G743C and G743A point mutation of HBV P gene in hepatitis B virus infected patients resistant to lamivudine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the point mutation in hepatitis B virus polymerase (HBV P) gene in HBV-infected patients resistant to lamivudine.</p><p><b>METHODS</b>HBV P gene was amplified by PCR and the products was sequenced to analyze the YMDD mutation. Then the variants were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the following restriction enzymes: Fok I, Ssp I, Alw441 and were separated by 8.0% polyacrylamide gel electrophoresis.</p><p><b>RESULTS</b>Comparing with the sequences of standard HBV genome, there were 16 patients with G743C mutation and 1 patient with G743A mutation, and the codon ATG turned to ATC and ATA, YMDD motif changed into YIDD. But this kind of YIDD mutation was not proved by PCR-RFLP assay in the 17 patients.</p><p><b>CONCLUSIONS</b>The G743C and G743A mutations in HBV P gene, resulting in YMDD motif changed into YIDD, are detected only by direct sequencing, not by PCR-RFLP. The new kind of G743C and G743A point mutations in HBV P gene is important for the detection of HBV P gene YMDD mutation.</p>

Construction of HBV S gene recombinant and its immunity induced in mice / 浙江大学学报·医学版

OBJECTIVE: To investigate the feasibility of HBV DNA vaccines. METHODS: HBV S gene was obtained by PCR and the PCR product was cloned into pcDNA3. The recombinant was screened by antibiotics, identified by digestion and confirmed by sequencing. The plasmid was then transfected into mammalian cell COS-7 for transient expression. Then the recombinant was injected into mice and the immune responses induced in mice were investigated. RESULTS: The sequence of HBV S gene was correct and HBsAg could be detected in cells transfected with pcDNA3-S. After immunization, the positive rate in mice immunized with pcDNA3-S and pCMV-S was 70%(7/10) and 80% (8/10). The mean levels of anti-HBs were (32.14+/-13.79)mIU/ml and (28.50+/-11.87)mIU/ml respectively. There was no statistically significant difference between them P 0.05 . The mean levels of anti HBs in the control group and blank groups were both less than 10 mIU/ml. In mice immunized with pcDNA3-S and pCMV-S the results were (35.40+/-4.85)% and (38.20+/-7.69)% when E/T was 20:1, or (23.95+/-3.98)% and (24.55+/-3.59)% when E/T was 10:1, again showing no difference statistically (P>0.05). The specific CTL cytotoxicity rate of control and blank groups was both less than 5%. CONCLUSION: A specific humoral and cellular immune response can be induced in mice by intramuscular injection of pcDNA3-S.

Serum levels of sFas, sICAM-1, IL-18 in patients with chronic hepatitis C and their clinical significance / 浙江大学学报·医学版

OBJECTIVE: To investigate the serum levels of soluble Fas antigen (sFas), soluble intercellular adhesion molecules-1 (sICAM-1), interleukin-18 (IL-18) in patients with chronic hepatitis C and to study their roles in pathogenesis of chronic hepatitis C. METHODS: Serum sFas, sICAM-1, IL-18 levels were measured in 30 cases of chronic hepatitis C before and after treatment of interferon-alpha by enzyme-linked immunosorbent assay (ELISA), serum titer of HCV-RNA was detected by quantitative PCR and serum ALT activity was also detected. RESULTS: Serum levels of sFas sICAM-1 IL-18 in chronic hepatitis C patients were significantly higher than those in normal controls (P<0.01), showing correlation with serum HCV-RNA titer (r=0.915, r=0.795, r=0.757, respectively, P<0.01), Serum levels of sICAM-1, IL-18 showed correlation with serum ALT level(gamma=0.952, gamma=0.969, respectively, P<0.01), but no relationship was observed between serum sFas and serum ALT level(P>0.05). Serum levels of sFsa sICAM-1 IL-18 markedly decreased in responsive patients while no change was observed in patients with no response after treatment. CONCLUSION: Soluble Fas, soluble ICAM-1, IL-18 may participate in the pathogenesis of chronic hepatitis C and show correlation with the severity of histological inflammation and viral titer.