Establishment and genotype identification of hepatic stellate cell-specific Grk2 gene knockout mouse model / 中国药理学通报

Aim To establish a stable hepatic stellate cell ( HSC ) -specific G protein-coupled receptor kinase 2 ( GRK2 ) knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC. Methods The loxP-labeled Grk2 gene mouse (Grk2

Structural distribution and mechanism of elastic fibers and collagen fibers in ventricular interstitium of aged rats / 解剖学报

Objective To investigate the structural distribution features and mechanism of elastic fibers and collagen fibers in ventricular interstitium of aged rats. Methods Five young SD rats (24 weeks) and five old SD rats (104 weeks) were used,and their cardiac function was examined by echocardiography. Modified Weigert elastic fiber staining, immunohistochemistry, immunofluorescence and Western blotting techniques were used to detect the expression changes of type I and IH collagen fibers and their proteins, elastic fibers and their proteins, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 2 (TIMP-2), respectively. Results The type I and type IH collagen in the ventricular interstitium of aged rats was very sufficient and wrapped around the cardiomyocytes. Compared with the young rats, the content of collagen protein in the ventricular interstitium of the aged rats significantly increased (P<0. 05). Elastic fibers in the ventricular interstitium of the aged rats were and widely distributed. Compared with the young rats, the number of elastic fibers and the level of elastin in the ventricular interstitium of the aged rats significantly decreased (P<0. 05), and the expression levels of MMP-2 and MMP-9 in ventricular muscle of aged rats increased, and the)' were correlated with the level of elastin. The level of TIMP-2 in ventricular muscle of aged rats decreased with age. Conclusion The number of collagen fibers and elastic fibers in ventricular interstitium of aged rats is fluctuated with each other. With the increase of age, the contents of TIMP-2 and elastic fibers in the ventricular interstitium gradually decreased, and the ratio of collagen fibers to elastic fibers is out of balance.

Secondary metabolites of endophyte Alternaria sp. W-1 from Laminaria japonica / 药学学报

Six compounds were isolated from the crude extract of the liquid culture of Alternaria sp. W-1 by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and HPLC. They were identified as 6-iso-tricycloalternarene 6a (1), tricycloalternarene 6a (2), tricycloalternarene B (3), uracil (4), 5-methyluracil (5), and lumichrome (6) through HR-MS, NMR and literature comparison. 6-iso-Tricycloalternarene 6a (1) is a new compound which has never been reported in the literature. In cytotoxicity assay, compounds 1-3 showed weak inhibition activity to human hepatoma cell line SMMC-7721 and human gastric cell line SGC-7901.

Genotyping of Klebsiella Pneumonia Strains Isolated from Eldly Inpatients by Multiple-locus Variable-number Tandem-repeat Analysis / 中国医学科学院学报

Objective To investigate the genotype of klebsiella pneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiella pneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiella pneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiella pneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiella pneumonia infection remained the nosocomial infection.

A Chinese herbal formula, Wuzi Yanzong pill, improves spermatogenesis by modulating the secretory function of Sertoli cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of the Chinese herbal formula Wuzi Yanzong Pill (, WYP) on the spermatogenesis and specific secretory functions of Sertoli cells in rat model and to investigate the underlying mechanism.</p><p><b>METHODS</b>Five groups of male Sprague-Dawley rats including the control group, the model group, the low-dose WYP group, the medium-dose WYP group and the high-dose WYP group (5 in each group) were treated daily with vehicle, multiglycosides of Tripterygium wilfordii Hook f (GTW) either alone (20 mg/kg) or followed by WYP (0.5, 1.0, or 2.0 g/kg daily), respectively for 30 days. Serum levels of follicle-stimulating hormone (FSH), inhibin B (INHB) and testosterone (T) were evaluated using enzyme-linked immunosorbent assay. Androgen-binding protein (ABP) gene expression and transferrin (TF) protein expression in testis tissue specimens of all rats were determined using real-time reverse transcriptase polymerase chain reaction and Western blotting analysis, respectively. Histopathological alterations in the testis were determined using Johnsen's score.</p><p><b>RESULTS</b>The toxicity of GTW towards Sertoli cell secretory functions and spermatogenesis was accompanied by increased serum FSH concentrations and decreased INHB and T concentrations. Upregulated ABP mRNA levels, and decreased TF protein expression and Johnsen's scores were detected in the model group compared with the control group P<0.05 or P<0.01). Oral high-dose WYP administrations to GTW-treated rats effectively alleviated all of the GTW-induced changes in specific secretory functions of Sertoli cells (ABP, INHB and TF). Furthermore, serum T level and Johnsen's score of the testis increased greatly compared with the model group (P<0.01).</p><p><b>CONCLUSION</b>WYP has the ability to improve the spermatogenesis, possibly through modulating the secretory proteins expression of Sertoli cells.</p>

Anti-fibrosis effects of fenofibrate in mice with hepatic fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the anti-fibrosis effects and mechanisms of fenofibrate on hepatic fibrosis using a mouse model of fibrosis induced by carbon tetrachloride (CCl4).</p><p><b>METHODS</b>Twenty-six male C57BL mice were divided into the following three groups: CCL4-induced untreated model control (n = 10), CCl4-induced fenofibrate-treated model (n = 10), and uninduced/untreated normal control (n = 6). All animals were sacrificed after the 5 weeks of induction and treatment. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA) and procollagen III amino-terminal peptide (PIIINP) were determined by routine biochemistry assays. Liver content of hydroxyproline (HYP) was measured by spectrophotometry. Liver content of malonic aldehyde (MDA) and superoxide dismutase (SOD) was measured by enzymatic assays. mRNA expression levels of liver fibrosis-associated factors were determined by PCR, and included alpha-smooth muscle actin (a-SMA), transforming growth factor-beta1 (TGFbeta1), type I collagen-alpha (Collagen1a), peroxisome proliferator-activated receptor-alpha (PPARa), and the inflammatory cytokines tumor necrosis factor alpha (TNFa) and interleukin-6 (IL-6). Finally, the degree of inflammation and fibrosis were assessed by histological analysis using hematoxylin-eosin and Sirius red staining.</p><p><b>RESULTS</b>Compared to the untreated model group, the fenofibrate-treated model group showed significantly lower levels of serum ALT (55.72+/-1.20 vs. 38.72+/-1.25 IU/L), HA (236.20+/-17.57 vs. 152.9+/-13.06 mug/L) and PIIINP (41.66+/-1.89 vs. 34.32+/-1.53 mug/L) (all P less than 0.05). The fenofibrate-treated group also showed a significantly higher level of hepatic SOD content (untreated model: 67.00+/-4.65 vs. 101.1+/-5.32) but significantly lower level of hepatic MDA content (14.67+/-0.93 vs. 10.17+/-0.60 nmol/mg) and lower level of hepatic HYP content (0.67+/-0.80 vs. 0.41+/-0.50 mg/g) (all, P less than 0.05). In addition, the fenofibrate-treated group showed significantly reduced mRNA expression levels of a-SMA (6.83+/-0.88 vs. untreated model: 11.57+/-1.31), TGFbeta1 (67.83+/-4.65 vs. 112.30+/-4.81), Collagen1a (67.83+/-4.65 vs. 112.30+/-4.81), TNFa (17.43+/-2.32 vs. 37.83+/-4.69), and IL-6 (4.00+/-0.49 vs. 5.62+/-0.54), but significantly increased PPARa (0.30+/-0.03 vs. 0.18+/-0.03) (all, P less than 0.05). Finally, the degree of CCL4-induced hepatic fibrosis was attenuated by the fenofibrate treatment.</p><p><b>CONCLUSION</b>Fenofibrate can reduce the degree of liver fibrosis in mice induced by CCl4. The mechanism may involve up-regulation of PPARa, inhibition of the inflammatory response, and enhancement of SOD antioxidant activity.</p>

The 2002 AJCC TNM classification is a better predictor of primary small cell esophageal carcinoma outcome than the VALSG staging system / 癌症

Small cell carcinoma of the esophagus (SCCE) is a rare and aggressive malignant tumor with a poor prognosis. The optimal disease staging system and treatment approaches have not yet been defined. This study aimed to evaluate the prediction of different staging systems for prognosis and treatment options of SCCE. We retrospectively accessed the clinicopathologic characteristics, treatment strategy, and prognosis of 76 patients diagnosed with primary SCCE between 2001 and 2011. The 1-, 2-, 3-, and 5-year overall survival rates were 58%, 31%, 19%, and 13%, respectively. Univariate analysis showed that the 2002 American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) classification (P = 0.002), Veterans Administration Lung Study Group (VALSG) stage (P = 0.001), predisposing factors (P < 0.001), T category (P = 0.023), and M category (P < 0.001) were prognostic factors for overall survival. Multivariate analysis showed that the 2002 AJCC TNM stage (P < 0.001) was the only independent prognostic factor for survival. The value of the area under the receiver operator characteristic (ROC) curve (AUC) of the 2002 AJCC TNM staging system was larger than that of VALSG staging system with regard to predicting overall survival (0.774 vs. 0.620). None of the single treatment regimens showed any benefit for survival by Cox regression analysis. Thus, the 2002 AJCC TMN staging system improved the prediction of SCCE prognosis; however, the optimal treatment regimen for SCCE remains unclear.

Construction and transfection experiment of a goose circovirus infectious clone / 病毒学报

A pair of primers with BamH I restriction site were designed to amplify the complete genome of goose circovirus. Two copies of the genome were ligated in tandem and cloned into pGEM-T Easy vector to construct an infectious clone named as pGEMT-2GoCV. The pGEMT-2GoCV linearized with EcoR I was transfected to negative embryos and gosling with Lipfectamine. PCR detection verified the proliferation of GoCV in geese. Some sera of the embryo transfected group were detected to be positive at 2 and 4 weeks after hatching and one bursa was detected to be positive at 4 weeks. Some sera of the gosling transfected group were also detected to be positive at 2 weeks after transfection. Furthermore, the mark in the PCR products were identified by BamH I digestion and the GoCV in positive tissue and sera were quantitated by Real-time PCR. The results showed that the virus load in positive bursa was 1.57 x 10(6) copies/mg, the virus load in positive sera were 3.52 x 10(4)-5.92 x 10(5) copies/microL. In conclusion, the infectious DNA clone constructed with two copies of full-length GoCV genome in tandem can transfect embryo and gosling and propagate the marked goose circovirus.

Metabolism-based interaction of diphenytriazol and flavone compounds / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To observe the metabolism-based interaction of diphenytriazol and flavone compounds.</p><p><b>METHODS</b>Flavone compounds kaempferol, isoharmnten and Elsholtzia blanda benth extract were chosen as the substrate of glucuronidation in the phase II metabolism. The metabolism was investigated in different rat liver microsome incubates pretreated with beta-naphthoflavone (BNF), diphenytriazol and tea oil (control). The concentrations of residual substrate were determined by HPLC. Quercetin and kaempferol were coincubated with diphenytriazol in control microsome to evaluate the inhibition for phase I metabolism. The concentration of diphenytriazol was determined by HPLC.</p><p><b>RESULT</b>The phase II metabolic activity of kaempferol, isoharmnten and Elsholtzia blanda benth extract in diphenytriazol-treated microsome was more potent than that in BNF-treated microsome (P<0.01). The phase I metabolism of diphenytriazol was markedly inhibited by quercetin and kaempferol, with the inhibition constants (Ki) (12.41 +/-0.26)microg . ml(-1) and (7.97 +/-0.08)microg . ml(-1), respectively.</p><p><b>CONCLUSION</b>Diphenytriazol demonstrates metabolism-based interaction with flavone compounds in vitro.</p>

Characteristics of human adrenal microvascular endothelial cells in morphology, phenotypes, and function / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the phenotypic and functional characteristics of human adrenal microvascular endothelial cells (AdrEC).</p><p><b>METHODS</b>AdrEC were isolated and purified from a sample of human adrenal tissue by sub-cell clone method. The cells identified by flow cytometry for classical endothelial markers von Willebrand factor (vWF) and CD31, uptake of Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL), as well as phenotypes. The cell fenestrations were checked by scanning electron microscopy. The expressions of endogenous vascular endothelial growth factor (VEGF) mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The glucocorticoid-induced cytotoxicities in different organs-derived microvascular endothelial cells were compared.</p><p><b>RESULTS</b>Human AdrEC expressed those classical endothelial markers such as vWF, CD31, and uptake of Dil-Ac-LDL. The phenotypic analysis indicated that alpha-1 proteinase inhibitor, tumor necrosis factor receptor p55, and intercellular adhesion molecule-1 were expressed in human AdrEC. Scanning electron microscopy demonstrated that there were many microvilli and fenestrations on cellular surface. RT-PCR and immunocytochemistry showed that there was expression of endogenous VEGF in AdrEC. In response to glucocorticoid-induced cytotoxicity, microvascular endothelial cells (MVEC) derived from human brain were highly susceptible, MVEC derived from human lung and human liver sinusoidal endothelial cells were sub-sensitive, while AdrEC were highly resistant.</p><p><b>CONCLUSION</b>Human AdrEC are specially differentiated and have characteristics that are different from other organ-derived MVEC in phenotypes and functions.</p>

Advances in the study of precise radiotherapy for non-small-cell lung cancer / 国外医学(肿瘤学分册)

With the development of computer techniques and medical imaging examining methods , precise radiotherapy is becoming the major direction of radiotherapy for tuomors. Both of tumor control probability and normal tissue complication probability are improved with precise radiotherapy. This paper critically review the value of PET-CT and breathing control in precise radiotherapy for non-small-cell lung cancer (NSCLC).

Treatment and survival analysis of pyriform sinus cancer / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of surgery for the patients with pyriform sinus carcinoma and analyze the prognostic factors related to the cancer.</p><p><b>METHODS</b>Between December 1995 and December 2002, 62 patients with pyriform sinus cancer were treated in Zhejiang Tumor Hospital. There were 13 patients staged T1, 17 T2, 12 T3, 20 T4. Four patients received preoperative radiation and 40 patients had post-operative radiation. Among 62 patients, 33 patients were treated by partial laryngectomy, 29 patients were treated by total laryngectomy.</p><p><b>RESULTS</b>The survival rate was calculated with Kaplan-Meier method. The overall 3- and 5-year survival rates were 42.3% and 27.8%, respectively. The 3-year survival rate between partial and total laryngectomy was 51.9% and 29.9%. The 5-year survival rate between partial and total laryngectomy was 39.5% and 11.2% (chi2 = 4.14, P<0.05). Early stage and combined modality therapy were the independent favorable prognostic factors.</p><p><b>CONCLUSIONS</b>Early diagnosis with treatment and combined treatment are the most important factors influencing the survival of patients with pyriform sinus carcinoma. Partial laryngopharyngectomy is a suitable treatment for early and selected advanced pyriform sinus carcinoma with a good function and oncologic outcome.</p>

Detection of four human herpesviruses DNA and virus-specific IgM antibody in blood specimens of infants / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis.</p><p><b>METHODS</b>A pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3'). DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP. At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses. Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA.</p><p><b>RESULTS</b>The PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique. The specificity and sensitivity of this PCR-RFLP were examined. There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA. Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3%. All ELISA-positive specimens were likewise positive by PCR. Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR. An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively. The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth. None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA.</p><p><b>CONCLUSIONS</b>This PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.</p>

HPLC fingerprint of the tablets of Ginkgo biloba L / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish the method for determination of the fingerprint of tablets of Ginkgo biloba L.</p><p><b>METHODS</b>HPLC-DAD was used to determine the constituents in tablets. Diamonsil C18(200 mm x 4.6 mm, 5 microm) was used as analysis column and acetonitrile/KH(2)PO(4) as mobile phase with gradient elution. The column temperature was at 24 degree. The profile of chemical constituents in control sample and tablets obtained from the chromatograms were analyzed by similarity software.</p><p><b>RESULT</b>The method developed for components analysis of the standard extracts was linear within certain concentration (r>0.999). There was no difference between the fingerprints of 3 batches of products. The fingerprints of tablets and the extract showed a good similarity(>0.965).</p><p><b>CONCLUSION</b>This method is accurate simple and can be used for the quality control of Ginkgo biloba L. preparations.</p>

Relationship among serum cortisol and insulin and blood glucose in the asphyxiated neonates / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the dynamic change of serum cortisol and insulin levels, and their relation with blood glucose concentration in asphyxiated neonates.</p><p><b>METHODS</b>The levels of serum cortisol and insulin at d1,d3 and d7 of birth were measured by radioimmunoassay and the concentration of blood glucose was measured with glucose oxidase method in 43 asphyxiated neonates.</p><p><b>RESULTS</b>The levels of serum cortisol at d 1, d 3 and d 7 of birth were gradually decreased (P<0.01). At d1, the incidence of hyperinsulism (>20 mIU/L) was 60.5%. The level of serum insulin reached normal level (<or=20 mIU/L) at d 3. At d 1, the level of serum cortisol was positively related with the level of blood glucose (r=0.432, P<0.01), the ratio of serum insulin and cortisol in hypoglycemia group (0.71+/-0.71) was significantly higher than that in normal blood glucose group (0.19+/-0.36, P<0.01). The incidence of hypoglycemia in hyperinsulism group was markedly higher than that in normal serum insulin group (P<0.05).</p><p><b>CONCLUSION</b>There are temporary hyperinsulism in asphyxiated neonates. Hypoglycemia in asphyxiated neonates is related with hyperinsulism and low serum cortisol level.</p>

Bone turnover and early parenteral calcium supply in preterm infants / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the dynamic change in biochemical markers of bone turnover in preterm infants and the effect of early parenteral calcium supply.</p><p><b>METHODS</b>Forty preterm infants were divided into parenteral calcium supply group and control group. Blood and urine samples were collected at 24 h and 11 d after birth. Serum osteocalcin (OC) was measured with ELISA, serum carboxyterminal telopeptide type I collagen (ICTP) with radioimmunoassay, serum alkaline phosphatase (AKP), calcium, phosphate, and urine calcium, phosphate, creatinine with automatic biochemical analyzer. Blood samples were also collected from 22 term infants as control. Calcium gluconate (10%, 4 ml/kg x d(-1)) was administered intravenously in parenteral calcium supply group.</p><p><b>RESULT</b>At 24 h, serum AKP, ICTP [(147.86+/- 44.87)IU, (57.36+/- 6.34)micro g/L] in preterm infants were significantly higher than those [(147.86+/- 44.87)IU, (57.36+/- 6.34)micro g/L] in term infants, and negatively correlated with gestational age and birth weight (r =-0.528, P<0.01; -0.614, P< 0.01), but serum OC [(648.77+/- 238.89) nmol/L] in preterm infants was lower than that [(851.68+/- 238.69)nmol/L] of term infants, and positively correlated with gestational age and birth weight (r=0.359, P< 0.05; 0.376, P< 0.01). At 11 day, serum OC [947.25+/- 335.47)nmol/L] in preterm infants was markedly elevated and reached the level of term infants [(941.65+/- 297.28)nmol/L], but serum ICTP [(65.44+/- 6.24)micro g/L] in preterm infants was higher than that [(57.10+/- 3.48)micro g/L] in term infants all along. Serum AKP [(246.00+/-66.64)IU] in parenteral calcium supply group was higher than that [(206.53+/- 53.9)IU] in the control group. There were no significantly differences in serum OC and ICTP between parenteral calcium supply group and the control group. Calcium in serum and urine was elevated, phosphate in serum and urine was reduced in the parenteral calcium supply group. Urine analysis and kidney ultrasounds were normal.</p><p><b>CONCLUSION</b>There is active bone formation and bone resorption in preterms as compared with terms. Alone parenteral calcium supply during early life can not increase formation of bone protein or decrease degradation of bone collagen, but can elevate serum calcium and urine calcium levels. Hematuria and renal calcification were not found in short duration.</p>