Objective To explore the effect of age on neurogenesis of dentate gyrus granule cell and the impact on differentiation of newborn cells in rats.Methods SD rats were selected and divided into 5 groups according age of 7,14,28,60,180 d(n=8),and the neurogenesis of dentate gyrus granule cell in hippocampus with normal development was detected,using 5-bromo-BrdU(BrdU) labled newborn neuron and ?-tubulin protein(TuJ1) and glial fibrillary acidic protein(GFAP) labeled glial cells,and understood the newborn cells to neurons and glial cell differentiation ratio.Results Neurogenesis was found in dentate gyrus granule cell layer with hippocampus of all different age rats.Various forms of cells with a larger nucleus that were round,oval,diamond were distributed over the entire granule cell layer.BrdU-positive cells within each group were 158.07?5.37,141.28?7.27,116.93?9.24,76.56?6.88,41.42?4.45,the number of BrdU-positive cells were reduced with the growth of rats(P0.05);4%-5% newborn cells expressed GFAP.In addition,some of the BrdU-positive cells at the same time did not express TuJ1 or GFAP.Conclusions There are neurogenesis in dentate gyrus granule cell in rats of different age.The new born cells mostly differentzate into granule neuron cell.The capability of cell proliferation are decreased with the growth of age.

The 3T3-L1 preadipocytes were used as carriers in the investigation of total extract, n-butanol extract, CB-1 and CB-2 of Coreopsis tinctoria Nutt. on cell proliferation and differentiation. Three groups at different doses were set for each of the four extract regions of C. tinctoria Nutt., respectively. MTT assay was used to detect 3T3-L1cell proliferation by four extract regions of C. tinctoria Nutt. Oil Red O staining was used to analyze the formation and accumulation of cytoplasmic lipid during cell differentiation. The results showed that compared with the control group, there were significant inhibition on cell proliferation when thetotal extract of C. tinctoriaNutt. at 100 μg·mL-1, n-butanol extract at 0.5, 5, and 50 μg·mL-1, CB-1 and CB-2 at 50 μg·mL-1 (P< 0.01). N-butanol extract showed certain dose-dependent manner (r = -0.903). Oil Red O staining showed that compared with the control group, thetotal extract of C. tinctoria Nutt. at 1, 10, 100 μg·mL-1 can obviously inhibit cell differentiation, reduce the formation of cytoplasmic lipid (P< 0.01). N-butanol extract can inhibit cell differentiation in a dose-dependent manner (r= -0.779). CB-1 and CB-2 obviously inhibited cell differentiation at the concentration of 50 μg·mL-1 (P < 0.01). It was concluded that thetotal extract, n-butanol extract, CB-1 and CB-2 of C. tinctoria Nutt. can inhibit the proliferation and differentiation of 3T3-L1 preadipocytes and reduce the formation of cytoplasmic lipid.

Objective To study the effect of Kanglaite combined with comprehensive therapy on advanced non-small cell lung cancer.Methods Sixty-one patients with advanced non-small cell lung cancer were randomly divided into treatment group ( n=31 ) and control group ( n=30 ).Both groups were given comprehensive therapy.Treatment group were additionally treated with intravenous injection of 200 ml Kanglaite.Clinical efficacy,quality of life,pain relief and adverse reactions of the two groups were observed.Results ( 1 ) Quality of life was improved in 20 cases (64.5％ ),stabled in 8 cases (25.8％),declined in 3 cases ( 9.7％ ) of treatment group,and in the control group there were 9 cases ( 30.0％ ) improved,9 cases ( 30.0％ ) stabilized,12 cases (40.0％ ) declined respectively.Quality of life in treatment group was higher than in control group ( U=2.91,P＜0.01 ).( 2 ) Pain relief:the number of patients with complete remission,partial remission,no change,and progression were 5 cases ( 16.1％ ),16 cases ( 51.6 ％ ),16 cases (51.6％ ),6 cases (19.4％) and 4 cases (12.9％ ) in treatment group,and in control group they were 2 cases(6.7％ ),9 cases (30.0％ ),11 cases(36.7％ ) and 8 cases(26.7％ ) respectively.The effect of treatment on pain relief in treatment group was better than that in control group ( U=2.32,P＜0.05 ).(3) Clinical efficacy:in the treatment group there were 12 cases (38.7％) with partial remission,14 cases (45.2％) stabilized,and 5 cases (16.1％ ) progressed,and in control group the numbers were 8 cases (26.7％ ),8 cases (26.7％ ) and 14 cases (46.7％ ) respectively.The clinical efficacy in treatment group was better than that in the control group( U=2.04,P＜0.05).(4) There were significant difference on the change of white blood cell count and gastrointestinal reactions Ⅲ and Ⅳ degrees between treatment group and contrl group [22.6％ (7/31) vs.53.3％(16/30),x2=6.139 P＜0.05;19.4％ (6/31) vs.46.7％ (14/30),x2=5.161,P＜0.05].Conclusion Kanglaite injection combined with comprehensive therapy can improve clinical efficacy of therapy for advanced non-small cell lung cancer,reduce the toxic adverse reaction,protect immunity system and improve the quality of life of patients.

Objective To observe the formation of asymmetric dimethylarginine (ADMA)and the expression of dimethylarginine dimethylaminohydrolase 2 (DDAH-2) of human umbilical vein endothelial cells (HUVECs) stimulated by uric acid (UA), and to explore the role of ADMADDAH axis in the vascular endothelial dysfunction induced by uric acid. Methods HUVECs were cultured in M199 medium supplemented with 10% FBS. Cells were exposed to different concentrations of UA (0, 60, 120 mg/L) for 6 h and 24 h. Under different concentrations and times, the level of ADMA in cell suspension was detected by high performance liquid chromatography (HPLC) technique; the gene and protein expressions of DDAH-2 were detected by RT-PCR and Western blotting; the fluorescence intensity of intracellular 2',7'-dichlorofluorescein (DCF) which represented the productions of ROS was detected by the flow cytometry (FCM). The activity of DDAH-2 in HUVCEs which were exposed to different concentrations of UA (0, 60, 120mg/L) or UA (120 mg/L) +NAC (10 mmol/L) for 24 h was estimated by directly measuring the amount of ADMA metabolized by the enzyme and the role of NAC in the activity was studied.Results The expression of ADMA induced by urid acid was dose-depent and higher at 24 h than that at 6 h in the same dosage (all P＜0.05). The dosage and stimulation time of UA did not have any influence on the expression of intracellular DDAH-2 (all P＞0.05). When HUVECs exposed to UA (120 mg/L) for 24 h, the production of intracellular ROS was significantly increased while the activity of DDAH-2 was decreasesd (all P＜0.05) as compared to 60 mg/L stimulation. This effect could be inhibited by the intervention of anti-oxidant NAC. Conclusions The high UA stimulation on HUVECs can increase the expression of intracellular ROS and inhibit the activity of DDAH-2 which increases the concentration of ADMA by decreasing the degradation of ADMA as well as the formation of NO. DDAH-ADMA axis may participate in the vascular endothelial dysfunction induced by UA.

Objective To study the expression of the tumor suppressor gene TSHR and pl6 in papillary thyroid carcinoma (PTC) and explore the relationship of the tumorigenesis and the promoter aberrant methylation of the two above genes. Methods RT-PCR was used to detect the mRNA expression of two tumor suppressor genes in 50 cases of PTC, 20 cases of nodular goiter and 12 cases of thyroid adenoma tissue. The promoter methylation status of the two genes were detected by methylation-specific PCR technique (MSP) (which of p16 by nested PCR). The promoter hypermethylation of the two genes was tested by randomly gene sequencing. Results Hypermethylation of promoter region were detected from 68.0 % (34/50) TSHR gene and 54.0 % (27/50) pl6 gene in PTC, while 21.9 % (7/32) and 15.60 % (5/32) in controls. The rate of promoter methylation in PTC was significantly higher than that in controls (χ2 = 16.61, P <0.05 vs χ2 =12.08 P <0.05). The relative mRNA expression of TSHR gene and pl6 gene were (0.41±0.11) and (0.51±0.17) in PTC, respectively, while those were (0.63 ±0.08) and (0.72 ±0.22) in controls, respectively. The mRNA expression of the TSHR gene and pl6 gene was obviously lower in PTC than that in controls (t = 3.86, P < 0.05 vs t =3.66, P <0.05). By the sequencing, it was confirmed that the CG in methylated promoter of the two genes was not changed, while the CG in unmethylated promoter was changed into TG. Conclusion Methylation of the TSHR gene and p16 gene in promoter region is a common molecule event and may be invovled in the genesis and development of human PTC.

Objective To report the clinical and peripheral neuropathological findings in two patients with autosomal recessive Charcot-Marie-Tooth disease 2K(AR-CMT2K).Methods Case one was a nine year-old girl.She had distal weakness of lower limbs for six years, with calf atrophy and contracture of Achilles tendon for three years.Case two was an eight year-old boy.He had distal weakness of lower limbs with contracture of Achilles tendon and calf muscle atrophy for three years, and proximal weakness of low limbs for two years.The motor nerve conduction velocities in median nerves were 48.1 m/s in case one and 47.6 m/s in case two.The compound motor action potential amplitude of median nerves decreased by 46% in case one and 69% in case two.Sural nerve biopsies and gene targeted next-generation sequencing were performed in both patients.Results Density of myelinated fibers was 8 407/mm2 in case one and 7 714/mm2 in case two.The ratio of myelinated fibers with diameter over 8 μm was 2.6% in case one and 0 in case two.Both patients had small regenerating cluster of myelinated fibers.Thin myelinated fibers appeared in case one.In case two, atypical onion bulb formations with focal folded myelin appeared, and electromicroscopy revealed mitochondrial aggregate in axons.Compound heterozygous mutations of ganglioside-induced differentiation associated protein 1 gene were detected in both patients, including c.767A>G(p.H256R) and c.466G>A (p.A156T) in case one and c.767A>G and 845G>A(p.R282H) in case two.Conclusions Contracture of Achilles tendon may appear in early childhood of AR-CMT2K patients.The main pathological changes in sural nerve are loss of large myelinated fibers, mitochondrial aggregate in axons and myelin abnormalities.

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology

Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.

Objective To understand the virology test characteristics of hepatitis B virus (HBV) for discuss the relation of HBV genotype and HBeAg, anti-HBc-IgM, HBV DNA and disease progression. Methods Two hundred cases of hepatitis B were detected by the ELJSA assay with two pairs of semi-markers (HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc) and anti-HBc-IgM, using fluorescence quantitative polymerase chain reaction (FQ-PCR) for detecting HBV DNA, using monoclonal antibody ELISA method (mAbs ELISA) for HBV genotyping and analysis of test results. Results In 200 patients with hepatitis B, the HBV genotype detected in 179 cases (89.5%), B-type 121 cases(60.5%), C-type 58 cases (29.0% ). There had no relationship with HBeAg, anti-HBc-IgM, HBV DNA and genotype. B-type HBV prevalent in asymptomatic carriers (ASC) and chronic hepatitis B (mild);C-type common in patients with liver cirrhosis (LC) and chronic hepatitis B (severe). Conclusions HBV genotype in Shenzhen mainly is B-type, C-type second;mAbs ELISA assay with HBV genotype is specific, sensitive, simple and practical features, HBV replication strength has nothing to do with the virus genotype. HBV genotype and HBeAg, anti-HBc-IgM, HBV DNA testing may complement each other, with the clinical application value.

Objective To study the expressions of the tumor suppressor gene TSH receptor( TSHR),P16, and RAS in papillary thyroid carcinoma ( PTC ) , and the correlation between the occurrence of tumor and the aberrant promoter hypermethylation of three tumor suppressor genes. Methods RT-PCR was used to detect the mRNA expression of three tumor suppressor genes in tissues of 50 cases of PTC ,20 cases of nodular goiter,and 12 cases of thyroid adenoma. The promoter methylation status of three tumor suppressor genes was examined by methylation-specific PCR technique( MSP). Gene sequencing was used to test if the hypermethylation existed in the promoter of three tumor suppressor genes. Results In 68.0% (34/50) TSHR gene, 54.0% (27/50) P16 gene, and 60.0% ( 30/50 ) RAS gene in PTCs, hypermethylation in promoter region was detected, the respective results 21.9% (7/32) , 15. 6% (5/32) ,and 31. 3% (10/32) were found in control tissues. The rates of the three genes with promoter hypermethylation in PTC were significantly higher than those in control tissues ( all P<0. 05). The mRNA expressions of TSHR,P16,and RAS were significantly lower in PTC than those in control tissues (0. 41 ± 0.11 vs 0.63±0. 08,0. 51±0. 17 vs 0. 72±0. 22,0. 56±0. 10 vs 0. 67±0. 16, all P<0. 05). The sequencing confirmed that there was CC to TC transmission in the promoters of three tumor suppressor genes. Conclusions The methylation of three tumor suppressor genes in promoter region is a common molecule event and may be involved in the genesis and development of human PTC.