1.Relationship between HBV genotypes/sub -genotypes of hepatitis B virus and antiviral therapy
Yanfang HU ; Miao WANG ; Qinying WANG
Chinese Journal of Primary Medicine and Pharmacy 2015;(13):1941-1943
Objective To investigate the relationship between the genotype /sub -genotype and antiviral treatment.Methods The clinical data of 159 patients with chronic HBV infections were collected,who were treated by adefovir dipivoxil (10mg/d)or entecavir(0.5mg/d)according to the disease′s conditions.The genotypes and subtypes of hepatitis B virus were determined by Nested polymerase chain reaction(nt PCR).The levels of serum HBVDNA replication,ALT levels and HBV serologic markers were detected pre or post -treatment 24 weeks, 48 weeks.Observed the relationship between the HBV genotypes/sub -genotypes and the antiviral efficacy of adefovir dipivoxil or entecavir treatment.Results After 24 weeks of adefovir dipivoxil therapy,ALT normalization rate of subgenotype Ba and subgenotype C2 was 66.7% vs 66.2%(χ2 =0.74,P >0.05),HBeAg negative conversion rate of two subgenotypes was 27.3% vs 23.1%(χ2 =0.10,P >0.05),HBVDNA negative conversion rate of two sub genotype Ba and subgenotype C2 was 33.3% vs 30.9%(χ2 =0.03,P >0.05),respectively.After treatment for 48 weeks,ALT normalization rates of subgenotype Ba and subgenotype C2 were 83.3% vs 82.4%(χ2 =0.01,P >0.05),HBeAg negative conversion rates of two subgenotypes were 45.5% vs 34.4%(χ2 =0.49,P >0.05 ),HBVDNA negative conversion rates of two subgenotypes were 58.3% vs 48.5%(χ2 =0.39,P >0.05).After 24 weeks of entecavir therapy, ALT normalization rates of subgenotype Ba and subgenotype C2 were 71.4% vs 69.6%(χ2 =0.02,P >0.05 ), HBeAg negative conversion rates of two subgenotypes were 33.3% vs 30.8%(χ2 =0.03,P >0.05 ),HBVDNA negative conversion rates of two subgenotypes were 42.9% vs 39.3%(χ2 =0.06,P >0.05 ),respectively.After treatment for 48 weeks,ALT normali zation rates of subgenotype Ba and subgenotype C2 were 85.7% vs 83.9%(χ2 =0.03,P >0.05),HBeAg negative conversion rates of two subgenotypes were 50.0% vs 44.9%(χ2 =0.10, P >0.05),HBVDNA negative conversion rates of two subgenotypes were 71.4%vs 67.8%(χ2 =0.07,P >0.05). Conclusion The study showed that genotype C(C2)is a predominant HBV genotype among people with chronic HBV infections in Shanxi province.HBV subgenotypes Ba and C2 have no significant difference in virologic,biochemi-cal,and immunologic response to ADV or ETV.
2.Construction of eukaryotic expression vector of hepatitis C virus NS5A gene
Xiufen SU ; Junqi NIU ; Yanfang JIANG ; Yulin HU
Journal of Jilin University(Medicine Edition) 2006;0(01):-
Objective To construct an eukaryotic expression vector of the hepatitis C virus(HCV)NS5A gene.and obtain a stable transfected Huh-7 cell line which provides a basis for further investigation of the hepatitis virus C NS5A protein.Methods The HCV NS5A gene from the pcDNA3.1(+)/HCV NS345 plasmid with HCV NS5A gene was amplified by PCR,and cloned to pGEM-T vector,and transformed into E.coli JM109.The positive colonies were first confirmed by restriction enzyme digestion and sequencing and then were inserted to eukaryotic expression vector pCI-neo,and verified by enzyme digestion and sequencing.Results After TA colon of the HCV NS5A was amplified by PCR,the positive colonies were finally verified by sequencing,which were totally in line with the designed coding sequence of HCV NS5A gene.Conclusion Eukaryotic expression vector of HCV NS5A gene has been successfully constructed.
3.Pharmacokinetics of dipfluzine hydrochloride in Beagle dogs after single intravenous dose
Huiqing HU ; Yongli WANG ; Jingwei SHENG ; Haiyan WANG ; Yanfang XU
Chinese Pharmacological Bulletin 2003;0(08):-
Aim To investigate the pharmacokinetics of dipfluzine hydrochloride,a novel piperazines calcium antagonist.Methods Eighteen Beagle dogs were randomly divided into three groups,which were administered with dipfluzine hydrochloride at iv single dose of 1.5,3.0 and 6.0 mg?kg-1,respectively.The blood was collected at different time.A RP-HPLC method was developed to determine the concentration of dipfluzine hydrochloride in plasma.The pharmacokinetic parameters were calculated by 3P97 software.Results The specificity,lowest limit of detection and quantification,extraction recoveries,the precision of intra-and inter-day and stability were qualified to the pharmacokinetic study.The concentration-time courses of dipfluzine hydrochloride were best fitted to a two-compartment open model at three doses.The main pharmacokinetic parameters at three doses were 24.7,24.2 and 29.6 h for T12?,0.44,1.12 and 2.86 g?min?L-1 for AUC,1.30,1.22 and 1.28 L?kg-1 for Vc,and 3.4?10-3,2.7?10-3 and 2.1?10-3 L?kg-1?min-1 for CL,respectively.Conclusions The developed RP-HPLC method for determination of dipfluzine hydrochloride in plasma can satisfy the requirement of pharmacokinetic study after iv dipfluzine hydrochloride.Analysis of plasma concentration-time curves indicates a biphasic decrease.There was a linear relationship between AUC and dosage.
4.The dynamic expression of Nrf2 and HO-1 in spinal cord tissues of experimental autoimmune encephalomyelitis of rats and the mechanism underlying neuroprotective effect of edaravone
Qiaolian ZHANG ; Li GUO ; Yanfang HU ; Zhen JIA ; Li LI
Chinese Journal of Immunology 1985;0(06):-
Objective:To study the effect of edaravone on EAE rats and the underlying mechanism.Methods:Wistar rats were immunized with GPSCH,and randomly divided into control group,EAE group,dexamethasone group,low dose of edaravone group and high dose of edaravone group.The morbidity of disease and clinical signs were observed.The pathological changes of spinal cord tissue sections were observed under light microscopy after HE staining and trichrome staining.The expression of Nrf2 and HO-1 was observed by immunohistochemistry.Results:Morbidity of high dose of edaravone group (8.33%) and DXM group (0%) was significantly lower than in EAE group (58.3%) (P
5.Study of the sex differences in experimental autoimmune encephalomyelitis of Wistar rat
Qiaolian ZHANG ; Yanfang HU ; Li GUO ; Ruichun LIU
Chinese Journal of Immunology 2001;0(07):-
Objective:EAE model of male and female Wistar rats was established respectively.The sex difference was evaluated.Methods:40 wistar rats were devided into male and female group (n=20).EAE model was established respectively in the two groups using Guinea pig spinal cord homogenate without PBS perfusion.30 days later,the spinal cord were taken and embedded in wax,and paraffin sections of the two groups were examined by histopathology.The differences in onset time,incidence rate,course of disease and neurologic score between the two groups were evaluated.Results:The onset time of female group was 13.67?3.50 days,its incidence rate was 60%.The onset time of male Wistar rat was 12.18?1.55 days,and its incidence rate was 85%.The feature of diseases course in female rat was relapse after recovery,but that in male rat was transient.The average clinical score of female group was 2.20?1.96,and that of male group was 3.46?1.61.Conclusion:Compared with female Wistar rat,the male had higher incidence rate and its diseases course was transient.The results of our study provided a useful foundation for further studies on the pathogenesis and the sex differences of multiple sclerosis.
6.Lenalidomide enhances anti-tumor effects of CIK cells against lymphoma
Kai HU ; Yanfang WANG ; Jing WANG ; Xiaoyan KE
Journal of Leukemia & Lymphoma 2012;21(5):257-260
ObjectiveTo investigate lenalidomide modulates anti-tumor effects of cytokine induced killer(CIK) cells against lymphoma cells in vitro.MethodsCIK cells were generated in vitro by stimulation of health donors' peripheral blood mononuclear cells subsets with interferon-gamma(INF-γ),IL-2 and anti-CD3 monoclonal antibody. On the different culture time CIK cells were treated with different concentrations of lenalidomide.CD3+CD56+ cell ratio was detected by flow cytometry and ELISA method was used for the detection of GM-CSF, INF-γ and TNF-α level of secretion. Inhibitory effects of CIK cells and CIK cells treated by lenalidomide on the three types of lymphoma cell lines were detected by cell counting kit-8(CCK-8).Results Amount of CD3+CD56+ cells were increased without inhibition by lenalidomide at 0.2-5.0 μmol/L(P>0.05).Lenalidomide led to significantly higher levels of GM-CSF,INF-γ and TNF-α secreted by CIK cells (P<0.05).On different culture time CIK cells treated by 1.0 μmol/L lenalidomide had higher killing effects on Z-138 [(42.53±2.19) % vs(15.5±3.82) %],Jurkat[(44.78±4.86) % vs(29.94±6.33) %],RPMI 8226 cells [(54.71±5.31) % vs(37.43±9.75) %]than that without lenalidomide treating(P<0.05).ConclusionLenalidomide can enhance killing effect of CIK cells on lymphoma cell lines in vitro.
7.Reconstruction of anti-hepatocellular carcinoma scFv pharmacy and its killing effect on hepatocardnoma cell in vitro
Guihong ZHANG ; Yanfang LIU ; Yong FU ; Haiyang HU ; Dawei CHEN
Cancer Research and Clinic 2008;20(12):802-804
objective To observe whether the killing effect on HCC SMMC-7721 cell of the antihepatocellular carcinoma scFv reconstructed by pharmacy was enhanced or not.Methods Prokarycytic expression vector containing PET32a-RC-RNase was induced to express by IPTG.The inclusion body purified and Western-blotting was used.PC.CHOL and CHS was added in chloroform.Dry membrane was formed after chloroform was removed.RC-RNase protein solution was added to dissolute the membrane.Then pass the solution over a Sephadex G-50 column after ultrasound and filtrated to detect the encapsulation efficiency of the liposome.The solution reacted in EDC.SSNHS and MES for 30 minutes.Then add hdscFv to the solution in 4 ℃ over night.MTT method was used to detect the killing effect on HCC cell of immunoliposome RC-RNase,immunotoxin RC-RNase and liposome RC-RNase in vitro.Resuits The killing effect on HCC cell of immunoliposome RC-RNase is the best.but that of Iiposome RC-RNase is the worst.The respective JC50 are:3.28μg/ml,22.44μg/ml and 98.26μg/ml.Conclusion The anti-hepatocellular carcinoma scFv relomtructed by pharmacy can promote the killing effect on HCC cell and may have potential in the treatment of hepatocarcinoma.
8.Structure Elucidation of Product of β-1,3-Glucosyltransferase Encoded by wfgD Gene in Escherichia Coli O152 Using Mass Spectrometry
Dawei ZHOU ; Bo HU ; Bin LIU ; Junli WU ; Yanfang HAN
Chinese Journal of Analytical Chemistry 2010;38(2):225-228
The O152 antigens of Escherichia coli contains a Glc-β-1,3-GlcNAc linkage within the repeating unit. The wfgD gene in E. coli O152 O antigen gene cluster had been demonstrated utilizing NMR technique to encode a glucosyltransferase which is responsible for the synthesis of Glc-β-1,3-GlcNAc linkage. In this study, a synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO_3-PO_3-(CH_2)_(11))-O-phenyl]) was used as an acceptor and UDP-Glc as a donor substrate, and electrospray ionization tandem mass spectrometry(ESI-MS-MS) was used for the detailed structural characte-)rization) of the enzyme product. A systematic study was conducted on product to allow rationalization of the fragmentation) processes. The major fragments observed in the ESI-MS-MS spectra result from cleavage of glycosidic) bond and diphosphate moiety. The fragment originating from the nonreducing end of the product yields information on sequence). Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting) the product, and "internal" cleavage ions which are derived from elimination of substituents from around) the pyranose ring, were also observed. This extensive fragmentation was shown that the expected Glc-β-1,3-GlcNAc linkage in the product, confirming that wfgD is in the form of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferase.)
9.Changes of plasma levels of sTRAIL and its DR4, DR5 in hypertension patients with left ventricular hypertrophy
Yan HONG ; Yanfang XIE ; Yanting WAN ; Lina WAN ; Jianhua HU
Chinese Journal of cardiovascular Rehabilitation Medicine 2013;22(2):105-108
Objective: To determine plasma levels of soluble tumor necrosis factor-related apoptosis inducing ligand (sTRAIL) and its soluble death receptor (sDR4, sDR5) in essential hypertension (EH) patients with left ventricular hypertrophy (LVH). Methods: Enzyme linked immunosorbent assay (ELISA) was used to measure plasma levels of sTRAIL, sDR4 and sDR5 in 50 EH + LVH patients (EH + LVH group), 50 EH patients without LVH (EH group) and 50 healthy subjects (healthy control group), and the results were compared and analyzed among three groups. Results: ① Compared with healthy control group and EH group, there were significant increase in plasma levels of sTRAIL [(0.95±0.11) ng/ml vs. (1.12±0.86) ng/ml vs. (1.74±1.19) ng/ml], sDR4[(2.38±0.32) pg/ml vs. (5.63±1.05) pg/ml vs. (8.72±1.14) pg/ml] and sDR5[(< 6 pg/ml) vs. (39.19±8.23) pg/ml vs. (78.21±11.2) pg/ml] in EH + LVH group, P<0.01 all; and levels of sDR4 and sDR5 in EH group were significantly higher than those of healthy control group (P<0.01 both), but there was no significant difference in sTRAIL level between the two groups (P>0.05); ② Pearson correlation analysis indicated that there were significant positive correlation among levels of sTRAIL, sDR4 and sDR5 in EH + LVH patients (r=0.325~0.410, P<0.05 or <0.01). Conclusion: Plasma levels of sTRAIL, sDR4 and sDR5 may be valuable indexes for prediction of left ventricular hypertrophy in patients with hypertension.
10.Oxided low density lipoproteins induce apoptosis with enhanced expression of Fas antigen in vascular endothelial cells
Ziwang HU ; Zhihua QUAN ; Yanfang CHEN ; Suizhan WANG ; Shuguang LIN
Chinese Journal of Pathophysiology 1989;0(05):-
AIM: To explore the effect of oxides of low density lipoprotein (OX - LDL)on apoptosis in vas- cular endothelial cells(EC) and its possible mechanisms. METHODS: Culted vascular ECS were incubated with OX - LDL(0.1?g/L or 0. 3?g/L) for 4 hours, flow cytometer with PI staining methed was used to determine the rate of cellular apoptosis, gel - electrophoresis was performed to observe foe DNA ladder of apoptosis, cellular RNA was isolated and reverse transcription-polymerase chain reaction (RT - PCR)was performed to determine Fas antigen ex- pression. RESULTS:OX - LDL treatment induced apoptosis in EC dose - dependently. No DNA ladder was ob- served in control EC. But it presented in EC beated with low dose of OX - LDL, and was even more when beted with higher dose of OX - LDL. RT - PCR revealed Fas antigen baldly had any expression in basic condition, OX - LDL treatment induced Fas antigen in EC in a dose dependent manner. CONCLUSION: OX - LDL can induce apoptosis in EC, upregulaton of Fas antigen expression maybe one of the possible mechanisms.