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Objective:To establish a HPLC method for determinating 9 components simultaneously in Swertia chirayita. Methods:By useing water Sunfire C18 column (4.6 mm× 250 mm,5 μm); Gradient elution was carried out with methanol-0.05% phosphoric acid solution as mobile phase. Setting the column temperature at 30 ℃, the flow rate at 1.0 ml/min, and the detection wavelength at 254 nm.Results:9 components showed good linear relationship within the injection quality range. The recovery rates of wertiamarin, Gentiopicroside, Angelica glycosides,Mangiferin, Isolysine, Gentianoside, Diol glycoside, 8-hydroxy-1,3,5 trimethoxyketone, and Daisy leaf gentinone were 95.38%, 92.41%, 95.14%, 91.87%, 92.24%, 92.51%, 95.08%, 91.72%, 95.74% ( n=6). Conclusion:The method is simple, efficient, sensitive, accurate, economical and practical, with repeatability and stability. It could provide reference for the quality control and comprehensive utilization of Swertia chirayita.
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Mixed lineage kinase domain-like protein (MLKL) is a pseudokinase with a kinase domain, which plays an important role in the regulation of necroptosis. After cerebral ischemia, MLKL, as the substrate protein of the receptor-interacting protein 3, undergoes oligomerization and phosphorylation, and then translocates from the cytoplasm to the plasmalemma, causing mitochondrial division and cell membrane rupture. MLKL can also mediate the inflammatory response after cerebral ischemia by inducing necroptosis and directly activating inflammasomes, thereby aggravating brain injury. Therefore, to clarify the biological characteristics of MLKL and its role and mechanism in cerebral ischemia is very important for the treatment of cerebral ischemia.
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Objective@#To establish a critical segmental bone defect model in a rabbit ulna.@*Methods@#Twenty male New Zealand white rabbits were randomly assigned into 5 groups according to the digital table, and then 14, 15, 16, 17 and 18mm segmental defects(contained periosteum) were created in the middle part of ulna on both sides, respectively.At 6 and 12 weeks after surgery, the repair of segmental bone defects was evaluated by naked eyes, X-ray and histological examination.@*Results@#At 12 weeks after surgery, all of the segmental defects with the length of 14 and 15mm were completely repaired.But none of the defects with the length of 16, 17 and 18mm was repaired.@*Conclusion@#The length of critical segmental bone defect in rabbit ulna was 16 mm.
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Objective To establish a critical segmental bone defect model in a rabbit ulna.Methods Twenty male New Zealand white rabbits were randomly assigned into 5 groups according to the digital table,and then 14,15,16,17 and 18mm segmental defects (contained periosteum) were created in the middle part of ulna on both sides,respectively.At 6 and 12 weeks after surgery,the repair of segmental bone defects was evaluated by na ked eyes,X-ray and histological examination.Results At 12 weeks after surgery,all of the segmental defects with the length of 14 and 15mm were completely repaired.But none of the defects with the length of 16,17 and 18mm was repaired.Conclusion The length of critical segmental bone defect in rabbit ulna was 16 mm.
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Deep venous thrombosis is one of the common complications after major surgery in the Department of Orthopedics. The selective knee replacement of the lower extremities is more likely to cause the occurrence of DVT. The most commonly used anticoagulants in the Department of Orthopedics now include low molecular weight heparin [LMWH], Rivaroxaban, ordinary heparin, aspirin and warfarin. At present, the clinical application of low molecular weight heparin is the most, and the effect is the most accurate. This study compared the efficacy and safety of three commonly used anticoagulants such as aspirin, LMWH and Rivaroxaban in preventing VTE after hip and knee arthroplasty, so as to provide a theoretical basis for selecting suitable anticoagulant drugs in clinic. It has been proved that LMWH has good efficacy and safety in the prevention of VTE after hip and knee arthroplasty and is a priority anticoagulant. Rivaroxaban can effectively control the occurrence of DVT and the drug is convenient, but it will increase the risk of bleeding and should be used carefully
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Objective:To investigate the high risk factors of pelvic lymph node metastasis in patients with type Ⅰ endometrial carcinoma in order to provide the basis for making reasonable operation scope.Methods:Risk factors of pelvic lymph node metastasis were analyzed in 136 cases of type Ⅰ endometrial carcinoma.Univariate analysis was performed with Chi square test or Fisher's exact probability method,and multivariate analysis was performed with a logistic regression mode.Results:The positive rate of pelvic lymph nodes in 136 patients with type Ⅰ endometrial carcinoma was 9.56％ (13/136).Univariate analysis showed that histological grade,size of lesion,depth of myometrial invasion and vascular invasion were related to lymph node metastasis(P ＜0.05);Multivariate Logistic analysis showed that low differentiation,deep muscular invasion,tumor diameter≥2 cm and LVSI were independent risk factors of pelvic lymph node metastasis in patients with type Ⅰ endometrial carcinoma(P ＜0.05).Conclusions:The rate of pelvic lymph node metastasis is low in type Ⅰ endometrial carcinoma.Patients with low differentiation,deep muscular invasion,tumor diameter≥2 cm and LVSI are more likely to occur pelvic lymph node metastasis in type Ⅰ endometrial carcinoma.
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Objective To detect the different expressions of Drosha between endometrial cancer (EC) tissue and other tissues and to explore correlation between Drosha mRNA transcription level and protein expression level with clinicopatho?logical characteristics of EC. Methods The mRNA transcription and protein expression levels of Drosha were examinaned by q-PCR and Western blot respectively in normal endometrial tissues (25 cases), atypical hyperplasia of endometrial tis?sues (20 cases) and endometrial cancer tissues (40 cases). Correlations between Drosha mRNA transcription and protein ex?pression with clinicopathological characteristics of EC were analyzed. Results The levels of Drosha mRNA and protein lev?els in EC were obviously lower than those in endometrial atypical hyperplasia and normal endometrium (P0.05). The protein expression levels of Drosha were consistent with transcription of mRNA transcription levels. Drosha mRNA expression does not differ significantly with differentiation, histological type, myometrial invasion, lymphatic metasta?sis and FIGO stages of EC (P>0.05). Conclusion The expression levels of Drosha in EC tissues were down-regulated, therefore the reduction of Drosha may contributed to tumorigenesis of EC.
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Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.
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Objective To explore the influence factors on hepatitis B virus (HBV) relapse after nucleos(t)ide analogues (NA) withdrawal in the chronic hepatitis B (CHB) patients who met NA cessation criteria. Methods Eighty-one consecutive CHB patients were treated with NA, 38 with lamivudine (LAM), 25 with adefovir dipivoxil (ADV), 12 with entecavir (ETV), 6 with LAM +ADV. Among recruited patients, 40 were hepatitis B virus e antigen (HBeAg) positive, 41 were HBeAg negative, 67 of them were initial treatment, 14 were retreatment due to resistance to NA at baseline. The treatment was discontinued after meeting China therapeutic end-point criteria. HBV DNA, HBV serological markers, alanine aminotransferase (ALT) were measured respectively at baseline, every month before virological response, every 3 months after virological response, every month within first 6 months and every 2 months over 6 months after drugs withdrawal. Twelve probable influence factors on relapse which were sex, age, HBV family history, baseline HBV DNA,baseline HBeAg status, baseline ALT, virological response time, total duration of treatment, duration of additional treatment, the level of hepatitis B virus surface antigen (HBsAg) at cessation therapy,initial treatment or retreatment, drug category were analyzed with univariate, multivariate Cox regression modle and stratified analysis. The cumulative relapse was calculated by the Kaplan-Meier method. Results A total of 36 patients (44. 4%) relapsed within 1 year. Initial treatment or retreatment, HBV family history, virological response time, the level of HBsAg at cessation therapy were independent risk factors. The relapse rate of retreatment was higher than that of initial treatment (78.6% vs 37. 3% , χ2 = 7. 983, P = 0. 005) , those of patients with HBV family history higher than without family history (64. 5% vs 15.0%, χ2 =12. 096,P = 0.002), those of patients obtained virological response within 3 months lower than after 3 months(34. 0% vs 64. 3% , χ2 =6. 823,P=0. 009) , those of patients with HBsAg≤150 μg/L at cessation therapy lower than >150 μg/L(27. 6% vs 53. 8%, χ2=5. 199,P=0. 023). Conclusions Retreatment, HBV family history, later virological response and higher HBsAg level at cessation therapy are risk factors of relapse after NA withdrawal. Such patients should be treated with prolonged duration after meeting end-point criteria to strengthen the efficacy.
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Objective To determine whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) could be detected in serum of patients with hepatitis B and evaluate the related factors and clinical significances. Methods Fifty-seven patients, including 26 with mild chronic hepatitis B (CHB) and 31 with severe hepatitis B (SHB) were enrolled. Prothrombin time (PT), hepatic biochemical indexes, serum markers of hepatitis virus, serum total HBV DNA and HBV cccDNA of every patient were detected after hospitalization. Factors associated with the detection rate of serum HBV cccDNA were analyzed using Logistic stepwise regression. Results Serum HBV cccDNA was detected in 13 patients with SHB and 1 with mild CHB, and serum levels of HBV cccDNA were varying from 1.25 × 103 to 4. 88 × 104 copy/mL. The detection rates were significantly different between the two groups (P=0. 0014). The sensitivity and specificity of SHB diagnose by serum HBV cccDNA detection were 41.94% and 96.15 %, respectively. Logistic regression analysis showed that the detection rate of serum HBV cccDNA was associated with PT (X2 = 7. 2192, P= 0. 0072), while not associated with age, sex, total serum HBV DNA, total bilirubin or alanine aminotransferase (ALT). Conclusion Serum HBV cccDNA could be detected in some of the patients with SHB, whic hmay be considered as one of the diagnostic indexes for SHB,
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Ohjective To observe the inhibition effect of total glucosides of Picrorhiza on hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG 2.2.15 cell line. Methods HepG 2.2.15 cells were incubated with culture medium containing 50 mg/L of picrosides or 5 mg/L of adefovir dipivoxil for 2 or 5 days. HBV DNA in the supernatant, intracellular cccDNA, relaxed circular DNA (rcDNA) and pregenomic RNA (pgRNA) were quantified by specific real-time polymerase chain reaction (RT-PCR) and inhibition rates were calculated. The means were compared by t test. Results After treated with picrosides for 2 and 5 days, the inhibition rates of HBV DNA in thesupernatant were 49. 74% (t=4.723, P<0.05) and 79.48% (t = 7.512, P<0.05), respectively. The inhibition rates of intracellular cccDNA were 43.55% (t = 5.216, P<0.05) and 56.43% (t=7.262, P<0.05), respectively, while those of intracellular rcDNA were 43.39% (t=4.137, P<0.05) and 63.86% (t=7.861, P<0.05), respectively, and those of intracellular pgRNA were 54.72% (t=4.532, P<0.05) and 56.08% (t=4.833, P<0.05), respectively. Comparatively, after treatment with adefovir dipivoxil for 2 and 5 days, the inhibition rates of HBV DNA in the supernatant were 25.56% (t=2.874, P<0.05) and 92.44% (t =10.276, P<0.05), respectively. Those of cccDNA were 18.54% (t=2.736, P<0.05) and 47.19% (t=6.852, P<0.05), respectively. Those of rcDNA were 21. 20% (t=3.206, P<0.05) and 71.47% (t=8.332, P<0.05), respectively, pgRNA were 11.14% (t=1.761, P>0.05) and 37.61%(t=3.632, P<0.05) respectively in HepG2.2.15 cells. Conclusions Pierosides may inhibit the replication cycle of HBV, including the formation of cccDNA in HepG 2.2.15 cells. The mechanism of pierosides on cccDNA may differ from adefovir dipivoxil's due to its earlier inhibition time phase.
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Objective To determine the effect of oxymatrine on the activity of HBV DNA polymerase in vitro. Methods Hepatitis B virus particles were purified from supernatant of cultured HepG2.2.15 cells by uhracentrifugation, and then were mixed with reaction buffer containing NP-40, β-mercaptoethanol, 32P-labelled nucleoside triphosphate (dCTP), MgCl2, and different concentrations of oxymatrine ( 1000 μg/ml, 800 μg/ml, 600 μg/ml, 400 μg/ml and 200 μg/ml) or adefovir dipivoxil ( 100 μg/ml, 80 μg/ml and 60 μg/ml, 40 μg/ml and 20 μg/ml). After incubation at 37 ℃ overnight, proteinase K was added to the reaction system for digestion and 35 μl of samples were spotted onto DE81 paper. Activities of endogenous polymerase in HBV particles were assessed by determining the radioactivity of 32P-labelled dCTP incorporated in the plus-strain of viral DNA. Results Compared with the blank control, the activity of endogenous polymerase in HBV particles treated with different doses of oxymatrine varied from 103% to 107%, and it varied from 91% to 101% when treated with different doses of adefovir dipivoxil. No significant difference was observed among treated groups and the control (P > 0. 05 ). Conclusion No direct inhibitory effect of oxymatrine on the activity of HBV polymerase was observed in vitro.
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In the course of emerging infectious disease learning,comprehensive methods including comparing the similarity of emerging infectious disease and classical infectious disease,uniting the general introduction and the typical examples explanation,strengthening the multimedia teaching and the case based teaching were adopted to strengthen the effect of teaching.
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Communion is an important way to study.Interaction is the most notable character of network.This paper introduces some experience on how to bring net interaction into play in the network-based medical teaching.
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To reduce the risk of 3′-terminal mismatch between primers and template and increase the sensitivity of polymerase chain reaction (PCR) in the detection of variable region of DNA. Methods: A pair of special primer(WU,WD) was designed to amplify a fragment of HBV DNA P gene by PCR. Other 2 similar pairs of primer (MU1, MD1, MU2, MD2) were obtained by knocking off 1 or 2 bases at the 3′-terminal of WU and WD. (1) Special primers (WU, WD) and degeneracy primers(WU, WD, MU1, MU2, MD1, MD2) were used to amplify 27 samples respectively by PCR under the same condition. The sensitivity of each PCR was compared. (2) Using degeneracy primers, serum HBV DNA was amplified from 4 patients who were resistant to lamivudine. The PCR products were sequenced to evaluate the effect of the 3′-terminal mismatch of primers upon PCR. Results: (1) The sensitivity of special primers and degeneracy primers were 70.4%(19/27) and 85.2%(23/27) respectively (P<0.05). (2) The sequencing analysis of the PCR products suggested that the 3′-terminal mismatch of primers caused false negative in the PCR detection. Conclusion: When amplifying the variable region of DNA, the false negative result can be avoided by using 3′-terminus shifted degeneracy primers.
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Objective To establish a method for quantitative detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA ) in infected cells. Methods The transfected cell line HepG2.2.15 which can consistently produce Dane particles was maintained in DMEM containing 380 ?g/ml G418 and 10% fetal bovine serum. Cells in the exponential period were harvested from flasks, then intracellular HBV cccDNA was extracted from pellet containing 1?10~6 cells with mini plasmid extraction kit (QIAGEN).The extraction product was further purified by mung bean nuclease to remove HBV relaxed circular DNA possibly remained. HBV cccDNA was quantitatively detected by fluorescent PCR with selective primer set and Taqman MGB probe. Culture medium before exponential period, HBV DNA positive and negative serum samples from patients with chronic hepatitis B (mild) were amplified simultaneously to test the specificity of the fluorescent PCR method. Plasmids containing whole HBV genome were amplified with the same primer set and fluorescent probe to determine the sensitivity of the method. Results HBV cccDNA was detectable in HepG2.2.15, and the average quantity was 18 copies per cell approximately. No detectable fluorescent signal was observed when culture and serum samples were amplified. The detectable HBV cccDNA was as low as 10~3 copies per ml at least by this method. Conclusions This method is convenient, highly specific and highly sensitive. It can be utilized in the quantitative detection of intracellular HBV cccDNA as well as in the screening and evaluation of antiviral agents.
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Objective:To evaluate the value of CK19 mRNA detection in diagnosis of breast cancer. Methods: Patients were categorized into 3 groups:Group 1 consisting of 77 stageⅠ/Ⅱbreast cancer patients, group 2 consisting of 77 stage Ⅲ/Ⅳ breast cancer patients, and control group consisting of 40 non-cancer volunteers. Total RNA was extracted from peripheral blood cells with the RNA Kit as described by the manufacturer, which was followed by one step RT-PCR of RNA. Agarose gel electrophoresis and sequencing were carried out to confirm the amplification of expected sequence (314 bp). ?~2 test was used to compare CK19 mRNA positive rates in different stages of breast cancer. Results: 15/77 (19.48%) of stage Ⅰ/Ⅱ breast cancers patients, 31/77 (40.26%) of stage Ⅲ/Ⅳ breast cancers patients and 2/40 (5.00%) of non-cancer volunteers demonstrated positive RT-PCR results. The positive rate of CK19 mRNA in peripheral blood of stage Ⅰ/Ⅱ or stage Ⅲ/Ⅳ breast cancer patients was significantly higher than that of non-cancer volunteers(P=0.037, P=0.001). The positive rate of CK19 mRNA in peripheral blood of stage Ⅲ/Ⅳbreast cancer patients was significantly higher than that of stageⅠ/Ⅱbreast cancer patients(P=0.005). Conclusion: CK19 RT-PCR is a promising method for detecting circulating tumor cells in patients with breast cancer, which may provide valuable information for tumor staging and treatment.
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Objective: To observe the effect of oxymatrine (OM) on the expression of HBV DNA in HepG2.2.15 cells and to investigate the anti-HBV mechanism of OM. Methods: The level of HBV DNA in HepG2.2.15 cells incubated with different concentrations of OM was quantified by equivalent competitive PCR combining with DNA hybridization quantitative detection technique (PCR-ELISA). The in vitro anti-viral effect of OM was evaluated by calculating the inhibiting rate. Results: OM inhibited the expression of HBV in HepG2.2.15 cells. The inhibiting rate increased with the drug concentration. The stable concentration of OM in medium was important in keeping the inhibiting rate. Conclusion: OM can inhibit the synthesis of HBV directly at the level of HBV DNA replication.
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Objective: To reduce the risk of 3'-terminal mismatch between primers and template and increase the sensitivity of polymerase chain reaction (PCR) in the detection of variable region of DNA. Methods: A pair of special primer(WU, WD) was designed to amplify a fragment of HBV DNA P gene by PCR, Other 2 similar pairs of primer (MU1, MD1, MU2, MD2) were obtained by knocking off 1 or 2 bases at the 3'-terminal of WU and WD. (1) Special primers (WU, WD) and degeneracy primers(WU, WD, MU1, MU2. MD1, MD2) were used to amplify 27 samples respectively by PCR under the same condition. The sensitivity of each PCR was compared. (2) Using degeneracy primers, serum HBV DNA was amplified from 4 patients who were resistant to lamivudine. The PCR products were sequenced to evaluate the effect of the 3'-terminal mismatch of primers upon PCR. Results: (1) The sensitivity of special primers and degeneracy primers were 70. 4%(19/27) and 85. 2% (23/27) respectively (P