Acupuncture Therapy ; methods ; Adult ; Aged ; Female ; Humans ; Intervertebral Disc Displacement ; therapy ; Male ; Middle Aged ; Young Adult

Objective To establish a novel and convenient method to study the phenotype of drug resistant hepatitis B virus (HBV) isolates,and to analyze the drug susceptibility by replacing the reverse transcriptase (RT) domain of wild-type HBV with that of the drug resistant HBV isolates.Methods Full length of HBV isolates was amplified and cloned from the sera of patients prior to nucleoside/nucleotide analogues (NA) treatment.Wild-type full-length HBV genomes was used to construct the recombinant expression plasmids PHY536207 (genotype B) and PHY97 (genotype C).The restriction enzyme sites were introduced in the upstream and downstream region of reverse transeription (RT) domain to construct plasmid,which were named as mPHY536207 and mPHY97,respectively.Lamivudine (LAM) resistant mutant and adefovir (ADV) resistant mutant were isolated and cloned to construct recombinant expression plasmids PHY634 and PHY6923,respectively.Subsequently,the RT domain of mPHY536207 was replaced by that of drug resistant mutant to construct the plasmids RT634 (LAM-resistant) and RT6923 (ADVresistant).The HBV constructs were transfected into Huh7 cells.The HBsAg levels in supernatant were determined by enzyme-linked immunosobent assay (ELISA),and the amount of intracellular HBV DNA was assayed by real-time polymerase chain reaction and Southern blot analysis.Results The plasmids PHY536207 and PHY97 containing genotype B and genotype C wild-type fulllength HBV genomes were constructed successfully,both of which could replicate in Huh7 cells.Intracellular HBV DNA extracted from cells in each of six-well culture plates was more than 1 × 107 copy/ mL,and the introduction of Pst Ⅰ restriction enzyme site did not affect the viral replication and HBsAg secretion.PHY634 and RT634,in which mutant RT domain was replaced into a wild type HBV expressing vector,exhibited the same HBV DNA replication under the treatment with different doses of LAM,the value of 50％ inhibitory concentration (IC50) was ＞100 μmol/L,while the IC50 of mPHY536207 was 0.18μmol/L.Moreover,wild-type isolate was sensitive to ADV (IC50 =1.2 μmol/L),while PHY6923 and RT6923 were resistant to ADV treatment (IC50 ＞100 μmol/L).Conclusion The phenotypic assay is successfully developed in this study based on replacing RT domain of wild-type HBV strains with that of clinical isolated drug resistant strain.

Objective:To investigate the effect of early acupuncture treatment on limb function in patients with acute cerebral infarction (ACI).Methods:A total of 114 patients with first-episode ACI in our hospital from March 2018 to March 2019 were selected and randomly divided into three groups, 38 cases in each group. The control group was treated with conventional western medicine therapy, while the experimental group A and group B were given acupuncture treatment on the basis of the control group. The group A started treatment at 3rd days and the group B started at 10th day after admission. All three groups were treated for 2 weeks. The National Institute of Health Stroke Scale (NIHSS) was used to evaluate the degree of neurological deficit. The Fugl Meyer assessment of motor function (FAM) was used to evaluate the motor function. The Barthel Index (BI) was used to evaluate the activities of daily living, and the rehabilitation status was evaluated according to muscle strength grading. The levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were detected by ELISA to evaluate the clinical efficacy.Results:The total effective rate was 94.7% (36/38) in group A, 84.2% (30/38) in group B and 73.7% (22/38) in control group. The difference was statistically significant ( χ2=14.748, P<0.01). After treatment, NIHSS scores of three groups were significantly decreased ( F=97.619, P<0.01), and the scores of FAM, BI and muscle strength in group A were significantly lower than those in the group B ( t=12.580, P<0.01); FAM, BI scores and muscle strength of three groups were significantly increased ( F=134.525, 35.100, 164.160, all Ps<0.001), and those of group A were significantly higher than those in group B ( t=8.634, 7.260, 20.305, all Ps<0.001). After treatment, serum NGF (148.80 ± 22.17 pg/ml, 132.14 ± 20.40 pg/ml vs. 125.82 ± 18.31 pg/ml, F=12.926), BDNF (5.01 ± 1.14 ng/ml, 4.10 ± 1.09 ng/ml vs. 3.92 ± 0.95 ng/ml, F=11.481) in the group A and group B were significantly higher than those in the control group ( P<0.01), and serum NGF, BDNF in group A were significantly lower than those in the group B ( t=5.046, 5.277, all Ps<0.01). Conclusions:Early acupuncture treatment can increase the levels of serum NGF and BDNF, promote the recovery of nerve factors and limb function, and the curative effect of acupuncture treatment starting at 3rd days after admission is better than 10th day.

OBJECTIVETo explore the autophagy of RPMI8226 cells induced by As(2)O(3) and its possible mechanisms.

METHODSRPMI8226 was incubated with different concentration of As(2)O(3) for different time, and the inhibiting rate was calculated by MTT method. The autophagic rate of RPMI8226 cells incubated with different concentration of As(2)O(3) was determined by FACS. The change of cells ultrastructure was observed by transmission electron microsopy (TEM). After incubation with different concentration of As(2)O(3), the expression of Beclin-1 on RPMI8226 was detected by RT-PCR and Western blot.

RESULTSDifferent concentration of As(2)O(3) could significantly inhibit the proliferation of RPMI8226 cells (P < 0.05), and the inhibitory effect was in dose- and time-dependent way in a certain range. the autophagic rate increased with the increasing of drug concentration and prolonging of action time (P < 0.05). TEM results revealed a typical autophagosome in RPMI-8226 cell treated by As(2)O(3) for 48 hours. Beclin-1 was up-regulated in RPMI 8226 cells when treated with different concentration of As(2)O(3) for 48h (P < 0.05).

CONCLUSIONAs(2)O(3) can induce autophagy of RPMI8226 cells, and the mechanism may be associated with the upregulation of Beclin-1.

Apoptosis Regulatory Proteins ; metabolism ; Arsenicals ; pharmacology ; Autophagy ; drug effects ; Beclin-1 ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Membrane Proteins ; metabolism ; Oxides ; pharmacology ; Up-Regulation

OBJECTIVETo study the expression of DNMT3b gene in myeloma RPMI8226 cells and its biological significance.

METHODSThe activity of DNA methyltransferase was detected by ELISA, and the expression of DNMT3b in RPMI8226 cells was analyzed by semi-quantitative RT-PCR and real-time fluorescent quantitative PCR. The proliferation and expression of DNMT3b gene in RPMI8226 cells intervened with capecitabine for 24 hours were detected.

RESULTSThe activity of DNMT and expression of DNMT3b in RPMI 8226 cells increased. The proliferation of RPMI8226 cells was inhibited, and the apoptosis occurred in RPMI 8226 cells intervened with capecitabine for 24 hours. The expression level of DNMT3b gene was decreased after being intervened with capecitabine for 24 hours.

CONCLUSIONThe expression level of DNMT3b in myeloma RPMI 8226 cells increase, and capecitabine can inhibit the proliferation of RPMI 8226 and induce apoptosis by inhibiting the expression of DNMT3b gene. Therefore, DNMT3b is expected to be a new target for myeloma therapy.