Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.

Purpose: To observe the curative effects of different treatments on genual osteoarthritis.Methods: The 148 cases were randomly divided into 4 groups: acupuncture group; Chinese herbs plus spectrograp irradiation group; comprehensive treatments group and western medicine group. Results:The total effective rate in these groups were 78.9%, 72.7%, 92.3% and 78.9% respectively, and there was not a significant difference among the groups (P＞0.05). The marked effective rate in comprehensive treatments group was higher than that in other three groups (P＜0.05). Conclusion: Acupuncture and Chinese herbs plus sepectrograp irradiation had certain effect on gonitis. Comprehensive therapies could coordinate the actions of acupuncture, and Chinese herbs plus spectrograp irradiation, and raise the curative effect.

In order to search for a new pathway to improve the yield of ginseng through growing at the full sun shine accompanied by salicylic acid (SA), the net photosynthetic rate (P(n)), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), malondialdehyde (MDA) in Panax ginseng leaves, and the content of ginsenosides in roots were compared under various concentrations of SA and full sun shine with the traditional shade shed. Under the full sun shine, 0.05, 0.2 mmol x L(-1) SA increased net photosynthetic rate to a great extent. Under the cloudy day, the average net photosynthetic rate increased by 127.8% and 155.0% over the traditional shade shed, 13.9% and 27.5% over the treatment without SA respectively; under the clear day, 23.5% and 30.4% over the traditional shade shed, 8.6% and 14.6% over the treatment without SA, particularly obvious in the morning and late afternoon. With such concentration, SA increased activities of SOD, CAT, POD, and decreased the contents of the MDA. This difference resulted from different light intensity, rise of light saturation point, and fall of compensation point. Full sun shine decreased ginsenosides contents, but with SA, the ginsenosides regained, the content of Rg1 and Re, Rb1, total six types of ginsenosides in SA 0.2 mmol x L(-1) group were higher than those in the control group (P < 0.05) and other groups. The application of 0.2 mmol x L(-1) SA under full sun shine during a short time has little threat to the P. ginseng in spring, and could enhance the resistance to the adversity, which would improve the yield of ginseng heavily.
Catalase ; analysis ; metabolism ; Ginsenosides ; analysis ; metabolism ; Light ; Malondialdehyde ; analysis ; metabolism ; Panax ; chemistry ; drug effects ; metabolism ; radiation effects ; Peroxidases ; analysis ; metabolism ; Photosynthesis ; drug effects ; Plant Proteins ; analysis ; metabolism ; Salicylic Acid ; pharmacology ; Seasons ; Superoxide Dismutase ; analysis ; metabolism

Female ; Humans ; Hyperacusis ; surgery ; Labyrinth Diseases ; surgery ; Middle Aged ; Otologic Surgical Procedures ; methods ; Retrospective Studies ; Semicircular Canals ; pathology ; surgery

Gindenosides are the active ingredients of Panax ginseng. 20 (S) -protopanaxadiolocotillol type epimers are the main metabolites of 20 (S) -protopanaxadiol. The previous studies showed that there are stereoselectivity difference in pharmacodynamics and pharmacokinetics between 24R-epimer and 24S-epimer. The purpose of this study was to explore the excretion of the epimers in bile, feces and urine of rat. Liquid chromatography tandem mass spectrometry method has been performed for determination of 24R-epimer and 24S-epimer in bile, feces and urine. 24R-epimer or 24S-epimer was intragastric administered to rats at a single dose of 10 mg x kg(-1). Results showed that after administration the recovery of 24R-epimer and 24S-epimer in feces was 17.69% and 17.09%, respectively, while both of the two epimers were hardly detected in urine. The 48 h cumulative biliary excretion rate of 24R-epimer was 8.01% after administration, while only 1.47% for 24S-epimer. It indicated that there are stereoselectivity in biliary excretion of the epimers with intragastric administration.
Animals ; Bile ; chemistry ; metabolism ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Feces ; chemistry ; Ginsenosides ; chemistry ; pharmacokinetics ; Male ; Mass Spectrometry ; Panax ; chemistry ; Rats ; Rats, Sprague-Dawley ; Stereoisomerism ; Urine ; chemistry

Objective:To observe the changes of macular microvessels in patients with retinal vein occlusion (RVO) and macular edema (ME) after intravitreal injection of aflibercept (IVA), and analyze its correlation with best corrected visual acuity (BCVA).Methods:A retrospective case study. Thirty patients (30 eyes) with monocular RVO with ME (RVO-ME) who were diagnosed in the clinical examination of Tianjin Eye Hospital from April 2019 to February 2020 were included in the study. Among them, there were 12 males (12 eyes) and 18 females(18 eyes); the average age was 54.30±13.17 years. The average course of disease was3.43±1.97 months. Both eyes were examined by BCVA and optical coherence tomography (OCTA). The on-demand injection was adopted after the first injection in IVA treatment regimen. The macular area 6 mm×6 mm in both eyes was scanned with an OCTA instrument, and the area of the foveal avascular area (FAZ), FAZ circumference (PERIM), and out-of-roundness were measured at baseline and 1, 3, and 6 months after treatment. Index (AI), blood flow density within 300 μm width of FAZ (FD-300), foveal retinal thickness (CMT), superficial retinal capillary plexus (SCP), deep retinal capillary plexus (DCP) blood flow density. The paired t test was used to compare the quantitative parameters of the affected eye and the contralateral healthy eye at baseline; the changes of the quantitative parameters at baseline and 1, 3, and 6 months after treatment were analyzed by repeated measures analysis of variance. Pearson correlation analysis was used to analyze the correlation between BCVA, retinal perfusion, and macular blood supply parameters at 6 months after IVA treatment. Results:At baseline, compared with the contralateral healthy eye, the FAZ area ( t=-4.091), PERIM ( t=-5.098) and AI ( t=-9.093) of the RVO-ME eye were enlarged, and FD-300 ( t=7.237) and overall SCP and DCP blood flow density ( t=8.735, 9.897) decreased, the difference was statistically significant ( P<0.001). Six months after treatment, the BCVA of RVO-ME eyes was significantly increased, CMT decreased, FAZ area expanded, and AI decreased ( t=8.566, 16.739, -6.469, 9.719; P<0.001), the difference was statistically significant. There was no significant change in the blood flow density of FD-300 and overall SCP and DCP, and the difference was not statistically significant ( t=1.017, 1.197, 0.987; P>0.05). Compared with baseline, the FAZ area of RVO-ME eyes gradually expanded at 3 and 6 months after treatment, and the difference was statistically significant ( F=21.979, P<0.001). Correlation analysis results showed that BCVA at 6 months after treatment was positively correlated with the overall SCP and DCP blood flow density at baseline and 6 months after treatment ( r=-0.538, -0.484, -0.879, -0.854; P<0.05). There was a negative correlation with the area of FAZ 6 months after treatment ( r=0.544, P=0.001). The number of ME recurrences was negatively correlated with BCVA and overall SCP and DCP blood flow density 6 months after treatment ( r=0.604, -0.462, -0.528; P<0.05), it was positively correlated with FAZ area ( r=0.379, P=0.043). Conclusion:Within 6 months of IVA treatment in RVO-ME eyes, ME is significantly reduced and visual acuity is improved; SCP blood flow density decreases, and FAZ area expands.

Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.

Actins ; metabolism ; Adult ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; Desmin ; metabolism ; Diagnosis, Differential ; Female ; Fibroadenoma ; metabolism ; pathology ; surgery ; Hamartoma ; pathology ; Humans ; Hyperplasia ; Leiomyoma ; pathology ; Microfilament Proteins ; metabolism ; Muscle, Smooth ; pathology ; Phyllodes Tumor ; pathology

ObjectiveTo investigate effects of pamidronate on the proliferation and differentiation of osteoblasts of rats in vitro.MethodsOsteoblasts isolated from newborn rat calvaria were treated with various concentrations of pamidronate, the proliferation of osteoblasts was evaluated with the method of methyl thiazole tetrazolium (MTT) and activity of alkaline phosphatase (ALP) in medium was measured with kit of ALP detecting.ResultsThe proliferation of osteoblasts increased under the stimulation of Pamidronate range 10-6-M-10-12 M(P<0.05), but was inhibited at the concentration of high level (10-4 M). The activity of ALP decreased in the experiment.ConclusionPamidronate can act on the osteoblasts directly and increase the proliferation of bone cells, but inhibit the differentiation of the same cells.