Objective To establish a specific HPLC method and a standard fingerprint for quality control ofZiziphi Spinosae Folium.Methods The samples were separated on a Kinetex C18 column (100 mm × 2.1 mm,2.6 μm) by gradient elution at the flow rate of 300 μL/min using acetonitrile and 0.1％ aqueous formic acid as the mobile phase.The column temperature was 30℃.The detection wavelength was 225 nm and the sample size was 10 μL.The fingerprint evaluation software (2012 edition) for Chinese materia medica (CMM) was used to evaluate the similarity of the 12 batches of samples.Results There were 13 characteristic peaks identified in the characteristic spectra ofZiziphi Spinosae Folium samples.Peak 6 was Rutin.The similarities of 13 batches ofZiziphi Spinosae Folium samples were proved to behigher than 0.850.Conclusion The method is available with a good reproducibility and accuraty which can control the quality standards effectively.

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.

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

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.

This study was aimed to investigate the effects of extract from Tibetan medicine Berberis Cortex (TMBC) on expressions of protein kinase C (PKC-β), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF-1α) in the retina of diabetic rats. Diabetic rats were established by one time intraperitoneal injection of strepto-zocin (STZ). Rats were randomly divided into the model group, low-dose TMBC group, medium-dose TMBC group, high-dose TMBC group, metformin group, calcium dobesilate group, berberine group and the normal control group. Intragastric administration was given. The medication amounts of TMBC in the low-dose, medium-dose and high-dose group were 5, 10, and 20 times to the adult medication dose. In the metformin group and the berberine group, 10 times of the adult medication dose were given. Distilled water was given in the model group and the normal con-trol group. After 6-week intragastric administration, all experimental rats were sacrificed. The expressions of PKC-β, VEGF and HIF-1α were examined by real-time fluorescence quantitative PCR assay and western blot. HIF-1α ex-pression was detected by immunohistochemistry. The results showed that compared with the normal control group, the mRNA and protein expression of PKC-β, VEGF and HIF-1α increased obviously in the retina of diabetic rats ( P<0.01). Compared with the model group, the mRNA expression of PKC-β, VEGF and HIF-1α in the high-dose and medium-dose TMBC group reduced obviously (P< 0.01). The protein expression levels of PKC-β, VEGF and HIF-1α were also obviously reduced (P< 0.05). The expressions of PKC-β, VEGF and HIF-1α in the low-dose TMBC group were obviously reduced (P < 0.05). It was concluded that TMBC can depress the expressions of HIF-1α, PKC-β and VEGF in the retina of diabetic rats, which can be served as a protective effect to prevent progress of di-abetic retinopathy.

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.

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.

Abdominal Pain/etiology* ; Acute Disease ; Appendicitis/diagnosis* ; Diabetes Mellitus ; Diabetic Ketoacidosis/diagnosis* ; Diagnostic Errors/adverse effects* ; Humans ; Insulin

Objective To investigate the impact of CD4+ CD25+ FOXP3+ regulatory T(Treg) cells on tumor recurrence in liver transplantation for hepatocellular carcinoma (HCC). Methods Im-munohistochemistry and flow cytometry were used for analysis of the frequency of Treg. Meanwhile,it was compared with that of non-cancer liver transplantation patients. Results The frequency of CD4+CD25+ FOXP3+ regulatory T cells in the blood of HCC liver transplantation was (10. 15 ±1. 00) % , which was significantly higher than that in the normal control group (3. 20±1. 18) %. Cir-culating CD4+ CD25+ FOXP3+ Treg frequency was increased significantly and correlated with the tumor recurrence in the HCC patients. An abundant accumulation of Treg concurrent with significant-ly reduced infiltration of CD8+T cells was found in tumor regions. Conclusion Increased CD4+ D25+FoxP3+ Treg may impair the effectors function of CD8+ T cells, promote the tumor recurrence and re-present a therapeutic target for HCC liver transplantation.

OBJECTIVETo investigate the temperature change of scrotum caused by varicocele(VC) and its significance.

METHODSOne hundred and sixty-six VC patients were examined by infrared ray imaging and colored ultrasonic scanning, and the results were analyzed. Comparisons of the infrared ray images and the routine semen analysis of 106 of the patients were made between before and after the operation.

RESULTSThe difference in the infrared ray images between before and after the operation on the VC patients was significant (P < 0.01), and so was the difference in the results of semen analysis(P < 0.01; some of the items P < 0.05).

CONCLUSIONSThe infrared ray imaging, together with the semen analysis and colored ultrasonic scanning, had a directive value for the diagnosis and treatment of VC patients.

Adolescent ; Adult ; Body Temperature ; Humans ; Infrared Rays ; Male ; Middle Aged ; Scrotum ; physiopathology ; Ultrasonography, Doppler, Color ; Varicocele ; diagnosis ; physiopathology