Objective:To detect the level of immune related cytokines in the serum of the patient with Parkinson’s Disease (PD) and to explore the influencing factors of the cytokines.Methods:51 patients with PD (PD group) and 35 healthy control (control group) were studied.The two groups were detected the serum concentration of IL-1?,IL-2,IL-6 and TNF-? by the way of radio immunity.Results:The serum level of IL-6 and TNF-? in the PD group is significantly higher than that of the control group (P

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.

AIM　To discuss the intraspecific relationship in Magnolia officinalis and the genuineness of Cortex Magnoliae officinalis, and to find some DNA characters of certified “Houpo”. METHODS　Thirty-three samples from eleven locations, which can represent most of the distribution of M.officinalis, were selected. The total DNA was extracted. Severty-four random primers were tried to get good amplification. RESULTS One hundred and sixteen bands amplified from seventeen primers, were clustered by NTSYS-pc software. Three branches were obtained. Some distinctive primers and bands, which represent certified species or fine breed, were obtained also. CONCLUSION　1) M.officinalis should be divided into three geographic clans instead of two subspecies or varieties, they are, a) typical officinalis, b) typical biloba and c) Middle type. This conclusion agrees with the leaf form and other characters. 2) The genetic difference between “Chuanpo” and “Wenpo” is evident and the difference is in correspondence with the quantities of their chemical constituents. So, the genetic difference is the main reason of the genuineness of Cortex Magnoliae officinalis. 3) These results may be used to establish DNA database for identification of Cortex Magnoliae officinalis.

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.

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 conduct extensive quality control tests on Madin-Darby Canine Kidney ( MDCK) cells used for the production of influenza vaccine .Methods Tests for characteristics , extraneous agents, endogenous agents and tumorigenicity were performed on MDCK cells according to Chinese Pharma -copeia Book III .Cell lysate and DNA of MDCK cells were tested for oncogenicity in the light of new interna -tional requirements .Results The MDCK cells extracted from canis were adherent cells with an epithelial morphology, whose average number of chromosome was 80±1.No bacteria, fungi and mycoplasma contami-nation were detected . The detection for extraneous and endogenous virus showed that there was no nonspecific virus causing cytopathic effect , hemadsorption , hemagglutination or animal death .Tests for re-verse transcriptase , bovine viruses and canine viruses were all negative .Each nude mouse was injected with 107 viable cells to observe their tumorigenicity .Twelve weeks after cell injection , no node was found at the injection site and in large organs by gross anatomy .There was no significant difference between test group and negative control group .The test for tumorigenicity of viable cells was negative .Cell lysate and cellular DNA collected from equivalent amount of cells were respectively injected into nude mice , and no node forma-tion was found.There was no significant difference between the test cells and negative controls in pathology indicating that the tested MDCK cells were non-oncogenic .Conclusion It showed the possibility of using MDCK cells for the production of influenza vaccine .

Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.

Objective To study the profiling and authentication of human cell lines in cell bank of our department using short tandem repeat (STR) loci and to analyze the situation of cell contamination and misjudgment. Methods Sixty-one human cells including cells collected and preserved by our cell bank and cells from the other departments were detected by the 16 STR loci-method. To analyze the cross contamina-tion between human cell lines, the results were aligned with profiles published by international cell banks. Isoenzyme detection was employed to authenticate the cell species when the STR signal can not be detected. Results Among the 61 cells, specific profiles were produced by 41 ceils and there was no cross contamina-tion. Thirty-six cells had the completely same STR profiles in 9 STR loci with the same cells preserved by ATCC or JCRB, while 5 cells have different profiling just in the vWA loci. The cells referred above can be recognized as correct cells; Eleven cells (18.0%) were the false cells. Among them, cancer cells of tongue named Tca8113 and cancer cell of liver named HHCC(changed to FHCC98 now) had the same profile with HeLa and HeLa S3 respectively; Two ceils both named HUT-102 have the completely different profiles with ATCC; The signal of 4 cells was not be detected, and all of them were determined as hamster cell lines by u-sing isoenzyme detection. Also 2 cells were identified to be mixed cells. Conclusion The phenomenon of cell misjudgment and cross contamination between cells is serious. Authentication of cell lines correctly, es-pecially for the re-authenticatian of domestic self-established cells, is very important for the guarantee of the reliability and reproducibility for scientific researches.

Objective:To explore the predictive value of renal resistive index (RRI) joint with semiquantitative power Doppler ultrasound (PDU) score to acute kidney injury (AKI) in non-septic critically ill patients.Methods:This prospective observational study enrolled non-septic critically ill patients admitted to the Emergency Intensive Care Unit of Cangzhou Central Hospital from January 2018 to August 2019. In addition to general data, RRI and PDU scores were measured with medical ultrasonic instrument within 6 h after admission. Renal function was assessed on the 5th day in accordance with kidney disease: Improving Global Outcomes criteria. The patients who progressed to AKI stage 3 within 5 days after admission were classified into the AKI 3 group, and the rest were classified into the AKI 0-2 group. The difference of each index was compared between the two groups in non-septic critically ill patients and patients with acute heart failure (AHF). Normal distributed continuous variables were compared using independent sample t-tests, whereas Mann-Whitney U tests were used to examine the differences in variables without a normal distribution. Categorical data were compared with the Chi-square test. Receiver operator characteristic curves were plotted to examine the values of RRI, PDU score, RRI-RDU/10 (subtraction of RRI and 1/10 of PDU score), RRI/PDU (the ratio of RRI to PDU score), and RRI+PDU (the prediction probability of the combination of RRI and PDU score for AKI stage 3 obtained by logistic regression analysis) in predicting AKI 3. Delong's test was used to compare the area under the curve (AUC) between predictors. Results:A total of 110 non-septic critically ill patients (51 patients with no AKI, 21 with AKI stage 1, 11 with AKI stage 2, and 27 with AKI stage 3) were recruited. Among them, there were 63 patients with AHF (21 patients with no AKI, 15 with AKI stage 1, 7 with AKI stage 2, and 20 with AKI stage 3). Among the non-septic critically ill patients as well as its subgroup of AHF, compared with the AKI 0-2 group, acute physiology and chronic health evaluation-Ⅱ score, sequential organ failure assessment score, arterial lactate concentration, mechanical ventilation rate, proportion of vasoactive drugs, 28-day mortality, serum creatinine, RRI, RRI-RDU/10, RRI/PDU, RRI+PDU, and rate of continuous renal replacement therapy were higher in the AKI 3 group, and urine output and PDU score were lower ( all P<0.05). As for non-septic critically ill patients, RRI/PDU [AUC=0.915, 95% confidence interval ( CI): 0.846-0.959, P<0.01] and RRI+PDU (AUC=0.914, 95% CI: 0.845-0.959, P<0.01) performed best in predicting AKI 3, and the AUCs were higher than RRI (AUC=0.804, 95% CI: 0.718-0.874, P<0.01) and PDU score (AUC=0.868, 95% CI: 0.791-0.925, P<0.01). The optimal cutoff for RRI/PDU was > 0.355 (sensitivity 92.6%, specificity 81.9%, Youden index 0.745). The predictive value of RRI-RDU/10 for AKI 3 (AUC=0.899, 95% CI: 0.827-0.948, P<0.01) was also better than RRI and PDU scores, but slightly worse than RRI/PDU and RRI+PDU, with statistically difference only between RRI and RRI-RDU/10 ( P<0.05). As for patients with AHF, RRI/PDU (AUC=0.962, 95% CI: 0.880-0.994, P<0.01) and RRI+PDU (AUC=0.962, 95% CI: 0.880-0.994, P<0.01) also performed best in predicting AKI 3, and the AUCs were higher than RRI (AUC=0.845, 95% CI: 0.731-0.924, P<0.01) and PDU score (AUC=0.913, 95% CI: 0.814-0.969, P<0.01) with statistically differences (all P<0.05). The optimal cutoff for RRI/PDU was > 0.360 (sensitivity 95.0%, specificity 90.7%, Youden index 0.857). The predictive value of RRI-RDU/10 for AKI 3 (AUC=0.950, 95% CI: 0.864-0.989, P<0.01) was also better than RRI and PDU score, but slightly worse than RRI/PDU and RRI+PDU, with statistically difference only between RRI and RRI-RDU/10 ( P<0.05). Conclusions:The combination of RRI and PDU score could effectively predict AKI 3 in non-septic critically ill patients, especially in patients with AHF. The ratio of RRI to PDU score is recommended for clinical application because of its excellent predictive value for AKI and its practicability.

his article expounds the concept, theory and mechanism of laughter therapy, introduces the process of laughter therapy, and analyzes and summarizes the effects of laughter therapy on elderly patients in other countries in order to provide a reference for studies on laughter therapy in China and to improve elderly people's physical and mental health as well as their quality of life.