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Objective To evaluate the feasibility of the novel staining protocol based on protein L in flow cytometry to detect the expression of chimeric antigen receptor (CAR) on the surface of T cells from patients.Methods The peripheral blood mononuclear cells(PBMCs) were collected from 2 patients with CD19 + lymphoma by hemapheresis and T cells were purified by magnetic bead selection.CAR was transfected with retroviruses targeting CD19 and CD22 into T cells to induce chimeric antigen receptor-modified T cells (CAR-T cells).The single-chain fragments of antibody molecules on the surface of CAR-T cells were labeled by biotinylated protein L-streptavidin-PE system.The proportions of activated CD19-CAR and CD22-CAR positive T cells in the culture were detected by flow cytometry.The results were compared with those detected by conventional flow cytometry method based on Anti-IgG staining.Results The expression of CAR on the surface of CAR-T cells was successfully detected by flow cytometry protocol based on the staining of single-chain variable fragment (scFv)-biotinylated protein L-streptavidin-PE.The percentages of CD19-CAR-T cells from the 2 patients were 71.6％ vs 64.2％ and 49.3％ vs 43.8％ in the protein L group and the Anti-IgG control group respectively,and the percentages of CD22-CAR-T cells were 53.1％ vs 46.3％ and 56.5％ vs 64.0％.The results of the both groups were similar.Conclusion The staining protocol based on protein L could be used as a routine staining method in flow cytometry for the detection of CAR-T cells.
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Objective To investigate the effect of minocycline on the cognition and expressions of brain-derived neurotrophic factor (BDNF), apoptosis related factor Bcl-2 and Bax in hippocampus of rats with Alzheimer’s disease (AD). Methods The rat model was established by microinjection of Aβ25-35 into lateral ventricle. Thirty healthy male SD rats were randomly divided into three groups:control group, model group and minocycline treatment group. Normal saline 1 mL/(kg·d) was intraperitoneally injected in control group and model group. The minocycline treatment group was intraperitoneally injected with minocycline 50 mg/(kg · d) for 14 days. Morris water maze was used to detect the behaviors of animals. The expressions of BDNF, Bcl-2 and Bax in hippocampus were measured by Western blotting and enzyme linked immunosorbent assay (ELISA). The apoptosis of neurons was detected by TdT-mediated dUTP nick-end labeling (TUNEL). Results Minocycline greatly improved the behaviors of AD rats, up-regulated the expressions of BDNF and Bcl-2, and down-regulated the expression of Bax in hippocampus, and reduced cell apoptosis. Conclusion Minocycline plays a protective role in neural function by promoting the growth of neurons and inhibiting the neuronal apoptosis.
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OBJECTIVE@#To evaluate the effect of chronic nicotine on contextual fear memory in rats.@*METHODS@#Rats were subcutaneously injected saline or nicotine for 14 days continuously to induce nicotine dependence. Somatic signs of spontaneous nicotine withdrawal were assessed on day 1 and 14 after the last injection. Locomotor activity was tested 13 days after the last injection. Contextual fear conditioning task was run 14 days after the last injection, which included 3 phases: habituation, fear conditioning, and test. Percent freezing was used as an index for fear memory during the conditioning and test phases. Finally, footshock sensitivity test was conducted.@*RESULTS@#Compared with the saline group, the global withdrawal scores significantly increased in the nicotine group when the somatic signs were assessed 1 day after the nicotine injection, while there was almost no difference in the 2 groups 14 days after the last injection. In the contextual task, the nicotine group showed a significant increase in the level of freezing response during the test phase but not in the conditioning phase. There was no significant difference in the locomotor activity and footshock sensitivity between the 2 groups.@*CONCLUSION@#Chronic nicotine enhances the contextual fear memory in rats. This effect is not attributed to the change of somatic signs, locomotor activity or footshock sensitivity.
Subject(s)
Animals , Male , Rats , Brain , Conditioning, Classical , Electric Stimulation , Fear , Freezing Reaction, Cataleptic , Learning , Memory , Nicotine , Pharmacology , Nicotinic Agonists , Pharmacology , Random Allocation , Rats, Sprague-DawleyABSTRACT
Objective To evaluate the significance of AFP-IgM, this is one of new tumor markers, in the diagnosis of primary hepatocellular carcinoma (PHC). Methods The contents of AFP-IgM and AFP in serum of 103 healthy subjects, 74 patients suffered primary hepatic carcinoma, 27 patients affected by liver cirrhosis and 63 patients affected by chronic hepatitis were detected by means of enzyme linked immunosorbent assay and electrochemiluminescence. No-PHC is comprised of liver cirrhosis,chronic hepatitis and health subjects as control group. Results The area under ROC curve of AFP was larger than that of AFP-IgM (0.85 vs 0.72, Z=3.21) and the best cut-off value of AFP-IgM and AFP was 3×105-AU/L and 10 ug/L respectively, which was determined by ROC curve. Under the cut-off value, the sensitivity of AFP- lgM and AFP for PHC were 64.9% and 79.7%, and the specificity were 75.6% and 80.3%, yet their efficacies were similar. However, for early diagnosis of liver cancer (stage Ⅰ and Ⅱ), the area under ROC curve of AFP-IgM was larger than that of AFP (0.91 vs 0.82,Z=1.73). The sensitivity of AFP-IgM andAFP were 94.4% and 72. 2%, and the specificity were 81.9% and 79.9%. The differences of AFP-IgMand AFP for early diagnosis of liver cancer were statistically significant. When both of the test results combined AFP-IgM with AFP are positive, it can be diagnosed as liver cancer. The specificity of combineddetermination of the two forms was 89.1%, and the efficacy was 79. 0%. Conclusions Both of thesensitivity and specificity of the AFP-IgM test were higher than that of the AFP for early diagnosis of livercancer. We also found that combined determination of the two forms significantly increased the specificityand the positive predictive value for the diagnosis of PHC, thus AFP-IgM was of especially significance forearly diagnosis of liver cancer.
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@#目的观察神经阻滞治疗颈源性头痛的效果。方法对颈源性头痛患者30例行星状神经节阻滞,伴颈椎旁神经或耳颞神经阻滞治疗,观察治疗前后疼痛视觉模拟评分(VAS)、每周头痛发作次数、服用止痛药物与睡眠改善例数。结果治疗前后VAS评分改善(P<0.05),治疗后头痛完全缓解15例,好转9例,有效4例,无效2例;治疗后头痛发作次数及持续时间均缩短(P<0.05);治疗前服用止痛药物28例,治疗后2周12例,4周4例(P<0.05);睡眠不佳治疗前29例,治疗后2周13例,4周为4例(P<0.05)。结论神经阻滞是治疗颈源性头痛是一种良好的治疗方法。