Application of TARP luciferase reporter system in function identification of CAR-T cells / 细胞与分子免疫学杂志

Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.

Influence of "Mental Health Law of the People's Republic of China" on psychological counseling in colleges and universities: An interview study on the heads of psychological counseling centers in colleges and universities in Beijing / 中国心理卫生杂志

Objective:To investigate the impact of China's Mental Health Law on the work of psychological counseling in colleges and universities, and to explore ways to improve the law. Methods: Totally 12 heads of college and university counseling centers in Beijing were conducted with semi-structured interviews. The average age of the interviewees was (40 ±7) years old, with master or doctor degrees in psychology or related disciplines. The method of content analysis was used to analyze the interviewees' understanding of the " Mental Health Law". Results: All of the 12 interviewees had gained some understanding of the "Mental Health Law", and accordingly amended the regulation of their counseling centers. Also, interviewees suggested the positive and negative impacts brought by the law, such as enhancing practitioners' legislative sense, clarifying their responsibilities and boundaries as college and university counseling, as well as difficulties to distinguish psychotherapy and psychological counseling, ambiguity in the legality of working with students who were diagnosed with mental disorders. Moreover, interviewees threw out suggestions on improving the law from the aspect of industry standard, supervision department and vocational qualification. Conclusion: The execution of "Mental Health Law" improves practitioners' legislative sense, clarifies their responsibility. Nevertheless, it does not clearly distinguish psychotherapy from psychological counseling, and be lack of regulation on the psychological counseling industry.

Inhibition of Notch signaling pathway promotes the differentiation of bone marrow mesenchymal stem cells into islet / 中华肾脏病杂志

Objective To investigate the effect of Notch signaling during bone marrow mesenchymal stem cells (BMSC) differentiating into islet in vitro.Methods The specific inhibitor of γ-secretase DAPT was used to inhibit the Notch signaling pathway.Mter induction,DTZ staining,indirect immunofluorescence staining,RT-PCR and Westenn blotting were used to detect the expression of insulin,glucagon,Pdx-1 and Ngn3.Results (1) Identification of BMSCs:Indirect immunofluorescence staining showed that BMSCs could express CD59 and CD90,which both were makrers of mesenchymal stem cells.Besides,BMSCs could express nerve culluar markers such as NSE,GFAP,suggesting multi-directional differentiation.(2) The result of MTT showed DAPT could inhibit the cell proliferation in a time-dependent manner and a dose-dependent mannar.Besides,DAPT could inhibit the expression of target gene of Notch signal pathway in a time-dependent manner and a dosedependent mannar.After treated by 1,5,20 μmol DAPT,the expression of Hes1 had reached to 92.06％,71.40％ and 46.89％ of controls respectively,suggesting efficiency of inhibition on Notch reached 7.94％,28.6％ and 53.11％ respectively (all P＜ 0.05).(3) Indirect immunofluorescence staining showed the expression of pancreas-specific markers such as insulin and glucagon were much higher in DAPT treated BMSCs than that in controls,which was confirmed by RT-PCR and Western blotting analyses.The proportion of insulin-producing cells differentiated from DAPT treated BMSCs was (74.03 ± 3.96)％,which was higher than that from controls[(36.49 ± 3.24)％,P＜ 0.05].(4)Furthermore,RT-PCR and Western blotting analysis showed that the expressions of Pdx-1 and Ngn3 were earlier than that of insulin and glucagon,and the expressions of Pdx-1 and Ngn3 were higher in DAPT treated BMSCs than that in controls.Conclusions Notch signaling pathway plays a role in the differentiation of BMSCs into islet in vitro.Pharmacological interference with Notch signaling pathway may provide a novel method to obtain islet for therapeutic use.

Progress in mesenchymal stem cells for treatment of atherosclerosis / 生物工程学报

Atherosclerosis is an inflammatory disease. However, its etiology has not been yet fully elucidated. Endothelial dysfunction is currently considered to be one of the most important steps in the initiation of atherosclerosis. In addition, vascular smooth muscle cells, which are the main cellular component of de novo and in-stent restenosis lesions, play an important role in the development of atherosclerosis. Promoting the regeneration of endothelial cells and inhibiting the proliferation of smooth muscle cells are pivotal for the prevention and treatment of vascular injury. Recently, some studies have demonstrated that mesenchymal stem cells can home to the site of injury and differentiate into endothelial cells to repair damaged blood vessels. On the contrary, other researches have revealed that mesenchymal stem cells can differentiate into vascular smooth muscle cells that are involved in the development of restenosis. Here, we review the fundamental researches of mesenchymal stem cell therapy for atherosclerosis and address the perspectives of mesenchymal stem cells in atherosclerosis treatment.

Changes in pattern of TCR V? repertoire in peripheral blood from CML patients / 第三军医大学学报

Objective To investigate the distribution and clonality of TCR V? repertoire T cells in peripheral blood mononuclear cells with chronic myeloid leukemia(CML)at chronic phase(CP)and complete remission(CR).Methods The CDR3 of TCR V? 8 subfamily genes were amplified in mononuclear cells of peripheral blood from CML patients in CP(8 cases)and CR(8 cases)phases for observation of the usage of TCR V? repertoire by RT-PCR.The positive PCR products were further labeled with fluorescence and analyzed by genescan technique for the CDR3 size for evaluation of the clonality of the detectable TCR V? T cells.A total of 10 cases of healthy adults served as controls.Results The mean values of detectable TCR V? subfamilies were(3.25?0.89)in patients with CML-CP,predominantly in V?1,2 and V?3,whereas(3.5?0.76)of 8 V? subfamily T cells were expressed in patients with CML-CR,predominantly in V?1,2,3 and V?8.There was no significant difference between the above groups and the healthy adults(3.5?0.52).In addition,a higher frequency expression of V?8 could be found from peripheral blood in CR phase(5/8)rather than from that in CP phase(2/8)and from that of the healthy adults(3/10).Genescan analysis showed that the majority of PCR products from both CML and healthy adults displayed clonal expansion,predominantly in V?1,V?2 and V?3.Clonal expanded V?7 subfamily T cells,however,could be identified from two patients in CR phase but not in all of 10 healthy adults.Conclusion The similar distribution and clonal pattern of TCR V? repertoire T cells can be found in peripheral blood from the three groups,suggesting that the cellular immunosuppression of CML patients might not be mainly involved in the alteration of TCR V? repertoire.In CML-CR groups,the expression and clonal pattern of some V? repertoires are different from those in other groups,which needs further studies.