Objective:To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods:Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results:Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions:This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.

Objective:To evaluate the feasibility of 8E5 cells and CD19-CAR-Jurkat cells used as reference cells in the detection of lentiviral vector integration sites with different methods.Methods:Single clones of 8E5 cells and CD19-CAR-Jurkat cells were selected using limiting dilution method. Digital PCR was established to detect the copy number of HIV-1 in 8E5 cells and the copy number of CAR in CD19-CAR-Jurkat cells. High-throughput sequencing techniques (whole-genome resequencing, modified genome sequencing and probe hybridization capture) were used to detect integration sites in 8E5 cells and CD19-CAR-Jurkat cells, and optical genome mapping (OGM) technology was used for further confirmation.Results:Three clones of 8E5-D8 cells and six clones of CD19-CAR-Jurkat 2-6 cells were selected using the limiting dilution method. 8E5-D8 and CD19-CAR-Jurkat 2-6 were chosen as candidate cells based on their gene copy numbers detected by digital PCR and flow cytometry. These cells were then expanded and cryopreserved. Digital PCR showed that 8E5-D8 cells contained approximately 1 copy per cell, while CD19-CAR-Jurkat 2-6 cells contained approximately 13 copies per cell. High-throughput sequencing revealed one integration site in 8E5 cells and 13 integration sites in CD19-CAR-Jurkat cells, which matched the copy number detection results. All these integration sites were further confirmed at the submicroscopic level of chromosomes using OGM.Conclusions:Based on the insertion copy numbers and integration sites, 8E5-D8 cells and CD19-CAR-Jurkat 2-6 cells could be used as reference cells in further development of methods for detecting integration sites in CAR-T cell lentiviral vectors.

Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.

Objective:To analyze the clinical characteristics of intestinal tuberculosis improving the diagnosis rate of intestinal tuberculosis.Methods:From January 2014 to June 2018, at Wuhan Pulmonary Hospital, the data of clinical symptoms, laboratory examination, imaging, endoscopy, surgery and pathological examination of 205 patients with intestinal tuberculosis were retrospectively analyzed. Descriptive analysis was performed for statistical analysis.Results:Among 205 patients with intestinal tuberculosis, 145 cases were male and 60 cases were female, aged 14 to 85 years old. A total of 189 cases (92.2%) were complicated with lung tuberculosis, of which 151 cases (79.9%) were positive for sputum acid fast staining. A total of 126 cases were tested for feces acid fast staining, of which 83 cases (65.9%) were positive. A total of 60 cases (29.3%) were tested for GeneXpert Mycobacterium tuberculosis/rifampicintablet (GeneXpert MTB/RIP), of which 49 cases (81.7%) were positive. A total of 44 cases of intestinal tuberculosis were diagnosed by biopsy under electronic enteroscopy, and 21 cases were pathologically diagnosed with intestinal tuberculosis after surgical resection. The 21 patients were tested for GeneXpert MTB/RIP, of which 19 cases (90.5%) were positive and 10 cases (47.6%) were positive for tuberculin test. Six patients were clinically diagnosed with intestinal tuberculosis after effective treatment of antituberculosis drugs. Conclusions:Combination of clinical symptoms and laboratory, imaging, endoscopic and pathological examination, as well as the therapeutic effect of diagnostic antituberculosis treatment could make comprehensive diagnosis of intestinal tuberculosis. The GeneXpert MTB/RIP examination is of great value in the diagnosis of intestinal tuberculosis.

The high-purity impurity C was isolated and purified from a new anti-allergic drug, rupatifen, by nucleophilic substitution reaction with bromobutaric acid, and its structure was confirmed by IR, UV, MS, 1H NMR, 13C NMR, DEPT135°,HSQC, HMBC, and 1H-1HCOSY. The preparation process of impurity C in this study was simple and easy to obtain under mild conditions, with the purity of 99.0% and the yield of 25%-30%;and the sample met the target compound by structural confirmation. The preparation process and structure confirmation provided sufficient impurity C reference substance for impurity research of raw materials and preparations of rupatifen fumarate, which laid a solid foundation for quality research of new drugs.

Objective To analezk thk charactkristics of drug-induckd livkr injure( DIFI)in childrkn with acutk lemphoblastic lkuckmia(LFF),so as to improvk thk phesician＇s undkrstanding of chkmothkrape DIFI,and to guidk clinical rational drug usk. Methods Onk hundrkd and forte-thrkk casks with LFF diagnoskd in thk Dkpartmknt of Hk-matologe and Oncologe in thk Pirst Lffiliatkd Hospital of Yhkngzhou Rnivkrsite from Januare 2012 to Dkckmbkr 2016 wkrk analezkd rktrospkctivkle. Baskd on DIFI diagnostic critkria and thk ARCLM scalk,thk casks with a scork of ≥3 points wkrk considkrkd to havk chkmothkrape DIFI. Groupkd be gkndkr,agk,immunoteping,risc and stagk of chkmo-thkrape,thk incidknck of DIFI was comparkd. Thk situation aftkr DIFI prkvkntion was comparkd bktwkkn two groups which was groupkd according to whkthkr thk application of hkpatoprotkctivk drugs. ResuIts Onk hundrkd and kight ca-sks(75. 52﹪)had DIFI,66 casks(61. 11﹪)showkd clinical manifkstations of livkr injure,and 42 casks(38. 89﹪) had no clinical semptoms. Lmong all thk casks 57. 41﹪(62 casks)wkrk mild livkr damagk,25﹪(27 casks)wkrk modkratk livkr injure and 17. 59﹪(19 casks)wkrk skvkrk livkr damagk. Thk clinical tepks which wkrk hkpatockllular accounting for 79. 63﹪(86 casks),cholkstatic 7. 41﹪(8 casks)and mixkd 12. 96﹪(14 casks). Malk wkrk 80 casks (79. 21﹪)and fkmalk 28 casks(66. 67﹪),but thk incidknck of DIFI bktwkkn diffkrknt gkndkr group had no statistical diffkrknck(χ2 ﹦2. 524,P﹦0. 112). Skvknte-fivk casks(77. 32﹪)wkrk ＜7 ekars agk and 33 casks(71. 74﹪)≥7 ekars agk,and thk incidknck of DIFI bktwkkn 2 groups was not statisticalle diffkrknt(χ2 ﹦0. 526,P﹦0. 468). Thkrk was no significant diffkrknck in T-LFF(8 casks,61. 54﹪)and B-LFF(100 casks,76. 92﹪)( χ2 ﹦0. 795,P﹦0. 372). Thk incidknck had significant diffkrknck in diffkrknt risc(P﹦0. 002). Thk incidknck of DIFI bktwkkn thk middlk risc group(60 casks,88. 24﹪)and standard risc(21 casks,58. 33﹪)had statistical diffkrknck( P ＜0. 05 ). Thk incidknck of DIFI bktwkkn thk middlk risc group and skvkrk risc(27 casks,69. 23﹪)had statistical diffkrknck( P﹦0. 015). Thk incidknck was diffkrknt in diffkrknt stagks of chkmothkrape(P＜0. 05). Thk incidknck of DIFI in induckd stagk was diffkrknt comparkd to othkr stagks(P＜0. 05). ARCLM scork ＞8 points accountkd for 21 casks(19. 45﹪), 6-8 points accountkd for 59 casks(54. 63﹪)and 3 -5 points accountkd for 28 casks(25. 92﹪). Eighte -nink patiknts(92. 71﹪)wkrk kffkctivk in thk hkpatoprotkctivk group and 8 patiknts(66. 67﹪)in thk no hkpatoprotkctivk thkrape group. Thk diffkrknck bktwkkn thk 2 groups was statisticalle significant(χ2 ﹦5. 317,P﹦0. 021). ConcIusions Thk clinical semptoms of drug-induckd livkr injure in childrkn with LFF chkmothkrape ark lacc of spkcificite. Thke ark mainle charactkrizkd be mild livkr injure. Thk clinical tepk of hkpatic injure is common in hkpatockllular. Thk ARCLM scork was mostle 6 to 8. Thkrk is no rklationship bktwkkn thk incidknck in LFF and gkndkr,agk,tepk of lkuck-mia. Thk incidknck with modkratk risc tepk is highkr than that of thk standard and high-risc tepk. Thk incidknck in induction rkmission stagk is highkst. Lpplication of hkpatoprotkctivk drugs is bknkficial to DIFI prognosis.

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.

Type 2 diabetes macrovascular disease is the main cause of death in type 2 diabetes mellitus. In recent years, the modern medical research and treatment of type 2 diabetes macrovascular disease has made some progress, but the international clinical trials suggest that the current treatment can not effectively reduce the incidence of this disease. Many clinical practices show that the effect of traditional Chinese medicine on this disease is exact, so that the clinical workers on the treatment of type 2 diabetes mellitus macrovascular disease of the status quo, now from the etiology and pathogenesis,clinical research, experimental research on the literature published in recent years, to provide reference for clinical treatment.

Objective To investigate the effect of triptolide (TP) on proliferation and apoptosis of cyst-lining epithelial cells with autosomal dominant polycystic kidney disease ( ADPKD). Methods Primary cultured cyst-lining epithelial cells were treated with TP at different concentrations for 12 h,24 h,48 h and 72 h, respectively.The proliferation activity of the cells was evaluated by Brdu assay. The cell cycle distribution was determined by flow cytometry. The apoptotic and apoptotic ratio were determined by FITC-AnnexinV binding/ PI. The morphological changes of cyst-lining epithelial cells were observed under transmission electron microscope. Results TP significantly inhibited the proliferation of cyst-lining epithelial cells and induced apoptosis in a dose- (10-40 ng?mL-1 )and time-dependent(12-48 h) manner. Typical ultrastructural changes of apoptotic cells were observed under electron microscope. Conclusion TP significantly inhibited the proliferation of cyst-lining epithelial cells and induced the apoptosis of cyst-lining epithelial cells, thus inhibited cyst forming and delayed cyst developing. The mechanism may involve several targets and pathways.

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.