Construction of luciferase-expressing pancreatic cancer cell lines and evaluation of their application in detecting cytotoxicity efficacy of immune cells / 中华微生物学和免疫学杂志

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.

Efficacy analysis of anti-platelet in the treatment of high-risk non-disabling ischemic cerebrovascular events guided by point-of-care testing of CYP2C19 gene / 中华神经科杂志

Objective:To explore the efficacy and safety of different anti-platelet regimens in the treatment of high-risk non-disabling ischemic cerebrovascular events (HR-NICE) guided by point-of-care testing of CYP2C19 gene. Methods:A single-centre, prospective, randomised, open-label, and blinded endpoint design was uesd in the study. From July 2020 to January 2022, HR-NICE patients were enrolled in the Stroke Green Channel and Department of Neurology of Xuzhou Central Hospital, and all patients were scraped the buccal mucosa for screening for CYP2C19 loss-of-function allele carriers by point-of-care testing . Patients with intermediate metabolism were defined as those who carried 1 loss-of-function allele and patients with poor metabolism were those who carried 2 loss-of-function alleles. This study reduced the test turnaround time to 1 hour by using a fully automated medical polymerase chain reaction analyzer for a point-of-care test of CYP2C19 genotype. CYP2C19 loss-of-function allele carriers were divided according to the random number table method into the conventional treatment group (clopidogrel 75 mg, once a day), the ticagrelor group (ticagrelor 90 mg, twice a day) and the intensive dose group (clopidogrel 150 mg, once a day) separately combined with aspirin (100 mg, once a day) dual antiplatelet for 21 days. Baseline information, Acute Stroke Org 10172 Treatment Trial staging, 90-day modified Rankin Scale score, occurrence of adverse events and severe adverse events were collected for all the 3 groups. The primary efficacy outcome was new stroke within 90 days, and the primary safety outcome was severe or moderate bleeding within 90 days. Results:A total of 716 patients were included: 240 in the conventional treatment group, 240 in the ticagrelor group and 236 in the intensive dose group. There was no statistically significant difference between the 3 groups at baseline (all P>0.05). There were 26 cases (10.8%) with new stroke events in the conventional treatment group, 11 cases (4.6%) in the ticagrelor group and 4 cases (1.7%) in the intensive dose group, with statistically significant differences among the 3 groups (χ 2=19.28, P<0.05), and the differences between the conventional treatment group and the ticagrelor group (χ 2=6.59, P=0.010) and between the conventional treatment group and the intensive dose group (χ 2=16.83, P<0.001) were statistically significant, whereas the difference between the ticagrelor group and the intensive dose group was not statistically significant ( P>0.05). In the 3 groups, there was 1 case (0.4%) of severe bleeding in the conventional treatment group, 6 cases (2.5%) in the ticagrelor group and none in the intensive dose group, which showed statistically significant differences (χ 2=7.23, P<0.05), and there was statistically significant difference between the ticagrelor group and the intensive dose group ( P=0.030). Among the patients with intermediate CYP2C19 metabolism, there were 13 cases (13/158, 8.2%) with 90-day recurrent stroke in the conventional treatment group, 4 cases (4/153, 2.6%) in the ticagrelor group, and 0 case (0/159) in the intensive dose group, with statistically significant difference (χ 2=16.04, P<0.001), and the differences between the intensive dose group and the conventional treatment group were statistically significant (χ 2=13.64, P<0.001), whereas there was no statistically significant difference between the intensive dose group and the ticagrelor group ( P>0.05). In the patients with 90-day recurrent stroke in the intensive dose group, there was 0 case (0/159) with intermediate metabolism and 4 cases (4/77,5.2%) with poor metabolism, with statistically significant differences ( P=0.011), whereas there were no statistically significant differences in the conventional treatment group and the ticagrelor group ( P>0.05). Conclusions:Screening carriers of CYP2C19 loss-of-function alleles by point-of-care testing can quickly and precisely guide the treatment of patients with non-cardiogenic HR-NICE. An intensive clopidogrel dose of 150 mg, once a day combined with aspirin was effective in reducing stroke recurrence with less occurrence of any bleeding and adverse events, and patients with intermediate CYP2C19 metabolism may be the best population to benefit.

Applicability of reference cells in lentiviral vector integration site detection with different methods / 中华微生物学和免疫学杂志

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.

Determination of Vitamin B2 in Weimeisu Tablets by HPLC-FLD / 中国药师

Objective:To establish a method for detecting the content of vitamin B2 in Weimeisu tablets by HPLC-FLD. Methods:A PAK C18(250 mmID × 4. 6 mm,5 μm) column was employed with methanol -0. 02 mol·L-1 ammonium acetate (35∶65) as the mobile phase, the flow rate was 1. 0 ml·min-1, and the injection volume was 10 μl. The detection wavelength λex and λem were 450mn and 522 mn, respectively. Results:Vitamin B2 was linear within the range of 1.0-20 μg·ml-1(r=0.999 9). The average recovery was 99. 20%(RSD=0. 63%, n=6). Conclusion:The method is specific, sensitive and accurate, and can be used to deter-mine vitamin B2 in Weimeisu tablets.

Selectively stimulating β1-adrenergic receptor attenuates rat myocardial ischemia/reperfusion injury in vivo by inhibiting high mobility group box 1 protein release / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate whether selectively stimulating β1-adrenergic receptor could inhibit high mobility group box 1 protein and attenuate myocardial ischemia/reperfusion(I/R) injury in rats.</p><p><b>METHODS</b>Eighty healthy male Sprague-Dawley (SD) rats were randomly divided into seven groups: (1) Sham operated group (SO); (2) Ischemia/reperfusion (I/R) group; (3) Dobutamine1 (5 µg×kg⁻¹ · min⁻¹) + I/R group; (4) Dobutamine2 (10 µg·kg⁻¹ × min⁻¹) + I/R group; (5) LY294002 (0.3 mg/kg) + Dobutamine2 + I/R group; (6) SB203580 (1 mg/kg) + Dobutamine2 + I/R group; (7) ZnPPIX (10 mg/kg) + Dobutamine2+I/R group. Rats were pretreated by saline, dobutamine, LY294002, SB203580 and ZnPPIX, respectively, then underwent myocardial I/R. Myocardial I/R injury and oxidative stress were assessed, and myocardial HO-1, NF-κB and HMGB1 expressions were measured by Western blot analysis.</p><p><b>RESULTS</b>Dobutamine significantly reduced the myocardial infarct size (P < 0.05), myocardial enzymes (LDH and CK) (P < 0.05) and proinfiammation cytokines (TNF-α and IL-6), reduced oxidative stress (MDA and SOD) in a dose-dependent manner (all P < 0.05). Meanwhile, dobutamine significantly and dose-dependently mediated the induction of HO-1 (P < 0.05), the expression of NF-κB (P < 0.05) and HMGB1 (P < 0.05). However, all the effects could be significantly reversed by co-treatment with LY294002, SB203580 and ZnPPIX (all P < 0.05).</p><p><b>CONCLUSIONS</b>Current study demonstrates that selectively stimulating β1-adrenergic receptor by dobutasmine could reduce rat myocardial I/R injury in vivo through promoting the induction of HO-1 and inhibiting HMGB1 release.</p>

Simultaneous Determination of Three Main Contents in Scrophulariae Radix Dispensing Particles by HPLC-UV Dual Wavelength Spectrometry Method / 中国中医药信息杂志

Objective To construct a simultaneous HPLC-UV dual wavelength spectrometry method for detecting three main contents - harpagide, harpagoside and cinnamic acid in Scrophulariae Radix dispensing particles. Methods Ultimate AQ-C18 column (250 mm×4.6 mm, 5μm) was used, with 1%acetic acid solution and acetonitrile as mobile phase of gradient elution. The flow rate was 1.0 mL/min, detection wavelength was 210 nm at former 13 min and 278 nm after 13 min. The column temperature was 30 ℃. Results The linear range of harpagide, harpagoside and cinnamic acid was 0.066 54-0.665 4 μg (r=1.000 0), 0.024 23-0.242 3 μg (r=0.999 9), and 0.100 28-1.002 8 μg (r=0.999 9), respectively. The average recovery (n=6) was 99.80%±1.22%, 100.31%±1.30% and 100.22%±1.24%, respectively. Conclusion The method is simple, repeatable, stable, and can be used for quality control and standardization of Scrophulariae Radix dispensing particles.