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.

ObjectiveTo investigate the effects of acupuncture combined with Du-Moxibustion (ADM) on peripheral blood cell count and levels of immune factors in patients with occupational chronic benzene poisoning. Methods A total of 70 patients with occupational chronic benzene poisoning (leukopenia and neutropenia) were selected as the research subjects by judgement sampling method. They were randomly divided into a control group and an ADM group using a random number table method, with 35 cases in each group. Patients in the control group were treated with conventional Western medicine such as leukocyte boosting and symptomatic treatment. While patients in the ADM group were treated with ADM treatment in addition to treatments of the control group, once per week for five consecutive weeks. Peripheral blood samples of patients were collected before and after treatment from both groups, to detect cell counts and serum levels of immune factors. Results The white blood cell count, red blood cell count, absolute lymphocyte count, absolute neutrophil count, platelet count, and levels of hemoglobin, immunoglobulins (Ig) A, IgM, IgG, complement C3 and complement C4 of patients in both groups improved after treatment compared with those before treatment (all P<0.05). The white blood cell count, levels of IgA, IgM, IgG, complement C3 and complement C4 of patients in the ADM group were higher than those in the control group after treatment (all P<0.05). Conclusion ADM treatment can increase peripheral blood white blood cells and serum levels of immune factor in patients with occupational chronic benzene poisoning (leukopenia, neutropenia), which helps improve patient recovery and can be promoted clinically.

Objective:To explore the effectiveness of limb motor rehabilitation program based on patient health engagement (PHE) model in patients with hemorrhagic stroke, and to provide reference for the limb motor rehabilitation management of hemorrhagic stroke patients.Methods:Through literature review and Delphi expert correspondence, a limb motor rehabilitation program for hemorrhagic stroke patients based on the PHE model was constructed. A non-contemporaneous controlled study was conducted, 45 hemorrhagic stroke patients hospitalized in the Department of Neurosurgery of Shanxi Bethune Hospital from March to August 2022 were selected by convenience sampling method as the control group, and routine exercise rehabilitation measure was given, 45 hemorrhagic stroke patients from September 2022 to February 2023 were selected as the intervention group, a limb motor rehabilitation program based on PHE model was implemented on the basis of control group. The functional exercise compliance, limb motor function, daily activity ability, emotional and social dysfunction of patients in the two groups were observed before intervention, 1 and 3 months after intervention respectively.Results:A total of 85 patients with hemorrhagic stroke were included. There were 42 patients in the intervention group, 25 males and 17 females, aged (52.07 ± 9.91) years old, and 43 patients in the control group, 21 males and 22 females, aged (53.93 ± 10.52) years old. There were no significant differences in the functional exercise compliance, limb motor function, daily activity ability, emotional and social dysfunction of patients before intervention between the two groups. At 3 months after intervention, the functional exercise compliance score in the intervention group was (40.83 ± 7.92) points, higher than that in the control group (37.14 ± 6.44) points, and the difference was statistically significant ( t = 2.36, P<0.05). At 1 and 3 months after intervention, the scores of limb motor function and daily activity ability in the intervention group were (27.12 ± 6.74), (33.67 ± 6.54) points and (61.31 ± 6.72), (74.40 ± 8.71) points, which were higher than (24.91 ± 6.03), (27.02 ± 6.59) points and (52.33 ± 9.78), (60.12 ± 10.03) points of the control group, the differences were statistically significant ( t values were 2.06-7.01, all P<0.05), the scores of emotional and social dysfunction were (75.52 ± 22.09) and (58.33 ± 18.88) points, which were lower than (86.02 ± 23.04), (78.51 ± 21.67) points of the control group, and the differences were statistically significant ( t = - 2.14, - 4.57, both P<0.05). Conclusions:The limb motor rehabilitation program based on the PHE model could improve the exercise compliance of patients with hemorrhagic stroke, improve the limb motor function and daily activity ability of patients, alleviate negative emotions, and reduce the level of social dysfunction.

【Objective】 To explore the intention of voluntary blood donation in students from a middle vocational school in Jinhua and its influencing factors. 【Methods】 A total of 400 students were enrolled from a middle vocational school in Jinhua by convenience sampling, and a self-designed questionnaire was issued to each student to investigate the cognition, intention and attitude of voluntary blood donation. The influencing factors of voluntary blood donation intention were analyzed by Logistic regression. 【Results】 A total of 394 valid questionnaires(98.50%) were collected. For 394 surveyed students, the average score of cognition of voluntary blood donation was (7.27±2.69) points, and the overall rate of intention to voluntary blood donation was 21.32%. Logistic regression analysis equation consisted of family support, better cognition, feeling happy about blood donation and worrying about blood donation, with risk ratios(OR) as 31.78, 188.69, 26.27 and 0.01, respectively(all P<0.05). 【Conclusion】 Students from a middle vocational school showed poor congnition and intention of voluntary blood donation.Family support for voluntary blood donation, better cognition of voluntary blood donation and happiness for blood donation were positive factors, and the fear of blood donation was a negative factor.It is necessary to strengthen the publicity of blood donation among students in middle vocational school to improve their congnition and intention to voluntary blood donation.

Objective:To analyze the difference of clinical characteristics and outcomes of infants with moderate and severe pediatric acute respiratory distress syndrome(PARDS)diagnosed according to baseline oxygenation index(OI) in pediatric intensive care unit(PICU).Methods:Second analysis of the data collected from the "Efficacy of pulmonary surfactant (PS) in the treatment of children with moderate and severe ARDS" program.Retrospectively compare of the differences in clinical data such as general condition, underlying diseases, OI, mechanical ventilation, PS administration and outcomes among infants with moderate and severe PARDS divided by baseline OI who admitted to PICUs at 14 participating tertiary hospitals from 2016 to December 2021.Results:Among the 101 cases, 55 cases (54.5%) were moderate and 46 cases (45.5%) were severe PARDS.The proportion of male in the severe group (50.0% vs.72.7%, P=0.019) and the pediatric critical illness score(PCIS)[72 (68, 78) vs.76 (70, 80), P=0.019] were significantly lower than those in the moderate group, while there was no significant difference regarding age, body weight, etiology of PARDS and underlying diseases.The utilization rate of high-frequency ventilator in the severe group was significantly higher than that in the moderate group (34.8% vs.10.9%, P=0.004), but there was no significant difference in PS use, fluid load and pulmonary complications.The 24 h OI improvement (0.26±0.33 vs.0.04±0.34, P=0.001) and the 72 h OI improvement[0.34 (-0.04, 0.62) vs.0.15 (-0.14, 0.42), P=0.029)]in the severe group were significantly better than those in the moderate group, but there was no significant difference regarding mortality, length of hospital stay and intubation duration after diagnosis of PARDS between the two groups. Conclusion:In moderate and severe(divided by baseline OI) PARDS infants with invasive mechanical ventilation, children in severe group have better oxygenation improvement in the early stage after PARDS identified and are more likely to receive high frequency ventilation compared to those in moderate group.Baseline OI can not sensitively distinguish the outcomes and is not an ideal index for PARDS grading of this kind of patient.

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.

Cellular immune responses as well as generalized and periarticular bone loss are the key pathogenic features of rheumatoid arthritis (RA). Under the pathological conditions of RA, dysregulated inflammation and immune processes tightly interact with skeletal system, resulting in pathological bone damage via inhibition of bone formation or induction of bone resorption. Single-cell omics technologies are revolutionary tools in the field of modern biological research.They enable the display of the state and function of cells in various environments from a single-cell resolution, thus making it conducive to identify the dysregulated molecular mechanisms of bone destruction in RA as well as the discovery of potential therapeutic targets and biomarkers. Here, we summarize the latest findings of single-cell omics technologies in osteoimmunology research in RA. These results suggest that single-cell omics have made significant contributions to transcriptomics and dynamics of specific cells involved in bone remodeling, providing a new direction for our understanding of cellular heterogeneity in the study of osteoimmunology in RA.
Humans ; Osteoclasts/physiology* ; Arthritis, Rheumatoid/pathology* ; Inflammation/pathology* ; Bone and Bones/pathology* ; Bone Resorption/pathology*

Objective:To study the effect of Wuzhi capsules on tacrolimus trough concentration in kidney transplant recipients with different CYP3A5 genotypes.Methods:From June 2015 to October 2019, 162 patients who underwent renal transplantation for the first time were retrospectively analyzed. The patients were divided into two groups, combined and uncombined, according to whether combined with Wuzhi capsules. There were 81 cases in the uncombined group (55 males and 26 females), and 81 in the combined group (62 males and 19 females). There was no significant difference between the two groups( P=0.219). The ages of the uncombined group and the combined group were (39.26±11.91) years old and (37.21±10.88) years old ( P=0.103), the weights were (62.39±11.64) kg and (66.18±13.89)kg ( P=0.298), systolic blood pressure were (147.28±20.24) mmHg and (145.00±16.42) mmHg (1 mmHg=0.133 kPa)( P=0.276), diastolic blood pressure were (92.25±13.87) mmHg and (92.20±12.53) mmHg ( P=0.886), alanine aminotransferase were (12.24±8.59) U/L and (17.06±13.11) U/L ( P=0.015), aspartate aminotransferase were (17.76±9.12) U/L and (16.57±8.37) U/L ( P=0.463), fasting blood glucose were (8.70±3.48) mmol/L and (7.18±2.74)mmol/L ( P=0.006), hemoglobin were (98.96±17.53) g/L and (101.05±18.67) g/L ( P=0.789), creatinine were (665.22±296.55) μmol/L and (797.32±279.32) μmol/L ( P=0.007), estimated glomerular filtration rate were (11.47±14.11) ml/(min·1.73m 2) and (8.85±3.71) ml/(min·1.73m 2) ( P=0.130)in the kidney transplant recipients before surgery. Among the 162 cases in this study, there were 86 cases (53.09%) of CYP3A5*1*3 genotype, 17 cases (10.49%) of CYP3A5*1*1 genotype, 59 cases (36.42%) of CYP3A5*3*3 genotype, and the minimum allele frequency of CYP3A5*1 was 37.04%. In the uncombined group, CYP3A5*1*3 genotype 39 cases (48.15%), CYP3A5*1*1 genotype 5 cases (6.17%), and CYP3A5*3*3 genotype 37 cases (45.68%). In the combined group, CYP3A5*1*3 genotype 47 cases (58.02%), CYP3A5*1*1 genotype 12 cases (14.81%), and CYP3A5*3*3 genotype 22 cases (27.16%), with statistically significant differences in the two groups ( P=0.024). The patients were treated with a triple immunosuppressive regimen (tacrolimus+ mycophenolate mofetil+ glucocorticoid) based on tacrolimus [initial dose: 0.15-0.30 mg/(kg·d)], combination of Wuzhi capsules in the combination group (11.25 mg, twice a day). The trough concentration of tacrolimus was detected by enzyme-linked immunosorbent assay, compare the difference in the trough concentration of tacrolimus between the two groups. The relationship between the effect of Wuzhi capsules and CYP3A5 gene polymorphism was compared, and compare the changes before and after the application of CYP3A5 genotype combined with Wuzhi Capsules. The influencing factors of tacrolimus trough concentration were analyzed by multiple linear regression. Results:In the combined with Wuzhi capsules, the dose corrected trough concentration (C 0/D) of tacrolimus was higher than that in patients without Wuzhi capsules, and the extent of increase was related to genotype. The C 0/D of tacrolimus in patients with CYP3A5*3*3 genotype in the combination and non-combination groups were (12.15±2.95) (ng·ml -1/0.1mg·kg -1·d -1) and (9.99±2.33) (ng·ml -1/0.1mg·kg -1·d -1) ( P=0.004), CYP3A5*1*3 genotype were (11.11±3.20) (ng·ml -1/0.1mg·kg -1·d -1) and (6.86±1.62) (ng·ml -1/0.1mg·kg -1·d -1) ( P<0.001), and there were significant difference. However, CYP3A5*1*1 genotype were(8.29±2.64) (ng·ml -1/0.1mg·kg -1·d -1) and (6.16±2.87) (ng·ml -1/0.1mg·kg -1·d -1) ( P=0.160), there was no significant difference. The tacrolimus C 0/D of the combined group before and after the Wuzhi capsule were as follows: CYP3A5*3*3 genotype: (7.18±2.33)(ng·ml -1/0.1mg·kg -1·d -1) and (13.33±3.09) (ng·ml -1/0.1mg·kg -1·d -1) ( P<0.001); CYP3A5*1*3 genotype: (5.14±2.14) (ng·ml -1/0.1mg·kg -1·d -1) and (10.61±3.20) (ng·ml -1/0.1mg·kg -1·d -1) ( P<0.001); CYP3A5*1*1 genotype: (5.17±3.75) (ng·ml -1/0.1mg·kg -1·d -1) and (8.31±2.74) (ng·ml -1/0.1mg·kg -1·d -1)( P=0.002), and the differences were statistically significant. The results of multiple linear regression showed that the combination of Wuzhi capsules (β=0.508, P<0.001) and CYP3A5 genotype(CYP3A5*1*3 and CYP3A5*3*3: β=-0.361, P<0.001; CYP3A5*1*1 and CYP3A5*3*3: β=-0.425, P<0.001)could influence the trough concentration. The sex (β=-0.100, P=0.124) and age (β=-0.003, P=0.967) of renal transplant recipients had no statistical significance to tacrolimus C 0/D. Conclusions:In the renal transplant patients, CYP3A5 genotype and combined use of Wuzhi capsules are the main factors affecting tacrolimus C 0/D. In order to achieve the expected trough concentration as soon as possible, the interaction between CYP3A5 genotypes and drug combination should be considered.

Objective:To evaluate the effect of evidence-based nursing practice for functional rehabilitation of post-stroke neurogenic bladder (PSNB) .Methods:From October 2020 to July 2021, 136 PSNB patients admitted to the Department of Neurology, Jiangyin People's Hospital were selected by convenience sampling as the research object. According to the random number table method, the patients were divided into the study group and the control group, with 68 cases in each group. The control group received routine rehabilitation nursing, while the study group was given evidence-based rehabilitation nursing based on the control group. The urodynamics, rates of bladder balance, urinary incontinence and urinary retention after intervention were compared between the two groups.Results:After three months of intervention, bladder compliance and bladder balance rate in the study group were higher than those in the control group, and the residual urine volume was less than that in the control group, with statistical differences ( P<0.05) . After three months of intervention, urinary retention rate in the study group was lower than that in the control group, and the difference was statistically significant ( P<0.05) . Conclusions:Evidence-based rehabilitation nursing can help to promote the rapid recovery of bladder balance in PSNB patients and improve the prognosis of patients.

【Objective】 To explore how the awareness and prevention status of COVID-19 epidemic, as well as anxiety affect blood donation behaviors of college students. 【Methods】 From June 7 to 13, 2020, students from two colleges in Jinhua were given self-made questionnaires through Wenjuanxing (a platform providing functions equivalent to Amazon Mechanical Turk), general demographic information, voluntary blood donation, core knowledge and prevention measures of COVID-19, as well as general anxiety were investigated. 466 valid questionnaires were collected, and the influencing factors of voluntary blood donation were analyzed by Logistic regression. 【Results】 College students donated(88.1±19.8)% were more likely to conduct qualified protections against COVID-19 than non-donation students(83.3±19.0)%(P<0.05), and be less anxious (9.68±4.80 vs 10.86±4.74, P<0.05). During COVID-19 outbreak, male donors was 3.732 times females(P<0.05). The donation rate of students with good awareness of COVID-19 protection, getting 60.0% correct or more, was 4.663 times students getting 60.0% below correct (P<0.05), and students with anxiety total score below 10 points was 2.227 times those above 10 points(P<0.05). 【Conclusion】 Gender, awareness of protections against COVID-19, and anxiety level are important factors affecting the blood donation behaviors of college students during COVID-19 epidemic, therefore strengthening psychological counseling and improving protection measures against COVID-19 are conductive to promote blood donation of college students during COVID-19 epidemic.