Development and verification of a rapid detection method for adalimumab biological activity based on U937-NF-κB-Luc cell line / 中国生物制品学杂志

@#Objective To develop and verify a rapid detection method for the biological activity of adalimumab based on U937-NF-κB-Luc cell line. Methods Using U937-NF-κB-Luc cell line as the detection cells,a method for detecting the biological activity of adalimumab was developed based on luciferase luminescence principle. The method was optimized for the concentration of tumor necrosis factor-α(TNF-α)(160 ng/mL as initial concentration,2 times serial dilution,10dilutions),the initial concentration of antibody(2 000 ng/mL,2 times serial dilution,20 dilutions),the dilution multiple of antibody(1. 5,2,3,4 times),the inoculation amount(8 × 103,2 × 104,4 × 104,6 × 104cells/well)and the incubation time(0. 5,1,2,3 h),and verified for the specificity,accuracy,precision and linear range. The relative potency of five batches of adalimumab was detected by using the optimized method and TNF-α neutralization activity method based on L929cells respectively. Results The dose-response curve of adalimumab international standard showed a typical S-type,and the data complied with the four-parameter equation y =(A-D)/[1 +(x/C)B]+ D,R2> 0. 99. The optimum concentration of TNF-α was 5 ng/mL,the initial concentration of antibody was 800 ng/mL,the dilution ratio for adalimumab was 1∶2,the inoculation amount was 2 × 104cells/well,and the induction time was 2 h. Three therapeutic monoclonal antibodies of TNF-α target,such as adalimumab,obtained good dose-response curves,while therapeutic monoclonal antibodies of other non-TNF-α targets did not show this curve. The linear regression equation of the logarithmic value of theoretical potency and the logarithmic value of the corresponding measured potency had a slope of 1. 037,and the relative bias was within the range of ± 12%. The geometric coefficient of variation(GCV)of the relative titer measured value of each sample was less than20%. The theoretical potency ranged from 64% to 156%,showing a good linear relationship with the measured values,and the fitting linear regression equation was y = 1. 037 4 x-0. 023 7,R2= 0. 998 4. There was no significant difference in the relative potency measured results of five batches of adalimumab by the two methods(t = 1. 198,P = 0. 265 1). Conclusion The developed detection method for adalimumab biological activity based on U937-NF-κB-Luc cell line has good specificity,accuracy and precision with short time consumption(3 h),which can be used as a rapid evaluation method for the biological activity of adalimumab.

Oncolytic Newcastle disease virus inhibits IL-6 induced migration and invasion of human glioblastoma U87MG cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨溶瘤新城疫病毒（NDV）对IL-6诱导的人胶质母细胞瘤U87MG细胞增殖、迁移和侵袭的作用及其可能的机制。方法：将U87MG细胞分为对照组、IL-6组、NDV组、NDV+IL-6组，其中IL-6组与NDV+IL-6组用75 ng/mL IL-6预处理1 h，其余组用DMEM预处理1 h，后分别用DMEM、75 ng/mL IL-6、1 HU NDV、1 HU NDV+75 ng/mL IL-6处理24 h。MTT法、细胞划痕实验和Transwell侵袭实验分别检测IL-6、NDV对U87MG细胞增殖、迁移和侵袭的影响，WB法检测各组细胞JAK2、p-JAK2、STAT3、p-STAT3和MMP2蛋白的表达水平。结果：与对照组相比，IL-6组细胞迁移率显著升高（P<0.05），侵袭细胞数目显著增多（P<0.01）；与IL-6组相比，NDV+IL-6组U87MG细胞增殖率显著降低（P<0.05），细胞迁移率和侵袭细胞数目均显著降低（均P<0.01）。WB实验结果显示，与对照组相比，IL-6组p-STAT3/STAT3比值显著升高（P<0.01），NDV组p-JAK2/JAK2、p-STAT3/STAT3比值显著降低（P<0.05，P<0.01），MMP-2蛋白表达量显著降低（P<0.01）；与IL-6组相比，NDV+IL-6组p-STAT3/STAT3比值、MMP-2蛋白表达量均显著降低（均P<0.05）。结论：NDV能抑制IL-6对人脑胶质瘤U87MG细胞迁移和侵袭的诱导作用，其机制可能与JAK2/STAT3信号通路的参与调控有关。

The efficacy of anti–TNF-? agents in the treatment of juvenile idiopathic arthritis-associated uveitis in a pediatric cohort

Purpose: Biologic therapy has shown promising control in children with often intractable juvenile idiopathic arthritis (JIA)?associated uveitis (JIA?U). Methods: This is a retrospective cohort study of 35 eyes of 35 children who received biologics for JIA?U. Pretreatment and posttreatment data (at 3, 6, 9, 12, 18, 24, and >24 months) were analyzed to determine functional success (stable/improved visual acuity), quiescence success (?0.5 cells in the anterior chamber), complete steroid success (termination of systemic, periocular therapy and decreased topical drops to ?2/day) or systemic steroid success (termination of systemic steroids only), and complete success (all of the above). Results: This study included 35 eyes up to 12 months and 21 eyes beyond 24 months. Steroid?sparing, functional, and quiescence success showed a rate of success of 52.43%, 77%, and 91%, respectively, at 12 months and 66.67%, 85.7%, and 76.2%, respectively, beyond 24 months. Complete success was 34.29% at 12 months, peaking at 18 months (65.62%) and reached 57.14% beyond 24 months. In their final follow?up, the best corrected visual acuity (BCVA) remained the same in 45.71%, improved in 37.14%, and worsened in 17.14% children. Conclusion: Biologic therapy is effective in JIA?U, especially in termination of systemic steroids, stabilization of vision, and maintaining quiescence

Prevalence of Smartphone Addiction and its Relation with Depression among School-going Adolescents

Background: Smartphone addiction among adolescents is an increasingly recognized problem worldwide. It affects the psychological well-being of an individual. Aim and objective: The current study aimed to assess smartphone addiction’s prevalence and its relation to depression among adolescents. Methods: This cross-sectional study was conducted among 400 school-going adolescents. Smartphone Addiction Scale - Short version (SAS-SV) and Patient Health Questionnaire (PHQ-9) were used to assess the prevalence of smartphone addiction and depression. Data were analyzed using Epi info software for windows (CDC, Atlanta). Statistical significance was set at p < 0.05. Results: The mean age of study participants was 14.4 years (SD=1.5 years). The prevalence of smartphone addiction was 23%, while depression was present among 45% of the study participants. Comparatively higher duration of smartphone use was significantly associated with smartphone addiction. Depression was significantly higher among smartphone addicts (77.2%) as compared to their counterparts (35.4%). Conclusion and Recommendation: The smartphone usage of adolescents, if not monitored, could lead to its addiction and thus increase the risk of depression among them. To prevent smartphone addiction, limiting children’s screen time is recommended. In this regard, parents can play a pivotal role by becoming responsible digital role models for their children.

Cloning and expression analysis of U6 promoters in Panax quinquefolius / 中国中药杂志

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Qiling Baitouweng Tang （QLBTWT） on proliferation and apoptosis， Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） signaling pathway and interleukin-10 （IL-10） in diffuse large B-cell lymphoma （DLBCL）. MethodWith human DLBCL cells OCI-LY10 and U2932 as research objects， cell proliferation was detected by cell counting kit-8 （CCK-8） assay. After treatment with 0， 4.6， 9.3， 18.7， 37.5， 75， 150 mg·L-1 QLBTWT for 24 h， the half-inhibitory concentration （IC50） of OCL-LY10 and U2932 cells was calculated to be 9.33， 16.13 mg·L-1， respectively， based on which， 9.5， 19， 38 mg·L-1 QLBTWT were selected for subsequent experiments. After 0， 9.5， 19， 38 mg·L-1 QLBTWT treatment for 24 h， the zymogen activities of Caspase-3， Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits （colorimetric）， and the IL-10 expression was detected by enzyme-linked immuno sorbent assay （ELISA）. The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly adenosine diphosphate ribose polymerase （cleaved PARP）， cleaved Caspase-3］， JAK2， STAT3， phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3） pathway proteins， and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0， 9.5， 19， 38 mg·L-1 QLBTWT were all tested by Western blot. ResultAfter QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， cell proliferation was inhibited in each QLBTWT group compared with that in the control group （P<0.05， P<0.01）. The zymogens of Caspase-3， Caspase-8 and Caspase-9 were activated （P<0.01）， and there was an increase in cell apoptosis （P<0.05， P<0.01） and cell cycle arrest at Gap phase1 （G1） phase in 9.5， 19 and 38 mg·L-1 QLBTWT group （P<0.05， P<0.01）. After 9.5， 19 and 38 mg·L-1 QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， the expressions of Bcl-2， p-JAK2 and p-STAT3 proteins were decreased （P<0.01）， and the expressions of Bax， cleaved PARP and cleaved Caspase-3 proteins were increased （P<0.01）， but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group， the expressions of c-Myc， p-JAK2， and p-STAT3 proteins were down-regulated in 19 mg·L-1 QLBTWT group and 19 mg·L-1 QLBTWT+10 μg·L-1 IL-10 group （P<0.05， P<0.01）， and up-regulated in 10 μg·L-1 IL-10 group （P<0.05， P<0.01）， while there was no difference in JAK2/STAT3 proteins. ConclusionQLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932， and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

Diffusion tensor field estimation based on 3D U-Net and diffusion tensor imaging model constraint / 南方医科大学学报

OBJECTIVE@#To propose a diffusion tensor field estimation network based on 3D U-Net and diffusion tensor imaging (DTI) model constraint (3D DTI-Unet) to accurately estimate DTI quantification parameters from a small number of diffusion-weighted (DW) images with a low signal-to-noise ratio.@*METHODS@#The input of 3D DTI-Unet was noisy diffusion magnetic resonance imaging (dMRI) data containing one non-DW image and 6 DW images with different diffusion coding directions. The noise-reduced non-DW image and accurate diffusion tensor field were predicted through 3D U-Net. The dMRI data were reconstructed using the DTI model and compared with the true value of dMRI data to optimize the network and ensure the consistency of the dMRI data with the physical model of the diffusion tensor field. We compared 3D DTI-Unet with two DW image denoising algorithms (MP-PCA and GL-HOSVD) to verify the effect of the proposed method.@*RESULTS@#The proposed method was better than MP-PCA and GL-HOSVD in terms of quantitative results and visual evaluation of DW images, diffusion tensor field and DTI quantification parameters.@*CONCLUSION@#The proposed method can obtain accurate DTI quantification parameters from one non-DW image and 6 DW images to reduce image acquisition time and improve the reliability of quantitative diagnosis.

A semi-supervised material quantitative intelligent imaging algorithm for spectral CT based on prior information perception learning / 南方医科大学学报

OBJECTIVE@#To propose a semi-supervised material quantitative intelligent imaging algorithm based on prior information perception learning (SLMD-Net) to improve the quality and precision of spectral CT imaging.@*METHODS@#The algorithm includes a supervised and a self- supervised submodule. In the supervised submodule, the mapping relationship between low and high signal-to-noise ratio (SNR) data was constructed through mean square error loss function learning based on a small labeled dataset. In the self- supervised sub-module, an image recovery model was utilized to construct the loss function incorporating the prior information from a large unlabeled low SNR basic material image dataset, and the total variation (TV) model was used to to characterize the prior information of the images. The two submodules were combined to form the SLMD-Net method, and pre-clinical simulation data were used to validate the feasibility and effectiveness of the algorithm.@*RESULTS@#Compared with the traditional model-driven quantitative imaging methods (FBP-DI, PWLS-PCG, and E3DTV), data-driven supervised-learning-based quantitative imaging methods (SUMD-Net and BFCNN), a material quantitative imaging method based on unsupervised learning (UNTV-Net) and semi-supervised learning-based cycle consistent generative adversarial network (Semi-CycleGAN), the proposed SLMD-Net method had better performance in both visual and quantitative assessments. For quantitative imaging of water and bone materials, the SLMD-Net method had the highest PSNR index (31.82 and 29.06), the highest FSIM index (0.95 and 0.90), and the lowest RMSE index (0.03 and 0.02), respectively) and achieved significantly higher image quality scores than the other 7 material decomposition methods (P < 0.05). The material quantitative imaging performance of SLMD-Net was close to that of the supervised network SUMD-Net trained with labeled data with a doubled size.@*CONCLUSIONS@#A small labeled dataset and a large unlabeled low SNR material image dataset can be fully used to suppress noise amplification and artifacts in basic material decomposition in spectral CT and reduce the dependence on labeled data-driven network, which considers more realistic scenario in clinics.

Effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis of human glioma cells / 中国药房

ABSTRACT OBJECTIVE To investigate the effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis in human glioma U87 and U251 cells. METHODS Using human glioma U87 and U251 cells as subjects， the proliferation and apoptosis， as well as the expression of histone deacetylase 1（HDAC1） protein and apoptosis-related proteins [B cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved caspase-3] were detected after being treated with different concentrations of ginsenoside Rh2. RESULTS The concentrations of 10，20，30，40，50，60，70，80 μmol/L ginsenoside Rh2 could generally significantly increase the proliferation inhibition rate of U87 and U251 cells （P＜0.05 or P＜0.01）， and the half inhibitory concentrations of this component after 48 hours of action were 51.34 and 55.84 μmol/L， respectively；30，50 μmol/L ginsenoside Rh2 could increase the total apoptotic rate of both types of cells， reduced the protein expressions of HDAC1 and Bcl-2， and increased the protein expressions of Bax and cleaved caspase-3 significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Ginsenoside Rh2 has a significant inhibitory effect on the proliferation of glioma cells and promotes the apoptosis of cells， which may be through reducing the expression of HDAC1 protein and activating the Bcl-2 family protein-mediated apoptosis pathway.

Effects of over-expression of E2F transcription factor 1 on radiosensitivity glioma U251 cells / 中华行为医学与脑科学杂志

Objective:To investigate the effects of over-expression of E2F transcription factor 1 (E2F1) on proliferation, invasion, apoptosis and radiosensitivity of glioma cell U251.Methods:Real-time quantitative PCR (qRT-PCR) were used to detect the differential expression of E2F1 mRNA in glioma cells LN18, SW1088, U251 and normal brain glial cells. The stable over-expression of E2F1 plasmid was constructed and transfected into U251 cells. qRT-PCR and Western blot test were used to detect the expression of E2F1, pituitary tumor transforming gene 1(PTTG1), C-Myc, B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax) mRNA and protein expression in the control group and E2F1 over-expression group.U251 cells were divided into control group(no X-ray irradiation), irradiation group(6 Gy dose of X-ray), and irradiation + E2F1 over-expression group(transfected with E2F1 first, then irradiated by 6 Gy of X-ray). Cell proliferation ability was detected by cell counting Kit-8(CCK-8) cell viability detection reagent, and cell invasion and migration ability were detected by Transwell chamber. Apoptosis and cell cycle were detected by flow cytometry.GraphPad Prism 8.0 was used for data analysis.The statistical methods were one-way ANOVA and independent sample t-test. Results:qRT-PCR showed that there was statistical difference in the mRNA levels of E2F1( F=201.92, P<0.05) in different cell lines.The expression levels of E2F1 mRNA in LN18(4.04±0.29), SW1088(3.19±0.16)and U251(4.66±0.20) cells were higher than those in HEB(1.02±0.07)cells ( q=27.00, 19.40, 32.52, all P<0.05). After successfully constructing U251 cells with stable over-expression of E2F1 plasmid, qRT-PCR and Western blot detection results showed that: the mRNA and protein levels of E2F1, PTTG1, C-Myc and Bcl-2 in E2F1 over-expression group were higher than those in control group ( t=77.16, 57.88, 4.63, 51.13, 7.50, 70.85, 8.38, 48.81, all P<0.05). Bax mRNA(0.20±0.01) and protein(0.66±0.01) levels were lower than those in control group((1.00±0.02), (0.94±0.01)), and the differences were statistically significant ( t=1.74, 54.65, both P<0.05). After X-ray irradiation (6 Gy), CCK8 detection results showed: the proliferation ability of the three groups at 24, 48, 72 and 96 h were significantly different ( F=95.41, 187.53, 1 158.49, 7 883.78, all P<0.05). The proliferation capacity of the irradiation group were lower than those of the control group at 24, 48, 72 and 96 h ( q=19.51, 27.20, 66.60, 174.9, all P<0.05). The proliferation capacity of irradiation + E2F1 over-expression group at 24, 48, 72 and 96 h were higher than those of irradiation group ( q=10.63, 10.81, 21.11, 60.90, all P<0.05). Transwell assay results showed that there were significant differences in cell invasion and migration ability among the three groups ( F=315.38, 681.10, both P<0.05). The invasion and migration ability of cells in the irradiation group were lower than those in the control group ( q=35.09, 12.76, both P<0.05), and the invasion and migration ability of cells in the irradiation + E2F1 over-expression group were higher than those in the irradiation group ( q=52.06, 22.81, both P<0.05). Flow cytometry showed that there were significant differences in apoptosis rate and percentage of cells in each cycle among the three groups ( F=667.63, 3 213.30, 3 011.26, 861.98, all P<0.05). The percentage of the apoptosis rate, S phase and G2 phase cells in the irradiation group were higher than those in the control group ( q=51.10, 89.39, 51.82, all P<0.05), while the percentage of G1 phase cells in the irradiation group was lower than that in the control group ( q=141.2, P<0.05). The apoptosis rate and percentage of S phase and G2 phase cells in the irradiation + E2F1 over-expression group were lower than those in the irradiation group ( q=18.87, 41.42, 29.31, all P<0.05), while the number of G1 phase cells in the irradiation + E2F1 over-expression group was lower than that in the irradiation group ( q=70.73, P<0.05). Conclusion:Over-expression of E2F1 can reduce the radiosensitivity of glioma U251 cells by regulating the expression of mRNA and protein of genes related to cell cycle and apoptosis, and E2F1 may be involved in the radioresistance of glioma cells.

Effects of butylphthalide combined with hyperbaric oxygen therapy on post-stroke cognitive impairment / 中国基层医药

Objective:To investigate the clinical efficacy of butylphthalide combined with hyperbaric oxygen therapy on post-stroke cognitive impairment in patients with acute ischemic stroke.Methods:A total of 90 patients with post-stroke cognitive impairment who were hospitalized within 72 hours of onset in Suining County People's Hospital from December 2019 to November 2020 were included in this study. They were randomly divided into a control group and an observation group ( n = 45/group). The control group was given conventional treatment and the observation group was given butylphthalide combined with hyperbaric oxygen therapy in addition to conventional treatment. The National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score were compared between the two groups before and after treatment. Results:Before treatment, there were no significant differences in the National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score between the two groups (all P > 0.05). At 14 days and 1 month after surgery, the National Institutes of Health Stroke Scale scores in the observation group were (4.02 ± 2.18) points and (3.21 ± 2.03) points, which were significantly lower than (5.21 ± 2.24) points and (4.62 ± 2.68) points in the control group ( t =2.55, 2.81, both P < 0.05). At 1 and 3 months after treatment, the Montreal Cognitive Assessment score in the observation group were (19.79 ± 5.67) points and (23.69 ± 2.67) points, which were significantly higher than (16.88 ± 5.12) points and (19.74 ± 2.29) points in the control group ( t = 2.56, 7.53, both P < 0.05). At 1 and 3 months after treatment, Activities of Daily Living scores in the observation group were (54.85 ± 5.69) points and (74.38 ± 4.98) points, which were significantly higher than (46.78 ± 6.24) points and (63.21 ± 5.24) points in the control group ( t = 6.41, 9.76, both P < 0.05). Conclusion:Butylphthalide combined with hyperbaric oxygen therapy for the treatment of post-stroke cognitive impairment in patients with acute ischemic stroke can alleviate neurologic deficits, and improve cognitive function and the ability of daily life.

FHL2 affects the resistance of glioblastoma U87 cells against temozolomide via MGMT / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨干扰四个半LIM结构域蛋白2（FHL2）的表达对胶质母细胞瘤U87细胞中O6-甲基鸟嘌呤DNA甲基转移酶(MGMT) 表达的影响，以及对U87细胞替莫唑胺（TMZ）耐药性的影响。方法：利用慢病毒感染技术分别将携带不同FHL2干扰序列的慢病毒（shFHL2-1#、shFHL2-4#）及其阴性对照（shN）感染U87细胞，分别命名为shFHL2-1#、shFHL2-4#和shN组；采用siRNA转染技术将siMGMT-1#、siMGMT-4#和siN转染至U87细胞，为siMGMT-1#、siMGMT-4#和siN组，qPCR和WB法验证FHL2或MGMT的敲低效果。用TMZ处理上述各组细胞（以DMSO处理组为对照），随后以CCK-8法和细胞克隆形成实验检测TMZ处理前后FHL2或MGMT敲低组细胞的增殖情况，FCM检测TMZ处理前后FHL2敲低组细胞的凋亡情况，WB法和免疫荧光法检测敲低FHL2对U87细胞中MGMT表达的影响，WB法检测TMZ处理对各组细胞中FHL2和MGMT表达水平的影响。结果：成功构建敲低FHL2或MGMT表达的U87细胞。与shN组相比，shFHL2-1#、shFHL2-4#组U87细胞的增殖能力减弱、凋亡水平升高（均P<0.01），MGMT表达水平明显降低（均P<0.01）。经TMZ处理后，与相应的DMSO处理组相比，shN组细胞中FHL2和MGMT的表达水平显著升高（均P<0.05），而细胞的增殖和凋亡均无显著变化（均P>0.05）；shFHL2-1#、shFHL2-4#组细胞中FHL2和MGMT的表达水平无显著改变（均P>0.05），但细胞增殖能力进一步显著降低、凋亡水平进一步显著升高（均P<0.01）。敲低MGMT使U87细胞增殖减慢（P<0.01），而siMGMT-1#、siMGMT-4#组细胞经TMZ处理后增殖能力进一步降低（均P<0.01）。结论：干扰FHL2表达使得U87细胞增殖减慢而凋亡加剧、MGMT表达下调，提示FHL2可能通过影响MGMT的表达调控U87细胞对TMZ的耐药性。

The Application and Pitfalls for Registering New Japanese Board of Cardiovascular Surgery / 日本心臓血管外科学会雑誌

The first examination of the new board of the Japanese cardiovascular surgery took place in 2022. As it is a transitional period for the new system, many doctors are not familiar with the changes and details of the new system, and some have their concerns. Here, we held a round-table discussion with doctors who actually took the new board of the Japanese cardiovascular surgery under the new system, and we summarized their opinion.

Application of U-Net network in automatic image segmentation of adenoid and airway of nasopharynx / 临床耳鼻咽喉头颈外科杂志

Objective:To explore the effect of fully automatic image segmentation of adenoid and nasopharyngeal airway by deep learning model based on U-Net network. Methods:From March 2021 to March 2022, 240 children underwent cone beam computed tomography（CBCT） in the Department of Otolaryngology, Head and Neck Surgery, General Hospital of Shenzhen University. 52 of them were selected for manual labeling of nasopharynx airway and adenoid, and then were trained and verified by the deep learning model. After applying the model to the remaining data, compare the differences between conventional two-dimensional indicators and deep learning three-dimensional indicators in 240 datasets. Results:For the 52 cases of modeling and training data sets, there was no significant difference between the prediction results of deep learning and the manual labeling results of doctors（P>0.05）. The model evaluation index of nasopharyngeal airway volume: Mean Intersection over Union（MIOU） s （86.32±0.54）%; Dice Similarity Coefficient（DSC）: （92.91±0.23）%; Accuracy: （95.92±0.25）%; Precision: （91.93±0.14）%; and the model evaluation index of Adenoid volume: MIOU: （86.28±0.61）%; DSC: （92.88±0.17）%; Accuracy: （95.90±0.29）%; Precision: （92.30±0.23）%. There was a positive correlation between the two-dimensional index A/N and the three-dimensional index AV/（AV+NAV） in 240 children of different age groups（P<0.05）, and the correlation coefficient of 9-13 years old was 0.74. Conclusion:The deep learning model based on U-Net network has a good effect on the automatic image segmentation of adenoid and nasopharynx airway, and has high application value. The model has a certain generalization ability.

The effect and mechanism of Xihuang pill inhibits the formation of vasculogenic mimicry in human glioblastoma cells by down-regulating HIF-1α/VEGFA/VEGFR2 signaling pathway / 药学学报

Our studies were aimed to explore the effect and mechanism of the inhibition of the formation of vasculogenic mimicry (VM) in human glioblastoma cells by Xihuang pill (XHP) medicated serum through regulating the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. The medicated serum of XHP was prepared by gavage for 7 days to male SD rats (approval number of animal experiment ethics: 202105A051). The hypoxia model of U251 cells was established using 200 μmol·L-1 of CoCl2. After treatment with XHP-medicated serum, cell viability and proliferation of U251 cells were detected by CCK-8 and cell cloning experiment. Cell apoptosis and cell cycle of U251 cells were determined by flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell invasion assay. The formation of VM was assessed by three-dimensional cell culture of U251 cells. The protein expression levels of HIF-1α, VEGFA, VEGFR2, phosphorylated-VEGFR2 (p-VEGFR2), vascular endothelial-cadherin (VE-cadherin), Eph receptor tyrosine kinases A2 (EphA2), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 14 (MMP14) and laminin γ2 in U251 cells were detected by Western blot. The results showed that 10% XHP-medicated serum had little effect on the cell viability, proliferation, apoptosis and cell cycle of U251 cells under hypoxia. Compared with the model group, 10% XHP-medicated serum at 1.0, 1.5 and 2.0 h significantly decreased the migration rate (P < 0.01) and the number of invading U251 cells (P < 0.01). 10% XHP-medicated serum at 2.0 h significantly suppressed the formation of VM tubular structures in U251 cells under the condition of hypoxia (P < 0.01). Western blot experiment showed that 10% XHP-medicated serum significantly down-regulated the expression of HIF-1α, VEGFA, phospho-VEGFR2, VE-cadherin, EphA2 and MMP14 proteins (P < 0.05). In conclusion, XHP could inhibit the formation of VM in human glioblastoma U251 cells to suppress the angiogenesis by down-regulating the HIF-1α/VEGFA/VEGFR2 signaling pathway.

Preliminary application of artificial intelligence in the pathological diagnosis of periapical cysts / 口腔疾病防治

Objective@#To study the effect of artificial intelligence in the pathological diagnosis of periapical cysts and to explore the application of artificial intelligence in the field of oral pathology.@*Methods@#Pathological images of eighty-seven periapical cysts were selected as subjects to read, and a neural network with a U-net structure was constructed. The 87 HE images and labeled images of periapical cysts were divided into a training set (72 images) and a test set (15 images), which were used in the training model and test model, respectively. Finally, the target level index F1 score, pixel level index Dice coefficient and receiver operating characteristic (ROC) curve were used to evaluate the ability of the U-net model to recognize periapical cyst epithelium.@*Results @# The F1 score of the U-net network model for recognizing periapical cyst epithelium was 0.75, and the Dice index and the areas under the ROC curve were 0.685 and 0.878, respectively.@*Conclusion@#The U-net network model constructed by artificial intelligence has a good segmentation result in identifying periapical cyst epithelium, which can be preliminarily applied in the pathological diagnosis of periapical cysts and is expected to be gradually popularized in clinical practice after further verification with large samples.

Targeted killing effect of bispecific CAR-T cells on EGFRvⅢ+/CD133+ glioma stem cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：制备双特异性CAR-T（bsCAR-T）细胞，观察其对表达表皮生长因子Ⅲ型突变阳性（EGFRvⅢ+，简称vⅢ+）和CD133+胶质瘤干细胞的靶向杀伤作用。方法：基于前期研制的vⅢ/CD133双特异性微抗体和二代CAR构建的双特异性CAR（bsCAR），制备慢病毒载体转染人外周血T细胞，FCM和WB法检测bsCAR转染效率和表达水平。bsCAR-T细胞和vⅢ+/CD133+ U87胶质瘤干细胞共培养，乳酸脱氢酶（LDH）释放实验、IFN-γ分泌实验检测其特异性杀伤作用和对IFN-γ分泌的促进作用。制备裸鼠vⅢ+/CD133+ U87干细胞移植瘤模型检测bsCAR-T细胞对移植瘤生长的抑制作用。结果：vⅢscFv和CD133scFv通过重叠PCR无缝连接入二代CAR表达框（S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3z）中，然后克隆入pCDH-MSCV-MCS-EF1-copGFP载体的EcoRⅠ和BamHⅠ位点（pbsCAR）。3种质粒（pVSV-G、pCMV-dR8.9和pbsCAR）共转染HEK293T细胞制备慢病毒载体，转染外周血T细胞，FCM检测bsCAR表达率为71.1%，WB法结果显示bsCAR表达正确。bsCAR-T细胞和vⅢ+/CD133+ U87干细胞共培养检测结果显示，bsCAR-T细胞对胶质瘤干细胞具有特异性杀伤作用，与效靶比呈正比；IFN-γ分泌量为（2 350.6±92） pg·mL-1，明显高于对照组（P<0.01）。裸鼠移植瘤动物模型显示，bsCAR-T细胞在体内具有明显的移植瘤抑制作用（P<0.01）。结论： bsCAR-T细胞能够特异性靶向杀伤vⅢ+/CD133+胶质瘤干细胞，实验结果为促进实体瘤的细胞免疫治疗提供了实验依据。

Current Status and Issues of Cardiovascular Surgery Institution in Community Medicine / 日本心臓血管外科学会雑誌

Lately, there has been a trend towards integration among cardiovascular surgery institutions. However, local institutions continue to play a crucial role in community-based medicine, given the emergent nature of cardiovascular diseases and the challenges involved in transporting patients with such conditions over long distances. We present the results of a questionnaire survey we conducted to examine the current status and issues faced by cardiovascular surgery institutions in community-based medicine.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Novel Pulmonary Nodule Position Detection Method Based on Multiscale Convolution / 中国医疗器械杂志

OBJECTIVE@#In order to improve the accuracy of the current pulmonary nodule location detection method based on CT images, reduce the problem of missed detection or false detection, and effectively assist imaging doctors in the diagnosis of pulmonary nodules.@*METHODS@#Propose a novel method for detecting the location of pulmonary nodules based on multiscale convolution. First, image preprocessing methods are used to eliminate the noise and artifacts in lung CT images. Second, multiple adjacent single-frame CT images are selected to be concatenate into multi-frame images, and the feature extraction is carried out through the artificial neural network model U-Net improved by multi-scale convolution to enhanced feature extraction capability for pulmonary nodules of different sizes and shapes, so as to improve the accuracy of feature extraction of pulmonary nodules. Finally, using point detection to improve the loss function of U-Net training process, the accuracy of pulmonary nodule location detection is improved.@*RESULTS@#The accuracy of detecting pulmonary nodules equal or larger than 3 mm and smaller than 3 mm are 98.02% and 96.94% respectively.@*CONCLUSIONS@#This method can effectively improve the detection accuracy of pulmonary nodules on CT image sequence, and can better meet the diagnostic needs of pulmonary nodules.