Objective: To identify an optimal back-flush solution for leukocyte-depleted filters that maximizes peripheral blood mononuclear cell (PBMC) recovery with high viability, long-term storage stability, and sterility of the harvested residues, thereby providing a clinically translatable strategy. Methods: Three sterile bag-packaged solutions—Saline, Solvent, and Hanks' balanced salt solution (HBSS)—were used to back-flush randomly assigned leukocyte-depleted filters. Nucleated cell recovery rate and viability of the harvested residues were compared. The optimal solution identified was applied to an expanded sample set. PBMC viability and yield were evaluated after 1h vs 48h storage of the residues. PBMCs isolated from the residues were cryopreserved in liquid nitrogen for 1 month, followed by post-thaw comparisons of viability and T-cell expansion capacity. Results: The Solvent group achieved the highest and most consistent nucleated cell recovery rate. Post-flush recovery rate from filters after 400 mL whole blood processing was (21.3±1.6)% for the Solvent group, significantly higher than Saline group (19.2±6.3)% and HBSS group (11.2±5.0)%, with residues from all groups maintaining viability >90%. No biologically significant difference in residue viability was observed between 48h vs 1h storage groups (93.3±2.3)% vs (95.7±1.8)%). PBMC recovery rates from residues showed no statistical difference between 48h vs 1h storage groups [(48.2%±9.5%)vs (40.41%±8.35%), P>0.05], with (17.7±2.6)×10 cells. After 1-month cryopreservation and 10-day expansion, PBMCs isolated from 48-hour-stored residues retained (91.2±3.2)% viability and achieved a (61.9±15.9)-fold expansion. Conclusion: The bag-packaged Solvent, as a back-flush solution, enables sterile acquisition of leukocyte-depleted filter residues through closed-system tubing connections. These residues maintained PBMC viability and recovery rates after 48h storage at 2℃-8℃, with post-cryopreservation (1-month liquid nitrogen) viability and expansion capacity remaining stable. This protocol complies with blood bank regulatory criteria, addresses the concerns about the infectious window period in cell therapy raw materials, and provides a clinically translatable strategy for PBMC-based applications.

[摘 要] 目的：探讨含SET结构域蛋白5（SETD5）对结肠癌细胞增殖、迁移和对5-氟尿嘧啶（5-FU）药物敏感性的影响及机制。方法：常规培养结肠癌细胞，用Lipofectamine 2000将siSETD5-NC、si-SETD5-1~3质粒转染至HT-29细胞中，将其分为对照组（未处理）、si-SETD5-NC组、si-SETD5组和si-SETD5+SC79组，si-SETD5+SC79组HT-29细胞转染质粒的同时用10 µmol/L SC79处理。qPCR法检测NCM460、HT-29和LoVo细胞中SETD5 mRNA表达，流式细胞术、细胞划痕法、WB法和CCK-8法分别检测各组HT-29细胞的凋亡情况、迁移能力、相关蛋白的表达，以及对5-FU的敏感性。结果：SETD5 mRNA在HT-29、LoVo细胞中均呈高表达（均P<0.01）。在HT-29细胞中成功地敲减了SETD5 mRNA（P<0.01）。敲减SETD5 mRNA可明显抑制HT-29细胞的增殖活性（P<0.01）、迁移能力（P<0.01）、相关蛋白（SETD5、p-PI3K、p-AKT1、p-mTOR蛋白）的表达（均P<0.01）、促进细胞凋亡（P<0.01），且提高其对5-FU的敏感性（P<0.01），这些作用均可被AKT激活剂SC79部分阻挡（P<0.05或P<0.01）。结论：SETD5在HT-29、LoVo细胞中高表达，SETD5通过PI3K/AKT1通路促进结肠癌HT-29细胞的增殖、迁移，且降低其对5-FU的敏感性，SETD5是结肠癌临床诊断、治疗的潜在靶点。

Objective To establish interferon-induced transmembrane protein 3(Ifitm3)knockout mice and to explore the effects of Ifitm3 on the proliferation and differentiation of adult neural stem cells of mice(aNSCs).Methods IFITM3 knockout mice were established by the CRISPR/Cas9 method and identified by genotype identification and Western Blot.The differences between Ifitm3-knockout mice and wild-type mice were analyzed by hematoxylin-eosin(HE)staining and flow cytometry.The aNSCs of wild-type mice and Ifitm3-knockout mice were isolated and cultured,the number and size of neurospheres were detected,The ability of aNSCs to proliferate and differentiate were detected by quantitative reverse-transcription polymerase chain reaction,Western Blot,and immunofluorescence.Results Ifitm3-knockout mice were successfully established.The mice developed normally,and there were no obvious abnormalities either histopathologically or the immune system.In vitro experiments showed that Ifitm3 knockout inhibited the self-renewal potential of aNSCs,led to a decrease in the proliferation ability of aNSCs,and inhibited the differentiation of aNSCs into immature neurons and astrocytes.Conclusions This study finds that a lack of IFITM3 result in the ability of aNSCs to proliferate and differentiate decreased,IFITM3 may regulate the function of aNSCs.

Objective:To investigate the regulatory effect of long non-coding RNA (lncRNA) FTX on gastric cancer cell proliferation through miR-22-3p/NOD-like receptor protein 3 (NLRP3) inflammasome pathway.Methods:The gastric cancer cell line NCI-N87 were divided into blank control group, si-FTX-NC group, si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group. Quantitative real-time fluorescent PCR was performed to analyze the expression levels of lncRNA FTX and miR-22-3p, clone formation assay was performed to analyze the proliferation ability of NCI-N87 cells, western blotting was performed to analyze the expressions of NLRP3 inflammasome pathway proteins, and dual-luciferase reporter assay was performed to analyze the targeting relationship between lncRNA FTX and miR-22-3p.Results:The relative expressions of lncRNA FTX in the blank control group, si-FTX-NC group, si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group were 1.03±0.09, 1.01±0.15, 0.42±0.08, 0.45±0.06 and 0.46±0.13 respectively, with a statistically significant difference ( F=52.19, P<0.001). The relative expressions of miR-22-3p were 1.04±0.12, 0.97±0.08, 2.26±0.15, 2.23±0.13 and 1.15±0.11 respectively, with a statistically significant difference ( F=178.53, P<0.001). Compared with the blank control group and si-FTX-NC group, the relative expressions of lncRNA FTX in the si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group decreased (all P<0.001). Compared with the blank control group, si-FTX-NC group and si-FTX+miR-22-3p inhibitor group, the relative expressions of miR-22-3p in the si-FTX group and si-FTX+miR-22-3p inhibitor-NC group increased (all P<0.001). The clones of the five groups were 115.50±7.25, 112.33±8.46, 54.83±5.17, 56.17±6.32 and 85.67±9.43, with a statistically significant difference ( F=91.67, P<0.001). The levels of NLRP3 protein in the five groups were 1.84±0.17, 1.86±0.12, 0.95±0.09, 0.97±0.11 and 1.28±0.19, with a statistically significant difference ( F=60.62, P<0.001). Compared with the blank control group and si-FTX-NC group, the number of clones and the level of NLRP3 protein of the si-FTX group, si-FTX+miR-22-3p inhibitor-NC group and si-FTX+miR-22-3p inhibitor group decreased (all P<0.05). Compared with the si-FTX+miR-22-3p inhibitor group, the number of clones and the level of NLRP3 protein in the si-FTX group and si-FTX+miR-22-3p inhibitor-NC group decreased (all P<0.05). The dual-luciferase reporter assay found that miR-22-3p was the target gene of lncRNA FTX. Conclusion:Silencing the expression of lncRNA FTX can inhibit the proliferation of gastric cancer cells, and the mechanism may be related to the regulation of lncRNA FTX on the miR-22-3p/NLRP3 inflammasome pathway.

The healing process after skin injury is a dynamic process of interaction between various cells, cytokines, and extracellular matrix. Fibrosis is one of the main ways of skin injury repair. The process of fibrosis involves the regulation of many factors. Studies have shown that nerve regeneration-related protein (NREP) plays a key role in the fibrosis of skin tissue and organs. Based on the mechanism of skin fibrosis, this paper discusses the construction of tertiary structure of NREP, summarizes the effects of NREP and different cells in the skin on skin fibrosis and the research progress of mechanism of NREP in skin fibrosis, thus providing new ideas for the treatment of skin fibrosis diseases.

The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.
Adsorption ; Autoantibodies ; Dimethylpolysiloxanes ; Protein Array Analysis ; Silica Gel ; Surface Properties

Objective:To observe the short-term effects of intravitreal injection of aflibercept (IVA) for initial treatment and dressing change on exudative age-related macular degeneration (eAMD).Methods:A retrospective clinical study. From June 2018 to February 2021, forty-nine eAMD eyes of 38 patients who underwent IVA treatment in Department of Ophthalmology of Central Theater Command Hospital of People’s Liberation Army were included in the study. Among them, there were 24 males with 29 eyes and 14 females with 20 eyes; the average age was 66.82±8.71 years. All affected eyes were treated with IVA. The initial loading dose was 2.0 mg, which was injected once a month for 3 consecutive months, followed by monthly review and treatment as needed. Of the 49 eyes, 26 eyes were initially treated (initial treatment group), they were diagnosed within 3 months of the first onset and followed by IVA treatment, and no intraocular drugs and surgery were performed from the onset to the first diagnosis. Twenty-three eyes were treated with drug exchange therapy (dressing change group), they received intravitreal injection of ranibizumab and/or conbercept more than 4 times 6 months before the replacement therapy, during which there was persistent interlaminar cystoid edema and/or subretinal fluid (SRF) in the macular area and no improvement in pigment epithelial detachment (PED). Before IVA treatment, there were no statistically significant differences in the best corrected visual acuity (BCVA), foveal thickness (CMT), PED height (PEDH), and PED volume (PEDV) of the two groups of eyes before IVA treatment ( P>0.05). The same equipment and methods as before treatment were used for related examinations, and the changes of BCVA, CMT, PEDH, PEDV and complications of the two groups of eyes were recorded in 1, 3, and 6 months after treatment. The comparison of BCVA, CMT, PEDH, and PEDV between the two groups were used repeated measures analysis of variance. Results:Six months after treatment, the number of IVA injections in the eyes of the initial treatment group and dressing change group were 4.15±0.73 and 4.39±0.72 times, respectively, and the difference was not statistically significant ( t=-1.141, P=0.260). The BCVA, CMT, PEDH, and PEDV of the the initial treatment group ( F=5.345, 22.995, 6.764, 5.425) and the dressing change group ( F=12.519, 15.576, 8.843, 9.406) were significantly improved compared before treatment with 1, 3, and 6 months. All were statistically significant ( P<0.05). There was no significant difference in BCVA, CMT, PEDH, and PEDV between the initial treatment group and the dressing group at each time point after treatment ( F=1.741, 0.069, 0.876, 3.455; P>0.05). During the follow-up period, none of the affected eyes had complications such as persistent intraocular pressure increase, endophthalmitis, and retinal pigment epithelial tear. Conclusions:IVA can improve eyesight of patients with eAMD and reduce CMT, PEDH, and PEDV. The initial treatment and dressing change have the same effect.

Objective:To observe the efficacy of intravitreal injection of aflibercept (IVA) in the treatment of exudative age-related macular degeneration (wAMD) combined with RPE detachment (PED).Methods:A retrospective case study. From June 2018 to June 2019, 32 eyes (overall group) of 27 wAMD patients with PED were included in the study. All eyes were treated with IVA. The initial loading dose was 2.0 mg, which was injected once a month for 2 consecutive months and and then use a PRN regimen after evaluation. According to the maximum height of PED (PEDH) 2 months after treatment, the overall group was divided into the response group and the partial response group, with 20 (62.50%) and 12 (37.50%) eyes respectively. The response group: PEDH decreased by ≥25% compared with before treatment. The partial response: PEDH decreased by <25%. The macular fovea was scanned with the 3D-OCT 2000 instrument from Topcon (Japan). PEDH, PED area (PEDA), PED volume (PEDV), and macular foveal retinal thickness (CMT) were measured. There was no significant difference in BCVA, CMT, PEDH, PEDA, and PEDV of the eyes in the response group and the partial response group ( t=-0.791, -0.488, -0.900, -1.130, -0.400; P=0.435, 0.630, 0.380, 0.270, 0.690). The changes of BCVA, PEDH, PEDA, PEDV, CMT in each group were observed before treatment and 1, 2, 4, and 6 months after treatment. The comparison of BCVA and PED-related indicators and CMT before and after treatment were performed by repeated measures analysis of variance. Results:Compared with before treatment, the BCVA, CMT, PEDH, PEDA and PEDV of the eyes in the overall group, the response group, and the partial response group were obviously improved after treatment. Among them, there were statistically significant differences in all indicators of the overall group and the response group ( FBCVA=5.871, 3.798; P=0.001, 0.019. FCMT=24.526, 14.109; P=0.000, 0.001. FPEDH=12.569, 12.091; P=0.000, 0.000. FPEDA=7.534, 6.286; P=0.000, 0.000. FPEDV=5.139, 4.104; P=0.004, 0.014); there was no statistically significant difference in PED-related indicators in the partial response group ( FPEDH=3.210, P=0.054; FPEDA=1.913, P=0.183; FPEDV=3.500, P=0.051), the difference between BCVA and CMT was statistically significant ( FBCVA=3.033, P=0.027; FCMT=11.140, P=0.001). Two months after treatment, the eye number of PEDH reduction rate <25%, 25%-<50%, 50%-<75%, and ≥75% were 12 (37.50%), 8 (25.00%), 9 (28.13%), and 3 (9.38%) in the overall group, respectively. And PED in one eye (3.13%) was completely eliminated. Six months after treatment, the proportion was 13 (40.23%), 5 (15.63%), 7 (21.88%) and 7 (21.88%), respectively, among which 4 eyes (12.50%) with PED were completely resolved. Conclusions:Aflibercept treatment of wAMD combined with PED can restore its anatomical indicators and improve visual function of patients in a short time; the efficacy of PED in the PRN stage is related to the efficacy of the loading dose stage.

Objective: To investigate the influence of tumor necrosis factor-related apoptosis-inducing ligant (TRAIL) deficiency on mice colitis and the gut microbiota composition by inclding the expermental colitis model in tumor necrosis factor-related apoptosis-inducing ligand gene knockout (TRAIL^-/-) mice. Methods: C57BL/6 TRAIL^-/- mice and wild type (WT) mice were selected and assigned into TRAIL^-/- control group (eight mice), TRAIL^-/- colitis group (16 mice), WT control group (eight mice) and WT colitis group (16 mice). The mice of two colitis groups were oral administrated with 3.5% dextran sulphate sodium (DSS) in drinking water for seven consecutive days to induce experimental colitis model. The severity of colitis was evaluated by clinical appearance and histopathological examination. The colonic tissue samples of mice were collected and microbiota profile was analyzed by 16S rDNA sequencing method. USEARCH software and R language were used to analyze the difference of gut microbiota among TRAIL^-/- control group, TRAIL^-/- colitis group, WT control group and WT colitis group. T test and Mann-Whitney U test were used for statistical analysis. Results: After modeling, the disease activity index (DAI) of WT colitis mice and TRAIL^-/- colitis mice both gradually increased over time. Furthermore, compared with colitis mice, TRAIL^-/- colitis mice developed body weight loss, diarrhea and hemafecia earlier. On the seventh day after modeling, the percentage of body weight loss of TRAIL^-/- colitis mice and WT colitis mice was (28.98±2.84)% and (17.87±3.70)%, respectively; and the difference was statistically significant (t=9.53, P<0.01). The length of colon of TRAIL^-/- colitis mice was shorter than that of WT colitis mice ((4.63±0.28) cm vs. (6.02±0.41) cm), and the difference was statistically significant (t=11.20, P<0.01). The DAI of TRAIL^-/- colitis mice was higher than that of WT colitis mice (3.00±0.00 vs. 2.32±0.05), and the difference was statistically significant (t=54.40, P<0.01). The histological score of TRAIL^-/- colitis mice was higher than that of WT colitis mice (6.19±0.25 vs. 3.87±0.22), and the difference was statistically significant (t=27.87, P<0.01). Under the microscope, colonic mucosal epithelial injury, crypt structure destruction and inflammatory cell infiltration were more obvious in TRAIL^-/- colitis mice than in WT colitis mice. The alpha diversity of colonic flora was more significant in TRAIL^-/- colitis group compared with that of WT colitis group. At the family level, the relative richness of Deferribacteraceae, Ruminococcaceae, Rikenellaceae, F16 and Paraprevotellaceae significantly increased in TRAIL^-/- colitis group, but the relative richness of Enterococcaceae obviously reduced ((19.839±19.991)% vs. (7.224±11.241)%, (3.564±2.543)% vs.(2.861±3.821)%, (0.123±0.066)% vs. (0.068±0.049)%, (0.032±0.033)% vs. (0.006±0.011)%, (0.153±0.098)% vs. (0.062±0.054)% and (0.013±0.027)% vs. (0.054±0.121)%, respectively; U=51, 69, 53, 35, 49 and 69, respectively; P<0.01 and 0.05, respectively). In addition, at the genus level the relative richness of Oscillospira, Mucispirillum and Cytophaga in TRAIL^-/- colitis group remarkably elevated, and the relative richness of Enterococcus significantly decreased ((2.363±2.147)% vs. (1.813±2.847)%, (19.839±19.991)% vs. (7.223±11.241)%, (0.104±0.153)% vs. (0.046±0.069)% and (0.076±0.049)% vs. (0.135±0.074)%, respectively; U=70, 51, 66 and 65, respectively; P <0.05 and 0.01, respectively). Conclusion TRAIL deficiency aggravate DSS-induced colitis, and increase the alpha diversity of colonic microbiota in colitis mice.

With "moxibustion" and "warm stimulation" as the keywords, the literature on moxibustion mechanism of warming and dredging from June 1st, 1995 to June 1st, 2016 was collected from PubMed, China National Knowledge Infrastructure (CNKI) and Wanfang database. The startup mechanism of moxibustion warming and dredging function was analyzed in terms of moxibustion warming stimulation. The results were found that moxibustion was based on local rising temperature of acupoint. It activated local specific receptors, heat sensitive immune cells, heat shock proteins and so on to start the warming and dredging function and produce various local effects. The warming stimulation signals as well as subsequent effects through nerve and body fluid pathways induced the effects of further specific target organs and body systems.