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.

OBJECTIVE: To observe the clinical effect of Fu's subcutaneous needling based on anatomy train theory for nonspecific low back pain (NLBP). METHODS: A total of 120 patients with NLBP were randomized into an anatomy train Fu's subcutaneous needling group (40 cases, 3 cases dropped out), a conventional acupuncture group (40 cases, 2 cases dropped out) and a conventional Fu's subcutaneous needling group (40 cases, 2 cases dropped out). Acupuncture was applied at ashi points and bilateral Shenshu (BL23) and Dachangshu (BL25) in the conventional acupuncture group, once every other day, 3 times a week. Fu's subcutaneous needling was applied at lumbodorsal myofascial trigger points (MTrPs) in the Fu's subcutaneous needling group, once every 3 days, twice a week. On the basis of the treatment in the Fu's subcutaneous needling group, Fu's subcutaneous needling was applied at MTrPs along the posterior superficial line and lateral line in the anatomy train Fu's subcutaneous needling group, once every 3 days, twice a week. All groups were treated for 2 weeks. Before and after treatment, the scores of numeric rating scale (NRS) and Oswestry disability index (ODI) were observed, the distance of Schober test was measured and the endurance of trunk extensors was assessed in the 3 groups. RESULTS: After treatment, in the 3 groups, the NRS and ODI scores were decreased compared with those before treatment (P<0.05), the Schober test distance was increased compared with that before treatment (P<0.05), the static and dynamic muscle endurance was increased compared with that before treatment (P<0.05). After treatment, in the anatomy train Fu's subcutaneous needling group, the NRS and ODI scores were lower than those in the conventional acupuncture group and the conventional Fu's subcutaneous needling group (P<0.05), the Schober test distance was longer than that in the conventional acupuncture group and the conventional Fu's subcutaneous needling group (P<0.05), the static and dynamic muscle endurance was superior to that in the conventional acupuncture group and the conventional Fu's subcutaneous needling group (P<0.05). CONCLUSION Fu's subcutaneous needling based on anatomy train theory can effectively relieve the pain symptom, enhance quality of life, improve lumbar motion and lumbar muscle function in patients with NLBP.
Humans ; Low Back Pain/physiopathology* ; Female ; Male ; Adult ; Acupuncture Therapy/methods* ; Middle Aged ; Acupuncture Points ; Young Adult ; Treatment Outcome ; Aged

OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

[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.

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.

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 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.