Objective: To investigate the impact and mechanism of NPM1 gene expression on acute myeloid leukemia (AML) cell lines. Methods: Human AML cell line U937 and HL-60 cells were transfected with NPM1 plasmid to establish stable clones, and the high NPM1 protein expression (NPM1^hi) clones were screened by Western blot. The cell proliferation was assayed by methylthiazolyl tetrazolium bromide (MTT) , cell cycle and cell apoptosis by flow cytometric, cell colony formation by microscope count, the molecular pathways related to cell cycle by Western blot. The expression of NPM1 gene in primary AML bone marrow mononuclear cells (BMMC) was investigated by RQ-PCR. Results: ①The proliferation of NPM1^hi U937 and HL-60 cells was similar with that of control cells (4.68±1.28 vs 3.89±0.81, 3.34±0.37 vs 2.68±0.29, P>0.05) . ②The percentage of S phase in NPM1^hi U937 and HL-60 cells was higher than that in control cells[ (50.22±3.42) % vs (39.78±3.80) %, (59.01±3.27) % vs (43.94±2.08) %, P<0.05]. ③The anti-apoptosis capacity and colony formation abilities of NPM1^hi U937 cells increased than that of control cells[ (68.8±10.2) % vs (48.7±3.22) %, and (772.7±24.0) vs (652.3±16.5) , P<0.05], but the above abilities of NPM1^hi HL60 cells were similar with that of control cells. ④ The expressions of CDK4, Cyclin D1, Cyclin D2 and Cyclin E in NPM1^hi leukemia cells were higher than that of control cells, but the expression of Cyclin D3 was lower. ⑤The NPM1 expression levels in AML patients with favorable cytogenetic prognosis were lower than that of patients with intermediate prognosis. Conclusions NPM1 protein could promote more cells to enter S phase, enhance the ability of antiapoptosis and colony formation in AML cell lines. The quantitative level of NPM1 may predict the cytogenetic risk of AML patients.

OBJECTIVE:To establish HPLC fingerprint of Bining nasal spray.METHODS:HPLC method was performed.The determination was performed on Neptune C18 column with mobile phase consisted of acetonitrile-0.2 ％ phosphoric acid (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set at 326 nm,and the column temperature was 25 ℃.The sample size was 10 μL.Using rutin as reference,HPLC chromatograms of 20 batches of samples were determined.Common peak identification and similarity evaluation were conducted by using Similarity Evaluation System for TCM Fingerprint (2012 edition).RESULTS:There were 40 common peaks in HPLC chromatograms of 20 batches of Bining nasal spray,with similarity ＞0.9.After validation,HPLC chromatograms of 20 batches of samples were in good agreement with the control fimgerprints of those.CONCLUSIONS:Established fingerprint can provide reference for identification and quality evaluation of Bining nasal spray.

Objective: To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Methods: Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot. Results: ①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (P<0.05) , respectively under ATRA exposure. ②The percentages of G₀/G₁ stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure. Conclusions ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G₀/G₁ stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.

Objective:Based on cognitive behavioral therapy, to construct a physical and mental adjustment intervention plan for the main caregiver of cancer patients through the network platform.Methods:Through evidence-based literature published from July 2012 to July 2022 screening and evaluation, combined with qualitative interviews for 10 primary caregivers of cancer patients, the intervention plan for physical and mental adjustment of the main caregivers of cancer patients was preliminarily formulated. After consultation with Delphi experts (15 cases) through two rounds, the intervention plan was finally determined.Results:In the two rounds of expert letter inquiries, 15 questionnaires were distributed and 15 valid questionnaires were recovered. The effective recovery rate was 100.00% and the expert authority coefficient were 0.89 and 0.90 in the two rounds of expert letter inquiries respectively, the Kendall harmony coefficients were 0.279 and 0.323 respectively, and the differences were all statistically significant ( P<0.01). The intervention plan for physical and mental adjustment ofthe main caregivers of cancer patients included 5 first-level indicators (basic knowledge, symptom education, home care knowledge, relaxation training, social support), 27 second-level indicatorsand 54 third-level indicators. Conclusions:The method of the psychosomatic regulation intervention program is scientific and practical, which can be initially applied to the psychological adjustment of the main caregivers of cancer patients, so as to provide a reference for improving their negative emotions.

Objective To construct recombinant human butyrylcholinesterase(rhBChE)knock-in goat fetal fibroblast cell lines(GFFs)by using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated homology-directed repair mechanism for subsequent production of goat expressing rhBChE.Methods The efficient sgRNA sites targeting goat[3-casein(CSN2)gene were designed and screened,and the targeting efficiency of the sgRNA in goat mammary epithelial cells(GMECs)was confirmed by electro-transfection,flow sorting,and sequencing of PCR products.The red fluorescent reporter gene homology repair vector(P2A-mCherry)targeting the sgRNA was constructed,and then the integration and expression efficiency was detected by flow cytometry.The rhBChE homology repair vector(P2A-rhBChE)targeting the sgRNA of CSN2 gene was constructed in GFFs,the rhBChE positive cell clones were obtained via electro-transfection and flow sorting,and the rhBChE knock-in cell lines was identified by sequencing of PCR products.Results The sgRNA4 was identified as an efficient target of goat CSN2 gene,which could be also used for targeted integration of other genes.Three rhBChE knock-in cell lines were successfully constructed.Conclusion The rhBChE knock-in GFFs targeting goat CSN2 gene lays the foundation for the production of mammary bioreactors expressing rhBChE.

Objective:To construct the core competency framework and content for cardiac rehabilitation specialist nurses so as to provide a basis for training, use and assessment for cardiac rehabilitation specialist nurses.Methods:From December 2018 to December 2019, this study initially formed the core competency framework and content for cardiac rehabilitation specialist nurses by literature review and expert interview. Delphi method was used to 17 experts and two rounds of consultations. Items were screened and revised, and item weight was determined by combining with Precedence chart.Results:Among two rounds of expert consultation, the core competency framework and content for cardiac rehabilitation specialist nurses included 5 first-level items, 27 second-level items and 107 third-level items. The valid response rates of two rounds of expert consultation were 100% and 94.12%, and authoritative coefficients were 0.894 and 0.900 respectively. In the second round of expert consultation, Kendall harmonious coefficients of the first-level, second-level and third-level items were 0.342, 0.244 and 0.212 respectively ( P<0.01) . Conclusions:There are high levels of expert activity, authority as well as coordination. The core competency framework and content for cardiac rehabilitation specialist nurses can provide a basis for training, use and assessment for cardiac rehabilitation specialist nurses.

Chronic myeloid leukemia (CML) is a slowly progressing hematopoietic cell disorder. Sphingosine kinase 1 (SPHK1) plays established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers, including leukemia.However, small-molecule inhibitors targeting SPHK1 in CML still need to be developed. This study revealed the role of SPHK1 in CML and investigated the potential anti-leukemic activity of hirsuteine (HST), an indole alkaloid obtained from the oriental plant Uncaria rhynchophylla, in CML cells. These results suggest that SPHK1 is highly expressed in CML cells and that overexpression of SPHK1 represents poor clinical outcomes in CML patients. HST exposure led to G2/M phase arrest, cellular apoptosis, and downregulation of Cyclin B1 and CDC2 and cleavage of Caspase 3 and PARP in CML cells. HST shifted sphingolipid rheostat from sphingosine 1-phosphate (S1P) towards the ceramide coupled with a marked inhibition of SPHK1. Mechanistically, HST significantly blocked SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways. In addition, HST can be docked with residues of SPHK1 and shifts the SPHK1 melting curve, indicating the potential protein-ligand interactions between SPHK1 and HST in both CML cells. SPHK1 overexpression impaired apoptosis and proliferation of CML cells induced by HST alone. These results suggest that HST, which may serve as a novel and specific SPHK1 inhibitor, exerts anti-leukemic activity by inhibiting the SPHK1/S1P/ S1PR1 and BCR-ABL/PI3K/Akt pathways in CML cells, thus conferring HST as a promising anti-leukemic drug for CML therapy in the future.

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers. Class I PI3Ks are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate (PIP2) at the 3-OH of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn activates Akt and the downstream effectors like mammalian target of rapamycin (mTOR) to play key roles in carcinogenesis. Therefore, PI3K has become an important anticancer drug target, and currently there is very high interest in the pharmaceutical development of PI3K inhibitors. Idelalisib has been approved in USA and Europe as the first-in-class PI3K inhibitor for cancer therapy. Dozens of other PI3K inhibitors including BKM120 and ZSTK474 are being evaluated in clinical trials. Multifaceted studies on these PI3K inhibitors are being performed, such as single and combinational efficacy, resistance, biomarkers, etc. This review provides an introduction to PI3K and summarizes key advances in the development of PI3K inhibitors.

BACKGROUNDIn-stent restenosis caused by airway granulation poses a challenge due to the high incidence of recurrence after treatment. Weekly applications of anti-proliferative drugs have potential value in delaying the recurrence of airway obstruction. However, it is not practical to subject patients to repeated bronchoscopy and topical drug applications. We fabricated novel pacilitaxel-eluting tracheal stents with sustained and slow pacilitaxel release, which could inhibit the formation of granulation tissue. And we assessed the quality and drug release behaviors of drug-eluting stents (DESs) in vitro.

METHODSStents were dipped vertically into a coating solution prepared by dissolving 0.5 g (2% w/v) of poly lactic acid-coglycolic acid (PLGA) and 0.025 g (0.1% w/v) of pacilitaxel in 25 ml of dichloromethane. DES morphology was examined by scanning electron microscopy (SEM). Pacilitaxel release kinetics from these DESs was investigated in vitro by shaking in PBS buffer followed by high performance liquid chromatography (HPLC).

RESULTSUsing an orthogonal experimental design, we fabricated numerous pacilitaxel/PLGA eluting tracheal stents to assess optimum coating proportions. The optimum coating proportion was 0.1% (w/v) pacilitaxel and 2% (w/v) PLGA, which resulted in total pacilitaxel loading of (16.380 6 ± 0.002 1) mg/stent. By SEM the coating was very smooth and uniform. Pacilitaxel released from DES was at (0.376 3 ± 0.003 8) mg/d, which is a therapeutic level. There was a prolonged, sustained release of pacilitaxel of >40 days.

CONCLUSIONSPaclitaxel-loaded PLGA coating tracheal stents were successfully developed and evaluated. Quality assessments demonstrated favorable surface morphology as well as sustained and effective drug release behavior, which provides an experimental reference for clinical practitioners.

Drug-Eluting Stents ; Glycolates ; chemistry ; Humans ; Lactic Acid ; chemistry ; Paclitaxel ; chemistry ; Polyesters ; Polymers ; chemistry

[摘 要] 嵌合抗原受体基因修饰T细胞（CAR-T细胞）疗法是一种肿瘤免疫治疗方法：来自人体的T细胞在体外经遗传学修饰、表达特异性嵌合抗原受体（CAR），然后将其回输入患者体内，用于靶向识别和消除肿瘤细胞。尽管CAR-T细胞疗法在血液系统肿瘤治疗中取得了较为显著的成功，其在实体瘤治疗中仍面临障碍。细胞因子释放综合征（CRS）等免疫相关不良反应（irAE）制约了CAR-T细胞的安全应用，肿瘤相关抗原（TAA）的异质性限制了单一CAR-T细胞的广谱适用性，也制约了其通用型开发。鉴于此，要使CAR-T细胞疗法在实体瘤临床治疗中得到应用，还需开展进一步的改良与提升研究。本文围绕实体瘤中CAR-T细胞疗法，从“CAR基因修饰策略”、“通用免疫受体的再靶向策略”及“抗原通用性‘赋靶’策略”三个方面对CAR-T细胞领域中为提高安全性和通用性所进行的探索进行述评，系统剖析各策略的研究路径、优势及局限性，并展望未来发展方向。通过综述CAR-T细胞安全性和普适性设计策略的进展，本文旨在为实体瘤的CAR-T细胞疗法研发提供创新思路。