OBJECTIVE To explore the improvement effect and mechanism of acacetin on juvenile asthma rats based on the silence information regulator 1 （SIRT1）/AMP-activated protein kinase （AMPK） signaling pathway. METHODS Juvenile SD rats were randomly divided into control group， asthma group， acacetin group （13.33 mg/kg， gavage）， SIRT inhibitor EX-527 group （1 mg/kg， intraperitoneal injection） and acacetin+EX-527 group （13.33 mg/kg acacetin， gavage+1 mg/kg EX-527， intraperitoneal injection）， with 12 rats in each group （half male and half female）. Except for the control group， the other groups were sensitized by intraperitoneal injection of ovalbumin and nebulized inhalation of ovalbumin to induce the asthma model. After modeling， rats in each drug group were orally administered or （and） intraperitoneally injected with the corresponding medication once a day for 2 weeks. After the last administration， the total number of cells， the proportion of eosinophils， and the levels of interleukin-5 （IL-5）， IL-4 and tumor necrosis factor-α （TNF-α） in the bronchoalveolar lavage fluid （BALF） were measured. The pathological changes and abnormal proliferation of goblet cells in lung tissue were observed， the levels of malondialdehyde （MDA） and superoxide dismutase （SOD） in lung tissue and the protein expressions of SIRT1, AMPK and peroxisome proliferator activated receptor-gamma co-activator factor-1α（PGC-1α） were detected. RESULTS Compared with control group， there was a large number of inflammatory cell infiltration and obvious goblet cell dysplasia in the lung tissue of rats in asthma group； the total number of cells in BALF， the proportion of eosinophils， the levels of IL-5， IL-4 and TNF-α in BALF， PAS score and MDA level in the lung tissue were significantly increased （P＜0.05）； the SOD level， protein expressions of SIRT1 and PGC-1α and protein phosphorylation level of AMPK in lung tissue were significantly decreased in asthma group （P＜0.05）. Compared with the asthma group， the pathological changes of lung tissue and goblet cell dysplasia of rats were reduced， and all quantitative indexes were significantly improved in acacetin group （P＜0.05）， while the pathological changes of lung tissue and goblet cell dysplasia of rats were increased， and all quantitative indexes were significantly worsened in EX-527 group （P＜ 0.05）. The combination of EX-527 could significantly reverse the effects of acacetin on oxidative stress and airway inflammation in juvenile asthma rats. CONCLUSIONS Acacetin can inhibit oxidative stress and airway inflammation in juvenile asthma rats，which may be related to the activation of the SIRT1/AMPK jinanlvshishen@163.com signaling pathway.

Objective:To explore the effect of acute and chronic glycemic ratio on the prognostic assessment in vulnerable phase of patients with acute heart failure (AHF).Methods:The clinical data of 98 AHF patients who treatment in Nanjing Pukou Hospital of Traditional Chinese Medicine from May 2019 to May 2022 were collected retrospectively, the patients were followed up for 3 months, according to whether adverse events occurred in the vulnerable phase, the patients were divided into adverse prognosis group (31 cases) and non-adverse prognosis group(67 cases). The acute and chronic glycemic ratio was calculated based on the intravenous blood glucose and glycosylated hemoglobin (HbA 1c). The influencing factors of adverse prognosis of AHF patients in vulnerable phase was analyzed by Cox risk proportion model, the predictive value of acute and chronic glycemic ratio on adverse prognosis was evaluated by receiver operating characteristic (ROC) curve. Kaplan-Meier method was used to draw the survival curve and compared the risk of adverse prognosis in patients with different acute and chronic glycemic ratio. Results:The severity of cardiac function grading as well as total cholesterol, urea nitrogen, blood glucose, acute and chronic glycemic ratio, highly sensitive C-reactive protein (hs-CPR), left ventricular anteroposterior diameter, right atrial anteroposterior diameter in the adverse prognosis group were higher than those in the non-adverse prognosis group: (3.88 ± 0.18)mmol/L vs. (3.76 ± 0.24) mmol/L, (9.39 ± 1.07) mmol/L vs. (8.68 ± 1.79) mmol/L, (10.49 ± 2.20) mmol/L vs. (7.64 ± 1.57)mmol/L, 1.37 ± 0.47 vs. 1.04 ± 0.35, (3.85 ± 0.36) mg/L vs. (3.68 ± 0.28) mg/L, (48.47 ± 7.86) mm vs. (45.37 ± 3.56) mm, (47.18 ± 5.04) mm vs. (44.05 ± 6.11) mm, there were statistical differences ( P<0.05). Cox multivariate analysis showed that hypertension, white blood cell count, blood sodium, low density lipoprotein cholesterol, acute and chronic glycemic ratio were the risk factors for adverse prognosis in vulnerable phase ( P<0.05). The area under the curve of acute and chronic glycemic ratio for predicting adverse prognosis in vulnerable phase was 0.718 (95 CI: 0.618 - 0.805, P<0.01), with specificity of 62.7%, sensitivity of 77.4%, and cut-off value of 1.07. According to the cut-off value of acute and chronic glycemic ratio, the patients were divided into acute and chronic glycemic ratio>1.07 group (43 cases), acute and chronic glycemic ratio≤ 1.07 group (55 cases), there was a statistically significant difference in the event free survival between the two groups ( P<0.01). Conclusions:AHF patients who with high acute and chronic glycemic ratio have a high risk of adverse prognosis in the vulnerable phase, which can be used as a predictor of the prognosis patients.

Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.

ObjectiveTo investigate the effect of killer cell immunoglobulin-like receptor (KIR) gene in immune killing of hepatoma cells.MethodsPeripheral blood mononuclear cell (PBMC) and hepatoma cells were co-cultured with different effector-target ratios. The expression of KIR gene family in PBMC, the content to interferon-γ (IFN-γ), the morphological change of hepatoma cell and the cytotoxicity to hepatoma cell by PBMC were observed after the co-incubation with different effector-target ratios. The comparison on cytotoxicity rates was conducted using one-way analysis of variance and LSD-t test.ResultsThe expression of activating KIR gene increased after 12 h of co-culture, but decreased after 24 h of co-culture. The expression of inhibitory KIR gene decreased after 12 h of co-culture. DAP12 maintained high expression all the time. The content of IFN-γ in PBMC decreased with the increase of effector-target ratio and reached the peak at 12 h of co-culture. Hepatoma cells co-cultured with different effector-target ratios were observed with increased chromatin condensation, rising proportion of cells with hemispherical or half moon shape and marginalized nucleus, and stagnant of active cell division. The cytotoxicity rate of effector-target ratio 1∶1, 10∶1 and 50∶1 was (8±3) %, (14±4) % and (32±6) %, respectively, with 50∶1 group significantly higher than 11∶1 and 10∶1 group (LSD-t=5.97, 4.61;P<0.05).ConclusionThe activating KIR gene plays an important role in immune killing of hepatoma cells.

OBJECTIVETo investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the proliferation and survival of Jurkat leukemia cells in vitro and explore the possible mechanism.

METHODSJurkat leukemia cells were co-cultured with hUC-MSCs isolated from human umbilical cord tissues by plastic adherence at a ratio of 10:1. The proliferation and survival of the co-cultured Jurkat cells, separated by immunomagnetic bead cell sorting on day 4, were evaluated by flow cytometry. Western blotting was performed to evaluate the activation of Notch signaling in the co-cultured Jurkat cells.

RESULTSJurkat leukemia cells co-cultured with hUC-MSCs for 4 days showed a lowered proliferation rate and cell cycle arrest at G0/G1 phase with a reduction in the cell apoptotic rate. Notch signaling pathway was activated in the co-cultured Jurkat cells as evidenced by an increased cellular expression of HES-1.

CONCLUSIONCo-culture with hUC-MSCs can inhibit the proliferation of Jurkat leukemia cells in vitro and protect the cells from apoptosis by activating Notch signaling, indicating a potential shielding effect of MSCs on leukemia cells.

Apoptosis ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Jurkat Cells ; Mesenchymal Stromal Cells ; cytology ; Receptor, Notch1 ; metabolism ; Signal Transduction ; Umbilical Cord ; cytology

The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.
Adenoviridae ; physiology ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Cytoplasm ; virology ; Endocytosis ; Endosomes ; virology ; Genetic Vectors ; Humans ; Microtubules ; Virus Internalization

Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.

Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .

AIM:To explore the role of survivin-2B in the process of tumor cell apoptosis .METHODS:The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained.Hu-man breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000.The cell cycle was determined by propidium iodide staining , and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection.Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes .RESULTS: Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF 7 cells.Compared with control group , totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48%down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated.CONCLUSION:Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G 2/M arrest of MCF7 cells.

Objective:To investigate effects of tumor specific TCR gene Vα12.2-Vβ7.1 modification on recognition of tumor antigen and activation of anti-tumor reactivity of T cells.Methods: T cells were transduced using recombinant Ad 5F35-TRAV-TRBV adenovirus ,and multiplicity of infection was optimized.Specific lysis of T cells was evaluated by calcein release assay.The frequency of apoptotic cells in target cells was detected by Annexin V /PI double-labeled FACS.The expression of FasL on T cells was analyzed by FACS.The secretion of cytokine IFN-γand IL-2 of T cells was determined by ELISA assays.Results: The highest tranduce efficiency was obtained at MOI 100 by recombinant Ad5F35-TRAV-TRBV adenovirus.The frequency of TCRVα12+Vβ7+cells reached above 25%3 days after transduction.TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells(P<0.001), the ability of T cells to induce HepG-2 apoptosis(P<0.001),and expression of FasL on T cells(P<0.001).TCR gene modification also enhanced T cells to secret IFN-γafter coculture with antigen positive tumor cells ( P<0.001 ).Conclusion: Specific TCR gene modification by recombinant adeno virus effectively promotes T cells to recognize antigen positive tumor cell and exert anti -tumor reac-tivity.