Objective To study the action of CNOT 7 (CCR4-NOT transcription complex subunit 7 human)gene and its mechanisms in the process of Vγ 9Vδ2T cell immunologic tolerance of HepG2 cells (Hepatoblastoma G2 Cell Line).Methods The shCNOT 7 (Recombinant plasmid of CNOT 7) and control vector shRNA were transfected into HepG2 cells.Vγ9Vδ2T cytokine stimulated each group before and after cell transfection,Cell apoptosis was detected by flow cytometry (FCM),CNOT 7 protein and STAT1,STAT3 expression level was detected by Western blot.CNOT 7,STAT1 and STAT3 protein expression levels of HepG2 liver cancer cell lines and L02 normal liver cell line was assayed by Western blot.Results When stimulated by Vγ9Vδ2T cytokine,the apoptosis rate of gene-knockdown group significantly improved from (7.55％ ±2.63％) to (20.59％ ±3.12％).Compared with L02 cells,the CNOT7 protein expression of HepG2 cells increased (F =28.76,P ＜ 0.01),STAT3 protein expression increased (F =110.29,P ＜ 0.01),while STAT1 protein expression was down-regulated (F =35.67,P ＜ 0.01).CNOT 7 knockout could induce HepG2 cells STAT1 expression (t =6.69,P ＜ 0.05).Conclusions CNOT 7 gene could induce HepG2 cells Vγ 9Vδ2T cellular immune tolerance.CNOT 7 knockout could reverse the Vγ 9Vδ2T cell immunologic tolerance of HCC.

Objective To investigate the percentage,mature classification and Immune killing function of Vγ9Vδ2T cell in peripheral blood of HCC patients.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from HCC patients (n =25) and healthy donors (n =20) by discontinuous density gradient centrifugation.Proportion,mature and differentiate subtypes and IFN-γ and CD107a expressing of the delta 2 T cells were detect by using flow cytometry,δ2Tcell were selectively cultured with zoledronate and human IL-2.After 12-14 days cells were collected and tested for the second time.Results While the percentage of Vγ9Vδ2Tcell of total T cell in peripheral blood of HCC patients is lower than healthy people before culture (t =4.505,P ＜ 0.001),after augmentation in vitro the proportion increased significantly (t =8.782,P ＜ 0.001),to a level similar to healthy group (t =1.644,P =0.109).There was no statistically significant difference when differentiation subtypes of patient's Vγ9Vδ2Tcell were compared with healthy group before culturing (all P ＞ 0.05),after culture the proportion of Tn,Tcm and Temra decreased [t(Tn) =2.081,t(Tcm) =2.478,t(Temra) =2.953,all P ＜ 0.05],and the proportion of Tem,Tem+ Temra increased [t(Tem) =12.6,t (Tem + Temra) =9.843,all P ＜ 0.001].Cell culture did not alter the proportion of IFN-γ and CD107a secreting Vγ9Vδ2T cells in the peripheral blood in both HCC patients and healthy people (all P ＞ 0.05).Conclusions While the percentage of Vγ9Vδ2T cell of HCC patients in peripheral blood was lower than healthy people,its matured subtypes are similar to those of healthy people,and functions of expressing IFN-γ and CD107a are not different with healthy people.Applying ZOL + IL-2 can amplifyVγ9Vδ2T cells of patients with HCC.

Herpes simplex virus type 1 (HSV-1) is a commonly occurring human pathogen worldwide. There is an urgent need to discover and develop new alternative agents for the management of HSV-1 infection. Tripterygium hypoglaucum (level) Hutch (Celastraceae) is a traditional Chinese medicine plant with many pharmacological activities such as anti-inflammation, anti-tumor and antifertility. The usual medicinal part is the roots which contain about a 1% yield of alkaloids. A crude total alkaloids extract was prepared from the roots of T. hypoglaucum amd its antiviral activity against HSV-1 in Vero cells was evaluated by cytopathic effect (CPE) assay, plaque reduction assay and by RT-PCR analysis. The alkaloids extract presented low cytotoxicity (CC_(50) = 46.6 μg/mL) and potent CPE inhibition activity, the 50% inhibitory concentration (IC_(50)) was 6.5 μg/mL, noticeably lower than that of Acyclovir (15.4 μg /mL). Plaque formation was significantly reduced by the alkaloids extract at concentrations of 6.25 μg/mL to 12.5 μg/mL, the plaque reduction ratio reached 55% to 75% which was 35% higher than that of Acyclovir at the same concentration. RT-PCR analysis showed that, the transcription of two important delayed early genes UL30 and UL39, and a late gene US6 of HSV-1 genome all were suppressed by the alkaloids extract, the expression inhibiting efficacy compared to the control was 74.6% (UL30), 70.9% (UL39) and 62.6% (US6) respectively at the working concentration of 12.5μg/mL. The above results suggest a potent anti-HSV-1 activity of the alkaloids extract in vitro.

Objective To study the regulation of dendritic cells by recombinant glycated polylysine-coupled MIP-3α-FL double-gene targeting expression vector in liver cancer immune microenvironment.Methods H22 hepatocarcinoma cells were transfected with recombinant plasmid of MIP-3α-FL (shMIP-3α-FL) and injected into hepatoma model mice.The survival time,tumor size were compared.Flow cytometry was used to measure the number and phenotype of tumor infiltrating DCs.Results Western blot and ELISA demonstrated that the secretion of MIP-3α and FL in H22 cells was significantly increased after transfection with MIP-3α-FL.The survival time of the mice in the experimental group was significantly prolonged,the tumor size decreased.Flow cytometry showed that the number of tumor-infiltrating DCs in the experimental group was significantly higher than that in the control group;the expression of CD80 and CD86 in the infiltrating dendritic cells (TIDCs) was significantly higher than that of the control group.Conclusions The co-action of MIP-3α and FL can significantly promote DC accumulation,maturation,and conjugate glycosylated polylysine carriers increase the precision of targeting and enhance the antigenpresentation of the DCs.

Objective To analyze tumor immune microenvironment and related mechanisms in liver cancer.Methods We included 10 cases of hepatocellular carcinoma,hepatitis B patients and healthy volunteers from January 2015 to December 2017 in Shanxi Grand Hospital.We first detected the peripheral and local GM-CSF level in each group,detected myeloid-derived suppressor cells (MDSCs) GM-CSF and pathway-related protein expression.from liver cancer,tumor margin and normal liver tissue through flow cytometry and immunohistochemistry,Finally,we transfected the CCR4-NOT transcriptional complex subunit 7 (CNOT7) recombinant plasmid in the hepatoma cell line,and then detected the related protein expression.Results There was no significant difference for peripheral blood GM-CSF level between liver cancer group,hepatitis group and control group (P＞0.05).The level of local GM-CSF was (32.2±8.9) ng/L,which was higher than that of hepatocellular carcinoma (9.7±2.7) ng/L and normal liver tissue (11.6±2.9) ng/L.The difference was statistically significant (P＜0.05).The proportion of MDSCs at the edge of the tumor was (9.9 ±3.6) ％,which was higher than that of liver cancer (4.0± 1.5) ％ and normal liver tissue (6.3±2.3) ％,and the difference was statistically significant (P＜0.05).Immunohistochemistrydata was consistent with previous data.Compared with normal liver tissue,CNOT7 and STAT3 were highly expressed in liver cancer tissues,while STAT1 was lowly expressed.HepG2 human hepatoma cells were selected for transfection.Compared with the empty plasmid group,CNOT7 expression was decreased in the knocking out group at the same time STAT1 expression was increased,STAT3 and GM-CSF expression was decreased.Conclusion In hepatocellular carcinoma,the secretion of GM-CSF increased and the number of MDSCs increased.Knocking out CNOT7 reduced GM-CSF secretion and activate the JAK/STAT signaling pathway.

Objective To study the effect of human CCR4-NOT transcription complex subunit 7 (CNOT7) gene knockdown on the immune microenvironment of HepG2 cells and explore its significance. Methods We designed a cell transfection protocol and performed the experiment with three groups:CNOT7-targeted knockdown group, control group, and CNOT7 overexpression group. The transfection efficiency was assessed using inverted fluorescence microscopy, and the expression level of CNOT7, transforming growth factor-β1 (TGF-β1), and nuclear factor-kappa B (NF-κB) p65 proteins was determined by Western blotting. The concentration of TGF-β1 secreted in the cell culture supernatant was measured by ELISA. The sensitivity of tumor cells to the killing function of natural killer (NK) cells was detected by flow cytometry. Results Compared with the control group, the expression level of TGF-β1 and NF-κB p65 proteins was significantly decreased in the CNOT7-targeted knockdown group, and the TGF-β1 concentration in the culture supernatant was also significantly reduced. However, in the CNOT7 overexpression group, the expression level of the two proteins and TGF-β1 concentration were significantly increased. NK cells were co-cultured with tumor cells, and the apoptosis rate of HepG2 cells transfected with CNOT7-specific shRNA was significantly increased. However, in the CNOT7 overexpression group, the apoptosis rate was significantly decreased. Conclusion CNOT7 forms the immune microenvironment of hepatocellular carcinoma. Targeted knockdown of CNOT7 can reduce TGF-β1 secretion and enhance the killing function of NK cells toward HepG2 cells.

Objective:To explore the effect of long-chain fat emulsion in parenteral nutrition therapy on the perioperative nutritional status of patients with low rectal cancer.Methods:A total of 204 patients who underwent rectal cancer surgery in the Third Hospital of Shanxi Medical University from January 2017 to June 2020 were retrospectively analyzed. The patients were divided into two groups according to the specific nutritional treatment methods, 100 cases in the study group used long-chain fat emulsion for parenteral nutrition support, and 104 cases in the control group used medium- and long-chain fat emulsion injection. After admission, the nutritional status of patients were evaluated according to the results of Scored Patient-Generated Subjective Global Assessment (PG-SGA) and related laboratory tests. At 7th day before the operation, the patients were treated with nutrition and electrolyte support. Parenteral nutrition and enteral nutrition combined treatment and early enteral nutrition were given after the operation. The albumin, prealbumin, retinol-binding protein, total cholesterol and body mass index (BMI) at 7th day before the operation, 1st day after the operation and 7th day after the operation and the patient's first exhaust time after surgery, occurrence of postoperative complications, postoperative fever and total hospital stay were recorded and compared between the two groups.Results:Postoperative first exhaust time [(42±11) h vs. (54±10) h], fever time [(48±8) h vs. (57±7) h], total hospital stay [(16.0±0.7) d vs. (18.0±0.9) d)], resting energy expenditure at the 7th day after surgery [(5 326±589) kJ/d vs. (5 840±599) kJ/d] and total cholesterol at the 7th day after surgery [(4.8±0.3) mmol/L vs. (5.0± 0.4) mmol/L] in the study group were lower than those in the control group, and albumin [(33±3) g/L vs. (28± 3) g/L], prealbumin [(0.189±0.041) g/L vs. (0.164±0.037) g/L] and retinol-binding protein [(0.039±0.016) g/L vs. (0.032±0.013) g/L] at the 7th day after surgery in the study group were higher than those in the control group, and the differences between the two groups were statistically significant (all P < 0.05). There was no statistical difference in other detection indexes between the two groups (all P > 0.05). Conclusion:The use of long-chain fat emulsion in low rectal cancer patients with malnutrition during the perioperative period may be more conducive to the recovery of the body.