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.

Objective To study the efficacy and safety of different rivaroxaban medications in prevention and treatment of thromboembolic disease in very old patients.Methods One hundred and ninety-eight very old thromboembolic disease patients were divided into low dose (2.5-7.5 mg)rivaroxaban treatment group (n =35),routine dose (10-20 mg) rivaroxaban treatment group (n=138),combined (10 mg) rivaroxaban and (75 mg) clopidogrel treatment group (n=25).Their baseline data were recorded.The incidence of major adverse thromboembolic disease and bleeding events in 3 groups was compared by Kaplan-Meiers survival analysis.Results No significant difference was found in gender,risk factors for and past history of thromboembolic disease and bleeding events,laboratory testing parameters in 3 groups (P＞0.05).The age was significantly older in low dose rivaroxaban treatment group than in routine dose rivaroxaban treatment group (90.43±5.02 vs 86.80±5.52,P=0.000).Kaplan-Meiers survival analysis showed no significant difference in the incidence of thromboembolic disease and bleeding events in 3 groups (P＞0.05).Conclusion Low dose rivaroxaban is effective in prevention and treatment of thromboembolic disease while combined clopidogrel and rivaroxaban is not only effective but also safe in prevention and treatment of thromboembolic disease in very old patients.

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.

After a description of the background and ideas to introduce the third party testing in information system construction of Sichuan grass-root medical and health institutions and the specific working contents of third party testing team, the problems to which importance should be attached in information system construction were analyzed with suggestions put forward for their solution .

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.

OBJECTIVETo explore the practicability of microskin grafting by spraying in burn management.

METHODSRazor thin autologous skin from pigs or burn patients was harvested and cut to pieces of 0.2 - 0.5 mm in size and suspended in normal saline. The suspension was put into a bottle with outlet and pumping device. The microskin suspended in the saline was sprayed to the burn wound and/or onto the alloskin sheets. The microskin distribution was detected by digital image analysis technique. In animal experiments, the burn wound development and pathomorphological changes after operation were observed. In burn patients who would receive microskin grafting, spraying method was used with the traditional flotation method as control. The treatment results and the operational procedures were compared between these two kinds of operation styles.

RESULTSThe microskin dispersion degree with spraying was much smaller than that with flotating method. In animal experiment with spraying method, the wound healing time was 23.2, 24.5 and 38.3 days in 3 groups, respectively, with the area ratio of donor to wound of 110, 120 and 150. In clinical study, The average operating time was 133.3 min with spraying method and 165.6 min with flotation method respectively (P < 0.05). The area ratio of donor to wound was 118.8 with spraying and 17.6 with flotation methods, respectively (P < 0.01). The one time wound coverage rate was 92.6% with spraying and 79.7% with flotation methods (P < 0.01). The wound healing time was 29.7 days with spraying and 37.3 days with flotating methods, respectively (P < 0.05).

CONCLUSIONSpraying method of microskin grafting might be a good method in major burn treatment. The advantages with this method included well-distributed microskin, simpler handling, saving of donor skin, shortening of operating time and less time needed for the wound healing. It might be recommended for other wound covering materials.

Animals ; Burns ; surgery ; therapy ; Culture Techniques ; Dermatologic Surgical Procedures ; Female ; Humans ; Male ; Rabbits ; Skin ; injuries ; Skin Transplantation ; methods ; Transplantation, Heterologous ; Wound Healing