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 inhibitory effect of immunosuppressive agent FRY720 on hepatocellular carcinoma Hepal-6. Methods Hepal-6 cells were cultured, and divided into 4 groups, namely control group and 0.1 μg/ml, 10 μg/ml, 100 μg/ml quality concentration groups. The cells were treated by the drugs for 24 to 48 hours respectively. The inhibitory rate of the cells was measured by MTT assay, and cell cycle and cell apoptotic rate were detected by flow cytometry (FCM). Results The ability of tumor cell growth were inhibited by FTY720 after 48 h. The maximal inhibition rate was 62.10 %, The apoptosis ratio was increased when FTY720 was 0.1-100 μg/ml, and it was 4.07 %, 8.16 %, 19.84 % respectively. FTY720 significantly prolonged cell G1 phase. Conclusion FTY720 could inhibit the growth of hepatocellular carcinoma, arrest the cell in G1 phase, and increase apoptosis.

Objective To study the relationship between Treg cell numbers and level of TGF-β1,IL-10 in the immune microenvironment of rat liver cancer.Method 40 male Wistar rats were randomly divided into three groups,liver cancer model group (n =20),saline control group (n =10) and blank control group (n =10).Liver cancer model was established by intraperitoneal injection of DEN (100 mg/kg).Percentage of Treg cells in cancer tissues was detected by fiow cytometry and the expression level of TGF-β1 and IL-10 by immunohistochemical SABC.Results The ratio of Treg cells in CD4 + T cells was 14.32％ ± 4.84％ in liver cancer tissues,significantly higher than that in the blank control group 5.64％ ±6.10％ and the saline control group 7.95％ ±3.55％.The difference was statistically significant (F =211.279,P ＜ 0.05).The expression level of TGF-β1 and IL-10 in cancer tissues was significantly higher than the blank control group and the saline control group (FTGF-β1 =250.740,FIL-10 =152.744,all P ＜0.05).The number of Treg cells was positively correlated with TGF-β1,IL-10 level (P ＜ 0.05).Conclusions TGF-β1,IL-10 and Treg cells significantly increased in rat experimental liver cancer tissues,and there was positive correlation between Treg cells,and TGF-β1 and IL-10 in liver cancer tissues.

OBJECTIVE: To study the mechanism and clinical significance of specific immunotherapy (SIT) on the expression changes of GM-CSF and IL-5 in the tissue samples of recurrent nasal polyps. METHOD: Perennial allergic rhinitis patients with recurrent nasal polyps were randomly divided into 2 groups. The experimental group of 19 patients was treated by SIT and standardized treatment (glucocorticoid nasal spray) , and the control group of 17 patients was only treated by standardized treatment (glucocorticoid nasal spray). We measured the expression levels of GM-CSF and IL-5 in the tissue samples of the nasal polyps by ELISA, and compared the results obtained before treatment with expression levels detected at 6 months and 1 year after the treatment. RESULT: The expression of GM-CSF and IL-5 in the recurrent nasal polyps reduced significantly (P < 0.05) in both groups after 6 months and 1 year post-treatment compared with pre-treatment, and the expression of GM-CSF and IL-5 in the experimental group was much lower than that of the control group. CONCLUSION SIT decreases the expression of GM-CSF and IL-5 and reduces the inflammatory reaction in the tissue samples of recurrent nasal polyps.
Enzyme-Linked Immunosorbent Assay ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Immunotherapy ; Inflammation ; drug therapy ; Interleukin-5 ; metabolism ; Nasal Mucosa ; pathology ; Nasal Polyps ; drug therapy ; metabolism ; Rhinitis, Allergic, Perennial ; drug therapy ; metabolism

OBJECTIVE To observe the efficacy of specific immunotherapy (SIT) with Alutard and NHD in children with allergic rhinitis due to different level of skin prick test (SPT) with dermatophagoides farinae (DF) and dermatophagoides pteronyssinus (DP). METHODS A total of 178 children with persistent allergic rhinitis were included in this study. Their age ranged from 6 to 12 years. They were divided into 3 groups according to level of SPT. Group 1: The level of skin index (SI) of DF is greater than that of DP, Group 2: The level of SI of DF is equal to that of DP and Group 3: The level of SI of DF is less than that of DP. The children in each group were randomly divided into 2 subgroups: Alutard group and NHD group. The children were given SIT with Alutard or NHD for one year. Symptom and medication scores were recorded and analyzed. RESULTS After receiving therapy for 3 months and 6 months, symptom and medication scores of the Group 1 and 2 in the NHD group were lower than those in the Alutard group (P<0.05); Symptoms and medication scores of the Group 3 in the Alutard group were lower than those in the NHD group (P<0.05). After receiving therapy for 9 and 12 months, the symptom and medication scores of the NHD and Alutard group in all the three groups showed no statistical difference (P>0.05). CONCLUSION The efficacy of SIT with Alutard and NHD is different in children with allergic rhinitis with different levels of SPT due to DF and DP after 3 and 6 months, but is similar after 9 and 12 months. SIT with Alutard and NHD is effective in treating children with allergic rhinitis.

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.

AIM:To study the influence of stimulation by LPS and CD40 ligandization in vitro on the TLR4-MD2 expression and IL-12 production in dendritic cells (DCs) modified by sCD40 gene and provide the experimental clues of inducing dornor-specific immune tolerance.METHODS: Plasmid pEGFP-N1/sCD40 and pEGFP-N1 was transfected into DC2.4 cell line with lipofectamine. After 6 h of treatment with LPS and anti-CD40mAb, the expression of TLR4-MD2 on DCs was determined with flow cytometry (FCM) and RT-PCR. IL-12p70 protein was detected by ELISA.RESULTS: LPS treatment of DCs down-regulated surface expression of TLR4-MD2, LPS treatment along with anti-CD40mAb significantly up-regulated TLR4-MD2 surface expression. CD40 ligandization did not affect TLR4-MD2 mRNA expression in DCs but partly increased its low level induced by LPS and markly enhanced IL-12p70 secretion after LPS stimulation. DCs modified by sCD40 gene inhibited the above effect.CONCLUSION: After treatment with LPS and anti-CD40mAb, DCs modified by sCD40 gene down-regulate surface expression of TLR4-MD2 and IL-12p70 secretion decreases significantly, which might be linked with the interruption of TLR4-MD2 transportation from cytoplasm.

Age impact in mouse model of secondary hepatic alveolar echinococcus was investigated in this research . Twenty-nine 8-week-old ,twenty-five 18-week-old and twenty-five 28-week-old female mice were anesthetized with 20% ure-thane by intraperitoneal injection and then transhepatically injected by Echinococcus multilocularis (E .m) tissue suspension through skin incision and abdominal muscle to liver in all three groups to establish mouse model of secondary hepatic alveolar e-chinococcus .Results showed that the survival rates for the three groups of mice were 62 .1% ,84% and 68% ,respectively (P>0 .05) .The E .m infection rates in liver were 72 .2% ,71 .4% and 76 .5% ,respectively (P>0 .05) .The diameter of E .m cysts in liver were 0 .915 ± 0 .103 cm ,1 .247 ± 0 .112 cm and 1 .215 ± 0 .197 cm ,respectively (P>0 .05) .The mass of E .m cysts in liver were 0 .332 ± 0 .035 g ,0 .532 ± 0 .155 g and 0 .382 ± 0 .085 g ,respectively (P> 0 .05) .HE stain showed no difference in pathology .Results indicated that the establishment of secondary hepatic alveolar echinococcus model by using transhepatic injection through skin incision and abdominal muscle of 18-week-old mice was capable of simplifying operation and improving the survival rate of the mice .

In order to observe the effects of different culture media and temperature on protoscoleces of Echinococcus multi‐locularis ,they were randomly divided into RPMI‐1640 group ,D‐MEM group and M199 group ,and cultured in three degrees of temperature (4 ,25 and 37 ℃) with 10% fetal calf serum (FCS) .Protoscoleces were counted by light microscope with 0 .1%eosin staining ,and calculated survival rate (per 100 protoscoleces) everyday until all the parasites died .At the same time ,the average number of the preservation days was observed .The experiment results showed that the survival rate of protoscoleces in RPMI‐1640 and D‐MEM groups were higher than that in M199 group (P<0 .05) and there’s no significant difference between RPMI‐1640 group and D‐MEM group (P>0 .05) .The survival rate of protoscoleces in RPMI‐1640 group at 4 ℃ and 37 ℃and D‐MEM group at 25 ℃ were higher ,but there was no significant effect of 4 ,25 and 37 ℃ on the survival rate of proto‐scoleces (P>0 .05) .Significant difference were found in the survival rate of protoscoleces on the 3rd day and the 9th day in these three groups (P<0 .05) .The average number of the preservation days were 34 days in RPMI‐1640 group at 4 ℃ ,36 days in D‐MEM group at 25 ℃ and 23 days in M199 group at 4 ℃ .It was concluded that the effects of different culture media and tem‐perature on protoscoleces are different ,and the RPMI‐1640 at 4 ℃ and D‐MEM at 25 ℃ are more suitable for culturing proto‐scoleces in v itro .

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.