Objective To explore the effect of dexmedetomidine on the proliferation,invasion and cell cycle of gallbladder cancer cells by regulating the C-C chemokine ligand 2(CCL2)-C-C chemokine receptor 2(CCR2)pathway.Methods GBC-SD cells were devided into the control group,the low concentration dexmedetomidine group(2 μmol/L),the high concentration dexmedetomidine group(4 μmol/L)and the high concentration dexmedetomidine+CCL2 group(4 μmol/L dexmedetomidine and 10 μg/L CCL2 protein).The clone formation experiment and Edu experiment were performed to measure cell proliferation.Transwell experiment was performed to measure cell invasion and migration.Flow cytometry was performed to measure cell cycle and apoptosis.Western blot assay was used to measure the proliferating cell nuclear antigen(PCNA),Cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,Bcl-2 associated X protein(Bax),cysteine-dependent aspartate-specific protease-3(Caspase-3),CCL2 and CCR2 proteins.The nude mouse transplant tumor experiment was used to determine the growth of gallbladder cancer transplant tumors.Results After treatment with low and high concentrations of dexmedetomidine,the number of cell clone formed,the positive rate of Edu,the numbers of invasions and migrations,the expression levels of PCNA,CyclinD1,MMP-2,MMP-9,CCL2 and CCR2 proteins,the proportions of G2/M and S phase cells decreased,the proportion of G0/G1 phase cells,apoptosis rate and expression levels of Bax and Caspase-3 proteins increased,and the effect of high-concentration dexmedetomidine was more significant(P＜0.05).The inhibitory effects of dexmedetomidine on the proliferation,invasion,migration and cell cycle of gallbladder cancer cells,as well as its promoting effect on cell apoptosis could be reversed by CCL2 protein(P＜0.05).In vivo experiments showed that dexmedetomidine could reduce tumor mass,tumor volume of nude mice and expression levels of CCL2 and CCR2 proteins(P＜0.05).Conclusion Dexmedetomidine inhibits the proliferation and invasion of gallbladder cancer cells,and blocks the cell cycle in the G0/G1 phase by suppressing the CCL2-CCR2 pathway.

Objective To explore the effect of dexmedetomidine on the proliferation,invasion and cell cycle of gallbladder cancer cells by regulating the C-C chemokine ligand 2(CCL2)-C-C chemokine receptor 2(CCR2)pathway.Methods GBC-SD cells were devided into the control group,the low concentration dexmedetomidine group(2 μmol/L),the high concentration dexmedetomidine group(4 μmol/L)and the high concentration dexmedetomidine+CCL2 group(4 μmol/L dexmedetomidine and 10 μg/L CCL2 protein).The clone formation experiment and Edu experiment were performed to measure cell proliferation.Transwell experiment was performed to measure cell invasion and migration.Flow cytometry was performed to measure cell cycle and apoptosis.Western blot assay was used to measure the proliferating cell nuclear antigen(PCNA),Cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,Bcl-2 associated X protein(Bax),cysteine-dependent aspartate-specific protease-3(Caspase-3),CCL2 and CCR2 proteins.The nude mouse transplant tumor experiment was used to determine the growth of gallbladder cancer transplant tumors.Results After treatment with low and high concentrations of dexmedetomidine,the number of cell clone formed,the positive rate of Edu,the numbers of invasions and migrations,the expression levels of PCNA,CyclinD1,MMP-2,MMP-9,CCL2 and CCR2 proteins,the proportions of G2/M and S phase cells decreased,the proportion of G0/G1 phase cells,apoptosis rate and expression levels of Bax and Caspase-3 proteins increased,and the effect of high-concentration dexmedetomidine was more significant(P＜0.05).The inhibitory effects of dexmedetomidine on the proliferation,invasion,migration and cell cycle of gallbladder cancer cells,as well as its promoting effect on cell apoptosis could be reversed by CCL2 protein(P＜0.05).In vivo experiments showed that dexmedetomidine could reduce tumor mass,tumor volume of nude mice and expression levels of CCL2 and CCR2 proteins(P＜0.05).Conclusion Dexmedetomidine inhibits the proliferation and invasion of gallbladder cancer cells,and blocks the cell cycle in the G0/G1 phase by suppressing the CCL2-CCR2 pathway.

N1-methyladenosine(m1A)RNA methylation is critical for regulating mRNA translation;however,its role in the development,progression,and immunotherapy response of head and neck squamous cell carcinoma(HNSCC)remains largely unknown.Using Tgfbr1 and Pten conditional knockout(2cKO)mice,we found the neoplastic transformation of oral mucosa was accompanied by increased m1A modification levels.Analysis of m1A-associated genes identified TRMT61A as a key m1A writer linked to cancer progression and poor prognosis.Mechanistically,TRMT61A-mediated tRNA-m1A modification promotes MYC protein synthesis,upregulating programmed death-ligand 1(PD-L1)expression.Moreover,m1A modification levels were also elevated in tumors treated with oncolytic herpes simplex virus(oHSV),contributing to reactive PD-L1 upregulation.Therapeutic m1A inhibition sustained oHSV-induced antitumor immunity and reduced tumor growth,representing a promising strategy to alleviate resistance.These findings indicate that m1A inhibition can prevent immune escape after oHSV therapy by reducing PD-L1 expression,providing a mutually reinforcing combination immunotherapy approach.

Objective To investigate the effect of sufentanil(ST)on lipopolysaccharide-induced acute lung injury(ALI)in rats by regulating the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)/stimulator of interferon gene(STING)signaling pathway.Methods Male SD rats were induced by lipopolysaccharide to construct an ALI model.The successfully modeled rats were randomly separated into ALI group,L-ST group,H-ST group,and H-ST+DMXAA group,with 6 rats in each group.Among them,the L-ST group and H-ST group were intraperitoneally injected with 2.5 μg/ml and 5 μg/ml ST after successful modeling immediately.The H-ST+DMXAA group was given 25 mg/kg DMXAA by gavage on the basis of intraperitoneal injection of 5 μg/ml ST.Six healthy and normal temperature rats were randomly selected as the control group,and an equal amount of physiological saline was injected intraperitoneally.The reagent kit was used to detect the SOD enzyme activity and MDA content in serum;ELISA was used to detect the levels of inflammatory factors such as interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),and IL-6 in bronchoalveolar lavage fluid(BALF);cell staining was used to detect the total number of cells and neutrophils in BALF;the wet to dry mass ratio of lung tissue was calculated;HE staining was used to observe pathological changes in lung tissue,while Western blot was applied to detect the expression of cGAS and STING proteins in lung tissue.Results After L-ST and H-ST treatment,the SOD enzyme activity and IL-10 level increased sequentially,the MDA,TNF-α,IL-6,W/D value,total number of cells and neutrophils,cGAS,and STING protein expression decreased sequentially(P<0.05),while lung tissue injury was effectively alleviated.DMXAA reversed the influence of H-ST on the above indexes(P<0.05).Conclusion ST may inhibit the cGAS-STING pathway,reduce inflammation and oxidative stress responses,and thereby alleviates lipopolysaccharide induced ALI in rats.

Objective To investigate the effect of sufentanil(ST)on lipopolysaccharide-induced acute lung injury(ALI)in rats by regulating the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)/stimulator of interferon gene(STING)signaling pathway.Methods Male SD rats were induced by lipopolysaccharide to construct an ALI model.The successfully modeled rats were randomly separated into ALI group,L-ST group,H-ST group,and H-ST+DMXAA group,with 6 rats in each group.Among them,the L-ST group and H-ST group were intraperitoneally injected with 2.5 μg/ml and 5 μg/ml ST after successful modeling immediately.The H-ST+DMXAA group was given 25 mg/kg DMXAA by gavage on the basis of intraperitoneal injection of 5 μg/ml ST.Six healthy and normal temperature rats were randomly selected as the control group,and an equal amount of physiological saline was injected intraperitoneally.The reagent kit was used to detect the SOD enzyme activity and MDA content in serum;ELISA was used to detect the levels of inflammatory factors such as interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),and IL-6 in bronchoalveolar lavage fluid(BALF);cell staining was used to detect the total number of cells and neutrophils in BALF;the wet to dry mass ratio of lung tissue was calculated;HE staining was used to observe pathological changes in lung tissue,while Western blot was applied to detect the expression of cGAS and STING proteins in lung tissue.Results After L-ST and H-ST treatment,the SOD enzyme activity and IL-10 level increased sequentially,the MDA,TNF-α,IL-6,W/D value,total number of cells and neutrophils,cGAS,and STING protein expression decreased sequentially(P<0.05),while lung tissue injury was effectively alleviated.DMXAA reversed the influence of H-ST on the above indexes(P<0.05).Conclusion ST may inhibit the cGAS-STING pathway,reduce inflammation and oxidative stress responses,and thereby alleviates lipopolysaccharide induced ALI in rats.

Objective:To observe the clinical efficacy of combining repeated transcranial magnetic stimulation (rTMS) at low frequency with xing nao kai qiao acupuncture in the treatment of upper limb motor dysfunction after a stroke.Methods:Forty stroke survivors with upper limb motor dysfunction were randomly divided into a control group and a combination group, each of 20. All received basic neurological medication, conventional rehabilitation treatment, and xing nao kai qiao acupuncture. The combination group additionally received rTMS. Both groups were treated once a day, 5 days a week for 2 weeks consecutively. Before and after the treatment, both groups were evaluated using the Brunnstrom motor function stages, modified Ashworth spasticity grades, Fugl-Meyer upper limb motor function (FMA-UE) scoring, the modified Barthel index (MBI), and motor evoked potential (MEP) latency and amplitude.Results:The upper-limb and hand motor functioning of both groups improved significantly compared with before the treatment. The hand motor function staging of the combination group was then significantly better than among the control group. The average FMA-UE and MBI scores of both groups improved significantly, with significantly greater improvement in the combination group than in the control group. MEP latency and amplitude also improved significantly in both groups, with the average MEP latency and amplitude of the combination group superior to the control group′s averages.Conclusion:Supplementing xing nao kai qiao acupuncture with low-frequency rTMS can significantly improve the motor functioning, spasticity, and skill in the activities of daily living of stroke survivors with upper limb motor dysfunction.

Objective To investigate the expression of CD4+CDhi25CDlow127 regulatory T cells (Treg cell), IL-2, IL-10 and IFN-γin peripheral blood of patients with different TNM stages of non-small cell lung cancer( NSCLC) , and to evaluate their correlation. Methods The proportion of CD4+CDhi25 CDlow127 Treg cells to CD4+cell in peripheral blood of 55 NSCLC patients were determined by flow cytometry, and levels of IL-2, IL-10 and IFN-γ were measured by ELISA. The proportion of CD4+CDhi25 CDlow127 Treg cells, IL-2, IL-10 and IFN-γ of patients with different TNM stages of NSCLC were compared, and correlation analysis was carried on. Results The proportion of CD4+CDhi25 CDlow127 Treg cells increased obviously together with the increasing of TNM stages of NSCLC(P<0. 05),so did the level of IL-10 (P<0. 05). On the other hand, the level of IL-2 and IFN-γdecreased apparently with the increasing of TNM sta-ges of NSCLC(P<0. 05). CD4+CDhi25CDlow127Treg cells and IL-10 were positively correlated(P <0. 05). IL-2 and IFN-γ were positively correlated too(P<0. 05). CD4+CDhi25CDlow127Treg cells and IL-10 were all negatively correlated with IL-2 and IFN-γ(P<0. 05). Conclusion The increasing of CD4+ CDhi25CDlow127Treg cells and IL-10 and the de-creasing of IL-2 and IFN-γ are possibly one of the reasons of antitumor immunity suppression in terminal NSCLC patients. The surveillance for them can be reference indicators to evaluate antitumor immunity status of terminal lung cancer patients.

OBJECTIVE:To explore the effect of alendronte sodium and vitamin D3 on the T cell immune indexes in patients with different genders and ages with advanced lung cancer. METHODS:76 patients with advanced lung cancer were divided into non-small cell lung cancer(NSCLC)group(n=40)and small cell lung cancer(SCLC)group(n=36),and another 38 healthy vol-unteers were control group. Based on palliative care,all patients received Alendronte sodium and vitamin D3 tablet 1 tablet,po,qd, for 1 month. T cell immune indexes(CD4+,CD8+and Treg levels)in 2 groups before and after treatment,and when volunteers enroll-ing the control group were compared;and the incidence of adverse reactions were observed. RESULTS:Compared with control group,there were significant differences in CD4+,CD8+,CD4+/CD8+and Treg levels(P<0.05);but there was no significant differ-ence between NSCLC group and SCLC group(P>0.05). After treatment,CD4+ and CD4+/CD8+ levels in NSCLC group and SCLC group increased,changes of male were greater than female,CD8+and Treg levels decreased,changes of male were greater than fe-male,except for CD8+ level in NSCLC group (P=0.33),there were significant differences in others (P<0.05);CD4+ and CD4+/CD8+ levels in patients ≤50 years old increased more than patients >50 years old,CD8+ and Treg levels decreased more than pa-tients >50 years old,the differences were statistically significant (P<0.05). No obvious adverse reactions were found. CONCLU-SIONS:Alendronte sodium and vitamin D3 can effectively improve the T cell immune indexes in patients with advanced lung cancer, especially for male and patients≤50 years old,which can be considered as conventional adjuvant drugs for patients with lung cancer.

Objective To observe the efficacy of CT guided implantation of iodine 125 particles combined with high-frequency hyperthermia in the treatment of the advanced lung cancer .Methods From 2011 November to 2013 March ,60 patients with ad-vanced lung cancer were randomly divided into treatment group :high frequency hyperthermia combined with iodine 125 particle im-plantation(30 cases) ,the control group :the iodine 125 particle implantation group(30 cases) .Thermotherapy was performed 3 -5 days after the implantation using NRL-004 type hyperthermia instrument made by Jilin Maida company in China ,every other day once time ,a total of six times ,60 minutes each time .The iodine 125 particle implantation was performed according to the treatment planning system(TPS) formulation by CT guided and the dosage was 90 Gy .The evaluation of treatment effect was conformed by CT scans after two months of treatment .The side effects was assessed too .Results The total effective rate was 96 .7% in the treatment group ,the control group was 80 .0% ,there was a statistically significant difference (P< 0 .05) .The local pain in chest wall remission rate was 75 .0% (6/8) and 50 .0% (5/10) in the treatment group and the control group(P>0 .05) .The side effects of the treatment was slight including pneumothorax and particle migration and so on .Conclusion Iodine 125 particles implantation is an effective treatment in the treatment of advanced lung cancer and can obtained better effects combined with high-frequency hy-perthermia .The side effects of the treatment is slight for two groups and no death related treatment .

BACKGROUNDp16INK4a and p14ARF, encoded by gene INK4a/ARF located at chromosome 9p21, are cyclin dependent kinase (CDK) inhibitors. Both p16INK4a and p14ARF are cell cycle regulatory proteins and play an important role in Rb and p53 passways respectively. In this study, wild-type INK4a/ARF gene was transfected into human lung adenocarcinoma cell line A549, in which this gene site was lost, and the effects on the cell's biological behavior were investigated.

METHODSThe recombinant eukaryotic expression plasmids pcDNA3-p16INK4a and pcDNA3-p14ARF were transfected into A549 by cationic liposome method. By RT-PCR, immunocytochemistry and Western blot after G418 selection, A549 cells that could stably express p16INK4a and p14ARF were obtained. As a control, the parental cell and negative control cell with plasmid pcDNA3-LacZ were used. Inhibition of proliferation was measured by MTT assay. The cell growth curve was drawn according to cell counts. Cell cycle distribution was measured by flow cytometry (FCM), the apoptosis indexes were observed at the same time. The colony formation rate was counted by staining the cells with Coomassie brilliant blue.

RESULTSThe introduction of exogenous INK4a and ARF caused significantly growth inhibition of A549. By FCM, more percentage of A549-p16INK4a-p14ARF cells couldn't pass through the checkpoint G1. The percentage of A549-p16INK4a-p14ARF cells inhibited at G0/G1 was 59.9%, 50.3% for A549-vector and 51.2% for A549. The statistical differences were significant between A549-p16INK4a-p14ARF cell and A549-vector cell (P=0.025) and between A549-p16INK4a-p14ARF cell and A549 cell (P=0.043). The apoptosis index of A549-p16INK4a-p14ARF cell was 8.0% and 2.7% for both A549-vector and A549 cell (P < 0.01). The colony formation ability of A549-p16INK4a-p14ARF was weaker than that of A549-vector and A549, they were 63%, 87% and 85% respectively.

CONCLUSIONSThe wild-type INK4a/ARF gene can be co-introduced effectively into A549 cell by cationic liposome method. The reexpression of p16INK4a and p14ARF in A549 can inhibit the growth and enhance the apoptosis. This trial will be helpful in using gene therapy of lung cancer in the future.