目的 通过生物信息学方法筛选和分析肿瘤数据库中胃癌突变基因,并分析其与患者预后及免疫功能的相关性,为胃癌患者的免疫治疗提供理论和数据参考.方法 从TCGA及ICGC数据库中下载胃癌患者的基因突变及临床数据,通过R软件筛选出与胃癌患者生存率和预后密切相关的共同高频突变基因.并对高频突变基因进行GSEA富集分析,运用CIBERSORT算法计算肿瘤浸润免疫细胞的相对丰度,探究共同高频突变基因对肿瘤浸润免疫细胞的影响.结果 在TCGA和ICGC数据库中发现21个共同的高频突变基因,其中FAT4与患者临床预后及肿瘤突变负荷相关.通过GSEA富集分析和CIBERSORT算法,发现FAT4突变与免疫系统相关信号通路的激活及免疫应答的增强相关.结论 胃癌中FAT4突变频率较高,且其突变与患者较好的预后及抗肿瘤免疫功能的增强相关.因此,FAT4具有作为预测胃癌患者免疫应答的生物标记物的潜力.

目的:探讨术前等待间乳腺癌病人的信息需求.方法:采用质性研究中的现象学研究方法对15例术前等待间乳腺癌病人进行半结构式访谈,使用Colaizzi 7步分析法进行资料分析.结果:术前等待间乳腺癌病人的信息需求可归纳为5个主题:疾病及相关治疗需求、手术室环境需求、手术过程需求、心理社会支持需求、多形式的信息提供方式.结论:护理人员应重视术前等待间乳腺癌病人的信息需求,通过多种形式为其提供信息支持.

人巨细胞病毒（human cytomegalovirus,HCMV）能诱导肿瘤细胞恶性转化且抑制肿瘤细胞凋亡,但HCMV编码的主要即刻早期调控蛋白IE86在这一过程中是否发挥关键作用仍然未知。为探究IE86对基因修饰荷胶质瘤小鼠p53表达水平及恶性胶质瘤细胞凋亡情况的影响,本研究通过PCR技术鉴定基因修饰小鼠IE86表达情况;实时定量PCR技术检测IE86和p53mRNA表达水平变化;免疫组织化学方法检测p53和p21蛋白的表达水平;TUNEL检测肿瘤组织细胞凋亡情况。结果显示,成功构建了IE86基因修饰小鼠模型;与IE86阴性组相比IE86阳性组p53表达水平上升（P＜0.05）,但p21表达水平下降（P＜0.05）;IE86阳性组细胞抗凋亡能力增强（P＜0.05）。以上结果表明,在基因修饰的小鼠中IE86持续表达但p53转录活性的指示标志p21下调,且IE86可提高恶性胶质瘤细胞抗凋亡能力。

目的 了解莪术、冬凌草及白花蛇舌草复方提取物联合更昔洛韦对人巨细胞病毒(HCMV)阳性脑肿瘤干细胞移植瘤的作用.方法 用单克隆培养法从U251胶质瘤细胞系中分离出脑胶质瘤干细胞,用流式细胞仪检测其CD133阳性细胞比例;用HCMV AD169感染脑胶质瘤干细胞,接种到40只BALB/c裸鼠脑内.2周后,将荷瘤鼠随机分为空白对照组(C组)、复方中草药组(A组)、更昔洛韦组(B组)及复方中草药联合更昔洛韦组(D组).其中C组采用生理盐水灌胃,其余各组给予相应药液灌胃,连续给药2周.处死裸鼠,取出瘤组织做病理学检查及IE、CD34免疫组化检查,计算各组移植瘤微血管密度(MVD);采用qRT-PCR技术检测各组移植瘤中Bcl-2mRNA表达情况.结果 U251肿瘤干细胞在无血清培养基中成球生长,第二代肿瘤球中CD133阳性表达细胞比例为99.89％.各组移植瘤与正常组织分界明显,瘤内出血坏死,移植瘤IE、CD34表达阳性.复方中药提取物和更昔洛韦抑制移植瘤微血管生成的主效应差异有显著性(F=8.29、8.05,P＜0.05),联合用药抑制移植瘤微血管生成较单独用药组效果明显,二者无交互作用(P＞0.05).qRT-PCR检测结果显示,复方中草药提取物和更昔洛韦抑制胶质瘤Bcl-2 mRNA表达的主效应差异有显著性(F=9.43、6.69,P＜0.05),二者无交互作用(P＞0.05),A组和B组Bcl-2 mRNA相对表达量低于C组,D组低于A组、B组和C组(F=9.43、6.69,P＜0.05).结论 自拟复方中草药提取物联合更昔洛韦对HCMV阳性恶性胶质瘤生长有抑制作用.

Objective:To detect immunological effects of procyanidin ,gastrodin,baicalein and apigenin on antigen presenting cells of RAW264.7 and DC2.4 and search for new tumor vaccine adjuvant.Methods: After different concentrations of four kinds of Chinese herbal monomer and LPS were used to stimulate RAW 264.7 or DC2.4 cells for 48 hours,cells were obtained and stained with CD80,CD86,MHCⅠand MHCⅡ antibody.Then protein expression levels of CD80,CD86,MHCⅠ and MHCⅡ on RAW264.7 and DC2.4 cells were analyzed by flow cytometry.Results: Results showed that after 48 hours stimulated with different concentrations of four kinds of Chinese herbal monomer on RAW 264.7 or DC2.4 cells,the expression levels of CD80,CD86,MHCⅠ,MHCⅡ were all up-regulated in comparison with control , procyanidin and baicalein exhibited stronger immunological stimulating activity on a cellular level.Conclusion:Procyanidin and baicalein were found having potential application value in clinical treatment as tumor vaccine adju -vants.

Abstract] Objective To analyze the molecular characteristics of the full-length genome of a new-ly isolated genotypeⅤJapanese encephalitis virus (JEV) strain (XZ0934) in China and the first genotypeⅤJEV strain ( Muar) isolated in Malaya 60 years ago.Methods Several softwares including ClustalX 2.0.9, DNAStar 7.1, Bioedit 7.2.5 and MEGA6.06 were used to conduct sequence alignments and phylo-genetic analysis.Results The full-length genomes of XZ0934 strain (isolated in Tibet, China in 2009) and Muar strain (isolated in Malaya in 1952) were composed of 10 983 and 10 988 nucleotides, respective-ly.The XZ0934 strain was highly similar with the Muar strain showing the homology of 90.6%in nucleotide (nt) and 98.3%in amino acid (aa).The open reading frame (ORF) of the two genotype Ⅴ JEV strains encoded 3433 aa while the ORF of other four genotypes (Ⅰ-Ⅳ) (10 299 nt) encoded 3432 aa.Compared with JEV strains of other genotypes, a serine were inserted into the NS4A gene of JEV strains genotype Ⅴand 10-14 nucleotides were inserted into the downstream of the ORF stop codon in 3′-untranslated region. Phylogenetic analysis of E sequences of all JEV strains genotypedⅠ-Ⅴrevealed that in the cluster of geno-typeⅤ, XZ0934 and 10-1827 ( isolated from mosquitoes in South Korea, 2010) stains formed a branch and were divergent from that of Muar strain indicating that there were molecular genetic differences among geno-typeⅤJEV strains after a 60 years hiatus.Conclusion The two genotypeⅤJEV strains showed high lev-els of identity in nucleotide sequences and amino acid sequences with serine insertion in the NS4A gene. However, there were molecular genetic differences between genotypeⅤJEV strains isolated after a 60 years hiatus.

目的 研究人巨细胞病毒(HCMV)感染对宫颈癌细胞Siha发生上皮—间质转化的作用及意义. 方法 实验组用HCMV AD169(MOI=5)和5 ng/ml TGF-[31作用宫颈癌Siha细胞,同时将仅TGF-β1作用的Siha细胞作为对照组,分别在0、24、48、72 h观察细胞形态的变化,应用RT-PCR检测E-钙粘蛋白和波形蛋白的转录水平,采用Western blot检测E-钙粘蛋白、波形蛋白和MMP-2的表达水平. 结果 实验组细胞于48 h发生细胞形态学的变化,早于对照组的72 h;实验组上皮细胞标记物E-钙粘蛋白的转录和表达水平较对照组显著下调(P＜0.01),间质细胞标记物波形蛋白的转录和表达较对照组显著上调(P＜0.01)且表达提前,MMP-2的表达较对照组显著上调(P＜0.01)且表达提前.结论 HCMV感染可促进宫颈癌细胞Siha发生化上皮—间质转化.

Objective To investigate the expression of inflammatory factors of Toll like receptor(TLR) signaling pathway in colorectal carcinoma.Methods The expression of TLR2,nuclear factor κB(NF-κB) and interleukin-8(IL-8)was detected by immunohistochemistry in 56 samples of colorectal cancer,36 samples of colorectal adenoma tissue and 56samples of pericancerous normal colorectal tissue.The correlation of TLR2,NF-κB and IL-8 expression with clinicopathological features of colorectal cancer was analyzed.Results In colorectal carcinoma tissue,adjacent normal colorectal tissue and colorectal adenoma tissue,the positive expression rates of TLR2 were 71.4%,42.9% and 58.3%,the positive rates of NF-κB were 78.6%,46.4% and 83.3%,and the positive rates of IL-8 were 75.0%,46.4% and72.2%,respectively(all P 0.05).The expression of NF-κB was closely correlated with the expression of TLR2 and IL-8(P 0.05).In addition,the expression of TLR2 was correlated with tumor differentiation,lymph node metastasis and clinical stages(P 0.05).The expression of NF-κB and IL-8 was correlated with vascular invasion,lymph node metastasis and clinical stages(P 0.05).Conclusion Overexpression of inflammatory factors of Toll like receptor signaling pathway is closely correlated with the biological behavior of colorectal cancer,suggesting that TLR signaling pathway may be involved in the pathogenesis and development of colorectal cancer.

Objective To investigate the expression of nuclear factor kappa B( NF-κB),tumor necrosis factor α( IL-6) and Interleukin-8( IL-8) in human esophageal carcinoma tissues and their clinical action. Methods 48 samples of esophageal cancer tissues and corresponding samples of paracancerous normal esophageal tissues were collected,and. NF-κB,IL-6 and IL-8 were detected by immunohistochemistry SABC method. The correlation between the expression of NF-κB、IL-6 and IL-8 with the clinical pathologic features of esophageal cancer was analyzed. Results The positive expressions rates of NF-κB in esophageal carcinoma and adjacent normal esophageal tissues turned out to be 79. 2% and 45. 8% respectively,IL-675. 0% and 41. 7% and IL-8 83. 3% and 52. 1%. The difference was significant for all groups( P < 0. 05). The expression of NF-κB was closely associated with the expressions of IL-6 and IL-8 respectively. In addition,the expressions of NF-κB and IL-8 was correlated with lymphnode metastasis and stageing,IL-6 were with vascular invasion,lymphnode metastasis and stageing. Conclusions Highly expressed NF-κB and its induced inflammation factors had close relationship with clinical pathologic features of esophageal cancer,which suggest that the pathway of NF-κB play an important role in carcinogenesis and development of esophageal cancer.

Objective The objective of this study was to elucidate whether beta2-GPⅠ interacts with LPS to increase the activation of NF-κB and the expression of TNF-α, IL-1β and AFP in liver cancer cell SMMC-7721. Methods NF-κB activation was assessed by immunofluorescence assay in SMMC-7721 HCC cells exposed to various treatments. The cells were normal control cells (group A), transiently transfected with vectors expressing beta2-GPⅠ(group B), incubated with different concentrations (1,10,100 ng/ml) LPS (group C1-C3) and group D1-D3 were incubated with different concentrations (1,10,100 ng/ml) LPS and transfected with beta2-GPⅠvector. TNF-α, IL-1β, AFP expression were detected by ELISA in all groups. Results Group B, C and D had induced activation of NF-κB. However, the highest activity of NF-kB was observed in group D3. The mean TNF-α, IL-1β and AFP levels were significantly higher in group D3 than other groups with significant differences (P<0.05) on concentration-dependent manner. Conclusion Beta2-GPⅠ can interact with LPS and then play a role in the development of HCC by activating NF-κB.

Objective To discuss the formation condition of Candida albicans' s pseudohyphae and make into specimen slices for the experimental teaching of pathogeny microbiology.Methods Candida albicans was incubated into the medium of the improved Sabourand's and put in 37℃ fungus incubator for 1-2days,then transplanted into the medium of the improved 1%Tween-80corn meal for further incubation for 3-4days.Specimen slices were made,dyed and observed under microscope.Results Pseudohypha formed well and was in complete form.Conclusion This effective way shows a high positive rate.Candida albicans' s pseudohyphae cultured by this method is in a complete form and is easy to observe.

Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.

刺参粘多糖(stichopus japonicus acid muco-polysaccharide,SJAMP)是从中国刺参体壁中提炼出来的一类多糖类物质.该物质成分恒定,结构简单,易于克服空间位阻,增加了对黏膜上皮的穿透能力,容易被吸收.初步研究证实[1-4],刺参粘多糖具有广谱的抗肿瘤活性,能抑制肝癌、宫颈癌等恶性肿瘤细胞的增殖,诱导细胞分化.本研究观察SJAMP对胰腺癌细胞SW1990增殖、凋亡的影响,探讨其可能机制.

Objective: To investigate the effect and the clinical significance of Th cells in the pathogenesis of patients with lupus nephritis(LN).Methods:64 systemic lupus erythematosus patients and 28 healthy controls were enrolled.The concentrations of serum IL-17,IFN-γ,IL-4 were measured by enzyme-linked immunosorbent assay(ELISA).Results: 1.The expression of IL-17 in LN Group exhibited a significant increase compared with that in SLE Group and healthy controls(P0.001).2.The levels of IFN-γ in LN Group were higher than those in SLE Group and in healthy controls(P0.05,P0.01,respectively).3.The levels of IL-4 in SLE Group,LN Group were higher than those in healthy controls(P0.01).4.Th1/Th2(IFN-γ/IL-4) ratio in LN Group increased significantly than that in SLE Group(P0.01) and HC group(P0.05);Th1/Th2(IFN-γ/IL-4) ratio decreased in SLE Group compared with that in HC group(P0.01).5.The levels of IFN-γ were positively correlated with SLEDAI scores in SLE patients(P0.05).Conclusions: The significantly elevated serum IL-17,IFN-γ,IL-4 levels in lupus nephritis,suggesting that Th17/ Th1/ Th2 functional imbalance may be involved in the pathogenesis of renal damage in SLE patients.

【Objective】 To detect specific biomarkers in serum of hepatocellular carcinoma(HCC) patients using Surface Enhanced Laser Desorption Inhibition Time of Flight Ionization Mass Spectrometry(SELDI-TOF-MS),and establish a serum protein pattern for screening HCC.【Methods】 Serum samples of 30 HCC patients,20 HBV carriers and 20 healthy controls were detected by SELDI-TOF-MS.The collected data were analyzed by Biomarker Wizard and BPS software to establish a serum protein pattern for screening HCC.The pattern was evaluated by masked test.【Results】 Total 25 peaks showed significant differences between HCC patients and controls,among which 5 peaks(6 888,13 764,6 487,3 450,6 814 Da) were automatically chosen to set up a serum protein pattern for screening HCC.The sensitivity and specificity of the pattern was 96.7%(29/30) and 90.0%(36/40).When tested by masked samples,it was 95.5%(21/22) and 90%(27/30),respectively.【Conclusion】 SELDI-TOF-MS had high sensitivity and specificity.It would play a valuable role on the diagnosis of HCC and on the screening of tumor specific biomarkers.

Objective To study the expression of activating transcription factor 5(ATF-5) and its significance in human colorectal cancer tissues.Methods The tissue sample of colorectal cancer was collected in 38 cases,and that of normal rectal tissue 12 cases.RT-PCR was used to detect the expression of ATF-5 in both samples and compared between them.ResultsThe expression of ATF-5 was noted in 32 samples in cancer tissue,and without any of that expression in normal rectal tissue,being a significant difference between the two groups(χ2=24.53,P0.01).Conclusion The high expression of ATF-5 may play a key role in the occurrence and development of this malignancy.

[Objective]To investigate the pollution status of the bacteria in indoor air of university campus,so as to provide scientific basis for making solutions to improve classroom air quality.[Methods]In October 2010,the natural sinking method was applied to detect the total bacteria count of indoor air of student dormitories,cafeteria,classrooms and laboratories in a university in Qingdao.[Results]A total of 139 samples were investigated and 138 ones were found to be qualified,with a qualified rate of 99.3%.The total colonies qualified rate was 97.73% for 44 dormitories,and all were 100.00% for 50 dormitories,25 cafeteria and 20 laboratories.The air quality of all the samples was rather clear,among which,it was slightly polluted at student dormitories;it was rather clean at cafeteria and clean at classrooms and laboratories.The total air bacteria count(CFU/m3) was 2 029±826 at male dormitories and 3 008±1 868 at female dormitories(P0.05).At the five floors from one to five,this value was 1 966±1 415,1 809±909,2 752±1 128,3 676±1 966,2 949±1 757(P0.05),respectively.[Conclusion]The indoor air quality in public places of a university is rather clean,while it is slightly polluted at student dormitories.

Objective To investigate the apoptosis-inducing effect and anti-proliferative effect of epigallocatechin-3-gallate (EGCG) on human pancreatic cancer cell SW1990 in vitro. Methods The effect of proliferation was evaluated by MTT after the SW1990 cells in vitro were incubated with different concentrations of EGCG (6.25, 12.5, 25, 50, 100 μg/ml). The apoptosis-inducing effect was determined by flow cytometry after the cells were treated with 25 μg/ml of EGCG. The cell cycle of SW1990 cells was detected by flow cytometry after the cells incubated with different concentrations of EGCG (0, 10, 20, 30, 40, 50 μg/ml).Results After SW1990 cell were treated with different concentrations of EGCG (0, 25, 50 μg/ml), the values of A492 were 0.46 ±0.04,0.42 ±0.04,0.27 ±0.03 at 24 h; 0.48 ±0.02, 0.31 ±0.03,0.16 ±0.02at 48 h; 0.51 ±0.01,0.24 ±0.04,0. 14 ±0.04 at 72 h. EGCG inhibited the proliferation of SW1990 in a doseand time-dependant manner(P ＜0.01 ). The apoptotic rates at 24, 48, 72 h were (8.33 ± 1.15 )%, (19.77 ±0.81 )%, (29.17 ± 0.75 )% in the EGCG treatment group; while the corresponding values were (2.77 ±0.45 ) %, (3.20 ± 0.26 ) %, (3.67 ± 0.35 ) % in the control group; and the difference was statistically significant (P ＜0.01 ). After 0, 20, 50 μg/ml of EGCG treatment for 24 h, the percentages of SW1990 cellsin G0/G1 stage were (57.59 ±0.97)%, (62.99 ± 1.91 )%, (68.87 ± 1.88)%, and the percentages of SW1990 cells in G0/G1 stage increased with the increase of concentrations of EGCG, while the percentages of SW1990 cells in G2/M stage decreased with the increase of concentrations of EGCG (P ＜0.01 ). Conclusions EGCG can significantly inhibit the proliferation of SW1990 cells. The mechanism may be related to the apoptosis-inducing effect and the regulation of the cell cycle of the SW1990 cells.

Objective:To investigate the different expression of Th17 type cytokines in the peripheral blood of rheumatoid arthri-tis patients with rheumatoid factor positive(RF+) and rheumatoid factor negative(RF-).Methods:51 RA patients including RF+ and RF-and 20 healthy subjects were studied.The percentage of Th17 cell in single PBMC was determined by flow cytometry and the levels of IL-17 and IL-6 in plasma were detected by Enzyme-linked immunosorbent assay(ELISA) in all subjects.Results:Th17 cell and the con-centrations of IL-6 and IL-17 were significantly higher in RA patients than in healthy controls.Furthermore,Th17 and the concentra-tions of IL-6 and IL-17 in RF+ cases were higher than that in RF-cases.Conclusions:Different immune reaction and inflammation ex-pression in RA patients with or without RF may relate with the differences of the expression of Th17 type cytokine.

Objective To investigate the role of HCMV infection in the development of colorectal cancer and its possible mechanisms.Methods Tumor tissue samples were collected from 50 patients with colorectal cancer and samples of normal tissue 10 cm distant from surgical margins were collected to serve as controls.HCMV infection was detected by RT-PCR in tumor tissue and tumor-adjacent normal tissue,while the expression of Bcl-2 was evaluated with immunohistochemical straining.Results The rate of positivity for HCMV IE2 was 40%(20/50) in tumor tissue and 6%(3/50) in adjacent normal tissue;the difference in the two was statistically significant(P0.05).The rate of positivity for Bcl-2 was 36%(18/50) in tumor tissue and 8%(4/50) in adjacent normal tissue;the difference between the two was statistically significant(P0.05).Positivity for Bcl-2 was higher in the 50 samples of HCMV-positive tumor tissue than in the HCMV-negative tumor tissue,and the level of Bcl-2 in tumor tissue correlated with HCMV infection(χ2=5.834,P0.05).Conclusion HCMV infection is related to the development of colorectal cancer and a possible mechanism may be its effect on the expression of Bcl-2.