Fas-670基因多态性与青海地区胃癌发生风险的相关性

目的探讨凋亡基因Fas-670基因多态性在青海地区胃癌发生中的作用。方法采用PCR-RFLP法检测95例胃癌患者（胃癌组）和129例健康人（对照组）Fas-670基因多态性,分析基因多态性与胃癌易感性的关系。结果胃癌组Fas-670基因多态分布为：纯合野生型AA 29.5%（28/95）、杂合型AG 49.5%（47/95）、纯合突变型GG 21.0%（20/95）。对照组Fas-670基因多态分布为：纯合野生型AA频率41.9%（54/129）、杂合型AG频率47.3%（61/129）、纯合突变型GG频率10.8%（14/129）。Fas-670基因型与青海地区胃癌风险有统计学意义（P=0.047）。结论 Fas-670基因多态可能与青海地区胃癌发生风险有相关性。     

Fas-1377基因多态性与胃癌易感性的相关性

目的探讨Fas-1377基因多态性与胃癌易感性的关系。方法提取234例胃癌患者（观察组）及321例健康献血者（对照组）的外周血基因组DNA,用聚合酶链反应-限制性片段长度多态性法确定Fas-1377基因型。结果Fas-1377AA、GA、GG基因型在观察组和对照组中的分布频率分别为13．25％、44．44％、42．31％和12．15％、47．04％、40．81％,组问比较元统计学差异（P〉0．05）。Logistic回归分析表明,Fas-1377基因多态性与胃癌发生风险无关。结论Fas-1377基因多态性与华北地区胃癌发生风险无明显相关性。     

雌激素受体β基因RsaI和AluI多态性与原发性肝癌的关系

目的 探讨雌激素受体β(ER β)基因多态性与原发性肝癌的关系.方法 选择西南地区100例原发性肝癌患者为观察组,100例同期非肝病患者作为对照组.应用分子生物学的限制性片段长度多态性方法分析Rsa I和Alu I,观察ER β基因型的分布.结果 观察组R等位基因频率为35.0%,对照组为51.0%,OR值0.517(95%可信区间为0.346～0.773),P＜0.01.观察组A等位基因频率为20.5%,对照组为11.0%,OR值2.086(95%可信区间为1.191～3.654),P＜0.01;Rsa I和Alu I限制性片段长度多态性在两组中均呈多态性分布.结论 ERβ基因多态性与原发性肝癌有关,R等位基因可能是其保护因素,A等位基因可能是其危险因素.     

肝癌的基因靶向治疗

从遗传学的角度来讲，肝癌与其他恶性肿瘤一样是一种复合基因病，其涉及的基因包括：癌基因、肿瘤抑制基因、细胞周期调节基因、细胞凋亡基因以及维持细胞基因组稳定性的基因等。有鉴于此，针对病变基因的基因靶向治疗成为肝癌领域中的研究热点。目前开展的肝癌基因靶向治疗的策略包括：自杀基因治疗、免疫基因治疗、导入抑癌基因、抗肿瘤血管生成和转移、核酶以及RNA干扰等。但直到现在，大部分关于肝癌基因靶向治疗的研究仍停留在实验研究或前期临床研究阶段。     

肝癌组织中HBx基因的多态性及其突变体对QSG7701细胞生物学行为的影响

目的研究中国南部部分地区肝细胞癌患者中是否存在特征性HBx基因突变，以及基因变异在肝癌发病中的作用。方法采用巢式PCR，单链构象多态分析（SSCP）、异源性双链分析（HA）和DNA测序等方法对51份肝细胞癌组织石蜡包埋切片标本和25份HBV携带者血清HBx基因多态性进行分析，对有明显缺失变异的HBx基因进行克隆，转染肝细胞，通过体外和体内实验比较变异和野生型HBx基因转染对肝细胞QSG7701生物学行为的影响。结果肝癌组织中HBx基因存在点突变和缺失型突变，B和C基因型之间突变类型和数量有一定差异，C基因型点突变频率更高（t=-2．522，P〈0．05），而B基因型存在缺失变异（HBx—d382和HBx—d431）。获得了含HBx缺失变异的基因的稳定转染细胞株。该细胞株细胞大小形态不一、体积增大、核浆比例增大，生长速度更快，克隆形成率高（P〈0．05），在裸鼠体内成瘤机会和速度更快。结论HBx基因在肝细胞癌组织中频繁地发生突变，nt382～400位置上的缺失型突变体转染QSG7701后能促进肝细胞的生长速度，细胞凋亡减少，使之获得肿瘤性细胞特性，因此HBx—d382缺失型突变体可能在中国南部部分地区肝癌发生发展中发挥重要作用。     

肝细胞癌相关肿瘤抗原基因的筛选与分析

目的 筛选和分析肝细胞癌相关的肿瘤抗原。方法 用肝细胞癌组织构建cDNA表达文库，通过重组cDNA表达文库血清学分析法，用自体患者和异体患者的血清对文库进行筛选。将所得阳性噬菌体克隆体内剪切获得其pBK-CMV噬菌粒。通过限制性核酸内切酶鉴定插入cDNA片段，并作测序和生物信息学分析。同时将阳性克隆中的LIMS1插入片段进行原核表达。结果 自体血清筛选得到14种基因，异体筛选获得11种基因，两组中均筛选到kinectin，共有24种肝细胞癌相关的肿瘤抗原基因。其中有8种基因目前还不清楚其功能，其余16种基因根据确定的或者推断的功能可以分成8组。LIMS1原核重组蛋白表达成功。结论 本研究为肝细胞癌的免疫治疗提供了候选基因，并为理解肝细胞癌发生和发展的机制提供了线索。     

黑色素瘤抗原-A1基因多态性及其在肝癌组织中的表达

目的了解黑色素瘤抗原(MAGE)-A1基因在肝癌组织中的表达状况,探讨该基因是否存在单核苷酸多态性(SNP).方法用RT-PCR方法检测49例原发性肝细胞性肝癌(HCC)患者肝癌组织MAGE-A1基因mRNA的表达情况,同时提取其中43例HCC患者的肝癌组织、癌旁组织及外周血细胞基因组DNA,PCR扩增MAGE-A1 DNA,并对上述PCR产物进行测序.结果49例患者中MAGE-A1 mRNA阳性22例,阳性率44.9%.43例HCC患者癌组织、癌旁组织和外周血细胞中扩增的KAGE-A1 DNA序列测定显示MAGE-A1基因存在两类变异:TAG有16例,变异发生率为37.2%,包括3个位置的变异:C159T,A272G,G393A,GTG变异有7例,发生率为16.3%,亦包括3个位点的变异即:A272G,C991T,A1125G.因为C991T没有改变编码的氨基酸,A1125G不在编码区,故仅A272G引起一个氨基酸的替换(T32A).结论MAGE-A1在HCC中高表达,表达率为44.9%.MAGE-A1 mRNA的表达与AFP无关.MAGE-A1基因存在三种SNP,但不影响其mRNA的表达.     

SMART技术构建人肝癌抗原基因cDNA噬菌体表达文库

目的:构建肝癌抗原基因cDNA噬菌体表达文库.方法:从肝癌细胞提取总RNA,纯化mRNA.用逆转录酶链式反应(RT-PCR)反转录合成cDNA第1链,LD-PCR合成cDNA第2链,除去<500 bp小片段,与λTripLEx2噬菌体载体连接并体外包装,转化大肠感菌E.coli XL1-blue,测定文库的库容和重组率,PCR鉴定插入cDNA片段大小.结果:构建的肝癌抗原基因cDNA噬菌体表达文库,原始库容为3.4×10~6pfu/mL,重组率为98.2%;文库扩增后库容为9.6×10~8 pfu/mL,重组率为97.2%.重组子插入cDNA片段大小0.6-2(平均1.4)kb.结论:成功构建高质量的肝癌抗原基因cDNA噬菌体表达文库,为肝癌特异性抗原的筛选和鉴定奠定了基础.     

肝细胞癌患者IL-18基因-137G/C和-607A/C位点单核苷酸多态性变化及其意义探讨*

目的 探讨肝细胞癌（HCC）患者白细胞介素-18（IL-18）基因-137G/C和-607A/C位点单核苷酸多态性（SNP）的变化。方法 在178例伴有HBV感染的HCC患者和251例健康人,取外周静脉血提取DNA,采用聚合酶链反应-连接酶检测反应（PCR-LDR）行基因分型,测定IL-18基因-137G/C和-607A/C位点SNP。结果 HCC患者和健康人IL-18-37 G/C基因GG基因型、GC基因型和CC基因型分布频率分别为75.3%对47.0%（P<0.05）、20.8%对51.4%（P<0.05）和3.9%对1.6%（P>0.05）,G等位基因和C等位基因分布频率分别为93.6%对72.7%（P<0.05）和6.4%对27.3%（P<0.05）;HCC患者和健康人IL-18-607A/C位点AA基因型、AC基因型和CC基因型分布频率分别为37.6%对13.5%（P<0.05）、43.3 %对66.9%（P<0.05）和19.1%对19.5%（P>0.05）,A等位基因和C等位基因分布频率分别59.3%对47.0%（P<0.05）和40.7%对53.0%（P<0.05）;HCC组IL-18-137G/C位点的GG基因和G等位基因频率显著高于健康人（P<0.05）,HCC组IL-18-607A/C位点的AA基因和A等位基因频率也显著高于健康人（P<0.05）,提示携带IL-18-137G/C和IL-18-607A/C位点基因型和A等位基因者罹患HCC的风险增加。结论 携带IL-18基因-137G/C位点GG基因型和G等位基因以及-607A/C位点AA基因型和A等位基因者可能更容易发生HCC,对HBV感染者筛查这些基因可能有助于早期发现肝肿瘤,以利于早期处理和改善预后。     

Association of Fas-670 gene polymorphism with inflammatory bowel disease in Chinese patients

AIM: Recent studies suggest that Fas-mediated apoptosis is involved in the pathogenesis of inflammatory bowel disease (IBD). It has been hypothesized that either increased apoptosis of intestinal epithelium or decreased apoptosis of lamina propria lymphocytes may induce inflammation of' gut. The aim of this study was to determine whether the Fas gene promoter polymorphism at position-670 was associated with IBD in Chinese patients. METHODS: Fifty unrelated Chinese patients with IBD (38 patients with ulcerative colitis and 12 with Crohn's disease) and 124 healthy controls were genotyped for the Fas-670 polymorphism by PCR-restriction fragment length polymorphism method. The PCR product was digested by Mva I restriction enzyme. RESULTS: Distribution of the Fas-670 gene polymorphism was 33% for the AA genotype, 52% for the AG genotype and 15% for the GG genotype in 124 healthy subjects. In patients with IBD, 30% was for the AA genotype, 42% for the AG genotype and 28% for the GG genotype respectively. However, there was no significant difference in the genotype (P= 0.1498), allele frequencies (P= 0.3198) and carriage frequencies (P = 0.4133) between healthy controls and IBD patients. Furthermore, we did not find any difference between the left-sided colitis and total colitis (P = 0.8242). CONCLUSION: Fas-670 polymorphism is not associated with IBD in Chinese patients.     

Association of Fas/Apol gene promoter （-670 A/G） polymorphism in Tunisian patients with IBD

AIM： To detect a possible association between the polymorphism of the （-670 A/G） Fas/Apol gene promoter and susceptibility to Crohn＇s disease （CD） and ulcerative colitis （UC） in the Tunisian population. METHODS： The （-670 A/G） Fas polymorphism was analyzed in 105 patients with CD, 59 patients with UC, and 100 controls using the polymerase chain reaction restriction fragment length polymorphism method. RESULTS： Significantly lower frequencies of the Fas -670 A allele and A/A homozygous individuals were observed in CD and UC patients when compared with controls. Analysis of （-670 A/G） Fas polymorphism with respect to sex in CD and UC showed a significant difference in A/A genotypes between female patients and controls （P corrected = 0.004 ＂in CD patients＂ and P corrected = 0.02 ＂in UC patients＂, respectively）. Analysis also showed a statistically significant association between genotype AA of the （-670 A/G） polymorphism and the ileum localization of the lesions （P corrected = 0.048） and between genotype GG and the colon localization （P corrected = 0.009）. The analysis of IBD patients according to clinical behavior revealed no difference. CONCLUSION： Fas-670 polymorphism was associated with the development of CD and UC in the Tunisian population.     

Association of Fas/Apo1 gene promoter (-670 A/G) polymorphism in Tunisian patients with IBD

AIM: To detect a possible association between the polymorphism of the (-670 A/G) Fas/Apo1 gene promoter and susceptibility to Crohn's disease (CD) and ulcerative colitis (UC) in the Tunisian population. METHODS: The (-670 A/G) Fas polymorphism was analyzed in 105 patients with CD, 59 patients with UC, and 100 controls using the polymerase chain reaction restriction fragment length polymorphism method. RESULTS: Significantly lower frequencies of the Fas -670 A allele and A/A homozygous individuals were observed in CD and UC patients when compared with controls. Analysis of (-670 A/G) Fas polymorphism with respect to sex in CD and UC showed a significant difference in A/A genotypes between female patients and controls ( P corrected = 0.004 in CD patients and P corrected = 0.02 in UC patients, respectively). Analysis also showed a statistically significant association between genotype AA of the (-670 A/G) polymorphism and the ileum localization of the lesions ( P corrected = 0.048) and between genotype GG and the colon localization ( P corrected = 0.009). The analysis of inflammatory bowel disease patients according to clinical behavior revealed no difference. CONCLUSION: Fas-670 polymorphism was associated with the development of CD and UC in the Tunisian population.     

Effect of Fas -670 A/G Gene Polymorphism on Corneal Allograft Endothelial Rejection

Background: Human cornea expresses functional Fas-ligand capable of killing Fas+ activated lymphocytes. Fas expression is partly regulated by -670 A/G polymorphism in the promoter region of Fas gene. Objective: The aim of the present study is to determine the association between Fas-670A/G polymorphism and survival of corneal transplantation. Methods: In 276 graft recipients who mainly underwent penetrating keratoplasty because of keratoconus, bullous keratopathy and corneal opacity, Fas -670 A/G polymorphism was determined by allele specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques. Results: There was no statistically significant relationship between Fas -670 A/G polymorphism and rejection episode (p=0.35). Moreover, the relationship between this polymorphism and rejection episode outcome (transplant recovery vs failure) was not statistically significant (p=0.13). Conclusion: The results of the present study show no significant correlation between corneal graft rejection, rejection recovery and Fas -670A/G gene polymorphism.     

Association of FAS （TNFRSF6）-670 gene polymorphism with villous atrophy in coeliac disease

AIM:To investigate the association of FASgene polymorphism with coeliac disease (CD) development.METHODS: FAS-G670A gene polymorphism, located in a gamma interferon activation site, was studied in 146 unrelated CD patients and 203 healthy ethnically matched controls. The restriction fragment length polymorphism (RFLP) method was used to identify FAS-G670A gene polymorphism.RESULTS:No significant difference was found in genotype frequency between CD cases and controls. In controls,however, the frequency of the GGgenotype was significantly higher in women (26.5%) than in men (12.8%) (OR=2.44,95% CI1.15-5.20, P=0.020) and it was also higher in men with CD than controls (OR=2.60, 95% (CI0.96-7.05, P=0.061).The GG genotype frequency was significantly higher in patients with most severe villous atrophy (Marsh Ⅲc lesions) (OR=3.74, 95% CI 1.19-11.82, P=0.025). A significantly less proportion of men suffered from Marsh IIIc lesions than women (OR=0.20, 95% (CI0.06-0.68, P=0.01). The risk of having severe villous atrophy increased with the additive effect of the Gallele in women (P=0.027 for trend, age and gender adjusted).CONCLUSION: FAS-G670A gene polymorphism is associated with the severity of villous atrophy in CD. Female gender is also associated with the severity of villous atrophy.     

乙型肝炎肝组织中Fas抗原表达的研究

目的；探讨乙型肝炎肝组织Ｆａｓ抗原表达民细胞凋亡和肝细胞损伤关系，方法：用免疫组织化学技术，对１０９例乙型肝炎肝组织Ｆａｓ抗原进行了检测。结果：Ｆａｓ抗原总检出率７６．１６％，急性重症肝炎（ＡＦＨ）慢性迁延型肝炎（ＣＰＨ）慢性活动肝炎（ＣＡＨ），活动性肝硬化（ＡＣ）Ｆａｓ抗原检出率分别为９０．９％，４３．４８％，７８．１２％，８５．７１％，急性重症肝炎Ｆａｓ抗原阳性细胞分布于大片坏死区域中残留肝细     

Fas抗原在乙型病毒性肝炎肝组织中表达的研究

目的 检测乙型病毒性肝炎肝组织中Fas抗原,以探讨Fas抗原的表达与肝细胞损伤之间的关系。方法 以免抗—Fas多克隆抗体,采用免疫组织化学技术检测33例急性重型肝炎,23例慢性迁延型肝炎,32例慢性活动性肝炎,21例活动性肝硬化和13例慢性重型肝炎肝组织中Fas抗原表达情况。结果 上述5组分型中Fas抗原检出率分别为90.9％、43.5％、78.1％、85.7％和92.3％。急性重型肝炎Fas抗原分布于大片坏死区残留肝细胞内,慢性肝炎和活动性肝硬化Fas抗原阳性细胞分布于碎屑状坏死灶周围或假小叶周边部。肝组织病理改变越重,Fas表达越强。结论 Fas抗原的表达与肝细胞损伤密切相关。     

Purification and characterization of α-L-fucosidase from human primary hepatocarcinoma tissue

AIM: To purify and characterize alpha-L-fucosidase from human liver cancer tissue and to detect the localization of alpha-L-fucosidase in tumor tissue. METHODS: Cation exchange chromatography on CM-52 and ultrafiltration were used to separate alpha-L-fucosidase (AFU) from crude extract of liver cancer tissue. 4-methylumbelliferyl-alpha-L-fucopyranoside was used as a fluorescent substrate to quantify the purified AFU activity in each step. A polyclonal antibody (pAb) against the purified AFU was obtained by anion exchange chromatography on DEAE-52 after ammonium sulfate fractionation and ultrafiltration. Immuohistochemical staining was used to observe the expression of AFU in malignant and adjacent liver tissues. RESULTS: Human alpha-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecular weight of 55 Ku. The specific activity of AFU in pooled fraction by chromatography was 10085 IU/mg. Western blot analysis indicated that the pAb could recognize one protein band of molecular weight of 55 Ku. The expression of AFU was observed in cytoplasm membrane of liver cancer tissue but not in that of adjacent tissue. CONCLUSION: The purified alpha-L-fucosidase from primary hepatocarcinoma (PHC) is different in its properties from alpha-L-fucosidase in human other organs. The polyclonal antibody prepared in this experiment can be applied to the diagnosis of PHC.