构建7q32区域鼻咽癌和正常鼻咽上皮细胞部分基因的表达图谱

目的构建７ｑ３２区域鼻咽癌细胞和组织及原代培养人正常鼻咽上皮细胞的部分基因表达图谱。方法通过差异ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ杂交的方法检测定位于７ｑ３２区域的２０个ＥＳＴ在鼻咽癌细胞和鼻咽癌组织及原代培养人正常鼻咽上皮细胞ｍＲＮＡ的表达水平。结果８个ＥＳＴ在鼻咽癌细胞ＨＮＥ１和原代培养人正常鼻咽上皮细胞中表达量较一致，７个ＥＳＴ在两种细胞中均无表达，３个ＥＳＴ（Ｗ７２６８８、Ｈ１９８３０、ＡＡ１３０６３０）在鼻咽癌细胞株中表达上调，而２个ＥＳＴ（ＡＡ０７０４３７、Ｈ９０８８２）在原代培养人鼻咽上皮细胞中表达上调。在１３例鼻咽癌活检组织中３０．７％（４／１３）的ＡＡ０７０４３７表达下调，７７％（１０／１３）的Ｗ７２６８８和７７％（１０／１３）的Ｈ１９８３０表达上升。结论构建了７ｑ３２区域鼻咽癌细胞和组织及原代培养人正常鼻咽上皮细胞部分基因表达图谱，并初步认为Ａ０７０４３７的表达下调和Ｗ７２６８８、Ｈ１９８３０的过表达与鼻咽癌的发生有关。     

cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异cDNA序列的初步研究

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。     

八株鼻咽癌细胞株的Rb基因及其表达

为观察８株鼻咽癌细胞株（ＣＮＥ１、ＣＮＥ２、ＨＮＥ１、ＨＮＥ２、ＨＮＥ３、ＨＯＮＥ１、ＳＵＮＥ１和ＨＫ１）Ｒｂ基因的结构和表达情况。在Ｓｏｕｔｈｅｒｎ杂交，除ＨＫ１出现一微弱的额外条带外，其余７株未见异常。聚合酶链反应分析发现ＣＮＥ１和ＳＵＮＥ１的Ｒｂ基因外显子１０和ＨＯＮＥ１的外显子１３扩增阴性。免疫沉淀／免疫印迹可见８株鼻咽癌细胞株均出现分子量正常的Ｒｂ蛋白条带。免疫组织化学则见生长活跃的细胞中强阳性反应细胞数远较静止和衰老的细胞为多。综上所述，８株鼻咽癌细胞株中４株可能有微小的Ｒｂ基因变化。免疫组化强阳性可能表示８株鼻咽癌细胞株部分细胞Ｒｂ基因表达异常。     

鼻咽癌组织中bcl-2、bax和p53的表达及其与瘤细胞凋亡的关系

目的：了解鼻咽癌组织中瘤细胞ｂｃｌ２、ｂａｘ和ｐ５３的表达及其与瘤细胞凋亡指数（ａｐｏｐｔｏｓｉｓｉｎｄｅｘ,ＡＩ）的关系。方法：对３８例未经治疗的鼻咽癌组织,应用免疫组化ＬＳＡＢ法检测瘤细胞ｂｃｌ２、ｂａｘ和ｐ５３的表达;末端标记细胞死亡检测法（ＴＵＮＥＬ）计算癌细胞的凋亡指数。结果：（１）３７例（９７４％）中有９０％左右的瘤细胞呈ｂｃｌ２过表达;（２）３８例（１００％）中有７０％左右的瘤细胞过表达ｂａｘ;（３）２９例（７６３％）呈ｐ５３过表达;（４）３８例活检组织的平均ＡＩ为２６０２±２６４２／ＨＰＦ;（５）ＡＩ与ｂｃｌ２、ｂａｘ和ｐ５３的表达无相关性。结论：（１）本组大多数鼻咽癌组织的绝大部分瘤细胞均呈ｂｃｌ２和ｂａｘ高表达;（２）由于ｂｃｌ２和ｂａｘ的表达已在高水平达到相对平衡,因此在大多数人体鼻咽癌中它们可能并不起到介导瘤细胞凋亡的主导作用;（３）鼻咽癌组织中过表达的ｐ５３蛋白可能已失去了调节ｂｃｌ２和ｂａｘ的表达进而影响细胞凋亡的功能。     

鼻咽癌细胞株中Epstein—Barr病毒编码的RNAs的检测

采用地高辛标记探针对高分化和低分化鼻咽癌细胞中由ＥＢ病毒编码的ＲＮＡｓ进行了检测。结果显示，在高分化鼻咽癌细胞株ＣＮＥ－１和低分化鼻咽癌细胞株ＣＮＥ－２，ＣＮＥ－３细胞中均有ＥＢＥＲｓ，ＥＢＥＲｓ位于细胞核中，且含量丰富，用原位杂交技术检测ＥＢＥＲｓ可望作为鼻咽癌早期诊断工具。     

GST-人可溶性成纤维细胞生长因子受体1融合蛋白在酵母细胞中的表达及活性测定

目的：表达重组人可溶性成纤维细胞生长因子受体１（ｓｏｌｕｂｌｅ　ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ　ｒｅｃｅｐｔｏｒ　１，ｓＦＧＦＲ１），研究其对成纤维细胞生长因子（ＦＧＦ）生物学活性的拮抗作用。方法：采用逆转录－ＰＣＲ（ＰＴ－ＰＣＲ）技术自人肺成纤维细胞获得ｓＦＧＦＲ１　ｃＤＮＡ，测序确证后，将其克隆人酵母细胞表达载体ｐＹＥＸ４Ｔ－１；重组质粒转化入酵母细胞（ＤＹ１５０）中进行诱导表达，表达产物经ＳＤＳ－ＰＡＧＥ及 Ｗｅｓｔｅｒｎ　ｂｌｏｔ鉴定。利用 ＮＩＨ３Ｔ３细胞增殖抑制实验检测重组人 ｓＦＧＦＲ１的生物学活性。结果：经 ＣｕＳＯ４诱导，酵母细胞表达出重组谷胱苷肽转移酶（ＧＳＴ）－ｓＦＧＦＲ１融合蛋白，此蛋白在凝胶上表现为 １条约 ６０ｋＤ的阳性区带，在 Ｗｅｓｔｅｒｎ ｂｌｏｔ实验中可被ＧＳＴ特异性抗体识别。重组ＧＳＴ－ｓＦＧＦＲ１融合蛋白的粗提物在体外能够桔抗ＦＧＦ介导的促ＮＩＨ３Ｔ３细胞增殖的活性。结论：重组ＧＳＴ－ｓＦＧＦＲ１融合蛋白在酵母表达系统中得到有效表达，并具有很好的生物活性。     

cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异 …

寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因，方法应用ｃＤＮＡ代表性差异分析法，分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ＰＧＥＭ－Ｔｅａｓｙ载体中，并链终止法测序。     

鼻咽癌细胞株SUNE1中EB病毒LMP1全基因克隆及部分？…

目的 研究广东地区鼻咽癌（ＮＰＣ）细胞株ＳＵＮＥ１中ＥＢ病毒ＬＭＰ１基因的结构特征。方法 利用聚合酶链反应从ＳＵＮＥ１细胞基因组中扩增ＬＭＰ１全长ＤＮＡ,并克隆到ｐｃＤＮＡ３载体中,采用Ｓａｎｇｅｒ双脱氧末端终止法测定ＳＵＮＥ－ＬＭＰ１ ３个外显子及２个内含子覆盖的序列,并与病毒原型Ｂ９５－８－ＬＭＰ１进行比较分析。结果 克隆到ｐｃＤＮＡ３中的ＳＵＮＥ１－ＬＭＰ１全基因长度为２．６ｋｂ,测序长度为     

Heymann肾炎致病原受体相关蛋白在大肠杆菌中的表达

为研究Ｈｅｙｍａｎｎ肾炎致病原的病理表型和免疫复合物形成机理，应用ＤＮＡ重组技术，将Ｈｅｙｍａｎｎ肾炎（ＨＮ）致病原受体相关蛋白（ＲＡＰ）的ｃＤＮＡ克隆到表达质粒ｐＧＥＸ中，构建重组表达质粒ｐＧＥＸ－Ｒ９３，经限制性内切酶图谱分析，ＤＮＡ测序，鉴定插入子ＲＡＰ－ｃＤＮＡ正确克隆到ｐＧＥＸ表达框架后，转化大肠杆菌ＤＨ５α，经ＩＰＴＧ诱导，高效表达了谷胱甘肽巯基转移酶－受体相关蛋白（ＧＳＴ－ＲＡＰ）融合蛋白，蛋白产量占大肠杆菌总蛋白量的３９．４％。经ＧＳＴ－Ｓｅｐｈｏｓｅ４Ｂ亲和层析得到高度纯化的ＧＳＴ－ＲＡＰ。免疫印迹分析证明，针对ＧＳＴ－ＲＡＰ的抗血清能特异地识别肾皮质天然抗原４４０００α蛋白，免疫组化分析显示ＧＳＴ－ＲＡＰ抗血清特异性识别大鼠肾近曲小管刷状缘抗原。     

食管癌组织染色体位点特异性的杂合性丢失和微卫星DNA序列不稳定

为了寻找在食管癌中发生缺失的染色体位点，根据以往细胞遗传学的工作基础，选取２４个分别位于１、２、３、５、７、９、１１、１７号染色体的食管癌染色体畸变＂热点＂及已知基因的位点，以微卫星序列－ＰＣＲ法检测这些位点处的杂合性丢失和微卫星序列不稳定。对３６对来自北京和山西阳泉的散发食管癌标本和相应正常组织的检测结果显示：在对应于９ｐ２２－２３、３ｐ１４．２、２ｐ２２、３ｐ２４－２６、１１ｑ１３．１、３ｐ２３、３ｐ２１．３、７ｑ３５、３ｑ２１－２２等染色体位点处存在较高频率（＞２０％）的杂合性丢失。同时，发现了染色体位点特异的微卫星ＤＮＡ不稳定。杂合性丢失和微卫星ＤＮＡ序列不稳定可能与食管癌的发生、发展过程有关。     

低密度脂蛋白诱导下调的新基因cDNA的克隆及组织表达

用改进的mRNA差异显示．PCR技术分析ECV304在低密度脂蛋白的诱导下表达水平明显差异的cDNA。获得-差异表达的265bp新EST；以该EST为探针，从人主动脉cDNA文库中筛选到一个1726bp的cDNA克隆，其748-1266bp之间的519个碱基构成一个完整的开放阅读框架，编码172个氨基酸组成的蛋白质。其羧基端94-172区间氨基酸序列中含有三个重复的C2H2型锌指蛋白基序CX2CX3FX5LX2HX3H，Northen Blot证实两组ECV304中均出现一条1．7kb的区带，LDL诱导后其表达降低了2．2倍。与差异显示-PCR的结果一致，该基因被定名为LRZFG。LRZFG是多组织表达的基因，其在动脉粥样硬化早期病理反应的作用有待于进一步研究。     

CLDN23 gene,frequently down-regulated in intestinal-type gastric cancer,is a novel member of CLAUDIN gene family

Microarray analyses combined with laser-capture microdissection have been applied for risk assessments of gastric cancer as well as for identification of novel genes associated with gastric cancer. EST AA393089 derived from an unknown gene has been reported to be frequently down-regulated in intestinal-type gastric cancer. Here, we identified and characterized the gene corresponding to EST AA393089 by using bioinformatics. EST AA393089 overlapped with BC016047 cDNA, and BC016047 overlapped with EST BM821052. Because the mRNA determined by assembling BM821052 and BC016047 was derived from a novel Claudin (CLDN) family gene, the gene corresponding to EST AA393089 was designated CLDN23. Human CLDN23 mRNA was expressed in germinal center B cells, placenta, stomach as well as in colon tumor. Mouse AK009330 and AK037108 cDNAs were derived from mouse Cldn23 gene. Human CLDN23 (292 aa) and mouse Cldn23 (296 aa) were four-transmembrane proteins, showing 79.5% total-amino-acid identity. WWCC motif, defined by W-X(17-22)-W-X(2)-C-X(8-10)-C, was conserved among four-transmembrane proteins of CLDN family. CLDN23 gene, linked to MFHAS1 and PPP1R3B genes, was mapped to human chromosome 8p23.1. CLDN21, CLDN22, and CLDN24 genes were also identified in this study. CLDN21 and CLDN22 genes were located within human genomic contig NT_022792.13. CLDN24 gene on human chromosome 11q23 was located within human genomic contig NT_033899.3. Among 23 CLDN family genes within the human genome, CLDN1 and CLDN16 genes were clustered on human chromosome 3q28, CLDN3 and CLDN4 on 7q11, CLDN6 and CLDN9 on 16p13.3, CLDN8 and CLDN17 on 21q22.11, CLDN21 and CLDN22 on 4q35.1. This is the first report on comprehensive characterization of CLDN23 gene, a candidate tumor suppressor gene implicated in intestinal-type gastric cancer.     

Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach

Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. But the integrated biocontrol mechanism of Trichoderma harzianum is not well explored at the genetic level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for T. harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of T. harzianum and 3298 clones have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector, and identified by sequence similarity searches against gene sequences in international databases. Of the 3298 mycelium clones, 2174 exhibit similarity to known genes and 451 to known ESTs, while 673 represent novel gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified in the mycelium were involved in processes related to mycoparasitism and fungicidal metabolites, as would be expected in biocontrol fungus. These results present the successful application of EST analysis in T. harzianum and provide a preliminary indication of gene expression in mycelium.