The up-regulated differentially expressed genes in HL-60 cell line treated with nocodazole

Objective：Microtubule damaging agents such as nocodazole have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of apoptosis induction activity of these agents are still unclear. We previously demonstrated that nocodazole induced apoptosis in human leukemic HL-60 cells. In order to investigate the genes involved in this process, cDNA RDA was performed to identify upregulated genes in the apoptosis process induced by nocodazole. Methods ：Sixteen up-regulated genes were identified and confirmed to be differentially expressed, including ribosomal proteins, cytochrome b, Wilm＇s tumor related protein QM gene, alpha-tubulin isoform 1, heterogeneous nuclear ribonucleoprotein C enzymes, follicular lymphoma variant translocation 1, Ribophorin I , etc. Results：It was the first time for several genes reported to be related to apoptosis, including one functionally unkown gene, BING4. We also analyzed the expression of these genes in apoptosis induced by etoposide, and results showed 9 genes were also up-regulated, indicating they could be important genes involved in apoptosis. Conclusion：The study provides important clues to understand mechanism of apoptosis induced by nocodazole, and establishes foundation for further study.     

Characterization of a cDNA encoding the 20-kDa photosystem I light-harvesting polypeptide of Chlamydomonas reinhardtii

A cDNA encoding the precursor to a major 20-kDa thylakoid polypeptide of Chlamydomonas reinhardtii (P22), previously localized to the photosystem I light-harvesting complex (LHCI), was characterized. N-terminal sequencing of P22 identified the precursor cleavage site. Genomic Southern blots and polymerase chain reaction analyses show that the gene for P22 (Lhca1^*1) is single-copy and contains at least one intron. Northernblot analyses show that Lhca1^*1 mRNA is highly regulated in light-dark synchronized cells. The primary sequence and predicted topology of P22 has features characteristic of light-harvesting chlorophyll a/b-binding proteins from higher plants. Sequence comparisons indicate that P22 has significantly greater identity with the Type-I LHCI protein of tomato, compared to other LHC proteins. This result suggests that the divergence of LHCI proteins into the classes found in higher plants may have occurred early in evolution, prior to the separation of green algae and land plants.     

严重烧伤F344大鼠免疫细胞中一个EST全长序列的扩增

目的:探讨免疫细胞在大面积烧伤后全身免疫功能紊乱中的变化,对F344大鼠烧伤后血液淋巴细胞和单核细胞中的1个差异表达基因片段(AI764697.1)进一步研究。方法:根据该片段核苷酸序列设计双向引物和巢式引物,应用cDNA末端快速扩增技术(SMARTRACE)对该片段进行双向扩增,扩增后结果经测序,用Northernblot杂交验证。结果:设计的两对引物经扩增都得到了产物,克隆测序后得到1条长度为592bp的片段。Northernblot杂交在烧伤后得到1个长度约700bp的产物,在脾脏中表达水平较高,与RACE结果符合。该核苷酸序列已得到GenBank登录号AF244895。结论:在严重烧伤F344大鼠的血液淋巴细胞和单核细胞中分离并扩增出一个新的基因全长序列,其来源、定位和功能需要进一步研究。     

A new molecular biology approach in morphology: basic method and application of laser microdissection

Our understanding of the role played by specific genes in various diseases is advanced as elucidation of the human genome progresses. One important step in this process is profiling gene expression so as to understand the roles of specific genes and the role of the translated protein. Profiling of the gene should also include tissue generation and an explanation of the mechanism of the disease. A useful technique in achieving this goal is the laser microdissection (LMD) method, which can draw genetic information from tissues of individual patients. We describe herein our technique using a special film which we developed and laser microdissection by the Palm Co. With this LMD method, RNA was extracted from carcinoma tissue and genetic analysis was carried out. LMD is equivalent to pathological diagnosis, and it is necessary to do dissection for accurate diagnosis. It seems to make clear gene pathological diagnosis possible in the future.     

~(60)Coγ射线诱导的白血病小鼠中辐射致癌相关基因的筛选

目的：研究辐射诱导的白血病小鼠胸腺组织中的辐射致癌相关基因。方法：应用基因芯片技术对^60Coγ射线诱导的小鼠白血病胸腺和正常对照组织中的mRNA进行检测。结果：在8000条目的基因中共发现显著差异的表达基因(差异在3倍以上)319条，其中表达上升的有111条，下降的有208条，这些基因涉及到信号转导、细胞周期调节、免疫球蛋白、DNA修复等功能。结论：辐射致癌过程中涉及到多个种类肿瘤相关基因的改变。     

华支睾吸虫囊蚴cDNA表达文库的构建及鉴定

目的 构建华支睾吸虫囊蚴cDNA表达文库,为筛选特异诊断抗原和疫苗候选抗原奠定基础。方法提取华支睾吸虫囊蚴总RNA;用Clontech公司SMARTTM cDNA文库构建试剂盒并按其操作方法进行,进行反转录合成双链cDNA。PCR产物经蛋白酶K消化、纯化后,进行SfiI酶切;用ChromaSpin 400柱将酶切产物进行分级分离,经1.1％琼脂糖凝胶电泳鉴定,回收0.4～4kb的组分,并与λTriplEx2载体连接;连接产物经体外蛋白包装,产生未扩增文库;检测未扩增文库滴度和重组效率后,进行文库的扩增,并测定扩增文库的滴度;随机挑取9个噬菌斑,用载体克隆位点两端的引物进行PCR扩增,以检测所构建的cDNA文库的质量。结果 未扩增文库滴度达7.0×107pfu/ml,扩增文库滴度达2.5×108pfu/ml;用载体两端的引物进行PCR鉴定,结果显示:扩增文库中,含1kb以上的占30％左右,1kb～500bp占69％。结论已成功地获得一高质量的华支睾吸虫囊蚴cDNA表达文库。     

蒿属花粉 cDNA文库的构建和初步鉴定

目的构建蒿属花粉cDNA表达文库。方法应用定向克隆技术将相应dscDNA片段插入λExCell/NotI/EcoRI/CIP载体的NotI和EcoRI双酶切位点间,经包装、转染宿主菌,构建成cDNA文库。检测文库库容量和重组效率,并采用PCR方法对插入片段大小进行分析。结果成功构建了一个含有5.6×105个重组子的cDNA表达文库,重组率为100%,插入片段大小平均约1.3kb。结论所建文库容量及插入片段大小适合对蒿属花粉特异性重组变应原的进一步研究。     

The Th1/Th2 immune-type response of the recurrent aphthous ulceration analyzed by cDNA microarray

BACKGROUND: The reduced ability to activate oral tolerance plays a role in the pathogenesis of some gastrointestinal inflammatory diseases. This activation may reflect a preferential reduction of a T-helper (Th)2- or Th3-type response. In recurrent aphthous ulceration (RAU), genetic and environmental factors may contribute to low tolerance, permitting a cytotoxic reaction against the oral epithelium. The cytokine profile has not permitted the definition of RAU as resulting from enhanced Th1 or Th2 responses. A cDNA microarray study would allow the identification of differentially expressed genes and provide a basis for classification of the immune response. METHODS: The cDNA from 29 samples of aphthae and from 11 samples of normal mucosa from aphthae-free volunteers were hybridized on microarray membranes with 1176 genes. RESULTS: Forty-one differentially expressed genes were identified, and a higher expression level of the Th1 gene cluster in RAU was found. CONCLUSIONS: Microarrays permitted us definition of the gene expression profile of the lesion and identify an increased Th1 activity in RAU lesions.     

一种与人蛋白激酶CK2α'亚基相互作用蛋白的cDNA序列测定

目的：测定一种与人蛋白激酶CK2α’亚基相互作用蛋白的cDNA序列。方法：选择一个与人蛋白激酶CK2α’亚基相互作用蛋白的重组质粒pCADT7-Library cDNA，使用BigDyeTM荧光标记终止底物循环测序试剂盒，用载体pGADT7的T7-启动子为测序引物，在ABI PRISM310型基因分析仪上进行DNA测序。结果：该重组质粒的插入片段与泛素一核糖体蛋白S27a cDNA序列只有一个碱基的差别。结论：通过DNA测序，该种与人蛋白激酶CK2α’亚基相互作用蛋白为泛素一核糖体蛋白S27a的变体。     

cDNA微阵列和组织微阵列对卵巢上皮性肿瘤基因的表达

[目的]结合cDNA微阵列和RNA与冰冻组织微阵列原位杂交的方法以寻找新的特异性卵巢癌候选癌基因,为卵巢癌的早期诊断和治疗提供理论依据.[方法]利用cDNA微阵列筛选在所有3种上皮性卵巢肿瘤中(卵巢浆液性交界性肿瘤、卵巢浆液性腺癌和卵巢子宫内膜样腺癌)显示有意义表达的基因,由RNA与冰冻组织微阵列原位杂交证实其结果.[结果]28个基因在＞70%卵巢肿瘤中显示出高表达,18个基因在＞70%卵巢肿瘤中低表达;干扰素诱导的转膜蛋白1(ⅡTP1)被进一步用于RNA与冰冻组织微阵列原位杂交研究,其结果与cDNA微阵列研究结果相符合.[结论]将cDNA微阵列和RNA与冰冻组织微阵列原位杂交相结合是寻找卵巢癌候选癌基因的可行方法,研究中显示有意义表达的基因有可能作为新的上皮性卵巢癌候选癌基因.