应用重组技术构建野生型及缺失型CDK2基因的真核表达载体，分别使野生型及缺失型CDK2蛋白与增强型绿色荧光蛋白（Enhanced-green Fluorescent Prutein, EGFP）形成融合蛋白。通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO，经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达。结果表明，野生型CDK2基因的表达产物定位于细胞核，而两种缺失型CDK2基因分别编码的CDK2蛋白N-端1～201及98～298多肽均主要定位于细胞质。以上结果提示，CDK2蛋白序列中不含有与核定位直接相关的信号，其入核过程可能是由其N-端1～97及202～298多肽范围内的部分氨基酸共同形成高级结构，并依赖此高级结构与其他含有入核信号的蛋白形成复合物，从而被带动进入细胞核的。

A novel tumor suppressor gene, PTEN/MMAC1, has been recently shown to be mutated in gliomas, breast, prostate, kidney cancers and melanomas. Loss-of-heterozygosity studies in melanoma have suggested the presence of at least one chromosome 10q locus lost early in tumor progression. In this study, we screened 45 melanoma cell lines and 17 paired uncultured metastatic melanoma and peripheral blood specimens for PTEN/MMAC1 alterations using PCR-SSCP and direct sequencing. We found nine melanoma cell lines with homozygous deletions (

真核生物中存在两种主要的非编码RNA（non-coding RNA），在真核生物中发挥重要作用。一类为微小RNA（microRNA,miRNA），另一为小干扰RNA（siRNA）。miRNA大小为119～25nt,在体内与蛋白质形成核糖核蛋白复合体（miRNA），在真核基因的表达调控，生长发育中起重要作用。siRNA在RNA干扰（RNA interference,RNAi）途径中起定位特异mRNA的作用。miRNA与siRNA有联系也有区别。miRNA在真核生物中的调控机制具有保守性。

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of enerating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip "probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.hybridization of LCM-generated RNA to cDNA-based expression microarrays, no report has yet shown the successful hybridization to high-density oligonucleotide mircoarrays. The limited quantity and quality of RNA isolated using LCM continues to be a technical obstacle for in vivo analysis of gene expression using microarrays. While RNA isolated from LCM-harvested tissue can be converted into cDNA libraries, it is generally agreed that the most accurate representation of gene expression for DNA chips is achieved by making the target sample directly from the RNA or by a T7-based linear RNA amplification (2,4,5). Comprehensive gene expression analysis of LCM-isolated pure cell populations will represent a major biomedical advance in our understanding of the pathogenesis of human disease (7). A major challenge in this line of investigation is the ability to generate a sufficient amount and quality of the desireddesired macromolecule from biopsied material. The GeneChip"probe arrays (Affymetrix, Santa Clara, CA, USA) allow the generation of accurate and reproducible mRNA transcript-level data (3,6,9). The HuGeneFL probe array contains probes representing approximately 7000 full-length human genes. The ability to generate target samples from LCM-based cells and tissues for hybridization to DNA chips represents an important advance that demonstrates the feasibility of using this method of sample collection for gene expression analysis on DNA chips.

AIM: Survivin, a recently identified member of the inhibitor of apoptosis protein family, is expressed during development and in various human cancers. However, its expression in normal tissues and clinical relevance in cancers are still debated. In the present study, we analyzed the expression of the survivin gene in human primary and metastatic gastric cancer cells as well as in paired epithelial cells from normal gastric mucosa by means of a novel laser capture microdissection (LCM) technique coupled with reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Thirty patients who had undergone gastrectomy with lymph node dissection for gastric cancer without preoperative treatments were included. Neoplastic tissue, metastatic lymph nodes, and apparently uninvolved normal tissue were collected from each patient. LCM-captured "pure" cell groups were espectively subjected to RT-PCR analysis with primers specific for the survivin gene. RESULTS: Of the paired samples from 30 gastric cancer patients studied, 24 (80%) primary gastric cancer cell groups and 7 (23%) adjacent morphologically "normal" gastric epithelial cell groups were shown to have a detectable survivin expression. There was a statistically significant difference in suvivin expression between these two groups (P<0.01). Meanwhile, 95% (19/20) of the metastatic gastric cancer cell groups from lymph nodes had a clear expression of the survivin gene. However, no significant correlation between survivin expression and clinicopathological features of gastric cancer was bserved in the present study.CONCLUSION: Survivin expression is present in the majority of gastric cancer cell groups obtained by LCM techniques. The high expression rate in metastatic lesions suggests a possible role of survivin in cancer invasiveness and metastasis. It may contribute to the detection of gastriccancer micrometastasis as a potential molecular marker. In addition, the high expression percentage renders survivin a potential target in the therapy for gastric cancer.