Methods for mammalian single cell research - a review / 生物工程学报

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

New advances in the subtyping of systemic amyloidosis / 中国实验血液学杂志

Amyloidosis is a heterogeneous group of diseases caused by deposition of misfolded proteins, which usually leads to organ dysfunction. Accurate typing of amyloid deposits is of paramount importance because organ involvements and disease prognosis differ widely among different subtypes, and its treatments are type specific. Correct identification of amyloidogenic protein is crucial to proper treatment. Traditional antibody-based diagnostic methods such as immunohistochemistry and immunofluorescence are helpful in amyloid typing, but limitations of those approaches including antibody availability and serum protein contamination impair sensitivity and specificity of diagnosis. Sometimes misdiagnosis can lead to catastrophic therapeutic outcome. Genetic testing is important to confirm the diagnosis of hereditary amyloidosis. Nowadays proteomic analysis has been used as an advanced strategy for amyloid typing and the gold-standard today is laser microdissection followed by mass spectrometry (LMD/MS), which can identify causal protein without additional clinical information. Furthermore, LMD/MS is performed on formalin-fixed paraffin-embedded (FFPE) specimens, thus large scale retrospective studies based on archival material can be conducted. In recent studies, LMD/MS has been proven superior to traditional methods without the drawbacks mentioned above. This proteomic approach provides guarantee of appropriate clinical management and probability of new insights into the mechanism of amyloidosis.In this article the new advances of studies on subtyping of systemic amyloidosis are reviewed.

Laser microdissection and mass spectrometry based proteomics in the diagnosis of kidney diseases / 生物工程学报

In recent years, laser microdissection followed by mass spectrometry (LMD/MS) has been successfully applied to the proteomic studies of formalin-fixed paraffin-embedded (FFPE) renal tissues. This new technique improves the diagnosis of kidney diseases and has a better potential for future clinical application. The review focuses on the use of this methodology for exploring the mechanisms, diagnosis and classification of kidney diseases including renal amyloidosis and membrane proliferative glomerulonephritis.

Detection and clinicopathologic significance of microsatellite alteration and p53 mutation of neuroendocrine cells in colorectal adenocarcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the possible clonal origin of neuroendocrine cells in colorectal adenocarcinoma.</p><p><b>METHODS</b>Twenty-six microsatellite loci were screened using laser capture microdissection, DNA extraction and whole genome amplification. Microsatellite instability (MSI) and loss of heterozygosity (LOH) in adenocarcinoma cells and neuroendocrine cells amongst 30 cases of colorectal carcinoma with neuroendocrine differentiation were detected using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-silver staining. The mutation status of p53 was evaluated by PCR-sequencing. The clonal origin of neuroendocrine cells in colorectal adenocarcinoma was determined.</p><p><b>RESULTS</b>Amongst the 30 cases studied, the prevalence of MSI was 16.9% while that of LOH was 8.5%. The rate showed no statistically significant difference between adenocarcinoma cells and neuroendocrine cells. In 6 cases, the microsatellite alteration was entirely consistent. In 23 cases, the rate of microsatellite alteration consistency was greater than that of inconsistency. In 1 case, the consistency and inconsistency rates were identical. There was statistically significant difference between consistency and inconsistency of microsatellite alteration. The prevalence of p53 mutation was 16.7% which was the same for both adenocarcinoma cells and neuroendocrine cells.</p><p><b>CONCLUSIONS</b>Adenocarcinoma cells and neuroendocrine cells in colorectal adenocarcinoma with neuroendocrine differentiation have similar biologic changes. It is likely that they are of identical origin.</p>

Análise da participação das células neuronais e não-neuronais na Esclerose Lateral Amiotrófica em camundongos transgênicos para SOD1 humana utilizando técnicas de microdissecção a laser e PCR em tempo real / Analysis of neuronal and non-neuronal cells participation in Amyotrophic Lateral Sclerosis in transgenic SOD1 mice by means of laser microdissection and real time PCR

A Esclerose Lateral Amiotrófica (ELA) é a doença neurodegenerativa do neurônio motor que acomete indivíduos adultos e promove a perda progressiva das funções motoras. A evolução é rápida (2 a 5 anos) e culmina na morte por complicações e falência dos músculos respiratórios. Descrições recentes sugerem a contribuição de tipos celulares não neuronais, particularmente o astrócito e a microglia, para a morte do neurônio motor. O camundongo transgênico SOD1G93A, que carrega a SOD1 humana mutada, foi utilizado neste trabalho. Estudos comportamentais apontaram alterações motoras importantes no animal transgênico a partir de 90 dias de vida e permitiram selecionar, então, as idades pré-sintomáticas de 40 dias e 80 dias para os estudos moleculares. A análise da expressão gênica nos animais transgênicos e selvagens destas duas idades foi realizada por microarray utilizando-se a plataforma que contém o genoma completo do camundongo e detectou 492 e 1105 transcritos diferencialmente expressos nos animais de 40 e 80 dias, respectivamente. Estes resultados foram validados por PCR quantitativa (qPCR). As análises bioinformáticas dos resultados identificaram 17 e 11 vias moleculares super-representadas nas idades de 40 dias e 80 dias, respectivamente. Destas, as vias endocitose, sinapse glutamatérgica, proteólise mediada por ubiquitina, via de sinalização de quimiocina, fosforilação oxidativa, processamento e apresentação de antígeno e junção oclusiva foram comuns a ambas as idades. Ainda, as vias sinapse glutamatérgica e fagossomo foram sugeridas como potencialmente mais importantes em animais transgênicos de 40 dias e 80 dias, respectivamente. Transcritos específicos foram analisados em amostras enriquecidas de células (astrócito, microglia e neurônio motor) microdissecadas a laser do corno anterior da medula espinal dos animais. Os transcritos Cxcr4, Slc1a2 e Ube2i foram avaliados por qPCR nas amostras enriquecidas de astrócitos dos animais de 40 dias...

Amyotrophic Lateral Sclerosis (ALS) is an adult onset motor neuron neurodegenerative disease that leads to the progressive loss of muscular functions. It is a fast progression disorder (2 to 5 years) culminating in death by respiratory failure. Recent findings suggest that non neuronal cell types, especially astrocytes and microglia, might contribute to the neuronal death. The transgenic mouse SOD1G93A, carring human mutant SOD1, was used in this study. Behavioral studies pointed to the onset of the clinical symptoms occurring at 90 days in the animal model, thus, allowing the selection of the pre-symptomatic ages of 40 and 80 days to the molecular studies. Gene expression analysis of transgenic mice and their non-transgenic littermates at those ages was performed by using a microarray platform containing the whole mouse genome and has detected 492 and 1105 differentially expressed genes at 40 days and 80 days old mice, respectively. These results were validated by quantitative PCR (qPCR). Bioinformatic analysis of the results identified 17 and 11 over-represented molecular pathways at 40 days and 80 days, respectively. Of these, endocytosis, glutamatergic synapse, ubiquitin-mediated proteolysis, chemokine signaling pathway, oxidative phosphorylation, antigen processing and presentation and also tight junction were common to both ages. Furthermore, glutamatergic synapse and fagosome were suggested as potentially more important at 40 and 80 days, respectively. Specific transcripts were analyzed on enriched samples of cells (astrocytes, microglia and motor neuron) obtained by laser microdissection from the ventral horn of mouse spinal cord. The transcripts Cxcr4, Slc1a2 and Ube2i were evaluated by qPCR in enriched samples of astrocytes of the 40 days old mice, and Cxcr4 and Slc17a6 were analyzed in motor neuron samples at this age. Cxcr4 has been found decreased in astrocytes from transgenic mice and increased in the motor neurons of these animals...

Expression of neonatal Fc receptor on human nephritis and rat nephritis models / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models: anti-Thy1.1 nephritis and Heymann nephritis.</p><p><b>METHODS</b>Thirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010, including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy, 12 cases of IgA nephropathy and 6 cases of lupus nephritis. Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls. Laser capture microdissection and real-time RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides, and immunohistochemistry (IHC) of FcRn by SuperVision method was performed. In addition, rat models of mesangial proliferative nephritis (anti-Thy1.1 nephritis) and passive membranous nephropathy (Heymann nephritis) were established and FcRn was examined in renal tissues by IHC.</p><p><b>RESULTS</b>The FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls (P < 0.05). FcRn protein expression by IHC was seen in lupus nephritis (6/6), membranous nephropathy (6/9) and IgA nephropathy (7/12), significantly higher than that of normal controls (0/5), P < 0.05. Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8, 0/4 respectively) and not statistically difference from that of normal controls. Furthermore, FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls.</p><p><b>CONCLUSIONS</b>Expression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thy1.1 nephritis and Heymann nephritis. FcRn may play a role in the development of immune-induced glomerulonephritis.</p>

Laser capture microdissection enables cellular and molecular studies of tooth root development / 国际口腔科学杂志·英文版

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.

Research status and prospects of DNA test on difficult specimens / 法医学杂志

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.

Characterization of Invading Glioma Cells Using Molecular Analysis of Leading-Edge Tissue

OBJECTIVE: We have introduced a method of characterization of invading glioma cells by using molecular analysis of marginal invading tumor cells and molecular profiles of glioma tumor margin. METHODS: Each of tumor core and marginal tissues was obtained in 22 glioma patients. Tumor core cells and marginal cells from each glial tumor were collected by laser capture microdissection or intraoperative microdissection under the operating microscope. Expression of MMP-2, MMP-9, CD44 and RHAMM mRNA by invading glioma cells compared with tumor core was confirmed by realtime-PCR of twenty-four glioma specimens. Clinical data also were reviewed for invasion and recurrence pattern of the gliomas radiologically and invasive rim pattern microscopically. RESULTS: Overall results of the molecular analysis showed that relative overexpression of MMP-2, MMP-9 and RHAMM were noted at the invasive edge of human glioma specimens comparing to the tumor core but CD44 was highly expressed in the tumor core comparing to the margin. High marginal expression of MMP-2 and MMP-9 were noted in poorly ill-defined margin on the pathological finding. High marginal expression of CD44 and MMP-2 were demonstrated in the midline cross group on the radiological review, and that of RHAMM and MMP-2 were showed in the aggressive recurrence group. High expression of MMP-2 seems to be involved in the various invasion-related phenomenons. CONCLUSION: Up-regulation of MMP-2, MMP-9, CD44 and RHAMM was noted in invasive edge of gliomas according to the various clinical situations.

Isolating sperm cells by laser capture microdissection technique from mixture sample / 法医学杂志

OBJECTIVE@#To assess the application value of laser capture microdissection (LCM) technique for isolating a small number of sperm cells from mixture sample.@*METHODS@#Mixture samples were prepared with sperm cells and vaginal epithelia at different concentrations. Both LCM technique and the differential lysis method were employed to obtain sperm cells from the mixture samples, and DNA was extracted by magnetic beads method. STR genotyping was determined using Identifiler kit.@*RESULTS@#The successful STR genotype rate of sperm cells isolated from mixture samples with LCM technique was 92.86% (13/14). The rate of differential lysis method was 7.14% (1/14). The successful rates between the two methods were statistically different (P < 0.05).@*CONCLUSION@#LCM technique can effectively exclude the interference of female cell component and isolate a small number of sperm cells to obtain a single male STR genotyping. LCM technique is obviously better than the differential lysis method.

Laser Capture Microdissection Reveals Specific Genes Related to Purkinje Cell Death in the Leaner Mice / 한국실험동물학회지

The leaner mouse carries a mutation in the gene encoding the alpha1A subunit of P/Q-type calcium channels. Leaner mice exhibit extensive cerebellar granule and Purkinje cell loss that results in cerebellar dysfunction. A previous study suggested that a small population of leaner Purkinje cells undergo apoptosis, however the cell death mode of the rest of degenerating Purkinje cells has not been identified. In order to investigate the mechanisms underlying leaner Purkinje cell death, gene arrays that contain 243 cell death related genes were carried out. To increase the chance of detecting Purkinje cell specific genes, laser capture microdissection was employed to obtain Purkinje cell enriched samples. The gene array analysis revealed several potential genes that are involved in autophagic cell death pathway including cathepsin D, a key lysosomal protease that triggers autophagic degradation. Further analysis on LC3, which is a hallmark for autophagic cell death showed that leaner Purkinje cells are degenerating via autophagic process. The present study provides evidence that calcium channel defects trigger different modes of neurodegeneration in the cerebellum.

Laser Capture Microdissection Reveals Specific Genes Related to Purkinje Cell Death in the Leaner Mice / 한국실험동물학회지

The leaner mouse carries a mutation in the gene encoding the alpha1A subunit of P/Q-type calcium channels. Leaner mice exhibit extensive cerebellar granule and Purkinje cell loss that results in cerebellar dysfunction. A previous study suggested that a small population of leaner Purkinje cells undergo apoptosis, however the cell death mode of the rest of degenerating Purkinje cells has not been identified. In order to investigate the mechanisms underlying leaner Purkinje cell death, gene arrays that contain 243 cell death related genes were carried out. To increase the chance of detecting Purkinje cell specific genes, laser capture microdissection was employed to obtain Purkinje cell enriched samples. The gene array analysis revealed several potential genes that are involved in autophagic cell death pathway including cathepsin D, a key lysosomal protease that triggers autophagic degradation. Further analysis on LC3, which is a hallmark for autophagic cell death showed that leaner Purkinje cells are degenerating via autophagic process. The present study provides evidence that calcium channel defects trigger different modes of neurodegeneration in the cerebellum.

Expression Patterns of Superoxide Dismutase Genes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection / 한국실험동물학회지

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 micrometer) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages I-V, VII-VIII, and IX-XI. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage VII, and showed a lowest level on stage IX-XI. However, the expression of SOD3 mRNA was highest on stages VII-VIII. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.

The Aberrant Expression of FHIT Transcripts in Uterine Cervical Cancer using Laser-capture Microdissection / 대한산부인과학회지

OBJECTIVE: This study was performed to determine the exact pattern of FHIT expression of the cervical carcinoma cell per se by microdissection and to investigate the clinical significance of the FHIT alteration in cervical cancer. METHODS: RT-PCR for FHIT transcript was performed in 18 cervical cancer tissues. Microdissection was performed using laser capture microdissection device and RNA was extracted by RT-nested PCR. PCR products were compared with known aberrant FHIT transcripts. Immunohistochemical analysis was performed to evaluate correlation between the altered expression of FHIT protein and clinical parameters. RESULTS: Six different size of aberrant FHIT transcripts were observed in cervical cancer tissues. Six of 18 (33.3%) cervical cancer sections exhibited full-length normal FHIT transcript only. Aberrant FHIT transcripts with normal one were observed in 9 (50%) and only aberrant transcripts in 3 (16.7%) frozen sections. Five normal cervical tissues expressed only a normal FHIT transcript. The sequences of the 6 different sizes of aberrant FHIT transcripts showed (1) deletion of exons 4-8, (2) deletion of exons 4-7, (3) deletion of exons 5-8, (4) deletion of exons 5-7, (5) deletion of exons 5-7 and insertion of intronic sequences, 153 bp, (6) deletion of exons 5-7 and insertion of intronic sequences, 84 bp. Microdissection of paired cervical tumor and normal stroma showed expression of aberrant FHIT transcripts only in tumor tissues. CONCLUSION: Aberrant FHIT expression was observed frequently in cervical carcinoma and they were observed mainly in cervical cancer cells by microdissection, but not in normal stromal cells. However, absence of FHIT expression did not correlate with clinical prognostic factors in cervical carcinoma.

The significance of pre-S1 mutants as a cause of groundglass hepatocyte during HBV infection / 대한내과학회지

BACKGROUND: Ground glass hepatocytes are unique histological feature of chronic hepatitis B viral infection. The pre-S1 region of large surface protein has been shown to regulate assembly, processing, and secretion of HBsAg. The purpose of this study was to elucidate that a mutant form of pre-S1 affects this normal secretory pathway and is responsible for ground glass hepatocyte. METHODS: We examined HBV sequences spanning the pre-S region from a patients with HBeAg positive chronic HBV infection. HBV DNA was extracted from serum, cloned, and sequenced and determined the intrahepatic viral composition by extracting HBV DNA from paraffin embedded liver tissue. To analyze the viral population of single groundglass hepatocytes, we used the technique of laser capture microdissection to isolate individual hepatocytes from biopsy specimen. Groundglass hepatocytes that stained positively with anti-HBs and normal hepatocytes were harvested individually and their subjected HBV DNA sequences were analyzed. To define the responsible mutations for the HBsAg secretion, we introduced the mutant gene into molecular clone of wildtype (adwR9) and assayed their HBsAg amounts in the transfected cell supernatants by ELISA. RESULTS: Of 12 clones in serum analyzed, 9 clones had identical wild type sequences in the N-terminal region of the pre-S1 protein which plays an important role in the secretion and retention of HBV envelope proteins. One of the wild type clones has deletion within pre-S2 region. 3 identical mutant clones were isolated. Mutant type clones were predominant groundglass hepatocytes. CONCLUSIONS: We speculate that a mutant form of the HBV pre-S1 protein may result in the formation of ground-glass hepatocytes. Expression of abnormal pre-S1 may lead to its retention and accumulation within hepatocytes.

Laser microdissection of a single cell from colon tissue for gene analysis / 中国应用生理学杂志

<p><b>AIM</b>To investigate the method of detecting gene expression in colon tissue at a single cell level.</p><p><b>METHODS</b>Individual cell(s) were picked up from colon frozen section using laser microdissection. RNA was extracted, reverse transcribed to complementary DNA (cDNA). cDNA was then analyzed by nested reverse transcription polymerase chain reaction (nested RT-PCR) using two pairs of primers.</p><p><b>RESULTS</b>Single cell(s) were selectively picked up using an ultraviolet laser micromanipulator. RNA was extracted, reverse transcribed and used for nested RT-PCR. Amplification products of cDNA from down to a single cell could be clearly visualized in the agarose gel.</p><p><b>CONCLUSION</b>The combined utilization of laser microdissection and nested RT-PCR provides an opportunity to analyze gene expression at single cell(s) level in colon tissue.</p>

Global Analysis of Estrogen-Regulated Genes in Mouse Uterus using cDNA Microarray and Laser Capture Microdissection / 대한불임학회지

No abstract available.

Global Analysis of Estrogen-Regulated Genes in Mouse Uterus using cDNA Microarray and Laser Capture Microdissection / 대한불임학회지

No abstract available.

Comparative Study of p53 Mutation and Oncoprotein Expression in Gastric Adenocarcinoma

PURPOSE: The p53 tumor suppressor gene is believed to play a pivotal role in preventing the uncontrolled cellular growth characteristic of cancer. Mutation of the p53 gene represent one of the most common genetic alterations in human cancers, and the acquisition of such defects is strongly associated with tumor progression and metastasis. The aim of this study was to evaluate the relation between p53 immunoreactivity and the mutation of p53 gene in gastric adenocarcinoma obtained by laser capture microscope. MATENRIALS AND METHODS: Formalin fixed paraffin embedded tissue specimens were obtained from 20 patients who underwent surgery for gastric cancer. According to UICC TNM system, 3 of the cases were Ia, 2 cases II, 4 cases IIIa, 5 cases IIIb, and 6 cases IV. RESULTS: Immunohistochemical staining revealed eight cases as negative (less than 10%), twelve cases as postive (more than 10%). The locations of mutations were as follows; 7 cases had point mutation at exon 4, and 3 cases point mutation at exon 8. There was no mutation at exon 5, 6, 7 and 9. The mutation was observed in 1 case out of 8 p53 oncoprotein negative cases, and 7 cases out of 12 p53 positive cases. The mutation was more common in p53 positive cases (P<0.05), However, there was no significant correlation between p53 mutation observed by DNA sequencing after laser capture microdissection and expression of p53 oncoprotein. CONCLUSION: These result suggest that he expression of p53 oncoprotein not to be related to the mutation of p53 gene at exons 4 through 9 in gastric cancer.

Comparative Study of p53 Mutation and Oncoprotein Expression in Gastric Adenocarcinoma

PURPOSE: The p53 tumor suppressor gene is believed to play a pivotal role in preventing the uncontrolled cellular growth characteristic of cancer. Mutation of the p53 gene represent one of the most common genetic alterations in human cancers, and the acquisition of such defects is strongly associated with tumor progression and metastasis. The aim of this study was to evaluate the relation between p53 immunoreactivity and the mutation of p53 gene in gastric adenocarcinoma obtained by laser capture microscope. MATENRIALS AND METHODS: Formalin fixed paraffin embedded tissue specimens were obtained from 20 patients who underwent surgery for gastric cancer. According to UICC TNM system, 3 of the cases were Ia, 2 cases II, 4 cases IIIa, 5 cases IIIb, and 6 cases IV. RESULTS: Immunohistochemical staining revealed eight cases as negative (less than 10%), twelve cases as postive (more than 10%). The locations of mutations were as follows; 7 cases had point mutation at exon 4, and 3 cases point mutation at exon 8. There was no mutation at exon 5, 6, 7 and 9. The mutation was observed in 1 case out of 8 p53 oncoprotein negative cases, and 7 cases out of 12 p53 positive cases. The mutation was more common in p53 positive cases (P<0.05), However, there was no significant correlation between p53 mutation observed by DNA sequencing after laser capture microdissection and expression of p53 oncoprotein. CONCLUSION: These result suggest that he expression of p53 oncoprotein not to be related to the mutation of p53 gene at exons 4 through 9 in gastric cancer.