Biomedical subjects

Helmut Friess

Publications and source records attributed to Helmut Friess.

At least 19 recordsLinked to original sources

PubMed2026

Pancreatic ductal adenocarcinoma: The Vision of Heracles.

Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest malignancies, as tumors evolve faster than therapies. Resistance is ecological, not merely KRAS driven, involving overlooked players like high-grade pancreatic intraepithelial neoplasias (PanINs), peripancreatic fat, stromal mechanics, myeloid-neural circuits, metabolic rewiring, and systemic host responses. We propose precision interception targeting PanIN/intraductal papillary mucinous neoplasm (IPMN) biology, spatial-functional-proteogenomic classification beyond transcriptomics, the Heracles Protocol (measure, prime, strike, and adapt), and integrated technologies from AI pathology to exosomal delivery and CRISPR-based synergy mapping, together making PDAC more tractable.

Humans↗

PubMed2003

Evidence-based surgery in chronic pancreatitis.

BACKGROUND: In the past two decades our knowledge of the pathophysiology and surgical treatment options in chronic pancreatitis has improved substantially. Surgical treatment in chronic pancreatitis has evolved from extending to organ-preserving procedures. DISCUSSION: The classical Whipple resection is no longer a standard procedure in chronic pancreatitis, and is continuously being replaced by operations such as the duodenum-preserving pancreatic head resection and pylorus-preserving Whipple. These procedures allow the preservation of exocrine and endocrine pancreatic function, provides pain relief in up to 90% of patients, and contributes to an improvement in life quality. CONCLUSIONS: In addition to presently available results from randomized controlled trials, new studies comparing available surgical approaches in chronic pancreatitis are needed to determine which procedure is the most effective in the treating chronic pancreatitis.

Chronic Disease↗

PubMed2003

Invasion and metastasis in pancreatic cancer.

Pancreatic cancer remains a challenging disease with an overall cumulative 5-year survival rate below 1%. The process of cancer initiation, progression and metastasis is still not understood well. Invasion and tumor metastasis are closely related and both occur within a tumour-host microecology, where stroma and tumour cells exchange enzymes and cytokines that modify the local extracellular matrix, stimulate cell migration, and promote cell proliferation and tumor cell survival. During the last decade considerable progress has been made in understanding genetic alterations of genes involved in local and systemic tumor growth. The most important changes occur in genes which regulate cell cycle progression, extracellular matrix homeostasis and cell migration. Furthermore, there is growing evidence that epigenetic factors including angiogenesis and lymphangiogenesis may participate in the formation of tumor metastasis. In this review we highlight the most important genetic alterations involved in tumor invasion and metastasis and further outline the role of tumor angiogenesis and lymphangiogenesis in systemic tumor dissemination.

Animals↗

PubMed2003

Down-regulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells.

BACKGROUND & AIMS: In both pancreatic cancer and chronic pancreatitis, there is enhanced expression of mitogenic growth factors and their tyrosine kinase receptors, which have the capacity to activate mitogen-activated protein kinase (MAPK). In view of the important role of MAPK kinase phosphatase (MKP)-1 in the regulation of MAPK activation, the expression and functional role of MKP-1 was analyzed. METHODS: Pancreatic tissues were analyzed by Northern blotting, Western blotting, and immunohistochemistry. Pancreatic cancer cells were transfected with a full-length MKP-1 antisense construct. Growth characteristics and tumorigenicity in vivo and the effects of mitogenic growth factors on cell growth and MAPK activation were determined in transfected and control cells. RESULTS: MKP-1 messenger RNA (mRNA) levels were increased in pancreatic cancer and chronic pancreatitis (CP) tissues. Moderate to strong MKP-1 immunoreactivity was present in the cancer cells, ductal cells of pancreatic intraepithelial neoplasia, and in tubular complexes in CP. Down-regulation of MKP-1 resulted in decreased anchorage-dependent and -independent growth of pancreatic cancer cells, and decreased tumorigenicity in a nude mouse tumor model. MKP-1 down-regulation led to decreased proliferation and sustained MAPK activation in response to mitogens. CONCLUSIONS: Suppression of MKP-1 expression reduces the tumorigenicity of pancreatic cancer cells in vivo, suggesting that MKP-1 contributes to enhanced mitogenic signaling in pancreatic cancer cells.

Adolescent↗

PubMed2003

Distribution of CCK1 and CCK2 receptors in normal and diseased human pancreatic tissue.

BACKGROUND & AIMS: The localization and functional role of cholecystokinin (CCK) receptor proteins in normal and diseased human pancreas, particularly in ductal pancreatic carcinomas, remain unclear. METHODS: Tissue samples of normal human pancreas, chronic pancreatitis, and ductal pancreatic carcinomas were investigated under carefully controlled conditions for expression of CCK1 and CCK2 receptor messenger RNA (mRNA) and proteins using in situ hybridization and in vitro CCK receptor autoradiography by means of subtype-selective analogues. Synaptophysin immunohistochemistry was used concomitantly for optimal identification of islets, nerves, and tumor areas with neuroendocrine features. RESULTS: CCK2 receptor mRNA and proteins were found abundantly in human pancreatic islets in normal pancreas and chronic pancreatitis. CCK1 receptor proteins were found occasionally in small-sized pancreatic nerves, whereas acini expressed a low density of CCK2 receptors in a few cases of chronic pancreatitis. Ductal pancreatic carcinomas rarely expressed CCK receptors; a few receptor-positive tumors, often characterized by neuroendocrine differentiation, expressed the CCK2 receptor at the mRNA or protein level. However, the main source of CCK receptors in the pancreatic tumor samples consisted of CCK2-expressing islets and/or CCK1-expressing nerves rather than neoplastic tissue. CONCLUSIONS: These data indicate that the presence of CCK receptors in human ductal pancreatic tumor samples is mainly due to CCK2 expression in residual pancreatic islets and CCK1 in pancreatic nerves. Pancreatic acini and ductal pancreatic tumor cells very rarely express CCK2 receptors. These observations suggest that CCK analogues may not be of clinical use to target most of these cancers.

Adult↗

PubMed2003

Primary human hepatocytes--a valuable tool for investigation of apoptosis and hepatitis B virus infection.

BACKGROUND/AIMS: Apoptosis is a key event in the pathophysiology of many liver diseases. Primary human hepatocytes (PHH) provide a useful model to study physiological and pathophysiological processes in the liver. Our aim was to optimize PHH cultures to allow studies on induction of apoptosis and of hepatitis B virus (HBV) infection. METHODS: PHH were isolated from human liver tissue by two-step collagenase perfusion. PHH and hepatoma cells were treated with different apoptosis-inducing agents in parallel. PHH cultures were infected with wild type HBV and transduced with HBV genomes using adenoviral vectors. RESULTS: PHH were successfully isolated from 40 different tissue samples with high viability and purity. Perfusion time and seeding density turned out to be critical parameters for optimal cell yield and culture conditions, respectively. Serum addition to the medium reduced viability of PHH. PHH allowed reproducible studies of CD95-dependent and -independent apoptosis. Sensitivity towards CD95-mediated apoptosis was markedly higher than in hepatoma cells. PHH could efficiently be infected with HBV, but infection did neither induce apoptosis nor prevent CD95-induced cell death. CONCLUSIONS: Our data show that PHH provide an excellent tool for the investigation of apoptosis induced by agents like death receptor-ligands and hepatotropic viruses.

Apoptosis↗

PubMed2003

Heparanase expression in hepatocellular carcinoma and the cirrhotic liver.

The endoglycosidase heparanase plays an important role in tumor cell invasion, angiogenesis and metastasis. In this study, we analyzed heparanase expression in primary hepatocellular carcinoma (HCC), liver cirrhosis and normal liver tissues to further evaluate its role in tumor pathogenesis. Heparanase mRNA expression was measured by real-time quantitative RT-PCR and localized at the cellular level by in situ hybridization. Heparanase protein levels and its localization were determined by Western blotting and immunohistochemistry. Expression of heparanase mRNA in primary HCC was increased 2-fold compared with liver cirrhosis and 2.2-fold in comparison with normal liver tissues, and this overexpression was even more pronounced in advanced stage HCC. In contrast, heparanase expression levels between cirrhotic tissues and normal liver tissues were not significantly different. In HCC the increased heparanase expression was localized in hepatic tumor cells and was only weakly present or absent in normal hepatocytes, bile duct epithelial cells and the connective tissues. These results suggest that increased heparanase expression is involved in the pathogenesis and progression of HCC. Its specific up-regulation in HCC but not in liver cirrhosis indicates that it might be used as a molecular marker for the differentiation of these disorders.

Journal Article↗

PubMed2003

Up-regulation of p75 neurotrophin receptor (p75NTR) is associated with apoptosis in chronic pancreatitis.

Activation of apoptosis in chronic pancreatitis has been demonstrated. The low-affinity neurotrophin receptor p75 (p75NTR) mediates apoptosis in many cell types in vivo and in vitro. The aim of this study was to examine whether p75NTR is involved in the apoptotic process in chronic pancreatitis. The quantity and localization of the receptor was evaluated using northern blot analysis, in situ hybridization, immunohistochemistry, and western blot analysis. Apoptosis was determined by TUNEL assay. By northern blot analysis, p75NTR mRNA levels were increased 40-fold in chronic pancreatitis compared with normal pancreas (P < 0.01). In situ hybridization revealed weak p75NTR mRNA expression in some ductal cells in the normal pancreas. In contrast in chronic pancreatitis moderate p75NTR expression was present in acinar cells next to fibrosis, ductal cells, and cells of ductular structures as well as in some islet cells. Immunostaining of p75NTR in normal pancreas and chronic pancreatitis tissue samples showed a similar intensity and distribution pattern as found by in situ hybridization. Higher p75NTR protein levels could be confirmed by western blot analysis, which revealed an 8.6-fold increase of p75NTR in chronic pancreatitis. TUNEL staining showed, in chronic pancreatitis samples, positivity in some acinar cells next to fibrosis, some ductal cells, and cells of ductular structures. Also some islet cells were positive by TUNEL staining. The presence of p75NTR immunoreactivity was positively correlated (P < 0.05) with the apoptotic index in the exocrine and endocrine pancreas. In conclusion, p75NTR, the low-affinity receptor of neurotrophins which mediates apoptosis, is up-regulated in CP and is involved in the apoptotic process of the exocrine and endocrine pancreas.

Adolescent↗

PubMed2003

Up-regulation, nuclear import, and tumor growth stimulation of the adhesion protein p120 in pancreatic cancer.

BACKGROUND & AIMS: Cell adhesion proteins have been implicated as tumor suppressors because they prevent malignant cells from dissociating their cell contacts. We have studied the role of p120(ctn), a recently discovered member of the cadherin/catenin family, in human pancreatic cancer. METHODS: In 32 resection specimens of pancreatic adenocarcinoma and 10 control samples the expression of p120(ctn) was studied by Northern blot, immunocytochemistry, and immunogold electron microscopy. Patient survival data, tumor grading, and staging were correlated to the experimental results. In PaTu 8889 T pancreatic cancer cells, p120(ctn) expression was suppressed with 21-nucleotide small interfering RNA (siRNA) duplexes and proliferation was determined by bromodeoxyuridine (BrdU) incorporation. RESULTS: In pancreatic cancer p120(ctn) messenger RNA (mRNA) was increased 3- to 4-fold. Although p120(ctn) was localized exclusively at cell contacts in controls it was found in the cytosol and nucleus of pancreatic cancer cells. This redistribution correlated to the degree of tumor dedifferentiation but was independent of tumor stage. The mean survival of patients with predominant membrane localization of p120(ctn) was 24 +/- 7 (SEM) months vs. 9 +/- 2 months for patients with predominant cytoplasmic p120(ctn) expression (P < 0.05). Silencing of p120(ctn) with siRNA duplexes reduced pancreatic cancer cell growth by 40%. CONCLUSIONS: Up-regulation, cytoplasmic redistribution, and nuclear import of p120(ctn) are associated with a more malignant phenotype of pancreatic cancer. This study further represents conclusive evidence for a direct involvement of p120(ctn) in malignant tumor cell proliferation. Both p120(ctn)-defective tumor cell contacts and p120(ctn)-mediated growth signals appear to contribute to the aggressive spread of pancreatic cancer.

Active Transport, Cell Nucleus↗

PubMed2003

Computational dissection of tissue contamination for identification of colon cancer-specific expression profiles.

Microarray profiles of bulk tumor tissues reflect gene expression corresponding to malignant cells as well as to many different types of contaminating normal cells. In this report, we assess the feasibility of querying baseline multitissue transcriptome databases to dissect disease-specific genes. Using colon cancer as a model tumor, we show that the application of Boolean operators (AND, OR, BUTNOT) for database searches leads to genes with expression patterns of interest. The BUTNOT operator for example allows the assignment of "expression signatures" to normal tissue specimens. These expression signatures were then used to computationally identify contaminating cells within conventionally dissected tissue specimens. The combination of several logic operators together with an expression database based on multiple human tissue specimens can resolve the problem of tissue contamination, revealing novel cancer-specific gene expression. Several markers, previously not known to be colon cancer associated, are provided.

Colon↗

PubMed2003

Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer.

INTRODUCTION: The microenvironment of low oxygen that is present in human pancreatic cancer in vivo may actively influence tumor growth as well as neovascularization. AIMS: To determine whether hypoxia-inducible factor 1 (HIF-1) is specifically activated by hypoxia in vitro in pancreatic cancer cells and correlated these findings with tumor specimens. METHODOLOGY: Hypoxic regulation of vascular endothelial growth factor (VEGF) was studied by northern blot analysis and enzyme-linked immunosorbent assay. Electrophoretic mobility shift assays and western blot analysis were used to demonstrate hypoxic activation of HIF-1. The relationship between HIF-1 and VEGF in human pancreatic cancer specimens was studied by immunohistochemical analysis, northern blot analysis, and in situ hybridization. RESULTS: Studies in vivo of human pancreatic cancer tissue showed co-localization of VEGF mRNA, which is produced in ductal cancer cells, and HIF-1alpha protein, which was detectable in cell nuclei of the same cells. HIF-1alpha mRNA expression was dramatically upregulated (approximately 13-fold) in these specimens as well. In vitro, all pancreatic cancer cell lines increased VEGF production when exposed to low oxygen levels, by highly specific activation of HIF-1 DNA binding activity to the VEGF promoter. Cancer cell lines with high constitutive levels of HIF-1alpha protein were found to produce higher basal levels of VEGF. CONCLUSION: We conclude that HIF-1 is the regulatory link between tumor hypoxia and VEGF production in pancreatic cancer, thus establishing a biochemical pathway between tumor hypoxia and neoangiogenesis in this highly aggressive neoplasm.

Adenocarcinoma↗

PubMed2003

Impaired insulin action on phosphatidylinositol 3-kinase activity and glucose transport in skeletal muscle of pancreatic cancer patients.

INTRODUCTION: Glucose intolerance or overt diabetes occurs in 80% of patients with pancreatic cancer (PC). This associated metabolic disorder includes peripheral insulin resistance, which may be caused by factors produced by the PC. The mechanism underlying PC-associated insulin resistance has not been clearly defined. AIM: To characterize basal and insulin-stimulated glucose transport, phosphatidylinositol (PI) 3-kinase activity, and glucose transporter 4 (GLUT4) in skeletal muscles of PC patients. METHODOLOGY: Skeletal muscle samples were obtained from the abdominal wall of 17 PC patients during surgery. Control muscles were sampled in the same way from 11 donors undergoing abdominal surgery for benign diseases. PI 3-kinase activity, glucose transport, and GLUT4 were assessed in vitro in these muscles. RESULTS: In the presence of physiologic concentrations of insulin, glucose transport and PI 3-kinase activity were significantly decreased in the PC group compared with controls. At supraphysiologic insulin concentrations, glucose transport was significantly decreased but PI 3-kinase activity was normalized. In the absence of insulin, these parameters were not significantly different between PC and control groups. Muscle GLUT4 contents were similar between PC and control groups. CONCLUSION: Defects in insulin-mediated PI 3-kinase activity and glucose transport contribute to the insulin resistance in patients with PC.

3-O-Methylglucose↗

PubMed2003

Prevention of metastatic pancreatic cancer growth in vivo by induction of apoptosis with genistein, a naturally occurring isoflavonoid.

INTRODUCTION: The critical need for novel therapeutic approaches to pancreatic cancer treatment is clear. Genistein, a naturally occurring isoflavonoid, is active against certain solid malignancies, but its effect on pancreatic cancer is unknown. AIMS: To investigate the bioactivity of genistein in experimental pancreatic cancer in vitro and in vivo. METHODOLOGY: The effect of intraperitoneal genistein administration on local tumor growth and metastatic disease was determined in an orthotopic nude mouse model. Apoptosis in tumor specimens was determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) technique. In vitro, the effect of genistein on cell growth was assessed by cell count and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay. Apoptosis was determined in vitro by DNA laddering and annexin-V. Caspase-3 and nuclear factor-kappaB activity were measured following genistein treatment. RESULTS: In vivo, genistein significantly improved survival, almost completely inhibited metastasis, and increased apoptosis in an orthotopic model of pancreatic cancer. In vitro genistein treatment resulted in apoptosis in all pancreatic cancer cell lines tested, and this appeared to be mediated by activation of caspase-3. CONCLUSION: These findings suggest that the antimetastatic effect of genistein treatment in vivo is mediated by induction of apoptosis. Genistein may have a therapeutic benefit for patients with pancreatic cancer, in particular after surgery, to prevent recurrence of metastatic disease.

Animals↗

PubMed2003

Neurokinin-1 receptor expression and its potential effects on tumor growth in human pancreatic cancer.

The neurokinin-1 receptor (NK-1R) and its ligand substance P (SP) are involved in the pathogenesis of certain neural tumors. Because nerves are significantly altered in pancreatic cancer, evidence for alteration of this pathway in human pancreatic cancer was sought. Expression of NK-1R was analyzed by real-time quantitative RT-PCR, in situ hybridization, immunohistochemistry, and Western blot analysis in normal human pancreatic and pancreatic cancer tissue samples and in pancreatic cancer cell lines. Furthermore, the influence of SP analogs and of the NK-1R antagonist MEN 11467 on pancreatic cancer cell growth was analyzed by sulforhodamine B (SRB) assay. By real-time quantitative RT-PCR, NK-1R mRNA was increased 36.7-fold (p < 0.001) in human pancreatic cancer samples compared with normal controls. Enhanced NK-1R expression levels were not related to tumor grade but were associated with advanced tumor stage and poorer prognosis. By in situ hybridization and immunohistochemistry, NK-1R mRNA and immunoreactivity were only occasionally weakly present in acinar and ductal cells in the normal pancreas. In contrast, moderate to strong NK-1R mRNA signals and immunoreactivity were present in most cancer cells. By Western blot analysis, NK-1R was increased 26-fold (p < 0.01) in pancreatic cancer samples in comparison to normal controls. NK-1R mRNA was detected in five pancreatic cancer cell lines by real-time quantitative RT-PCR, with the highest levels in CAPAN-1 cells and the lowest in ASPC-1 cells. SP analogs stimulated pancreatic cancer cell growth, depending on the NK-1R expression level, and this effect could be blocked by a selective NK-1R antagonist. These findings illustrate that the NK-1R pathway is activated in human pancreatic cancer and has the potential to contribute to cancer cell growth, thus suggesting the existence of a neuro-cancer cell interaction in vivo.

Blotting, Western↗

PubMed2003

Origin and development of the precursor lesions in experimental pancreatic cancer in rats.

Notwithstanding the importance of understanding how pancreatic ductal adenocarcinoma develops, the process remains controversial. A key question is whether the cells of origin of the tubular complexes that constitute precursor lesions are derived from a single cell type or from multiple types. Suggestions that they arise solely from centroacinar cells or ductal cells have been based on inference due to their morphologic appearance in tissue from patients or investigation of limited numbers of tubular complexes in animal models later in the carcinogenic process. The present study establishes clearly that two steps are involved; rapid transdifferentiation to produce tubular complexes followed later by transformation of the component cells. Animals were killed at intervals beginning 1 day after implantation of the carcinogen dimethylbenzanthracene. Transdifferentiation of acinar cells to ductal cells does not require cell division. Transition of lobules to tubular complexes begins by 2 days after implantation of carcinogen. Within 4 days after implantation well-developed tubular complexes are present. Islets participate in the process. Ductal adenocarcinoma is observed by 1 month after implantation of carcinogen. Chymotrypsin and cytokeratin localized by immunocytochemistry indicate acinar and ductal cell characteristics. Acino-ductal transdifferentiation persists in carcinogen-implanted animals, but not in controls implanted with sodium chloride crystals or subjected to sham implantation. The precursor lesions (tubular complexes) are formed by the transdifferentiation of acinar cells and to a lesser extent islet cells, with the incorporation of the duct cells pre-existing in the lobules. Therefore, cells that at one time were acinar cells, islet cells, and duct cells, provide the precursor cells for the ductal adenocarcinoma that develops from tubular complexes. The results raise the question whether the transdifferentiated cells in the tubular complexes of patients with chronic pancreatitis are more susceptible to carcinogenic influences, resulting in the increased rate of pancreatic cancer.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2003

Relationship between overexpression of NK-1R, NK-2R and intestinal mucosal damage in acute necrotizing pancreatitis.

AIM: To study the expression of neurokinin-1 receptor (NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum of acute necrotizing pancreatitis (ANP) and to evaluate the relationship between expression of these two receptors and intestinal mucosal damage. METHODS: A total of 130 adult Sprague-Dawley rats were randomly divided into two groups: the rats in ANP group (n=80) were induced by the retrograde intraductal infusion of 30 g.L(-1) sodium taurocholate. And the rats in normal control group (n=50) received laparotomy only. Sacrifices were made 6 h, 12 h, 24 h and 48 h later in ANP and normal control group after induction respectively. Intestinal mucosal permeability was studied by intrajejunal injection of 1.5 mCi radioactive isotope (99m)Tc-diethlene triamine pentacetic acid (DTPA) and the radioactivity of (99m)Tc-DTPA content in urine was measured 6 h, 12 h, 24 h and 48 h after induction. Then the pancreas and intestine were prepared for pathology. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, and Western blot was used to investigate the protein level of NK-1R and NK-2R. RESULTS: In ANP rats, serious histologic damages in intestinal mucosa were observed, and the radioactivity of (99m)Tc-DTPA in urine increased significantly in the ANP group. RT-PCR revealed that NK-1R and NK-2R mRNA level was overexpressed in the distal ileum of ANP as compared with the normal control group. Western blot discovered stronger NK-1R (14-fold increase) and NK-2R (9-fold increase) immunoreactivity in the intestinal mucosa of ANP rats. Moreover, the overexpression of NK-1R was associated with mucosal pathological score (r=0.77, P<0.01) and intestinal permeability (r=0.68, P<0.01) in ANP rats. CONCLUSION: NK-1R and NK-2R contribute to disrupted neuropeptides loop balance, deteriorate intestinal damage, and are involved in pathophysiological changes in ANP.

Amylases↗

PubMed2003

Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection.

AIM: The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology. METHODS: Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory. RESULTS: Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis. CONCLUSION: The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are involved in physiological and pathophysiological processes in a complex of variable cell phenotypes.

Colon↗

PubMed2003

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection↗