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      Generation and characterization of human insulin-releasing cell lines

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          Abstract

          Background

          The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.

          Results

          We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate.

          Conclusion

          We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

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          Most cited references41

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          Functioning insulinoma--incidence, recurrence, and long-term survival of patients: a 60-year study.

          For the 60-year period from 1927 through 1986, we assessed the incidence, recurrence, and long-term survival among all Mayo Clinic patients with histologically confirmed functioning insulinoma. With use of the complete medical record system at Mayo and the comprehensive epidemiologic data base of residents of Olmsted County, Minnesota, we found 224 patients in whom an initial pancreatic exploration at Mayo had confirmed the presence of insulinoma. The median age (and range) of these patients at surgical diagnosis was 47 (8 to 82) years, and 59% were female patients. During the study period, eight cases of insulinoma occurred among residents of Olmsted County; their age and gender distributions were similar to those of the total cohort. The incidence of insulinoma among residents of Olmsted County increased during the study period to a stable level during the last 2 decades of 4 cases per 1 million person-years. For the total cohort, 7.6% had multiple endocrine neoplasia type I (MEN I), and 5.8% had malignant insulinoma. The risk of recurrence was greater among patients with MEN I (21% at 10 and 20 years) than in those without MEN I (5% at 10 years and 7% at 20 years). Although survival of the total cohort was not significantly impaired, it was significantly worse than expected for patients with malignant insulinoma (29% versus 88% expected at 10 years postoperatively). We conclude that insulinoma is less rare than previously suspected. After successful surgical removal, the long-term risk of recurrent insulinoma is relatively high in patients with MEN I; for patients with benign disease, the long-term survival is normal.
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            Endocrine tumours of the pancreas.

            Endocrine pancreatic tumours (EPTs) are uncommon tumours occurring in approximately 1 in 100,000 of the population, representing 1-2% of all pancreatic neoplasms. Some of the tumours may be part of multiple endocrine neoplasia type one (MEN-1) syndrome or von Hippel-Lindau (vHL) disease. EPTs are classified as functioning or non-functioning tumours on the basis of their clinical manifestation. The biochemical diagnosis of EPT is based on hormones and amines released. Besides specific markers such as insulin, there are also general tumour markers such as chromogranin A, which is the most valuable marker and has been reported to be increased in plasma in 50-80% of patients with EPTs and correlates with tumour burden. The location of endocrine tumours of the pancreas includes different techniques, from endoscopic investigations to scintigraphy (e.g. somatostatin receptor scintigraphy) and positron emission tomography. The medical treatment of endocrine pancreatic tumours consists of chemotherapy, somatostatin analogues and alpha-interferon. None of these can cure a patient with malignant disease. In future, therapy will be custom-made and based on current knowledge of tumour biology and molecular genetics.
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              Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.

              The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using SYBR green I and the LightCycler that better permits accurate quantification of mRNAs. Omission of dithiothreitol from the cDNA synthesis reaction was found to be crucial. This resulted in a lower cycle number at which the cDNA level is determined (C(T) value), steeper amplification curves, and removal of background fluorescence in the subsequent PCR. In addition, the choice of the cDNA priming oligo can improve detection sensitivity even further. In contrast to hexamer primer usage, both gene-specific and oligo-dT(VN) priming were very efficient and accurate, with gene-specific priming being the most sensitive. Finally, accurate quantification of mRNAs by real-time PCR using SYBR green I requires verification of the specificity of PCR by both melting curve and gel analysis.
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                Author and article information

                Journal
                BMC Cell Biol
                BMC Cell Biology
                BioMed Central
                1471-2121
                2009
                19 June 2009
                : 10
                : 49
                Affiliations
                [1 ]Nucleo de Terapia Celular e Molecular (NUCEL), Universidade de São Paulo, São Paulo, SP, Brazil
                [2 ]"Angel. H. Roffo" Institute of Oncology, Research Area, Universidad de Buenos Aires, Buenos Aires, Argentina
                [3 ]Instituto de Química, USP, São Paulo, Brasil
                [4 ]Cirurgia, Faculdade de Medicina, USP, São Paulo Brasil
                Article
                1471-2121-10-49
                10.1186/1471-2121-10-49
                2706802
                19545371
                2dd06c9b-1171-4f99-958f-f93579a259e5
                Copyright © 2009 Labriola et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 21 January 2009
                : 19 June 2009
                Categories
                Research Article

                Cell biology
                Cell biology

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