“Everyone should do research”




Скачать 352.03 Kb.
Название“Everyone should do research”
страница6/9
Дата конвертации28.10.2012
Размер352.03 Kb.
ТипДокументы
1   2   3   4   5   6   7   8   9

Role of Islet-Associated Heparan Sulfate (HS) and Heparanase in Development of Type 1 Diabetes (T1D) in NOD/Lt Mice



CJ Simeonovic, AF Ziolkowski, C Freeman, CR Parish


The John Curtin School of Medical Research, The Australian National University, GPO Box 334, Canberra, ACT, 2601

Charmaine.Simeonovic@anu.edu.au


Introduction:

T1D development in NOD/Lt mice correlates with a shift from non-destructive to destructive insulitis and the demise of insulin-producing islet beta cells. We investigated whether destructive insulitis correlates with degradation of islet-associated HS by heparanase and whether autoimmune damage of islets can be prevented by inhibiting heparanase activity.


Methods:

RNA was isolated from islets of young (4-5 week), prediabetic and diabetes-onset female NOD/Lt mice, NODscid and CBA/H mice. Heparanase, CD45R and UBC (house-keeping gene) transcripts were analysed quantitatively using real-time RT-PCR. Islet-associated HS was identified histologically by staining with alcian blue (0.65M MgCl2/ pH 5.8). Heparanase protein was identified in pancreas sections by immunohistochemistry. Beta cell-associated HS was removed by in vitro treatment with bacterial heparinase (I, II and III (Sigma), 0.25U/ml) and cell viability was assessed by fluorescence microscopy using Sytox (Invitrogen) to distinguish dead cells. Prediabetic NOD/Lt female mice (from 10.5 weeks of age; n=23) were treated with the heparanase inhibitor PI-88 (10mg/kg/day i.p.); control mice were treated i.p. with saline (n=24).


Results:

Heparanase mRNA was increased 7-fold in prediabetic and diabetes-onset NOD/Lt islets, compared to young NOD/Lt islets. Histologically, destructive insulitis correlated with loss of islet-associated HS and insulitis leukocytes expressed heparanase. Heparinase treatment of isolated beta cells significantly increased beta cell death from 11.0 ± 1.0% to 79.5 ± 5.1% (n=3; P<0.0001). PI-88 treatment delayed T1D onset by 10 weeks (P=0.0039) and reduced T1D incidence from 60% (controls) to 30% at 253 days.


Conclusions:

This study demonstrated that beta cell viability is dependent on cell-associated HS. Destructive autoimmunity correlated with peak expression of heparanase mRNA and loss of islet HS. Inhibition of heparanase activity protected NOD/Lt mice from T1D.


Notes:

…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………………………………………………………………………

……………………………………………………………………………………………………


SESSION 4: Chair Geoff Farrell




A16


ENDOVASCULAR INTERVENTION: A POTENTIALLY DETRIMENTAL ALLURE FOR STEM CELLS


1C O’Meara, 2P Little, 3D Hardman, 4R Dilley

1Department of Medicine, University of Melbourne, St Vincent's Hospital, Fitzroy, 3065, 2Cell Biology of Diabetes Laboratory, Baker Heart Research Institute, Prahran 3 Department of Vascular Surgery, ANU, The Canberra Hospital, Canberra 2900,

4Australian Tissue Engineering Centre, 42 Fitzroy Street, Fitzroy, Vic 3065, C O’Meara: u4181881@anu.edu.au


Objective:

Stem cells recruited to injured tissues during repair mechanisms may be important contributors to the regeneration process. Little is known regarding mechanisms of recruitment of stem cells to vascular repair after angioplasty or retention of these cells to contribute to subsequent restenotic lesions.


Aims

The present study examined interactions between expression of the chemokine stromal derived factor-1 (SDF-1) and the adhesive matrix molecule hyaluronan produced by vascular smooth muscle cells. SDF-1 is chemotactic for mesenchymal stem cell populations via interaction with the CXCR4 receptor, and we propose here that cells thus attracted to regenerating tissues may become attached via a CD44 receptor dependent adhesion to hyaluronan (HA).


Methods & Results

We examined rat aortic tissue during the development of a neointimal layer after balloon catheter injury. SDF-1 was localised to rat aorta media and was present during neointima formation. Neointimal SDF-1 expression was regulated by re-endothelialisation of the damaged intimal surface. HA distribution after injury was also localised to the neointima. In vitro studies showed that degradation of HA with hyaluronidase, or inhibition of CD44:HA interaction with anti-CD44 antibodies, significantly (p<0.05) decreased human mesenchymal stem cell (MSC) adhesion to human aortic and internal mammary artery smooth muscle cell cultures and rat aortic tissue, while pre-activation of stem cells with SDF-1 significantly increased their hyaluronan-dependent adhesion.


Conclusions

We conclude that SDF-1 contributes to regulation of HA-dependent adhesion mechanisms and may play an integral role in the formation of vascular restenotic lesions. Interventions to inhibit MSC contribution to neointimal hyperplasia may reduce morbidity associated with restenosis.


Notes:

…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

A17


Does maternal disease state affect blood pressure in the early neonatal period?


1,6AL Kent, 2, 6 B Shadbolt, 3E Hu, 4S Meskell, 5MC Falk, 3,6JE Dahlstrom.


1Dept of Neonatology, 2Clinical Epidemiology Unit, 3Dept of Anatomical Pathology, 4Centre for Newborn Care, 5Dept of Nephrology, Canberra Hospital, PO Box 11, Woden, Canberra, 2606, ACT, Australia, 6Australian National University Medical School, Canberra, 2601, ACT, Australia.


Objective

To determine whether maternal disease state affects blood pressure (BP) in the early neonatal period.


Methods

Neonates greater than 27 weeks gestation not ventilated or requiring inotropes for more than 24 hours enrolled. Other exclusion criteria included chromosomal or congenital anomaly and illicit maternal drug use. Study group were neonates of mothers with hypertension (pregnancy induced or essential) or diabetes of any kind, requiring treatment. BP measurements taken until discharge on day 1, 2, 3, 4, 7, 14, 21 and 28. Placental histopathology was performed.


Results

190 infants enrolled, 104 in the control and 86 in the study group. 65 infants were born between 28-31 weeks and 125 infants between 32-41 weeks gestation. Those born between 28-31 weeks with a history of diabetes had a statistically higher systolic, mean and diastolic BP throughout the first 28 days of life (p=0.001; p=0.007; p=0.02). Those born between 32-41 weeks gestation with reduced uteroplacental perfusion had a higher systolic BP (p=0.005).


Conclusions

Maternal disease state appears to influence BP in the early neonatal period. Diabetes and altered placental perfusion were associated with higher BP readings. Whether this elevation in BP is persistent in later life is unknown and requires further study.


Notes:

…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

…………………………………………………………………………………………………………………………………………………………………………………………………………

A18


GENOMIC ALTERATIONS IN A DNA REPAIR-DEFICIENT MOUSE MODEL OF ACCELERATED LIVER CARCINOGENESIS


1Pyakurel P, 2Alsop A, 3Gu Y and 1Teoh NC.


1Gastroenterology and Hepatology Laboratory, ANU Medical School at The Canberra Hospital, ACT; 2Gene Regulation Laboratory, Peter MacCallum Cancer Institute, Victoria; 3Dept of Radiation Biology, University of Washington, Seattle, USA.

narci.teoh@act.gov.au


Introduction

Defects in non-homologous end joining (NHEJ) DNA repair, such as Ku70 deficiency, enhance hepatocarcinogenesis by causing chromosomal instability (CIN), a hallmark of hepatocellular carcinoma (HCC). Ku70 -/- mice injected with diethylnitrosamine (DEN) develop accelerated HCC, present at 6 mth vs 9 mth in wildtype (wt) mice. Further, chromosomal aberrations are striking in Ku70 -/- HCCs and recapitulate the karyotypic changes in human HCCs.


Objective

To investigate how NHEJ deficiency and CIN contribute to hepatocarcinogenesis.


Method

We employed array- comparative genomic hybridization (CGH) using genomic DNA from normal Ku70 -/- liver and HCC tissue to identify changes in copy number of DNA sequences.


Results

Array CGH identified 7 loci where changes in copy number of DNA sequences were significant in HCCs from NHEJ-deficient animals compared to Ku70 -/- normal liver. Consistent deletions of genomic DNA were found on chromosomes 4, 6, 7, 10, 13, 16 and 19 in all tumours from Ku70 -/- mice. Specific genes that were located within these altered chromosome loci include Ccne1, Fancb, Mbt-1, plekhf1, Sumo1, Abi3bp and genes involved in ubiquitination and chromosomal segregation. Real-time PCR analysis of genes overexpressed (≥ 2 fold) in Ku70 -/- tumours compared to surrounding liver confirmed upregulation of genes involved in proliferation (Ccne1), DNA repair (Fancb), epigenetic silencing (Mbt-1), p53 regulation (Sumo1) and mitosis (Aurora kinase, Tpx2). Interestingly, liver from DEN-treated Ku70 -/- mice before the onset of HCC (these livers contain pre-malignant, dysplastic foci) already showed significant upregulation of Mbt-1 and downregulation of the apoptosis gene, plekhf1 compared to wt pre-neoplastic liver. When compared to early tumours, advanced HCCs from Ku70 -/- mice (9 mth vs 6 mth) displayed a distinctly different gene regulation profile. Further, Ku70 -/- HCCs were associated with loss of p53 protein (by immunoblotting), and showed high proliferative activity with increased cyclins A, D1, PCNA expression and mitotic index.


Conclusion

Genomic alterations in DEN-treated Ku70 -/- mouse liver and subsequent HCCs may implicate a link between defective DNA repair, structural chromosomal integrity, and dysregulation of genes that regulate cell cycle, mitosis and cell death. By identifying a potential origin for CIN in HCC, further studies in this animal model will greatly enhance our understanding of the molecular pathogenesis of human HCC.


Notes:…………………………………………………………………………………………………………………………………………………………………………………………………

1   2   3   4   5   6   7   8   9

Похожие:

“Everyone should do research” iconResearch Goal I. Enhanced Capacity to Solve Problems Using System Research Approaches. 4

“Everyone should do research” iconThe aim of this course is to explore the most common research methods employed in sociological research and in related disciplines. The course will include an

“Everyone should do research” iconMaize Research Department, Field Corps Inst., Agricultural Research Center, Giza, Egypt

“Everyone should do research” iconSenior Research Associate, 1998-2000 National Research Council, Washington, dc 20418, usa

“Everyone should do research” iconResearch Fellow, Centre for Economic Policy Research (cepr)

“Everyone should do research” iconResearch Fellow, Centre for Economic Policy Research (cepr)

“Everyone should do research” iconIntroduction to Qualitative and Quantitative Research Methods for Research Students

“Everyone should do research” iconThis course focuses on research as the basis for practice. An introduction to the basic research methods is designed to prepare the student to understand

“Everyone should do research” iconDeveloping Foresight for the development of Higher Education/Research relations In the perspective of the European Research Area (era)

“Everyone should do research” iconWas the movies based on accurate research that can be substantiated from research or previous studies that you have participated in. Were there any attempts to tell the story from a first-person narrative?


Разместите кнопку на своём сайте:
lib.convdocs.org


База данных защищена авторским правом ©lib.convdocs.org 2012
обратиться к администрации
lib.convdocs.org
Главная страница