2011 - CTS-IXA


This page contains exclusive content for the member of the following sections: TTS, CTS, IXA. Log in to view.

Parallel Session 5- Islet Xenotransplantation - Preclinical Models (Xeno Track)

16.175 - The impact of heme oxygenase-1 (Ad-HO-1) on apoptosis of neonatal porcine cluster cell (NPCC)s and generation of human HO-1 transgenic pigs

Presenter: Hye Jung, Yeom, Seoul, Korea
Authors: Hye-Jung Yeom1, OK Jae Koo1,2, Jong-Ik Hwang3, Bumrae Cho1,2, Sunghoon Hurh1, Hwajung Kim1, Sol Ji Park1,2, Eun Mi Lee1, Eun Won Lee1, Juho Hong1, Kyu Hyun Han1, Byeong-Cheon Lee2, Jaeseok Yang1, Curie Ahn1

175

The impact of heme oxygenase-1 (Ad-HO-1) on apoptosis of neonatal porcine cluster cell (NPCC)s and generation of human HO-1 transgenic pigs

Hye-Jung Yeom1, OK Jae Koo1,2, Jong-Ik Hwang3, Bumrae Cho1,2, Sunghoon Hurh1, Hwajung Kim1, Sol Ji Park1,2, Eun Mi Lee1, Eun Won Lee1, Juho Hong1, Kyu Hyun Han1, Byeong-Cheon Lee2, Jaeseok Yang1, Curie Ahn1

1Transplantation Center, Seoul, Korea; 2Dept. of Theriogenology & Biotechnology, College of Veterinary Medicine, Seoul National University; 3Graduate School of Medicine, Laboratory of G Protein Coupled Receptors, Korea University; Seoul, Korea

Background: Heme oxygenase-1 (HO-1) is an inducible protein capable of cytoprotection via radical scavenging and apoptosis prevention against cellular stress during inflammatory disease. Islets are exposed to various cellular stresses such as operation, isolation and engraftment processes during transplantation period. Therefore, we investigated whether introduction of human HO-1 (hHO-1) to Neonatal porcine cluster cells (NPCCs) can reduce their apoptosis and tried to generate hHO-1 transgeneic pigs for xenogeneic islet transplantation.

Methods: NPCCs weretransduced with adenovirus vectors containing hHO-1gene, or mock gene containing GFP (50 and 200 MOI). Flow cytometric analysis were performed using simultaneous Annexin-V/7-AAD staining in orer to detect apoptotic cells after treatment of either recombinant human tumor necrosis factor a (r-hTNF-α, 25ng/ml)/cycloheximide (CHX, 20 μg/ml)or hydrogen peroxide (H2O2, 400uM) for 24 hours. hHO-1 cDNA (867bp) was inserted into EcoRI/XhoI sites of pcDNA3HA vector. Finally, we generated transgenic pigs expressing the hHO-1/HA using somatic cell nuclear transfer and transgenesis was confirmed by genomic DNA PCR, RT-PCR and western blot analysis.

Results: Transduction of the NPCCs with Ad-HO-1 significantly decreased apoptosis (16.1% for r-hTNF-α/CHX stimuli, 45.1% for H2O2stimuli), in comparison to the NPCCs with Ad-GFP control (26.5% for r-hTNF-α/CHX stimuli, 64.9% for H2O2stimuli).

Next, we successfully produced transgenic pig line with the hHO-1 gene under the control CMVH promoter. Three transgenic pigs were born and their hHO-1 expression was confirmed by both RT-PCR and western blotting. Immunohistochemical staining demonstrated that HO-1 was expressed in most tissues such as kidney, spleen, pancreas, testis, lung and heart in the transgenic pig.

When fibroblasts derived from a hHO-1 transgenic pig and a wild type pig were treated with r-hTNF-α (0 or 30ng/ml)/CHX (10ug/ml) for 15 hours, fibroblasts from the transgenic pig was relatively resistant to apoptosis (19.8%) compared with those from the wild type pig (38.00%). Treatment of H2O2(0, 400, 600, 800uM) induced significantly higher level of ROS in the wild type fibroblast than the hHO-1 transgenic fibroblasts.

Conclusion: We successfully generated healthy hHO-1 transgenic pigs. In parallel with antiapoptotic effect of hHO-1 transduction to NPCCs, fibroblasts from the hHO-1 transgenic pig showed antioxidant and antiapoptotic effects.


Important Disclaimer

By viewing the material on this site you understand and accept that:

  1. The opinions and statements expressed on this site reflect the views of the author or authors and do not necessarily reflect those of The Transplantation Society and/or its Sections.
  2. The hosting of material on The Transplantation Society site does not signify endorsement of this material by The Transplantation Society and/or its Sections.
  3. The material is solely for educational purposes for qualified health care professionals.
  4. The Transplantation Society and/or its Sections are not liable for any decision made or action taken based on the information contained in the material on this site.
  5. The information cannot be used as a substitute for professional care.
  6. The information does not represent a standard of care.
  7. No physician-patient relationship is being established.

Social

Contact

Staff Directory
+1-514-874-1717
info@tts.org

Address

The Transplantation Society
International Headquarters
740 Notre-Dame Ouest
Suite 1245
Montréal, QC, H3C 3X6
Canada