대한핵의학회지 (The Korean Journal of Nuclear Medicine)

대한핵의학회 (The Korean Society of Nuclear Medicine)
권호 표지

신경줄기세포(HB1.F3)에서 나트륨옥소 공동수송체 도입유전자 발현

Expression of Sodium/iodide Symporter Transgene in Neural Stem Cells

  • 김윤희 (서울대학교 뇌과학 협동과정) ;
  • 이동수 (서울대학교 뇌과학 협동과정) ;
  • 강주현 (서울대학교 의과대학 핵의학교실) ;
  • 이용진 (서울대학교 의과대학 핵의학교실) ;
  • 정준기 (서울대학교 의과대학 핵의학교실) ;
  • 이명철 (서울대학교 의과대학 핵의학교실)
  • Kim, Yun-Hui (Program in Neuroscience, Seoul National University) ;
  • Lee, Dong-Soo (Program in Neuroscience, Seoul National University) ;
  • Kang, Joo-Hyun (Department of Nuclear Medicine, Seoul National University College of Medicine) ;
  • Lee, Yong-Jin (Department of Nuclear Medicine, Seoul National University College of Medicine) ;
  • Chung, June-Key (Department of Nuclear Medicine, Seoul National University College of Medicine) ;
  • Lee, Myung-Chul (Department of Nuclear Medicine, Seoul National University College of Medicine)
  • 발행 : 2004.02.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

목적: 생체 내로 이식한 신경 줄기 세포의 이동과 증식을 비침습적으로 추적하는 것은 기초와 임상에서 중요한 것으로 알려져 있다. 신경줄기 세포주(F3)를 생체내로 이식 후, 비침습적으로 추적하기 위해 사람의 hNIS 유전자를 F3 세포에 안정적으로 형질 도입하여 세포 배양 시간 및 조건에 따른 F3-NIS 세포 내에서 hNIS 유전자의 발현 변화를 알아보았다. 방법: HB1.F3는 태아 종뇌에서 신경 줄기 세포를 분리한 후 v-myc유전자로 불멸화한 신경줄기 세포주이다. CMV 프로모터 조절 받도록 hNIS와 하이그로마이신 저항 유전자를 IRES(internal ribosomal entry site)를 이용하여 재조합하였다(pIRES-NIS/Hyg). pIRES-NIS/Hyg를 리포좀을 이용하여 HB1.F3 세포를 형질전환 하였다. 탈메틸화시약(5-Azacytidine)와 히스톤탈아실화효소저해제(trichostatin; TSA)을 세포주에 24시간 처리한 후, hNIS 발현을 I-125 섭취율과 역전사효소 중합효소연쇄반응(RT-PCR)으고 측정하였다. 결과: pIRES-NIS/Hyg 재조합 유전자를 HB1.F3에 형질도입 후, 2주 동안 하이그로마이신 B를 처리해 hNIS 유전자를 안정적으로 발현하는 HB1.F3 세포를 얻었다(F3-NIS III). I-125 섭취율은 HB1.F3에 비해 F3-NIS가 12.9배 높았으며, $KClO_4$를 처리 했을 때 F3-NIS의 I-125 섭취가 완전히 저해되었다. F3-NIS를 계대 배양하면 hNIS 유전자의 발현이 1.9배 까지 서서히 감소하였다. 5-Azacytidine과 TSA를 F3-NIS에 24시간 처리한 결과, I-125 섭취율이 5-Azacytidine과 TSA 농도에 따라 증가되었다. 또한 같은 방법으로 F3-NIS 세포에 5-Azacytidine과 TSA를 처리한 후 hNIS 프라이머로 RT-PCR을 수행한 결과 hNIS mRNA가 농도에 따라 증가 되었다. 결론: hNIS 유전자 이입된 F3 세포는 계대 배양하는 동안 생물학적인 특성이 변화되는 것으로 관찰되었으며, 이는 줄기 세포에 이입된 외래 유전자의 발현이 DNA 탈메틸화나 히스혼아세틸화를 통한 에피지네틱 조율 때문이라고 생각한다.

Purpose: The ability to noninvasively track the migration of neural progenitor cells would have significant clinical and research implications. We generated stably transfected F3 human neural progenitor cells with human sodium/iodide symporter (hNIS) for noninvasively tracking F3. In this study, the expression patterns of hNIS gene in F3-NIS were examined according to the cultured time and the epigenetic modulation. Materials and Methods: F3 human neural stem cells had been obtained from Dr. Seung U. Kim (Ajou University, Suwon, Korea). hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of CMV promoter. This construct was transfected to F3 with Liposome. To investigate the restoration of hNIS gene expression in F3-NIS, cells were treated with demethylating agent (5-Azacytidine) and Histone deacetylase inhibitor (Trichostatin A: TSA). The expression of hNIS was measured by I-125 uptake assay and RT-PCR analysis. Results: The iodide uptake of the F3-NIS was higher 12.86 times than F3 cell line. According to the cell passage number, hNIS expression in F3-NIS gradually diminished. After treatment of 5-Azacytidine and TSA with serial doses (up to $20{\mu}M$, up to 62.5nM, respectively) for 24 hours, I-125 uptake and mRNA of hNIS in F3-NIS were increased. Conclusion: These results suggest that hNIS transfected F3 might undergo a change in its biological characters by cell passage. Therefore, the gene ex[ressopm of exogenous gene transferred human stem cell might be affected to the epigenetic modulation such as promoter methylation and Histone deacetylation and to the cell culture conditions.

키워드

참고문헌

  1. Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci. 2000;3:537-44
  2. Zhang ZG, Jiang Q, Zhang R, Zhang L, Wang L, Zhang L, et al. Magnetic resonance imaging and neurosphere therapy of stroke in rat. Ann Neurol. 2003;53:259-63
  3. Blasberg RG, Gelovani J. Molecular-genetic imaging: a nuclear medicine-based perspective. Mol Imaging. 2002;1:280-300 https://doi.org/10.1162/153535002760235472
  4. Chung JK. Sodium iodide symporter: its role in nuclear medicine. J Nucl Med. 2002;43:1188-200
  5. Momparler RL. Cancer epigenetics. Oncogene. 2003;22:6479-83
  6. Cho T, Bae JH, Choi HB, Kim SS, McLarnon JG, Suh-Kim H, et al. Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport. 2002;13:1447-52 https://doi.org/10.1097/00001756-200208070-00020
  7. Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke. 2003;34:2258-63
  8. Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, et al. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol. 2000;18:410-4
  9. Bulte JW, Ben-Hur T, Miller BR, Mizrachi-Kol R, Einstein O, Reinhartz E, et al. MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain. Magn Reson Med. 2003;50:201-5 https://doi.org/10.1002/mrm.10511
  10. Stroh A, Lorenz P, Marschinke F, Grune T, Pilgrimm H, Zimmer C. Detection of transplanted stem cells in a parkinson rat model by molecular magnetic resonance imaging. Mol Imaging. 2003;2:266. (Abstract)
  11. Arbab AS, Bashaw LA, Miller BR, Jordan EK, Lewis BK, Kalish H, Frank JA. Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging. Radiology. 2003;229:838-46
  12. Anderson S, Glod J, Arbab A, Noel M, Fine H, Frank J. Tracking of labeled endothelial precursor cells into neovasculature in a mouse malignant glioma model by in vivo MRI. Mol Imaging. 2003;2:266. (Abstract)
  13. Daldrup-Link HE, Rudelius M, Oostendorp RA, Settles M, Piontek G, Metz S, et al. Targeting of hematopoietic progenitor cells with MR contrast agents. Radiology. 2003;228:760-7
  14. Daldrup-Link H, Rudelius M, Piontek G, Oostendorp R, Metz S, Pichler B, Schlegel J, et al. Gadophrin-2 labeled hematopoietic progenitor cells can be depicted in vitro and in vivo with MR imaging and optical imaging. Mol Imaging. 2003;2:268. (Abstract)
  15. Magnitsky S, Watson D, Walton R, Pickup S, Bulte J, Glickson J, Wolfe J, et al. In vivo and Ex vivo magnetic resonance imaging of transplanted stem cells in the mouse brain. Mol Imaging. 2003;2:270. (Abstract)
  16. Rudelius M, Daldrup-Link HE, Heinzmann U, Piontek G, Settles M, Link TM, Schlegel J. Highly efficient paramagnetic labelling of embryonic and neuronal stem cells. Eur J Nucl Med Mol Imaging. 2003;30:1038-44
  17. Rudelius M, Daldrup-Link H, Piontek G, Settles M, Rummeny E, Schlegel J. In vivo tracking of genetically modified neuronal stem cells. Mol Imaging. 2003;2:268. (Abstract)
  18. Rosenqvist N, Hard Af, Segerstad C, Samuelsson C, Johansen J, Lundberg C. Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation. J Gene Med. 2002;4:248-57
  19. Kim YH, Lee DS, Kang JH, Lee YJ, Chung J-K, Jeong JM, et al. Neural stem cell tracking with NIS (Sodium/iodide synporter) reporter gene. Mol Imaging. 2003; 2:269 (Abstract).
  20. El Kharroubi A, Piras G, Stewart CL. DNA demethylation reactivates a subset of imprinted genes in uniparental mouse embryonic fibroblasts. J Biol Chem. 2001;276:8674-80
  21. Kierszenbaum A. Genomic imprinting and epigenetic reprogramming: unearthing the garden of forking paths. Mol Reprod Dev. 2002;63:269-72 https://doi.org/10.1002/mrd.90011
  22. Shaker S, Bernstein M, Momparler LF, Momparler. Preclinical evaluation of antineoplastic activity of inhibitors of DNA methylation (5-aza-2'-deoxycytidine) and histone deacetylation (trichostatin A, depsipeptide) in combination against myeloid leukemic cells. Leuk Res. 2003;27:437-44
  23. Reik W, Dean W, Walter. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089-93
  24. Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, Csankovszki G, et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet. 2001;27: 31-9
  25. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat Genet. 1999;21:103-7
  26. McBurney MW, Mai T, Yang X, Jardine K. Evidence for repeat-induced gene silencing in cultured Mammalian cells: inactivation of tandem repeats of transfected genes. Exp Cell Res. 2002;274:1-8 https://doi.org/10.1006/excr.2001.5443
  27. Bartoli A, Fettucciari K, Fetriconi I, Rosati E, Ianni MD, Tabilio A, et al. Effect of trichostatin a and 5'-azacytidine on transgene reactivation in U937 transduced cells. Pharmacol Res. 2003;48:111-8
  28. Niki T, Rombouts K, De Bleser P, De Smet K, Rogiers V, Schuppan D, et al. A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. Hepatology. 1999;29:858-67
  29. Travers H, Spotswood HT, Moss PA, Turner BM. Human CD34+ hematopoietic progenitor cells hyperacetylate core histones in response to sodium butyrate, but not trichostatin A. Exp Cell Res. 2002;280:149-58
  30. Schinstine M, Iacovitti. 5-Azacytidine and BDNF enhance the maturation of neurons derived from EGF-generated neural stem cells. Exp Neurol. 1997;144:315-25
  31. Marin-Husstege M, Muggironi M, Liu A, Casaccia-Bonnefil P. Histone deacetylase activity is necessary for oligodendrocyte lineage progression. J Neurosci. 2002;22:10333-45
  32. Condorelli DF, Dell'Albani P, Conticello SG, Barresi V, Nicoletti VG, Caruso A, et al. A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene. Dev Neurosci. 1997;19:446-56