Original Articles Korean Circulation J 1999;292:182-191 레트로바이러스를이용한배양된심근세포로의유전자전달 허정은 1 김덕경 1 변종회 1 박선진 1 전은석 2 최윤혁 1 정은아 1 권현철 1 박승우 1 김준수 1 이상훈 1 홍경표 1 박정의 1 François-Loïc Cosset 3 서정돈 1 이원로 1 Gene Transfer into Cultured Cardiac Myocytes Mediated by Retrovirus Jeong-Eun Huh, MS 1, Duk-Kyung Kim, MD 1, Jong Hoe Byun, PhD 1, Sun-Jin Park, BS 1 Eun-Suk Jeon, MD 2, Yoon-Hyuk Choe, PhD 1, Eun-A Jung, MS 1, Hyeon-Cheol Gwon, MD 1 Seung Woo Park, MD 1, June Soo Kim, MD 1, Sang Hoon Lee, MD 1, Kyung Pyo Hong, MD 1 Jeong Euy Park, MD 1, François-Loïc Cosset, PhD 3, Jung Don Seo, MD 1 and Won Ro Lee, MD 1 1 Division of Cardiology, Department of Medicine, Sungkyunkwan University Scool of Medicine, Samsung Cardiac and Vascular Center, Samsung Medical Center, Samsung Biomedical Research Institute, Seoul, 2 Department of Medicine, Chungnam University, College of Medicine, Chungnam, Korea 3 Vectorologie Retrovirale et Therapie Genique, INSERM U412-Unit de Virologie Humaine, Ecole Normale Superieure de Lyon, Lyon Cedex, France ABSTRACT BackgroundTransplantation of cardiac myocytes CMs into the injured heart emerges as a potential alternative for the treatment of heart failure. Genetic modification of CMs could enhance andor modify its therapeutic effects. The characteristics of retroviral gene delivery, which is most commonly used in human trial, has been minimally studied in CMs due to its low efficiency in non-dividing cells. In this study, using newly developed high-titer retrovirus, we evaluated 1 the efficiency of gene transfer into CMs, 2 whether S phase during infection is necessary for the transduction, and 3 characteristics of gene delivery to mononucleated vs binucleated CMs. MethodsEnriched CMs were cultured from the ventricles of 1 day-old rat hearts. The cells were transduced by MFG-nls-LacZ retroviruses 510 7 IU/ml in the presence or absencce of polybrene. 3H-thymidinewas added to label cells in S phase. The cells were stained for -galactosidase activity and then immunostained using MF20Ab to identify CMs. The cells were subsequently processed for in vitro autoradiography. Results1 With 3 rounds of infection, 5.9% of total cultured cells were LacZ-positive. The efficiency of transduction reached upto 7.4% in the presence of polybrene 8 g/ml. 2 Nuclear morphology of LacZpositive CMs was pleomorphic from mononucleated to multinucleated, mostly binucleated. 3 Among mononucleated CMs, 17% of cells were labelled with thymidine. Transduction efficiency TDE of thymidinepositive and -negative mononucleated CMs were 37.9% and 3.1%, respectively. Among binucleated cells, 28.9% of cells were labelled with thymidine. TDE of thymidine-positive and -negative binucleated CMs were 182
75.4% and 13.6%, respectively. 4 In total, TDE of binucleated cells were 3.5 times compared to the one of mononucleated cells 31.5% vs 9.0%. ConclusionTDE of CMs using high-titer retrovirus is relatively low. S phase of cells during retroviral infection is not mandatory for the retroviral transduction. Binucleated CMs are susceptible to retroviral gene delivery and their TDE is higher than the one of mononucleated CMs. Korean Circulation J 1999;292:182-191 KEY WORDSCardiac myocytes Retrovirus Binucleation Gene therapy. 서론 183
방법 시약및재료 심근세포배양 184 nls-lacz 바이러스액의생산 Fig. 1. Structure of the MFG-nls-LacZ retroviral vector. Arrows indicate approximate location of promoters and direction of transcription. LTRMolony derived murine leukemia virus MoMuLV LTR, nls-laczescherichia coli -galactosidase gene of nuclear localization, SDsplicing donor, SAsplicing acceptor, papolyadenylation signal. Korean Circulation J 1999;292:182-191
Transduction 효율 X-gal staining 3 H-thymidine incorporation과 in vitro autoradiography Immunostaining 결과 185
2) 레트로바이러스에 의하여 nls-lacz 유전자가 심 개 이상인 다핵세포도 관찰되었으나 이들 다핵세포는 근세포에 전달되어 X-gal 염색된 세포의 핵의 형태는 1% 미만이었다(Fig. 3). 따라서 세포질분열(cytokinesis) 다양하여 단핵심근세포, 이핵심근세포 이외에도 핵이 3 없이 핵분열(karyokinesis)만 일어나는 이핵세포와 다 핵세포도 레트로바이러스에 의하여 유전자가 전달이 가능함을 알 수 있었다. 3) Thymidine 표지 유무와 X-gal염색 유무에 따라 세포들을 4군으로 분류할 수 있는데 단핵심근세포, 다핵 심근세포 모두에서 이들 4군의 세포, 즉 thymidine 양성 이면서 X-gal 염색 양성인 세포와, thymidine 음성이 지만 X-gal 염색 양성인 세포, thymidine 양성이면서도 X-gal 염색 음성인 세포, thymidine과 X-gal 염색이 모두 음성인 세포들을 관찰할 수 있었다(Figs. 4 and 5). 4) 전체 심근세포 중 단핵심근세포는 90.2%이었고 이핵심근세포는 9.8% 이었다. 단핵심근세포 중 thy- Fig. 2. Line graphs showing the transfer efficiency of nlslacz gene by high-titer retrovirus in cardiac myocytes. To find whether transduction efficiency is increased by polybrene (PB) as policationic material, cells were exposed to LacZ (5 107 IU/ml) supernatant with or without 8 μg/ml polybrene for 4 hours at 37. Fresh medium was changed and cells were X-gal stained for LacZ activity 48h later. Transduction efficiency (%) was measured by number of X-gal (+) cell/number of total cell 100. Data are mean±sem. ** p<0.01 vs LacZ. midine 표지 양성인 세포는 17% 이었으며, 감염시 thymidine 표지 양성인 세포와 음성인 세포의 유전자 전달 효율은 각각 37.9%와 3.1% 이었다. 이핵심근세 포 중 thymidine 표지 양성인 세포는 28.9% 이었으며, 감염시 thymidine 표지 양성인 세포와 음성인 세포 의 유전자 전달 효율은 각각 75.4%와 13.6% 이었 다(Table 1). 따라서 단핵세포, 다핵세포 모두에서 레 Fig. 3. Cardiac myocytes showing polymorphic pattern of nuclei in a culture infected with MFG-nls-LacZ. Cells were X-gal stained and subsequently counter stained with nuclear fast red. 186 Korean Circulation J 1999;29(2):182-191
트로바이러스 감염시 DNA 합성이 없어도 레트로바이 다. 또한 이핵세포가 단핵세포에 비하여 thymidine 표 러스에 의해 유전자가 전달될 수 있음을 알 수 있었으 지율이 높음을 알 수 있었다. 전체적으로 이핵심근세포 며, 예상되었던 바와 같이 thymidine 음성인 세포들도 의 유전자 전달 효율은 단핵심근세포의 3.5배(31.5% 외부 유전자가 전달되나 그 효율은 낮음을 알 수 있었 vs 9.0%) 이었다. Fig. 4. Morphologic analysis of mononucleated-cms according to the positivity of Xgal staining or thymidine labeling. Representative examples of LacZ (+) or (-) cell with or without active DNA synthesis during retroviral infection. Fig. 5. Morphologic analysis of binucleated-cms according to the positivity of X-gal staining or thymidine labeling. Representative examples of LacZ (+) or (-) cell with or without active DNA synthesis during retroviral infection. 187
Table 1. Comparisons of thymidine incorporation and transduction efficiency between mononucleated cardiac myocytes and binucleated cardiac myocytes. 188 고 Cell number % Thymidine Cell type Incorporation number % Mononucleated 309 17.1 CMs 1815 90.2 1506 82.9 Binucleated CMs 197 9.8 57 28.9 140 71.1 찰 X-gal staining Cell number % 117 37.9 192 62.1 47 3.1 1459 96.9 43 75.4 14 24.6 19 13.6 121 86.4 Korean Circulation J 1999;292:182-191
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190 요약 연구배경 : 방법 : 결과 : Korean Circulation J 1999;292:182-191
결론 : 중심단어 감사문 REFERENCES 1) Crystal RG. Transfer of genes to humans Early lessons and obstacles to success. Science 1995270404-10. 2) Mulligan RC. The basic science of gene therapy. Science 1993260926-32. 3) Ferry N, Duplessis O, Houssin D, Danos O, Heard J. Retroviral mediated gene transfer into hepatocytes in vivo. Proc Natl Acad Sci USA 1991888377-81. 4) Gojo S, Kitamura S, Germeraad WT, Yoshida Y, Niwaya K, Kawachi K. Ex vivo gene transfer into myocardium using replication-defective retrovirus. Cell Transplant 19965S81-S84. 5) Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737-46. 6) Palsson B, Andreadis S. The physico-chemical factors that govern retrovirus-mediated gene transfer. Exp Hematol 19972594-102. 7) Springett GM, Moen RC, Anderson S, Blaese RM, Anderson WF. Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J Virol 1989633865-9. 8) Humphries EH, Glover C, Reichmann ME. Rous sarcoma virus infection of synchronized cells establishes provirus integration during S phase DNA synthesis prior to cell division. Proc Natl Acad Sci USA 1981782601-5. 9) Varmus HE, Padgett T, Heasley S, Simon G, Bishop JM. Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell 1977 11307-11. 10) Lewis PF, Emerman M. Passage through mitosis is required for oncoviruses but not for the HIV. J Virol 1994 68510-6. 11) Roe TY, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 1993122099-108. 12) Cosset FL, Takeuchi Y, Battini JL, Weiss RA, Collins MKL. Hightiter packaging cells producing recombinant retroviruses resistant to human serum. J Virol 199569 7430-6. 13) Koh GY, Kang MG, Soonpaa MH, Field LJ. Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest 1993921548-54. 14) Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 199426498-101. 15) Li RK, Mickle DAG, Weisei RD, Mohabeer MK, Zhang J, Rao V, et al. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 199796179-187. 16) Millano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, et al. Enhanced myocardial function in transgenic mice overexpressing the 2-adrenergic receptor. Science 1994264582-6. 17) d Apice AJF, Goodman DJ, Pearse MJ. Xenotransplantation An update. Trends Cardiovasc Med 19988319-25. 18) Kirshenbaum LA, MacLellan WR, Mazur W, French SA, Schneider MD. Highly efficient gene transfer into adult ventricular myocytes by recombinants adenovirus. J Clin Invest 199392381-7. 19) Maeda Y, Ikeda U, Shimpo M, Ueno S, Ogasawara Y, Urabe M, et al. Efficient gene transfer into cardiac myocyte using adeno-associated virus AAV vectors. J Mol Cell Cardiol 1998301341-8. 20) Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem 199092129-35. 21) Pasumarthi KBS, Kardami E, Cattini PA. High and low molecular weight fibroblast growth factor-2 increase proliferation of neonatal rat cardiac myocytes but have differential effects on binucleation and nuclear morphology. Circ Res 199678126-36. 22) Kajstura J, Cheng W, Reis K, Anversa P. The IGF-1- IGF-1 receptor system modulates myocyte proliferation but not myocyte cellular hypertrophy in vitro. Exp Cell Res 1994215273-83. 23) Marino TA, Walter RA, Cobb E, Palasiuk M, Parsons T, Mercer WE. Effects of norepinephrine on neonatal rat cardiocyte growth and differentiation. In vitro Cell Dev Biol 199026229-36. 24) Palmer JN, Hartogensis WE, Patten M, Fortuin D, Long CS. Interleukin-1 induces cardiac myocytes growth but inhibits cardiac fibroblast proliferation in culture. J Clin Invest 1995952555-64. 191