Radiation Oncology Journal

The Korean Society for Radiation Oncology (대한방사선종양학회)
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Expression of Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-2 in Radiation Exposed Small Intestinal Mucosa of the Rat

방사선조사를 받은 흰쥐 소장 점막의 손상과 재생과정 중 금속단백효소 및 억제자의 발현

  • Kwag, Hyon-Joo (Department of Diagnostic Radiology, Kangbuk Samsung Hospital , Sungkyunkwan University) ;
  • Lee, Kyoung-Ja (Department of Radiation Oncology Medical College, Ewha Womans University) ;
  • Rhee, Chung-Sik (Department of Diagnostic Radiology Medical College, Ewha Womans University)
  • 곽현주 (성균관의대 강북삼성병원 진단방사선과) ;
  • 이경자 (이화여자대학교 의과대학 방사선종양학교실) ;
  • 이정식 (이화여자대학교 의과대학 진단방사선과학교실)
  • Published : 2003.03.01
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Abstract

Purpose : The matrix metalloprotelnases (MMPs) are a family of enzymes whose main function is the degradation of the extracellular matrix. Several studies have revealed that MMPs and TIMPS are related to the wound heating process and in photoaging caused by ultraviolet Irradiation. However, the expressions of MMP and TIMP after irradiation have not, to the best of our knowledge, been studied. This study investigates the expressions of MMP-2 and TIMP-2 in rat Intestinal mucosa following irradiation. Materials and Methods : The entire abdomen of Sprague-Dawley rats was irradiated using a single dose method. The rats were sacrificed on day 1, 2, 3, 5, 7 and 14 following irradiation. Histopathological observations were made using hematoxilin & eosin staining. The expressions of MMP-2 and TIMP-2 were examined using immunohistochemistry, Irnrnunoblotting and ELISA. Results : Radiation induced damage associated with atrophic villi, and infiltration of inflammatory cell was observed from the first postirradiation day, and severe tissue damage was observed on the second and the third postirradiation days. An increase in mitosis and the number of regenerating crypts, as evidence of regeneration, were most noticeable on the fifth postirradiation day. From the immunohistochemlstry, the MMP-2 expression was observed from the first postirradiation day, but was most conspicuous on the third and the fifth postirradiation days. The TIMP-2 expression was most conspicuous on the fifth postirradiation day. From the irnrnunoblotting, the MMP-2 expression was strongly positive on the third postirradlatlon day, and that of TIMP-2 showed a strong positive response on the fifth postirradiation day. In ELISA tests, the expressions of MMP-2 and TIMP-2 were increased in the postirradiation groups compared to those of the normal controls, and showed a maximum increase on the fifth postirradiatlon day. These results were statistically significant. Conclusion : The expressions of MMP-2 and TIMP-2 were increased in the intestinal mucosa of the rats following irradiation, and these results correlated with the histopathological findings, such as tissue damage and regeneration. Therefore, this study suggests that MMP-2 and TIMP-2 play roles in the mechanisms of radiation-induced damage and regeneration of intestinal mucosa of rats.

목적 : 금속단백효소(MMPs)는 세포외 기질의 분해가 주 기능이며 금속단백효소와 억제자(TIMPS)가 상처 치유 과정과 자외선 조사에 의한 피부노화에 관여한다는 것이 알려져 있다. 그러나 방사선조사 후 금속단백효소와 억제자의 발현에 관한 연구는 보고된 바 없다. 본 연구는 흰쥐의 소장에 방사선을 조사하여 금속단백효소와 억제자의 발현을 시간적으로 관찰하여 방사선에 의한 소장 점막 손상 및 재생과정과 금속단백효소와 억제자의 관계를 밝히고자하였다. 대상 및 방법 : 실험동물로 암, 수 구별 없이 생후 4~5개월, 체중 250~300 gm의 흰쥐(Spraque-Dawley) 30마리를 대상으로 하여 실험군으로 전복부에 8 Gy의 방사선을 조사한 후 1일, 2일, 3일, 5일, 7일, 14일에 각각 5마리씩 희생시켜 소장을 적출하여 사용하였고, 정상대조군은 각 시기별로 1마리씩 사용하였다. H&E 염색을 하여 조직병리검사를 하였고, MMP와 TIMP끌의 발현에 대해서는 면역조직화학염색, 면역블로팅, ELISA법으로 확인하였다. 결과 : 조직병리 소견상 방사선조사에 의한 소장 조직손상은 1일부터 관찰되어 2일과 3일에 현저하였고 재생은 3일에 관찰되어 5일에 현저하였다 면역조직화학염색 결과 MMP는 1일에 발현되어 3일, 5일에 가장 강하게 나타났으며 TIMP를는 1일에 나타나기 시작하여 5일에 가장 강하게 나타났다. 면역블로팅 결과 MMP◎는 3일과 5일에강하게 나타났으며 TIMP끌는 1, 3, 5, 7일에 같은 정도의 양성반응을 보였다. 혈액에서의 MMP와 TIMP의 ELISA 검사법에서는 대조군에 비해 방사선조사군에서 통계적으로 유의한 증가가 있었고 방사선조사 후 1, 2, 3, 5, 7, 14일 각각에서 유의한 차이가 있었으며 5일에 가장 높은 수치를 보였다. 결론 : 방사선조사 후 조직병리학적 검사에서 손상의 척도인 염증 반응과 재생의 척도인 유사분열 수의 변화가 면역조직화학염색, 면역블로팅, ELISA검사에 의한 MMP-2, TIMP의 증가와 일치하는 것으로 나타나 이들이 방사선 조사 후 조직 손상과 재생 기전에 역할을 하고 있음을 보여주었다.

Keywords

References

  1. Park KR, Rhee CS, Kim SS, Lee YH, Ryu SH, Suh PG. The influence of 5-fluorouracil administration mode on the expression of phospholipase C and Ras oncoprotein associated with regeneration of rat intestinal mucosa following radiation. J Korean Soc Ther Radiol 1994;12(3):271-284
  2. Yoo JH, Kim SS, Lee KJ, Rhee CS. The signal transduction mechanisms on the intestinal mucosa of rat following irradiation. J Korean Soc Ther Radiol 1997;15(2):79-95
  3. Kim SS, Woo YJ, Huh J, et al. Expression of phospholipase C isoenzymes in radiation-induced tissue damage and subsequent regeneration of murine small intestine. Korean J Pathol 1998;32:155-161
  4. Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: relevance to radiotherapy. Int J Radiat Oncol Biol Phys 1995;33(4):781-796 https://doi.org/10.1016/0360-3016(95)00214-8
  5. Kim SS, Woo YJ, Huh J, Rhee CS, Choi IP. The relationship between radiation-induced apoptosis and the expression of cytokines in the small intestine of the rat. J Korean Cancer Assoc 1997;29(6):921-929
  6. Kureshi SA, Hofman FM, Schneider JH, Chin LS, Apuzzo MLJ, Hinton DR. Cytokine expression in radiation-induced delayed cerebral injury. Neurosurg 1994;35(5):822-830 https://doi.org/10.1227/00006123-199411000-00004
  7. Kahari VM, Saarialho-Kere U. Matrix metalloproteinases in skin. Exp Dermatol 1997;6:199-213 https://doi.org/10.1111/j.1600-0625.1997.tb00164.x
  8. Parson SL, Watson SA, Brown PD, Collins HM, Steele RJ. Matrix metalloproteinase. Br J Surg 1997;84:160-166 https://doi.org/10.1002/bjs.1800840206
  9. Pyke C, Ralfkiaer E, Tryggvason K, Dano K. Messenger RNA for two type IV collagenases is located in stromal cells in human colon cancer. Am J Pathol 1993;142(2):359-365
  10. Visscher DW, Hoyhtya M, Ottosen SK, Liang C M, Sarkar FH, Crissman JD. Enhanced expression of tissue inhibitor of metalloproteinase-2 (MMP-2) in the stroma of breast carcinomas correlates with tumor recurrence. Int J Cancer 1994;59:339-344 https://doi.org/10.1002/ijc.2910590308
  11. Hong SI, Park IC, Hong WS, et al. Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer. J Korean Med Sci 1996;11(8):474-479 https://doi.org/10.3346/jkms.1996.11.6.474
  12. Kim SS, Woo YJ, Huh J, Yoon H, Park JM, Kim MY. Immunodetections of the metalloproteinase (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinases (TIMP-2) in prostatic adenocarcinomas. J Korean Cancer Assoc 1997;29 (3):445-453
  13. Furukawa A, Tsuji M, Nishitani M, et al. Role of the matrix metalloproteinase and tissue inhibitors of metalloproteinase families in noninvasive and invasive tumors transplanted in mice with severe combined immunodeficiency. Urology 1998;51(5):849-853 https://doi.org/10.1016/S0090-4295(98)00010-7
  14. Ko BK, Cho HR, Choi DW, et al. Reduced expression of tissue inhibitor of metalloproteinase in nodal metastasis of stomach cancer. J Korean Med Sci 1998;13:286-290
  15. Pardo A, Barrios R, Maldonado V, et al. Gelatinases A and B are up-regulated in rat lungs by subacute hyperoxia: pathogenetic implications. Am J Pathol 1998;153(3):833-844 https://doi.org/10.1016/S0002-9440(10)65625-8
  16. Madlener M, Parks WC, Werner S. Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. Exp Cell Res 1998;242(1):201-210 https://doi.org/10.1006/excr.1998.4049
  17. Witte MB, Thornton FJ, Kiyama T, et al. Metalloproteinase inhibitors and wound healing: a novel enhancer of wound strength. Surgery 1998;124(2):464-470 https://doi.org/10.1016/S0039-6060(98)70154-0
  18. Roeb E, Purucker E, Breuer B, et al. TIMP expression in toxic and cholestatic liver injury in rat. J Hepatol 1997;27(3): 535-544 https://doi.org/10.1016/S0168-8278(97)80359-5
  19. Theret N, Musso O, L'helgoualc'h A, Campion JP, Clement B. Differential expression and origin of membranetype 1 and 2 matrix metalloproteinases (MT-MMPs) in association with MMP2 activation in injured human livers. Am J Pathol 1998;153(3):945-954 https://doi.org/10.1016/S0002-9440(10)65636-2
  20. Hirohata S, Kusachi S, Murakami T, et al. Time dependant alterations of serum matrix metalloproteinase-1 and metalloproteinase-1 tissue inhibitor after successful reperfusion of acute myocardial infarction. Heart 1998;78(3):278-284
  21. Bassiouny HS, Song RH, Hong XF, Singh A, Kocharyan H, Glagov S. Flow regulation of 72-kD collagenase IV (MMP-2) after experimental arterial injury. Circulation 1998;98 (2):157-163 https://doi.org/10.1161/01.CIR.98.2.157
  22. Shofuda K, Nagashima Y, Kawahara K, Yasumitsu H, Miki K, Miyazaki K. Elevated expression of membrane-type 1 and 3 matrix metalloproteinases in rat vascular smooth muscle cells activated by arterial injury. Lab Invest 1998;78(8): 915-923
  23. Heo JH, Lucero J, Abumiya T, et al. Matrix metalloproteinase increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab 1999;19:624-633
  24. Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin aging and retinoid antagonism. Nature 1996;379(25):335-339 https://doi.org/10.1038/379335a0
  25. Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang SW, Voorhees JJ. Pathophysiology of premature skin aging induced by ultraviolet light. N Eng J Med 1997;337:1419-1428 https://doi.org/10.1056/NEJM199711133372003
  26. Ito H, Meistrich ML, Barkley HT, Thomas HD, Milas L. Proliferation of acute and late radiation damage of the gastrointestinal tract. Int J Radiat Oncol Biol Phys 1986;12:211219
  27. Wither HR, Elkind MM. Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int J Radiat Biol 1970;17:261-267 https://doi.org/10.1080/09553007014550291
  28. Lesher J, Lesher S. Effects of single-dose partial-body Xirradiation on cell proliferation in the mouse small intestinal epithelium. Radiat Res 1974;57:148-157 https://doi.org/10.2307/3573763
  29. Stricklin GP, Liying L, Jancic V, Wenczak BA, Nanney LB. Localization of mRNAs representing collagenase and TIMP in sections of healing human burn wounds. Am J Pathol 1993;143:1657-1666
  30. Creemers LB, Jansen ID, Docherty AJ, Reynolds JJ, Beertsen W, Everts V. Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue. Matrix Biol 1998;17(1):35-46 https://doi.org/10.1016/S0945-053X(98)90123-8
  31. Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC. Matrix metalloproteinase expression increases after cerebral focal ischemia in rats:inhibition of matrix metalloproteinase- 9 reduces infarct size. Stroke 1998;29(5):1020-1030 https://doi.org/10.1161/01.STR.29.5.1020
  32. Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP. Tis- sue Inhibitors of metalloprotelnases: structure, regulation and biological functions. Eur J Cell Biol 1997;74(2):111-122
  33. Tryggvason KH, Yhty M, Pyke C. Type IV collagenase In invasive tumors. Breast Cancer Res Treat 1993;24:209-218 https://doi.org/10.1007/BF01833261
  34. Bramhall SR, Neoptolemos JP, Stamp GW, Lemoine NR. Imbalance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of the matrix metalloprotelnases (TIMPs) In human pancreatic carcinoma. J Pathol 1997;182(3):347-355 https://doi.org/10.1002/(SICI)1096-9896(199707)182:3<347::AID-PATH848>3.0.CO;2-J
  35. Gohji K, Fujimoto N, Ohkawa J, Fujii A, Nakajima M. Imbalance between serum matrix metalloproteinase-2 and Its Inhibitor as a predictor of recurrence of urothelial cancer. Br J Cancer 1998;77(4):650-655 https://doi.org/10.1038/bjc.1998.104
  36. Alexander JP, Samples JR, Acott TS. Growth factor and cytokine modulation of trabecular meshwork matrix metalloproteinase and TIMP expression. Current Eye Res 1998;17(3):276-285 https://doi.org/10.1076/ceyr.17.3.276.5219