KOR J CEREBROVASCULAR SURGERY March 2OO9 Vol. 11 No 1, page 12-8 경희대학교동서신의학병원신경외과 1 서울대학교의과대학신경외과학교실분당서울대학교병원신경외과 2 방재승 1 권오기 2 오창완 2 Radiation Induced Senescence-like Changes of Endothelial Cells and Blood-brain Barrier Disruption Jae Seung Bang, MD 1, O-Ki Kwon, MD 2, Chang Wan Oh, MD 2 Department of Neurosurgery, East-West Neo Medical Center of Kyung Hee University 1 Department of Neurosurgery, Bun-dang Seoul National University Hospital, Department of Neurosurgery, Seoul National University College of Medicine 2 ABSTRACT Objectives : This study was performed to evaluate the relationship between radiation induced senescence-like changes of endothelial cells and blood-brain barrier disruption. Materials and Methods : Radiation of 15 Gy was applied to a monolayer culture of bovine aortic endothelial cells (BAEC). The morphological changes were observed over 19 weeks. An artificial blood-brain barrier (BBB) model was constructed using the Transwell and co-culture of the BAEC with C6 glioma cells. After treatment with the same dose of radiation, changes in the BBB were observed by measurement of the trans-endothelial electrical resistance (TEER). Results : Senescence-like changes of the endothelial cells appeared 1 week after irradiation; it was most prominent during the third week and replacement by normal endothelial cells was noted from the seventh week. The recovered normal endothelial monolayer was maintained until the 19th week. Senescence-like endothelial cells showed positive staining with senescence-associated β-galactosidase (SA-β-gal). In the Transwell, the TEER began to decrease 1 week after irradiation, and the decreased resistance reached its peak 18 days after irradiation, and then began to recover to some degree. Conclusion : After application of radiation (15 Gy), senescence-like changes of the endothelial cells were observed in the monolayer culture. These findings demonstrated good correlation with the disruption of blood-brain barrier in an in-vitro model of the BBB. (Kor J Cerebrovascular Surgery 11(1):12-8, 2009) KEY WORDS : Senescence Endothelial cells Radiation Blood-brain barrier. 서 론 논문접수일 : 2009 년 02 월 16 일심사완료일 : 2009 년 03 월 09 일교신저자 : 오창완, 463-707 경기도성남시분당구구미동 300 분당서울대학교병원신경외과전화 : (031) 787-7162 전송 : (031) 787-4059 E-mail : wanoh@snu.ac.kr 연구비수혜사항 : 본연구논문은서울대학교병원신진연구비 ( 과제번호 05-2001-004-0) 의일부지원으로작성되었음. 이온화방사선조사 (ionizing radiation) 는양성및악성뇌종양, 뇌혈관기형등중추신경계질환의치료에자주사용된다. 방사선조사의치료효과는조사후수미리초 (several milli-seconds) 이내에발생하는자유기 (free radical) 가염색체를파괴하여세포를사망하게함으로써이루어지는것으로여겨지고있으며, 4) 이러한기전이정상조직에도작용하여부작용을유발할수있다. 12
방재승 권오기 오창완 뇌및척수에대한방사선손상중일부는정상혈관손상의결과이다. 21) 이온화방사선조사는장기적으로뇌혈관협착및폐색, 뇌동맥류, 뇌괴사, 모야모야증후군, 동맥경화, 뇌출혈, 혈뇌장벽장애등을야기시킬수있으며, 22) 이러한혈관에대한영향이중추신경계방사선치료의제한요인중하나이다. 본연구에서는방사선조사후발생하는내피세포의유사-노화현상과혈뇌장벽장애의관련성에대하여실험을시행하였다. 재료및방법 1. 내피세포배양내피세포는소대동맥내피세포 (BAEC; bovine aortic endothelial cells; Cell Applications, Inc.) 를사용하였다. 동결된소대동맥내피세포를일반배양플라스크에서배양을시작하여양을늘린후, 9~21계대배양된세포를실험에사용하였다. 배양액은 DMEM/F12 배지에 10% fetal bovine serum, penicillin, streptomycin 및 fungizone을혼합하여사용하였다. 내피세포에대한방사선조사후형태학적변화를관찰하기위하여, 60 mm 페트리디쉬 (petri-dish) 에내피세포 (BAEC) 를배양하여, 세포가전체표면을모두덮은후 2일경과하여방사선을조사하였으며, 배양액은격일로교환하였다. 실험군과대조군각각 4개씩을유지하며대표적인변화소견을관찰하였다. 2. 교세포배양실험에사용할교세포는, 장기적실험을위하여배양시장기적세포생존이가능한 C6 교종세포를사용하였다. 배양에앞서배양면에대한 fibronectin 도포를시행하였고, 배양은 glutamine, penicillin, streptomycin, fungizone 등을함유한 DMEM (1.7g/L glucose) 배지를사용하였다. 3. 노화관련 senescence-associated β- galactosidase (SA-β-gal) 염색세포노화 (senescence) 감별을위하여, 거대세포로변형된내피세포에대하여 SA-β-gal 염색을시행하였다. 6) 세포를 PBS로세척후상온에서 2% formaldehyde/0.2% glutaraldehyde 용액으로 3 5분간고정한후다시 PBS로세척하였다. 이후 SA-β-gal 염색액으로핵주변세포질이확실하여질시기까지 4 5시간염색하였다. SA-β-gal 염색액은신선한것을사용하여야되며, 그성분은다음과같다 : 5- bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) 1 mg/ml, citric acid 100 mm, sodium phosphate dibasic 200 mm, potassium ferrocyanide 5 mm, potassium ferricyanide 5 mm, NaCl 150 mm and MgCl2 2 mm; ph 6.0. 4. Transwell 혈뇌장벽모델 Costar Transwell membrane insert (polyester, 12 mm, 0.4 μm pore size) 를사용하여그상부에내피세포를 Fig. 1. Sequential changes of endothelial monolayer culture after irradiation of 15 Gy. A : Before irradiation. B : 1 week after irradiation, showing appearance of senescence-like changes. C : 3 weeks after irradiation, demonstrating prominent senescence-like cells. D : 5 weeks after irradiation, with many dead cells. E : 7 weeks after irradiation, dead cells being replaced by normal cells. F : 19 weeks after irradiation, showing typical cobble stone appearance of normal endothelial cells. Kor J Cerebrovascular Surgery 11(1):12-8, 2009 13
배양하고하부에교세포를배양하였다. Fibronectin 도포후먼저 insert를뒤집어놓아바닥면을위로향하게하여그위에교세포를 4일간배양하여부착시켰다. 이때 insert를 100 mm 세포배양플라스크에놓고배양액이배양막까지차도록유지하였다. 교세포부착후 insert를 Transwell 에다시넣고배양막상부에내피세포를배양하여부착시켰다. 내피세포는 9계대배양된것을사용하였으며, 배양액은내피세포배양시와같은것을사용하였다. 내피세포가전체표면을모두덮은후 2일경과하여방사선을조사하였으며, 배양액은격일로교환하였다. 실험군과대조군각각 8개씩의 Transwell 을유지하였다. 5. 내피세포및 Transwell 방사선조사형태학적변화의관찰을위한페트리디쉬에배양한내피세포단막 (endothelial monolayer) 에대하여, 실험군의경우 15 Gy를조사하였고, 동일조건에서대조군을유지하였다. Transwell 의경우에서도, 실험군에는 15 Gy를조사하였고동일조건에서대조군을유지하였다. 모든경우방사선조사 24시간전에배양액을교환하였다. 6. 혈뇌장벽의검사방사선조사후혈뇌장벽의변화는경내피세포저항 (transendothelial electrical resistance; TEER) 을측정하여추정하였다. EVOM epithelial voltohmmeter (World Precision Instruments, Sarasota, Florida) 를사용하여 Transwell 상부와하부사이의저항치를측정하였으며, 상대적인변화를관찰하는것이므로표면적에따른보정은하지않았다. 정확도를높이기위하여 ENDOHM 전극 (12 mm, World Precision Instruments) 을이용하여저항을측정하였다. 7. 방사선조사후검사내피세포배양에대한방사선조사후형태학적변화는위상대조현미경 (phase contrast microscope) 을이용하여매 1 2주간격으로시행하였다. 세포의형태학적변화가뚜렷한시점에서, 실험군과대조군각 1개씩의페트리디쉬를 SA-β-gal 염색처리하여세포노화현상을확인하였다. TEER 측정은대조군과 15 Gy 방사선조사군각각 8개씩의 Transwell 에서측정하여그평균치를구하였다. 측정은방사선조사직후및이후 2 3일간격으로, 세포폐사로 TEER이급격히감소하는시점까지지속하였다. 결과 1. 방사선조사후내피세포의시간경과에따른형태변화배양 9계대내피세포를 15 Gy 방사선조사하고, 형태학적변화를위상대조현미경 (phase contrast microscope) 을이용하여관찰하였다. 방사선조사 1주후, 3주후및이후 2 주간격으로 19주까지관찰하였다. 대조군은 19주까지형태학적변화가없었다. 대조군 (Fig. 1-A) 에비하여, 실험군의경우 1주후일부세포의크기가커지는양태를보였으며 (Fig. 1-B), 3주후에는뚜렷한거대세포들이나타났다 (Fig. 1-C). 거대세포는세포핵과세포질모두그표면적의증가를보였으며, 특히세포질의양이크게증가하였다. 방사선조사 5주후에는전체적으로많은세포들이죽어가는양태를보였으나 (Fig. 1-D), 7주후부터다시건강한세포들이증식하는소견이나타나서 (Fig. 1-E) 이후 19주까지대부분의영역에서건강한세포들로대체되어있는양상을유지하였다 (Fig. 1-F). 2. 배양계대수에따른방사선조사후반응배양 9계대 (Fig. 2-A), 12계대 (Fig. 2-B) 및 21계대 (Fig. 2-C) 내피세포에 15 Gy의방사선조사후 3주경과하여형태의변화를위상대조현미경으로관찰하였다. 계대수가증가될수록방사선조사후유사-노화현상을보이는거대세포의출현이현저하게증가되는경향을보였다 (Fig. 2). Fig. 2. Effect of the number of passage on the senescence-like changes, 3 weeks after irradiation of 15 Gy. A : Passage number 9 cells. B : Passage number 12 cells. C : Passage number 21 cells. 14 Kor J Cerebrovascular Surgery 11(1):012-018, 2009
방재승 권오기 오창완 3. 노화관련 SA-β-gal 염색배양 9계대내피세포를 15Gy 방사선조사하고, 3주경과하여 SA-β-gal 염색을시행하여, 거대세포의노화를검사하였다. 형태학적변화를위상대조현미경을이용하여관찰하였고 (Fig. 3-A), 동일부위의 SA-β-gal 염색소견을일반현미경으로관찰하였다 (Fig. 3-B). 거대세포대부분에서핵주변세포질의 SA-β-gal 염색양성소견이관찰되어, 이러한거대세포가노화현상을보이고있음을확인하였다 (Fig. 3- B). 4. 방사선조사후경내피세포저항의변화 Transwell 에내피세포와교세포를부착하여혈뇌장벽모델을형성한후 15 Gy 방사선을조사하고경내피세포저항 (TEER) 을 2 3일간격으로측정하였다. 대조군은방사선조사후 25일까지비교적일정한 TEER 수치를유지하다가이후급격한감소추세를보였다. 방사선조사후 7일경과하여실험군 Transwell 의 TEER이대조군에비하여감소하기시작하였으며, 이후그격차가점차증가되어 13일에는유의한차이를보였고 (p=0.04), 18일에그격차가최고에도달하였다. 이후 22일, 25일에다소격차가감소하는추세를보이다가 27일경대조군과실험군모두 TEER의감소를보이며 29 일에는급격한감소를보였다 (Fig. 4). TEER의급격한감소를보인시점에서현미경검사상대부분의세포가사망한소견이관찰되었다. 고찰 혈관의구성세포중특히내피세포는방사선에대하여취약하며, 방사선조사후급성기의여러반응에대한연구가있었다. 22) 특히, 소대동맥내피세포 (bovine aortic endothelial cells; BAEC) 를배양하여실행한실험에서이러한변화가자연적세포노화현상과유사함을관찰할수있었다. 즉, 방사선조사후계대배양 (subculture) 을하지않아 도계대배양에의해유도된자연적세포노화현상과동일한변화를관찰할수있었다. 23) 세포체의크기가증가하는형태학적변화를보였으며, 세포의표면적이방사선조사량에비례하여증가하였고, 이러한세포들은 SA-β-gal에양성반응을보여자연적노화세포와유사한생화학적특성을보였다. 이러한유사-노화세포는 p21의지속적상승을보여, 본현상이방사선조사후일시적으로나타나는세포괴사와는달리자연적세포노화 (natural cellular senescence) 와유사함을확인할수있었다. 23) 본연구에서는이러한유사-노화현상의장기적변화를관찰하기위하여, 계대배양수가적은젊은 BAEC의단층배양상태에서 15 Gy의방사선조사후 19주이상형태학적변화를관찰하였다. 계대배양수가적은젊은 BAEC를사용한이유는세포배양이비교적쉽고, 다음단계의혈뇌장벽실험시교세포와동시배양하면혈뇌장벽을잘형성하기때문이다. 12)33) 본실험결과, 내피세포의유사-노화현상은방사선조사후 1주경과하여발현되기시작하여 3 주후절정에도달하였으며, 5주경에는많은세포들이사망하는양태를보였으나 7주경에는건강한세포들이증식하여빈공간을채웠다. 이러한정상내피세포의상태는 19주까지도잘유지되었다 (Fig. 1). 방사선조사후단층배양에서발현된유사-노화현상은계대배양수가높은노화세포에서더강하게나타났으며, 이는방사선에의한유사-노화현상과자연적노화현상의유관성을시사하여주는것으로생각된다 (Fig. 2). 유사-노화현상을보인세포들은 SA-β-gal에양성염색되어, 역시생화학적으로자연적노화세포와유사한특성을보였다 (Fig. 3). 이러한유사-노화현상의발현및회복은방사선치료후혈뇌장벽장애의발생및회복경과와매우유사하다. 즉, 뇌의일반적방사선치료및정위적방사선수술후 3 12개월경과하여발생하는혈뇌장벽장애는수주 수개월지속된후회복을보이는데, 본실험의관찰결과에서도방사선조사후유사-노화현상을보이는세포들의수가 1주후부터점차증가되었다가, 이러한세포들이사라지면서 7주경부터는정상내피세포로교체되어회복되는현상이관찰되었 Fig. 3. Morphology of senescencelike endothelial cells after staining with SA-β-gal. A : Photograph by phase-contrast microscope. B : Photograph by usual microscope. Kor J Cerebrovascular Surgery 11(1):12-8, 2009 15
다. 생체에서의변화는그시간경과가더느리게나타나지만, 이는생체외실험에서세포의변화가통상적으로더빠르게나타나는현상에기인하는것으로생각된다. 내피세포의방사선조사후관찰된유사-노화현상과뇌방사선치료후발생하는혈뇌장벽장애의관련성을확인하기위하여, BAEC와 C6 교종세포를 Transwell 에동시배양하여 인공혈뇌장벽모델 을만들어실험을시행하였다. 혈뇌장벽은혈액과중추신경간의물질교환을선택적으로조절하여주는역동적경계이며, 이러한기능은주로내피세포에서이루어지고교세포및호르몬등의영향을받는다. 1) 뇌의미세혈관내피세포는세포흡수작용 (pinocytosis) 이적고천공 (fenestration) 이없으며세포외물질투과를막는폐쇄소대 (tight junction) 가있다. 1) 생화학적으로는특이한선택적물질이동기전과관련된 gamma-glutamyltranspeptidase, glucose transporter type 1 (GLUT-1), P-glycoprotein, alkaline phosphatase, Na + -K + -ATPase, Ca ++ -ATPase, and 5 -nucleotidase 등의표식자가특징적으로나타난다. 25) 이러한혈뇌장벽표식자는뇌혈관및비-뇌혈관내피세포모두에서발현될수있으며, 25)35) 교세포의세포막구조물혹은용해성분비물이내피세포에작용하여이러한혈뇌장벽특성을유도하는것으로추정된다. 1)30) 따라서실험실에서 인공혈뇌장벽모델 을만들기위해서는내피세포와교세포의동시배양이필요하며, 이러한목적으로가장간단한것은 Transwell 을사용하여, 배양막의상부에는내피세포를하부에는교세포를배양하는것이며, 본실험에서는이러한모델을사용하였다. 방사선조사후혈뇌장벽의변화는배양막상부와하부간의경내피세포저항 (trans-endothelial electrical resistance; TEER) 을측정하여추정하였 Fig. 4. Changes of trans-endothelial electrical resistance (TEER) after irradiation. Endo : Endothelial Cell. Astro : Astrocyte (C6 glioma cell). 다. 9)11)12)16) 실험결과, 방사선조사후 1주경과하여실험군의혈뇌장벽장애가관찰되기시작하였으며, 18일경장애가절정에도달하였고, 이후다소회복을보이는추세를보였다 (Fig. 4). Transwell 의경우배양세포의생존기간이상대적으로짧아, 방사선조사 4주경과하여대부분의세포들이사망하여좀더장기적인변화를관찰할수는없었다. 그러나관찰기간중내피세포의단층배양에서의유사-노화현상발현과유사한경과를보이는혈뇌장벽장애의발생을확인할수있었다. 방사선이교세포에미치는영향이혈뇌장벽장애에기여할가능성도있으나, 방사선조사후동물교세포의조직학적변화는미미하고, 17) 본연구자의예비실험에서도동일량의방사선조사후교세포는내피세포에비해큰변화를보이지않아그기여도는적을것으로추정되며, 이러한혈뇌장벽장애발생의주원인은내피세포의변화일것으로추정된다. 본연구의결과는, 방사선조사후관찰되는배양된내피세포의유사-노화현상이방사선치료후발생하는일과적혈뇌장벽장애와관련성이있음을시사하고있다. 세포노화현상 (cellular senescence) 이란생체노화현상 (aging) 과대비된생체외배양세포의노화현상을지칭한다. 10)15)27) 노화세포 (senescent cell) 는영구적으로세포분열능력이없으며, 특징적인세포기능변화를보인다. 24)29) 생화학적으로는 telomere의단축, 8) telomerase의억제, 29) 산화성 DNA 손상, 5) 각종종양억제유전자 (p53, prb, p16 INK4a and p21 WAF1 ) 의발현 2)28)34)36) 및발암유전자 (proto-oncogenes) 의이상발 현 13)31) 등이나타난다. 노화세포와단순히분열을멈춘세포 (quiescent cell) 를감별할수있는표식자로는 SA-β-gal, 6) 변형된 fibronectin 발현, 14) interleukin-1α 7) 등이있다. 한편, 방사선조사된세포중세포분열능력을상실한세포 (clonologically dead cells) 들은특징적으로노화세포와유사한형태로변화되며, 이러한세포중일부는노화세포에특이하게반응하는항체에대해서도양성반응을보이는것으로알려져있다. 26) Bey-Dih 등 3) 은방사선및항암제로처치한종양세포가노화세포와유사한특징을보이며, 노화세포표식자인 SA-β-gal에반응하고영구적으로분열이불가능한것으로보고하였다. 이러한현상은방사선에의한염색체의영구적손상이, 반복세포분열에의한 telomere 손실이유발하는자연적세포노화 (natural senescence) 현상과유사한작용을하는것으로설명할수있다. 이러한방사선조사세포들은염색체손상으로세포분열능력은잃었으나상당기간생존이가능한것으로알려져있다. 본연구에서도배양중인내포세포에방사선조사후유사-노화세포가생성되며, 이러한세포들이정상내피세포로교체되기까지수주간생존하는것을확인할수있었다. 한편, 노화된동물은혈뇌장벽의변 16 Kor J Cerebrovascular Surgery 11(1):12-8, 2009
방재승 권오기 오창완 화를보여선택적투과성이상실되며특히 choline, 18) glucose, 20) triiodothyronine 19) 등에대하여그러하다. 인간태정맥내피세포 (human umbilical vein endothelial cell; HUVEC) 를이용한실험에서내피세포노화는여러유전자발현의변화를보였으며, 이들중일부는혈관기능과중요한관련이있을것으로추정된다. 32) 본연구의결과는, 방사선조사로유발된내피세포의유사-노화현상역시혈뇌장벽의장애발생과관련이있음을시사하고있으며, 이는자연적노화에서관찰되는혈뇌장벽장애현상과유사성이있다. 결 배양된내피세포에방사선조사후장기간관찰한결과일시적으로유사-노화세포들이발생하며, 이러한세포들은수주간생존하였다가점차정상세포로대체되어회복되는것을관찰할수있었다. 이러한내피세포의변화는방사선조사후발생하는일과성혈뇌장벽장애와관련성이있을것으로사료된다. 론 중심단어 : 세포노화 내피세포 방사선조사 혈뇌장벽. REFERENCES 11) Abbott NJ, Revest PA. Control of brain endothelial permeability. Cerebrovasc Brain Metab Rev 3:39-72, 1991 12) Atadja P, Wong H, Garkavtsev I, Veillette C, Riabowol K. Increased activity of p53 in senescing fibroblasts. Proc Natl Acad Sci USA 92:8348-52, 1995 13) Chang BD, Broude EV, Dokmanovic M, Zhu H, Ruth A, Xusan Y, et al. A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res 59:3761-7, 1999 14) Bump EA, Malaker K. 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