Original Article J Korean Geriatr oc 2008:12(4):235-245 성상세포종에서아밀로이드베타 (Aβ 1-42 ) 단백과 Interferon Gamma 투여후보체조절유전자들의발현변화 가톨릭대학교의과대학병리학교실 최영숙ㆍ김상호 Expression Change of Complement Regulator Genes of the Human Astrocytoma Cell Line after Aβ 1-42 and Interferon Gamma Administration Young-ook Choi, M.., ang-ho Kim, M.D. Department of Pathology, College of Medicine, The Catholic University of Korea, eoul, Korea Background: We determined the changes of complement regulator gene expression in the amyloid-β 1-42 (Aβ 1-42 ) and interferon-gamma (IFN-γ)-stimulated human astrocytoma cell line. Methods: The human astrocytoma cell line, U373MG, was stimulated with IFN-γ (62.5-1,000 U/ml) in the presence or absence of aggregated Aβ 1-42 (1-20 μm) for 24 hours. Messenger RNA expression of C1 inhibitor (C1-INH), complement factor I (CFI), clusterin, vitronectin, decay accelerating factor (DAF), membrane cofactor protein (MCP), and CD59 was measured by quantitative real-time reverse transcriptase-pcr. Results: IFN-γ (final concentration, 500 U/ml) markedly increased the expression of mrna for C1-INH in a timedependent fashion. Aβ 1-42 (final concentration, 2 μm) induced a slight increase in the expression of C1-INH. Messenger RNAs for CFI and clusterin were minimally increased, but other regulators were unchanged or decreased by either Aβ 1-42 or IFN-γ. IFN-γoverrode Aβ 1-42 -induced mrna expression of C1-INH when the cells were treated with these two reagents together. Conclusion: Among the complement regulator genes in the human astrocytoma cell line, U373MG, only C1-INH was significantly up-regulated by IFN-γ with or without Aβ 1-42 administration. Key Words: Alzheimer's Disease, beta-amyloid protein, C1 Inhibitor, Complement Factor I, Interferon-gamma 서 론 해마와대뇌피질에진행성변성질환을보이는알츠하이머병 (Alzheimer's disease, AD) 의병리조직학적특징은신경세포내에형성된신경섬유농축체 (neurofibrillary tangle) 와신경세포밖에형성된아밀로이드판 (amyloid plaque, 노 접수 : 2008 년 9 월 16 일승인 : 2008 년 12 월 1 일 Address for correspondence: ang-ho Kim, M.D. Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, eocho-gu, eoul 137-701, Korea Tel: 82-2-590-1189, Fax: 82-2-537-6586 E-mail: complt@catholic.ac.kr 인성반점, senile plaque) 의형성이다. 39-42개의아미노산 (Aβ 1-39, Aβ 1-42 ) 으로구성된섬유형태의불용성아밀로이드가판 (plaque) 을형성하고, 이러한구조물의뇌조직내축적은신경세포의손상과세포자멸사를초래한다 1). AD를염증이동반된만성신경변성질환으로간주하는이유는 Aβ( 아밀로이드베타 ) 펩티드의응집과축적이후만성염증세포및염증매개물질들의출현때문이다 2,3). AD의신경염증병소에서성상세포의역할이특히중요하여, 반응성세포증식이신경섬유농축체와아밀로이드판주위에서일어나고, 소교세포와함께성상세포는보체, 시토카인, 급성기반응단백등을생산, 분비한다 4). 아밀로이드베타자체는시험관내에서보체계의고전및교대경로의활성화를일으킨다 5). 뇌에서보체의주된 J Korean Geriatr oc 12(4) December 2008 235
최영숙외 : 성상세포종에서아밀로이드베타 (Aβ 1-42) 단백과 Interferon Gamma 투여후보체조절유전자들의발현변화 원천은혈류에서유입된것이아니라, 침윤된대식세포, 소교세포, 성상세포및신경세포들이며, 이들세포는대부분의보체성분이외에특이보체조절인자및보체수용체도합성할수있다 6). 보체조절인자들은보체성분의과도한소모를막아주는목적이외에숙주세포에활성화된보체성분의원하지않는결합에의하여, 또는혈장보체계의자동활성화에의한조직손상을막기위하여필요한것이다. 이를위하여숙주조직에는수용성보체조절인자로 C1-inhibitor (C1- INH), C4 binding protein (C4bp), complement factor H (CFH), complement factor I (CFI), clusterin, vitronectin이있고, 세포막결합보체조절인자로 decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), CD59, complement receptor 1 (CR1, CD35) 등이존재한다 7). AD병소에서는활성화된성상세포에서합성후분비되는각종염증매개물질에의하든지또는아밀로이드베타자체에의하여보체계가활성화되면이는신경세포의손상을동반한, 조절되지않는만성신경염증을초래하고, 이때보체계활성화를억제, 통제하는보체조절인자의역할을통하여주변의 innocent bystander 신경세포의파괴를억제해줄수있을것이다. 따라서본연구의목적은시험관내아밀로이드베타 (Aβ 1-42) 와 interferon gamma (IFNγ) 투여로활성화된성상세포에서각종보체조절인자유전자들의발현증감을관찰함으로서, 궁극적으로는 AD염증병소내성상세포에서보체조절인자들의역할에대한정보를얻고자한다. 대상및방법 1. 세포배양 세포는사람성상세포종세포주 U373MG를한국세포주은행에서구입하여사용하였다. 배지는 RPMI-1640 배양액 (BioWhittaker, Walkersville, MD, UA) 에불활성화시킨 10% 우태아혈청 (Gibco-BRL, Grand Island, NY, UA) 과 100 IU/ml penicillin-streptomycin (Gibco-BRL) 을첨가하여사용하였고, 세포는 37, 5% CO 2 배양기에서배양하였다. 배양된세포는세포생존율검사로 95% 이상의 viable cell 조건에서실험에사용하였다. 2. Aβ 1-42 제조 Aβ 1-42 (Bachem, Bubendorf, witzerland) 제조는증류수로 1mM 농도의 stock solution 을만든다음이미기술된방법 8) 에따라실온에서 1 주일간방치하여응집시킨후, 1-20 μm 을최종농도로사용하였다. 3. Aβ 1-42, Interferon gamma (IFNγ) 의단독투여 Aβ 1-42 투여는 Aβ 1-42 (1-20 μm, Bachem) 를 RPMI-1640세포배양액에첨가후 24시간까지시간별로배양한다음 total RNA를분리하였고, IFNγ (Thermo Fisher cientific, Rockford, IL, UA) 투여는 IFNγ (62.5-1,000 U/ml) 을 RPMI-1640 세포배양액에첨가후 24시간까지시간별로배양한후 total RNA를분리하여보체조절유전자각각의 mrna를제조하였다. 4. Aβ 1-42, IFNγ 의복합투여 Aβ 1-42 ( 최종농도 2 μm) 와 IFNγ ( 최종농도 500 U/ml) 복합투여군은 RPMI-1640 세포배양액에이들두가지세포자극제를동시에첨가한후 24시간까지배양한후 total RNA 를분리하여보체조절유전자각각의 mrna를제조하였다. 복합투여군에서보체조절유전자들의발현정도를대조군, Aβ 1-42 ( 최종농도 2 μm) 단독투여군및 IFNγ( 최종농도 500 U/ml) 단독투여군에서의결과와각각비교하였다. 5. 보체조절인자 전부 7가지로서, 수용성보체조절인자는 C1-INH, CFI, clusterin, vitronectin 유전자를, 세포막결합보체조절인자는 DAF, MCP, CD59 유전자를관찰하였다. 그러나예비실험에서 CR1, C4bp 및 CFH는 U373MG 세포에서전혀검출되지않아서관찰대상에서제외하였다. 6. 실시간역전자중합효소반응 (real-time reverse transcriptase-polymerase chain reaction, real-time RT-PCR) U373MG세포에서유전자발현의증감을보기위하여 Aβ 1-42 단독투여군, IFNγ 단독투여군, Aβ 1-42 +IFNγ 복합투 236 J Korean Geriatr oc 12(4) December 2008
Young-ook Choi, et al: Alzheimer's Disease, beta-amyloid protein, C1 Inhibitor, Complement Factor I, Interferon-gamma 여군각각에서세포 LH total RNA를 Trizol (Invitrogen, Carlsbad, CA, UA) 을사용하여분리하였다. RT-PCR은 oligo-(dt)12-18 primer (Table 1) 를사용하여 RNA Core kit (Applied Biosystems, Branchburg, NJ, UA) 로실시하였다. 50 ng cdna를 iq5 YBR Green supermix (Bio- Rad, Hercules, CA, UA) 와 12가지유전자의 primer ( 최종 Table 1. Primers and conditions used in the real-time reverse transcriptase PCR (polymerase chain reaction) Gene Primer sequence (5' to 3') GFAP GCA GGG CAT GAC TTT GTC CCA TTT TGT GTG AGT AAG AAG GGA CCG CAA C1 inhibitor CAA CCC ACC CAA CCA ACT AC TGA CTC TCC AAG TCA GAG CAG A CFI GAA ACG AAT TGT GGG AGG AA TGC AGC AGT CAG AAT CCA AC Clusterin TGC AGG AAT ACC GCA AAA A GGG CTG CAG CTC ATC TTG Vitronectin TCT TCT GGG GCA GAA CCT C TGC CAT GCC TGA GAT GTA GA DAF CAG CAC CAC CAC AAA TTG AC TGC TCT CCA ATC ATG GTG AA MCP ATC ATA CAT GGC TAC CTG TCT TGC ATC TGA TAA CCA AAC TC CD59 ATT TTG ATG CGT GTC TCA TTA TTT CTC TGA TAA GGA TGT CCC CR1 TGG CTA TCC ATT TAC ATT CTC AAG TTC AGC TGT ATA GTA CTT TGT CAA GGA C4BP GCC AAA AAG GTT TTG TTC TCA CAG GAA GCA TGT GGA ATG TC CFH TAT AAG GCG GGT GAG CAA GT GGA TTC ACA CAG GAG GTG TCT GAPDH CCA TGT TCG TCA TGG GTG TGA ACC A GCC AGT AGA GGC AGG GAT GAT GTT C Type A A A A A A A A A A A A Product length (bp) GeneBank accession # Anneal ( ) 199 NM002055 60 107 NM000062.2 62.5 127 NM000204 55.1 104 NM001831.2 55.1 122 NM000638.3 62.5 112 NM000574 62.5 124 NM002389 55.1 191 NM000611 55.1 109 NM000651.4 60 130 NM000715.3 61.3 131 NM000186.2 62.5 173 NM008084 60 J Korean Geriatr oc 12(4) December 2008 237
최영숙외 : 성상세포종에서아밀로이드베타 (Aβ 1-42) 단백과 Interferon Gamma 투여후보체조절유전자들의발현변화 농도 5pmol/μl) 를각각혼합한후, real-time PCR machine (iq5, Bio-Rad) 을이용하여이들유전자의 mrna 발현정도를측정하였다. 7. 세포생존율검사세포를자극하는 Aβ 1-42 와 IFNγ의세포독성및세포생존율검사는 tetrazolium salt (MTT) quantitative colorimetric assay 방법으로 microplate reader (molecular device, unnydale, CA, UA) 로파장 490 nm에서측정하였다. 8. 통계처리각실험결과는평균 ± 표준오차로표기하였고, tudent t-test를사용하여통계분석을하였다. 결 과 1. IFNγ 단독투여군에서의보체조절유전자들의발현 IFNγ을 500 U/ml 농도로세포배양액에넣은후 24시간까지 7가지보체조절유전자전체에대한발현정도를관찰한결과 C1-INH는대조군에비하여계속현저한증가를보였다 (4, 24시간, p<0.01; 8시간, p<0.05). CFI와 clusterin은 24시간까지그발현이소량증가하였다 (p<0.01). 그러나 vitronectin, DAF, MCP, CD59 모두 24시간까지대조군에비하여발현은감소하였다 (Fig. 1). IFNγ을 62.5-1,000 U/ml의농도로각각세포배양액에넣은후 24시간까지세포배양을한후 C1-INH mrna 를정량한결과 dose-dependent하게, 그리고시간별로점차증 Fig. 1. Effect of IFNγ on complement regulator expression in the human astrocytoma cell line, U373MG cell. U373MG cells (3 10 5 cells/well) were stimulated with either 500 U/ml of IFNγ, or medium alone for 0-24 hours. Total RNA was prepared and levels of complement regulators were determined by real-time RT-PCR. Results are mean±e of three replicate cultures. C1-INH mrna was remarkably increased for 24 hours. Expression of mrnas for CFI and clusterin were slightly increased but other regulator genes were unchanged or decreased. Each column represents mean±e for two independent experiments compared with control. * p<0.05 and p<0.01 versus control. tatistical analysis was performed by tudent t-test. 238 J Korean Geriatr oc 12(4) December 2008
Young-ook Choi, et al: Alzheimer's Disease, beta-amyloid protein, C1 Inhibitor, Complement Factor I, Interferon-gamma Fig. 2. IFNγ-induced increase of mrna expression of C1- INH gene in U373MG cell. Cells were incubated with IFNγ (62.5-1,000 U/ml) for 0-24 hours. Extracted mrna from the cell lysate was quantified by real-time RT-PCR. Note doseand time-dependent upregulated expression of C1-INH 24 hours after administration of IFNγ. Each graph represents mean±e for two independent experiments compared with control. * p<0.05 and p<0.01 versus control. tatistical analysis was performed by tudent t-test. 가하였고, 특히 24 시간째가장현저하였다 (p<0.01, Fig. 2). 2. Aβ 1-42 단독투여군에서의보체조절유전자들의발현 1-20 μm 농도로 Aβ 1-42 를세포배양액에투여하여 24시간배양한후 total RNA를분리하여세포내보체조절유전자각각의 mrna를정량한결과, 2, 10 μm 농도에서 C1- INH는대조군에비하여약간의발현증가를보였으나, 20 μm 농도에서는오히려감소하였다. Glial fibrillary acid protein (GFAP) 은 2 μm 농도에서가장많이발현하였다 (Fig. 3). 2 μm 농도에서 24시간세포를배양후 C1-INH를포함한보체조절유전자들을관찰한결과, IFNγ 단독투여군과비슷하게, CFI와 clusterin은둘다대조군에비하여그발현이소량증가하였으나 (p<0.05), DAF와 CD59는감소하였고, vitronectin과 MCP는약간증가하였지만통계적의의가없었다 (Fig. 4). 3. Aβ 1-42 와 IFNγ 복합투여군에서의보체조절유전자들의발현 Aβ 1-42 (2 μm) 와 IFNγ (500 U/ml) 을복합투여한후 24 시 Fig. 3. C1-INH expression of Aβ 1-42 -stimulated U373MG cell. Unstimulated and 1-20 um of Aβ 1-42 -stimulated U373MG cells were incubated for 24 hours after which total RNA was isolated and real-time RT-PCRs were performed using specific primers against C1-INH and GFAP. Increased expression of C1-INH mrna was not significant after 2-10 um Aβ 1-42 peptide stimulation. Each graph represents mean±e for two independent experiments compared with control. * p<0.05 and p< 0.01 versus control. tatistical analysis was performed by tudent t-test. 간배양한다음세포에서 total RNA를분리하여보체조절유전자들의 mrna 발현정도를측정하여대조군과비교하였고, 또한동시에실험한 Aβ 1-42 (2 μm) 단독투여군, IFNγ (500 U/ml) 단독투여군의결과와도함께비교하였다. 그결과, 복합투여군에서 C1-INH는대조군에비하여현저히높았고 (p<0.01), Aβ 1-42 단독투여군에비하여도역시현저하게증가하였으나 (p<0.01), IFNγ 단독투여군에비하여는그발현이오히려다소감소하였다. CFI는대조군보다는복합투여군에서약간증가하였으나 (p<0.05), Aβ 1-42 단독투여군및 IFNγ 단독투여군보다는다소감소하여통계적의의가없었다. Clusterin은복합투여군이대조군에비하여증가하였으나 (p<0.01), IFNγ 단독군과차이가없었고, Aβ 1-42 단독투여군보다약간감소하였으나, 통계적의의가없었다. DAF는복합투여군에서대조군, Aβ 1-42 및 IFNγ 단독투여군보다증가하였다. Vitronectin과 CD59는모두복합투여군에서대조군, Aβ 1-42 및 IFNγ 단독투여군보다는다소감소하였으나통계적의의가없었다. MCP는복합투여군에서대조군보다증가하였으나, 각각의단독투여군과는그발현정도에차이가없었다 (Fig. 4). J Korean Geriatr oc 12(4) December 2008 239
최영숙외 : 성상세포종에서아밀로이드베타 (Aβ 1-42) 단백과 Interferon Gamma 투여후보체조절유전자들의발현변화 Fig. 4. Effect of Aβ 1-42 plus IFNγ on the mrna expression of various complement regulators from U373MG cells in comparison with control, Aβ 1-42 and IFNγ alone group. Cells were cultured in 24-well plates and then stimulated for 24 hours with 2 μm Aβ 1-42, 500 U/ml IFNγ, 2 μm Aβ 1-42 + 500 U/ml IFNγ or medium alone. Aβ 1-42 plus IFNγ stimulation shows that IFNγ overrides Aβ 1-42 -induced mrna expression of C1-INH. Each column represents mean±e for two independent experiments compared with control. * p<0.05 and p<0.01 versus control, p<0.05 versus Aβ 1-42 alone. tatistical analysis was performed by tudent t-test. 고찰 아밀로이드베타는 transmembrane glycoprotein 인 amyloid precursor protein (APP) 에 α-secretase와 BACE1 (β-secretase) 가작용후 γ-secretase에의해형성된 39-42개의아미노산 cleavage product이다. 아밀로이드베타펩티드의원천은과량생산된신경세포로부터또는혈관으로부터이며 9), 이불용성의응집된원섬유들이아밀로이드판을형성하여신경세포밖과소혈관주위에지속적으로축적된다. Aβ 1-42 가 Aβ 1-40 보다도더응집이잘되고신경독성을가진다고한다 10). AD환자대뇌에응집된아밀로이드베타의축적에따라 innate immunity에의하여성상세포및소교세포가변성신경세포주위를둘러싼다. 이들세포수의증가및염증관련보체의검출등은 AD가염증매개기전임을의미한다 5). AD에서신경세포의소실은아밀로이드베타단백의 직접적인신경독성작용 1) 및주위를둘러싸는성상세포및소교세포들에의한염증반응 2) 때문일것이다. 중추신경계내신경아교세포중가장많은수를차지하는성상세포는 AD병소에서활성화되었을경우 IL-1, TGFβ, CCL-2, CCL-3 등의시토카인과 α 1 -antichymotrypsin, protease, 유착분자, inducible nitric oxide synthase, cyclooxygenase-2, 활성산소종등을생산하여국소염증을조절하며, 동시에아밀로이드베타를파괴시켜신경세포를보호하는능력도가진세포이다 11). 자극받지않은사람성상세포종에서보체조절인자 DAF, MCP, CD59, clusterin 및 vitronectin mrna가각각검출된다 12). 시험관내에서성상세포를자극, 활성화시키는방법으로아밀로이드베타, IFNγ, LP 13) 등이이용된다. 응집된아밀로이드베타펩티드에의하여성상세포는 IL-Iβ, TNFα, macrophage inflammatory protein, monocyte chemoattractant 14) protein (MCP), PGE 2 등을생산할수있고, U373MG 세포주는 IFNγ (100 U/ml) 자극또는 30 μm Aβ 42 +IFNγ (100 240 J Korean Geriatr oc 12(4) December 2008
Young-ook Choi, et al: Alzheimer's Disease, beta-amyloid protein, C1 Inhibitor, Complement Factor I, Interferon-gamma U/ml) 자극으로 MCP-1을생산한다 15). IFNγ은주로조력 T림프구 1(TH1), 자연세포독성세포또는세포독성 T림프구에서생산되고, 그기능중특히대식세포를활성화하여포식작용을증가시키고, MHC II 항원의발현증가와항세균, 항바이러스기능이있음이이미잘알려져있으나 16), 성상세포에대한활성화기전은보고된바없다. 본실험에서성상세포종세포를자극하는두가지물질을단독또는복합투여하여세포내에서체질적 (constitutional) 으로가지고있던보체조절인자들의유전자발현의증감을비교관찰하였다. 그결과 U373MG세포자체는아밀로이드베타및 IFNγ에의하여각각활성화되었음을 GFAP mrna의발현증가를통하여확인할수있었다. 뇌의소교세포, 성상세포, 희소돌기아교세포및신경세포에서모두대부분의보체, 보체조절인자및보체수용체의합성이가능하다 17). 특히성인의성상세포에서는보체 C1-C9, factor B, factor D와보체수용체 CR1, C3aR, C5aR 를생산하고, 세포막결합보체조절유전자는 CD59, MCP, DAF의순으로많이발현되며, 수용성보체조절유전자는 C4bp을제외하고모두발현되고, 그러나신경세포에서는 MCP, CD59, C1-INH 및 CFH가모두매우낮은수준으로발현된다고보고되었다 6). 현재까지유핵세포에서 10가지의보체조절인자가알려져있으며, 본실험에서사용한 U373MG세포에서는관찰된 7가지유전자이외에 C4bp 유전자가관찰되지않은점은 Gasque 등 6) 의보고와일치하였다. 그러나 CR1과 CFH는검출되지않아서이는아마도이들의 primer와증폭과정의차이때문일것으로추정된다. AD환자의뇌에서 C1q-C9 모두그발현이증가되었고 18), 특히대뇌피질, 노인성반점과신경섬유농축체에서는 C5b-9 복합체가관찰된다 19). 아밀로이드베타는고전, 교대보체경로모두를직접활성화시키고, 그결과소교세포의이동과활성화및성상세포증가를초래한다 20). 보체계의부적절한활성화와숙주세포의파괴를막기위하여숙주내에서보체계는잘조절되어, 활성화된보체는병원균에대한숙주의방어를도와주지만, 또한편으로는자기조직에대한원치않은손상을줄수도있다. 해마나대뇌피질의신경세포들은보체조절인자들에의해보호되지만, 그들대부분의발현이약하기때문에보체매개손상이쉬워진다 21). 본실험에서보체조절유전자중 C1-INH만이 IFNγ으로 자극된 U373MG 세포에서그발현이의의있게증가됨을알수있었고 (Fig. 1, 2), 아밀로이드베타는 C1-INH 발현을경하게증가시켰다 (Fig. 3, 4). IFNγ과아밀로이드베타의복합투여시 IFNγ에의한 C1-INH 발현증가가아밀로이드베타의 C1-INH 발현증가를압도함을볼수있었다 (Fig. 4). C1-INH는 serine protease 억제제의하나로, 그기능은혈장에서활성효소인 C1r2C1s2에붙어서 C1q로부터이들을떨어뜨림으로 C1의자발적인활성화를제한한다. 본실험에서아밀로이드베타또는시토카인 IFNγ을 24시간까지의단기투여에의하여세포활성화초기에 C1복합체의활성을조절하는 C1-INH의 upregulation 현상의의미는생체에서세포방어에중요한의의를지닌다고해석된다. 즉 C1-INH의현저한발현증가는고전경로보체계활성화의첫단계를직접억제하여보체계전체구성인자들의과도한소모를조절하고, 보체매개염증의억제효과를통하여생체보호기능의유지를의미한다. 그러나이현상은차후, AD모델실험동물등을이용하여생체조직병소내에서직접증명해야할것으로판단된다. C1q와아밀로이드베타의결합을억제하면해마의신경세포는보체공격으로부터보호되어, 보체에의해매개되는아밀로이드베타의세포독성을막을수있다고하였다 22). 본실험과관련하여, 저자들은사람신경아세포종과마우스소교세포에서도아밀로이드베타의단기투여에의하여 C1-INH의발현증가를이미확인한바있다 23,24). AD환자뇌조직의성상세포와 neuritic plaque에서 C1-INH 가오히려빨리소모되기때문에효율적으로보체활성화를억제하지못한다고하였고 25-27), C1-INH와 glycophosphoinositol-anchored 세포막단백인 CD59 둘다정상인과 AD 환자의뇌에서모두그 level이낮다고보고되었다 26). 그러나정상성인사체의뇌에서분리, 배양한성상세포또는신경아세포주배양액에 IFNγ을첨가하여이들세포를자극하면 C1-INH의발현이유도되었다 28). CFI는고전및교대경로에모두관여하며, DAF, MCP, CR1, C4bp 및 CFH를보조인자로이용하여 C4b와 C3b를분해시키는 serine protease이다. 황반변성환자에서아밀로이드베타가보체를활성화시키고, 동시에 CFI를불활성화시켜그결과망막하조직에만성염증이지속됨이최근에알려졌다 29). 본실험에서아밀로이드베타나 IFNγ 에의하여 CFI의발현증가가대조군에비하여경하게유 J Korean Geriatr oc 12(4) December 2008 241
최영숙외 : 성상세포종에서아밀로이드베타 (Aβ 1-42) 단백과 Interferon Gamma 투여후보체조절유전자들의발현변화 도됨을알수있었지만 (Fig. 1, 4), 아밀로이드베타또는 IFNγ의복합투여시는단독투여에서와유전자발현의뚜렷한차이는볼수없었다 (Fig. 4). AD환자에서 clusterin은해마의 pyramidal neuron과 entorhinal cortex에서 mrna 발현이증가되어있고 30), 노인성반점의아밀로이드축적부위에서 vitronectin과함께검출된다 31). Clusterin은세포변성과세포사를막아주는기능 32) 과사람성상세포에축적된아밀로이드펩티드를용해, 청소해주는기능 33) 이알려져있다. 본실험에서 CFI와비슷하게 clusterin은아밀로이드베타나 IFNγ 단독투여에의하여경한발현증가를볼수있었지만, 이들의복합투여시와그차이가뚜렷하지않았다 (Fig. 1, 4). Vitronectin은 AD환자의뇌에서 clusterin과함께 upregulation 되어있어서 30) 이들이세포막공격보체복합체에대한비특이적방어기능을가진다고하였다 34,35). 본실험에서는아밀로이드베타나, IFN의자극후이유전자의증감을볼수없어서그역할을알수없었다. 한편세포막결합보체조절인자의경우 DAF, MCP, CD59, CR1 등은혈관과적혈구에서양성반응을보였으나, 아밀로이드판이나, 신경섬유농축체에서는검출이안되어보체활성화의조절기능이불완전하다고하였다 36). 사람태아의성상세포에서는 DAF (CD55), MCP (CD46) 및 CD59 는양성이나 CR1 (CD35) 은음성이었고, 신경세포와신경아세포종에서는 CD46과 CD59는양성이나 CD55와 CD35 는음성이었다 37,38). DAF의기능은 C3 전환효소의형성을억제하고, C4b2a, C3bBb의분해를촉진시킨다 39). 사람의신경능선 (neural crest) 유래신경세포주를 phorbol 12-myristate 13-acetate로분화시키면 DAF의발현이 upregulation되지만, CD59와 MCP는변화가없었다고하였다 40). 본실험에서 DAF는대조군에비하여아밀로이드베타와 IFNγ복합투여에의하여서만발현증가가유도됨을알수있었다. MCP는 C3 전환효소와 C5 전환효소를구성하는 C3b와 C4b를각각분해하는 serine protease인 CFI의보조인자로서, 사람 herpes virus-6와홍역바이러스의수용체역할을하며, 사람성상세포, 소교세포및희소돌기아교세포의세포표면에서검출된다 41). 본실험에서는아밀로이드베타와 IFNγ 복합투여시에만대조군에비하여약간의발현증가를보일정도였다. AD환자의뇌아밀로이드판에서 CD59가검출되지만 42), AD환자의해마와전두엽피질에서는그 level이현저히낮고, CD59 결핍시에는신경세포소실이초래된다 21,27). 아밀로이드베타자체는 CD59의발현을 down-regulation시켜세포막공격보체복합체형성이억제되지않도록한다고하였다 21). 본실험에서도 CD59유전자는아밀로이드베타와 IFNγ로자극된 U373MG 세포에서그발현이감소됨을확인하였다. 이상의결과를종합하여볼때사람성상세포종세포주 U373MG세포의보체조절유전자중 C1-INH만이 IFNγ의단기자극에의하여현저하게그발현이증가됨을알수있었다. 또한아밀로이드베타나 IFNγ 자극은 C1-INH, CFI 및 clusterin을제외하고는나머지보체조절유전자들의발현에별다른영향을미치지못함을알수있었다. 요약 연구배경 : 사람성상세포종세포를 Aβ 1-42 와 IFNγ 투여로자극후세포내보체조절유전자들의발현변화를보고자하였다. 방법 : 사람성상세포종세포주 U373MG세포에 IFNγ (62.5-1,000 U/ml), 응집 Aβ1-42 (1-20 μm) 단독투여, 또는이들의복합투여후 24시간까지시간별로배양한다음보체조절유전자 C1-INH, CFI, clusterin, vitronectin, DAF, MCP 및 CD59의 mrna 발현증감을실시간역전사중합효소반응법으로정량하여대조군과비교하였다. 결과 : IFNγ ( 최종농도 500 U/ml) 은 7가지의보체조절유전자중시간별로 C1-INH만을현저하게증가시켰다. Aβ 1-42 ( 최종농도 2 μm) 에의한 C1-INH의증가는경미하였다. Aβ 1-42, IFNγ의단독투여후 clusterin과 CFI는모두약간증가되었으나, vitronectin, DAF, MCP, CD59는감소내지양적변동이없었다. IFNγ과 Aβ 1-42 의복합투여시 IFNγ에의한 C1-INH 발현증가가 Aβ 1-42 유도 C1-INH의발현변화를압도하였다. 결론 : 사람성상세포종 U373MG세포에 IFNγ 단독또는 Aβ 1-42 와의복합투여는보체조절유전자들중 C1-INH 만을현저하게상향조절시켰다. 중심단어 : 알츠하이머병, 아밀로이드베타, C1억제제, 보체 I 인자, 감마인터페론 242 J Korean Geriatr oc 12(4) December 2008
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