工學碩士學位請求論文 신규미백제로서의귀전우의특성규명 Characterization of Euonymus alatus Sieb as a depigmenting agent 年 2 月 仁荷大學校大學院 生物工學科 ( 生物工學專攻 ) 李婑离

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1 저작자표시 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 이차적저작물을작성할수있습니다. 이저작물을영리목적으로이용할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 工學碩士學位請求論文 신규미백제로서의귀전우의특성규명 Characterization of Euonymus alatus Sieb as a depigmenting agent 年 2 月 仁荷大學校大學院 生物工學科 ( 生物工學專攻 ) 李婑离

3 工學碩士學位請求論文 신규미백제로서의귀전우의특성규명 Characterization of Euonymus alatus Sieb as a depigmenting agent 年 2 月 指導敎授 金殷基 이論文을工學碩士學位論文으로提出함 仁荷大學校大學院 生物工學科 ( 生物工學專攻 ) 李婑离

4 Characterization of Euonymus alatus Sieb as a depigmenting agent. by Yu Ri Lee A THESIS Submitted to the faculty of INHA UNIVERSITY In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE Department of Biological Engineering February 2010

5 이論文을李婑离의碩士學位論文으로認定함 年 2 月 主審 : 副審 : 委員 :

6 요약 피부색은 melanocyte내의 melanin의합성에의해결정되며, UV등의자극에의해유발된다. 이런 melanin의특성으로는햇빛에의한피부손상보호, 피부색을결정하는역할을한다. 피부색은 melanin이라는피부결정색소에의해결정이되며, melanin의양과타입에따라결정되어진다. melanin의기능은 UV빛에의한피부손상을보호한다. 이 melanin은 melanocyte 내의 melanosome이라는특별화한기관에의하여합성되며, 이후 melanin이 melanocyte에서각질층까지이동되어피부색을결정한다. 특히동양인의경우흰피부에대한욕구가높고, 현재의미백제의제품안정성의문제로신규물질의필요한시점이다. 그리하여, 중국유래의천연약재인귀전우를선택하여, silica open chromatography 와 HPLC를통하여활성물질분리를하였고, 동물세포실험, Western blot을통하여 Melanogenesis 관련단백질들의발현을확인, DNA와단백질간의결합확인을위해 Electrophoretic mobility assay(emsa) 를수행하였다. 결과적으로귀전우는 Melanogenesis 관련단백질을조절하는것을확인하였으며, MITF 단백질과 E-box의결합을저해하여미백제로서의가능성을제시하고자한다. I

7 ABSTRACT Skin pigmentation results from melanin synthesis by melanocytes and is caused by exposure to UV radiation. Melanin plays an important role in the prevention of sun-induced skin injury, and is a major determinant of skin color. Euonymus alatus Sieb is one kind of medical plan grown in the korea. In order to find the active constituents from this plant, the water extracts were chromatographed by using solvent-solvent partition, silica gel column chromatography and high performance liquid chromatography (HPLC).The depigmneting activity of Euonymus alatus Sieb was evaluated by melanin assay, electrophoretic mobility assay (EMSA), and Western blot. In treated B16F10 melanoma cells, the separated compound demostrated high potential for inhibition of melanin production in a dose dependent manner. Finally, it is suggested that melanin inhibition is cause by reducecing the expression of tyrosinase, MITF, CREB. From these results, I suggest that these extracts might be useful as a new whitening agent in cosmetics. II

8 CONTENTS 요 약 Ⅰ ABSTRACT Ⅱ CONTENTS Ⅲ 1. INTRODUCTION Melanin PKA pathway 기존미백제의문제점및신규미백원료의필요성 5 2. OBJECTIVES 8 3. MATERIALS AND METHODS 천연약재 귀전우 ( Euonymus alatus Sieb ) 천연약재. 추출및분리 HPLC 동물세포실험 세포및시약 세포생존도측정실험 (MTT assay) 12 III

9 Melanin 양측정 Western blot Electrophoretic Mobility Shift Assay(EMSA) Results Skin cell test Crude Extract Solvent-Solvent Extract Silica open chromatography Western blot Melanogenesis Pathway MAPK Pathway EMSA ( Electrophoretic Mobility Shift Assay ) MITF specific inhibition CONCLUSIONS REFERENCE 29 IV

10 1. INTRODUCTION Melanin 인종별로피부색이다른이유는주로피부에존재하는멜라닌 (Melanin) 색소의차이에기인한것으로멜라닌은표피의기저층에위치한피부세포의한종류인 Melanocyte에의해합성되어다른피부세포인각질형성세포 (Keratinocyte) 로이동되어이세포가각질형성과정을통해피부밖으로나와피부로부터분리될때까지지속적으로존재하여피부색을결정하게되는요인이된다. Melanocyte는성숙되면서돌기를형성하게되고이돌기를통해주변의각질형성세포에멜라닌을전달하게되며피부색뿐만아니라머리카락등의색깔도결정하게된다. 이멜라닌은검정또는갈색계열의색소인 eumelanin과붉거나노란계열의색소인 pheomelanin 두종류가존재한다. 인체의내외부적인요인에의해멜라닌생성과정에이상이생기면백반증이나백색증및기미, 주근깨, 검버섯등의색소침착, 심각하게는피부암까지발생하는것으로알려져있다 (Fig. 1) 1

11 Fig.1 The melanin biosynthetic pathway 2

12 1.2. PKA pathway UV와같은호르몬등에의해자극을받게되며 α-msh(melanocyte stimulate hormone) 같은호르몬이분비되어 cell membrane상에존재하며, MC1R(melanocortin-1 receptor) 와결합을하게되면, 세포질내에서cAMP를증가시킨다. 이렇게증가된 camp는 PKA를활성화시키고, 이는세포핵내로들어가 MITF Promotor상의발현을유도하여 MITF ptrotein이대량으로발현하게된다. MITF는연결된 pathway상에서 Tyrosinase, TRP-1, TRP-2 라는 melanogenesis Key enzyme의발현을유발시키고, 이와같은단백질들이핵밖으로방출이되어색소유발을한다. 또한 ckit receptor에의해자극되어진 MAPK pathway에의해, MITF를 degradation을유발시켜, melanogenesis를저해하는 pathway가있다. (Fig. 2) 3

13 Fig. 2 Signaling pathways of melanogenesis 4

14 1.3. 기존미백제의문제점및신규미백원료의필요성 피부색은 melanin, 피부결정색소에의해결정이되며, melanin의양과타입에따라결정되어진다. UV, 호르몬등의자극에의해만들어진다고알려져있다. 이 melanin 합성은기질인 tyrosin이 tyrosinase, TRP1 (tyrosinase related protein 1), TRP2(tyrosinase related protein 2) 에의해산화되어결국 pheomelanin과 eumelanin이만들어진다. 그중 melanogenesis를야기시키는데 key enzyme으로알려진 tyrosinase는대부분미백소재에서 target으로잡고있으며, 기존에알려진미백제의 target은멜라닌생선전, 중, 후세가지로구분할수있다. 현재까지미백제로는 Ascorbic acid, Arbutin, Kojic acid, Hydroquinone 등이사용되어왔으나강한미백효과에도불구하고제품안정성이낮다는문제가있으므로세포독성이낮고멜라닌활성이높은저해물질의개발이필요한시점이다. (Fig. 3,4) 5

15 Before melanin synthesis During melanin synthesis After melanin synthesis Pigment Cell Res 16: , 2003 Fig. 3 Skin depigmentation mechanism 6

16 Material Mechanisms Remarks Hydroquinone Cytotoxic effect on melanocytes High toxicity to the skin Arbutin Inhibition tyrosinase activity Obscure effect Kojic acid Interrupts intermediates in melanin biosythesis Slight skin irritation & allergy reaction Stimulates melanin elimination Azealic acid through Obscure effect the keratinocytes AHA Vitamin A Enhance cell regeneration Skin irritation Glucosamine Gakactosamine Inhibites tyrosinase synthesis Potent cytotoxic effect SOD Glutathione Scavenges free radicals Not stable Fig. 4 Mechanism of depigmenting agents 7

17 2. OBJECTIVE 동양인의경우맑고흰피부에관한동경은오랫동안계속되어왔고이러한요구에의해미백시장은불경기속에서도매년 10~15% 의성장률을보여왔다. 현재화장품과의약품의중간적인성격을갖는기능성화장품이각광을받고있으며, 이에따라점차바이오를접목한기능성사업으로전환되고있어, 피부구조에관한접근이필요한시점이다. 우선선행연구결과중국유래의천연약재를 Screening하였고, 그중효능이좋은귀전우를선택하여, 실질적으로천연물의성분이 animal cell test, western blot, EMSA를수행하여효능이피부기작중어느부분에서영향을미치는지를알아보고자하였다. 특히이미백제가어느부분을저해하는것은생리학적으로중요한역할을할것으로예상되어, melanin 합성되는 melanocyte내의 signal을연구하고자메커니즘연구를수행하였다. 또한동시에귀전우를분리하여활성성분을확인하고자하였다. 8

18 3. MATERIALS AND METHODS 3.1. 천연약재 귀전우 ( Euonymus alatus Sieb ) 천연약재는오랜기간동안사람들에의해구전으로그효능이전해져왔으며, 그안에는이미아미노산, 단백질, flavonoids 등많은활성물질들이존재하고있다. 우리나라민간에서오래전부터암과피부병치료에쓰기도했었으며, 차로애용되고있는귀전우에대해초점을맞추고실험을진행하였다. (Fig. 5) Fig. 5 Euonymus alatus Sieb 9

19 3.2. 추출및분리 귀전우전초 700g을물 80도에서추출을하였으며, 그후 Rotary vacuum evaporator를사용하여물을날렸다. 이로얻은물추출물은 Hexane, Chloroform, Ethyl acetate, Water 순으로추출을진행하였으며, Ethyl acetate 층에서활성성분 0.5g을얻었다. Silica open chromatography를통해, 이동상은 chloroform과 methanol의비율을조절하여사용한결과 Chloroform: Methalnol 8:2 40mg얻었으며, 그수율은 0.06% 이다. (Fig. 6) Fig. 6 Isolation process of active compound from Euonymus alatus Sieb 10

20 3.3. HPLC ( High performance liquid chromatography ) 분석용 HPLC(Binary HPLC pump-waters 1525) 하였으며, Atlantis T3 3um 4.6 x150mm Column을사용하였고, 실험조건은 Flow rate 1ml/min, UV detector로는 210, 254nm 파장범위를선택하였으며, Mobile phase 로는 10%MeOH ~ 100%MeOH 를사용하여확인하였다. 11

21 3.4. 동물세포실험 (in vivo) 세포및시약 사용한세포는 B16F10 melanoma 세포로서, 배양에쓰인배지로 DMEM(GIBCO) 배지에 100unit/ml의 penicillinstreptomycin을 0.1%, fetal bovine serum 10% 가첨가되어사용되었고, 37, 5% CO2 조건의항온기를이용하였다. 세포생존도측정실험에첨가하는 MTT 시약은 Sigma Aldrich(USA) 사의제품을사용하였다. camp inducer 시약으로서 α -msh(50nm) 을첨가하였다. 대조군으로처리한세포와처리하지않은세포를각각실험에적용하였다 세포생존도측정실험 (MTT assay) B16F10 melanoma 세포 (1x10 3 cell/ml) 을 96well plate 에 200 μl주입 한후, 37, 5% CO 2 항온기에 24시간전배양시키고부착된세포에시험시료가포함된동일양의배양액을처리한후, 2일간같은조건에서배양하였다. 배양된세포에 MTT(50mg.ml) 시약을처리 (0.1mg) 하여형성된 formazan을 DMSO로녹인후 540nm에서 plate reader로측정하여대조군과비교하였다. 12

22 melanin 양측정 B16F10 melanoma 세포 (1x10 5 cell/ml) 을 6well plate에 2ml씩넣은후, 37, 5% CO 2 항온기에 24시간전배양시키고배양액을제거하여시험시료가포함된동일양의배양액을처리하였다. 2일간 37, 5% CO 2 항온기에서배양을거친후, 다시배양액을제거하여 PBS 1ml씩첨가하여 2번세척을한후, trypsin-edta를첨가하여세포수집을한다. 수집된세포를 1.5ml eppen tube에옮겨닮아원심분리하여배양액을제거후, 다시 PBS로현탁시켜원심분리를이용하여 cell pellet을얻어낸다. 이와같이수집한각각의세포에 10% DMSO가들어있는 1N NaOH 용액을 200μl씩넣어주고현탁시켜 80 에서 1시간동안반응시킨다. 반응하여용해된세포내의 melanin은 plate reder를이용하여 405nm 파장에서대조군과비교하여측정하였다. 13

23 3.5. Western blot 세포를 100mm dish에 1x10 5 cell을넣고배양하여 24시간이지나면시료물질을첨가하고 2일동안배양하였다. 배양이끝난후 scraper를사용하여 dish로부터세포를분리하여 1.5ml tube에옮겨담아원심분리를통해 cell down시킨후, 상등액을제거하고 lysis buffer[40 mm Tris (ph 8.0), 120 mm NaCl, 0.5% Nonidet P-40 (NP-40), 0.1mM sodium orthovanadate, 2 g/ml aprotinin, 2 g/ml leupeptin, and 100 g/ml phenylmethylsulfonyl fluoride (PMSF)] 를첨가하여 4 에서 30분간반응시켰다 rpm에서 10분간원심분리를실시해 cell debris등을제거하였다. 시료내단백질의정량은 bicinchoninic acid(bca) (Pierce, Rockford, IL, U.S.A.) 방법으로수행하였으며총단백질 100μg을이용하여 10% SDS-PAGE를실시하였다. 전기영동이끝난후, PVDF membrane(0.45μm, invitrogene) 을사용하여 electrotransfer(100v, 2시간 ) 하였다. 반응이끝난 PVDF membrane을 blocking solution(tbst with 5% skim milk) 으로실온에서 3시간반응시켰다. 1차항체는 Tyrosinase goat antibody(santa Cruz), MITF rabbit antibody(neomarkers), β-actin goat antibody(santa Cruz), CREB, ERK, p38, PI3K (cell signaling) 를사용하였으며, 2차항체로 HRP로 conjugat된 Rabbit anti-goat antibody(santa Cruz), Goat anti-rabbit antibody(santa Cruz) 를사용하였다. 14

24 3.6 EMSA (Electrophoretic Mobiltiy Shift Assay) Nuclear extract Monolayer의 B16F10 mouse melanoma cell과 fibroblast를 scraper를이용하여 1ml PBS에모은후, 차가운 PBS로 2번세척을하였다. 14,000rpm, 4 의조건으로원심분리후, pellet을 2배부피의 buffer A [10 mm HEPES (ph 7.9), 1.5 mm MgCl 2, 10 mm KCl, and 0.5 mm DTT plus protease inhibitors] 에넣은후 10분동안얼음에보관하였다. 다시같은조건으로원심분리하여상등액을제거하고 pellet 2배부피의 buffer C[20 mm HEPES (ph 7.9), 420 mm NaCl, 1.5 mm MgCl 2, 0.2 mm EDTA, 0.5 mm DTT, 25% (v/v) glycerol plus protease inhibitors] 에넣은후, 10분동안얼음에서보관하였다. 같은조건으로원심분리하여상등액을분리해실험에사용하였다 Electrophoretin Mobility Shift Assay LightShift EMSA kit(pierce, USA) 를사용하였다. Well당 10ug의 nuclear extract와 20fg의 probe(table. 1), 귀전우를넣고 30분의반응시간을준후, 100V를유지하면서 4% native gel을사용하여분리하였다. Supershift band를보기위해 4μg의 MITF rabbit antibody(neomarkers) 를넣고 37 항온기에서 1시간동안반응시켰다. 양전하를띤 nylon 15

25 membrane 에 transfer 한후, FLA-5000 scanner 와 X-ray film 을사용하여 밴드를측정하였다. Forward : (Biotin)5 -AAG TTA GTC ATG TGC TTT Tyrosinase GCA G-3 Reward : 5 -CTG CAA AGC ACA TGA CTA ACT T- 3 Table.1 E-box probe for EMSA 16

26 4. RESULTS 4.1. Skin cell test Crude Extract 귀전우를각각 100, 200, 300 ppm의농도로 B16F10 melanoma cell에처리한후, control 대비 melanin의양과 formazon의양을비교하여봤을때귀전우가농도에비례하여 cell에거의영향이없이 melanin 양을줄인다는것을알수있었다. 또한 melanogenesis를야기시킨다고알려진 α-msh를처리하였을때, α-msh에의해야기된 melanin은귀전우에의해효과적으로감소하였으며, 이와같은결과에서귀전우가 depigmenting effect를지니고있다는것을확인할수있었다. (Fig. 7 A, B) 17

27 A. B. Fig. 7 Melanin synthesis inhibition by Euonymus alatus Sieb a: Without a-msh b: With a-msh 18

28 Solvent-Solvent Extract 귀전우를분리과정단계를거쳐효과를측정하였다. 전초 700g에서 Hexane, Chloroform, Ethyl acetate, Water 순으로분리하여, 활성을얻은결과 Ethyl acetate 층 0.5g을얻었으며, 이를각각 0, 12.5, 25, 50ppm의농도에서 B16F10 melanoma cell에처리한후, control 대비 melanin의양과 formazon의양을비교하여봤을때귀전우가농도에비례하여 cell 에거의영향이없이 melanin 양을줄인다는것을알수있었다. (Fig. 8) Fig. 8 Effect of partition fractions of Euonymus alatus Sieb on Melanogenesis of cultured B16F10melanoma cells 19

29 Silica open chromatography of Ethyl acetate 활성을보이는 Ethyl acetate층을 Silica open chromatography를통해, Chloroform: Methalnol 8:2 부분 (Fraction 2) 의활성부분을얻었으며, 각각 0, 12.5, 25ppm의농도에서 B16F10 melanoma cell에처리한후, control 대비 melanin의양이감소한것을볼때, 귀전우가 depigmenting effect를지닌것을확인할수있었으며 (Fig. 9), 또한 HPLC를통하여활성부분을확인하고자하였다. (Fig. 10) Fig. 9 Effect of silica open chromatograph fractions on Melanogenesis of cultured B16F10melanoma cells 20

30 AU Minutes Fig. 10 HPLC chromatogram of Fraction 2 21

31 4.2. Western blot Melanogenesis Pathway Skin cell test 결과귀전우가 melanin의합성을저해하는것을확인할수있었으므로 protein level에서 MITF와 Tyrosinase을비롯한 melanogenesis와관련된단백질들의발현정도를확인하기위해 western blot을수행하였다. α-msh에의한영향을확인하기위해귀전우과 α-msh를넣고비교하였고, 그결과귀전우는 α-msh에의해야기되어, 다시귀전우에의해멜라닌이감소함을확인할수있었다. 또한 Tyrosinase protein을비롯한 melanogenesis와관련된 3가지의 Pathway(PKA, MAPK, p38 pathway) 에서모두귀전우가영향을미치는것을확인하였다. control로 actin을사용하여발현정도를확인하였다. (Fig. 11) 22

32 Fig. 11 Melanogenesis related protein expression of Euonymus alatus Sieb 23

33 MAPK Pathway Melanogenesis와관련된 3가지의 Pathway(PKA, MAPK, p38 pathway) 에서모두귀전우가영향을미치는것을확인후, 좀더심도있는실험을위하여, MAPK pathway의 signal을연구하고자귀전우 200ppm에서 0, 10, 30, 60, 180, 360분이지남에따라점차 ERK가활성이되고이로인해 MITF를 degradation시켜 melanogenesis를저해시키는효과를확인할수있었다. (Fig. 12) Fig. 12 MAPK pathway related protein expression of Euonymus alatus Sieb 24

34 4.3. EMSA ( Electrophoretic Mobility Shift Assay ) MITF specific inhibition of Euonymus alatus Sieb 귀전우에의한 depigmenting effect가 MITF protein과 E-box의결합을저해하여일어나는것인지확인하기위하여귀전우를농도별로 EMSA를수행하였다. 또한귀전우가 E-box probe와결합하는 protein이 MITF protein인지확인하기위해 MITF mab를이용하여 supershift assay를수행하였다. 그결과귀전우의농도에따라 MITF의 shift band가줄어들어 50ppm이상의농도에서는 band가보이지않는것을확인할수있었으며, B16F10 nuclear extract에서 E-box와결합하는 protein이 MITF protein임을확인할수있었다. (Fig. 13) 25

35 Fig. 13 MITF specific inhibition of Euonymus alatus Sieb 26

36 5. CONCLUSIONS 1. 귀전우가 depigmenting effect 를지니고있음을확인하고, 이러한효과가 Melanogenesis 와관련된 pathway 상에서 signal 의영향과 MITF 와 E- box 의결합을저해하는효과를확인하고자하였다. 2. 귀전우를분리과정단계를거쳐효과를측정하였다. 전초 700g에서 Hexane, Chloroform, Ethyl acetate, Water 순으순으로분리하여, 활성을얻은결과 Ethyl acetate 층 0.5g을얻었으며, Silica open chromatography를통해, Chloroform: Methalnol 8:2 부분 (Fraction 2) 의활성부분을얻었다. 각각 control 대비 melanin의양이감소한것을볼때, 귀전우가 depigmenting effect를지닌것을확인할수있었으며또한 HPLC를통하여활성부분을확인하고자하였다 3. 귀전우의영향을확인하기위해 skin cell test를통해 depigmenting effect를확인하고, 피부내작용기작의범위를알아보기위해 Western blot을시행한결과 Melanogenesis와관련된 pathway(pka, MAPK, p38) 에서모두그효능을확인할수있었다. 특히 MAPK pathway의 signal을연구하고자귀전우 200ppm에서시간이지남에따라점차 ERK가활성이되고이로인해 MITF를 degradation시켜 melanogenesis를저해시키는효과를확인할수있었다 27

37 4. 귀전우의 depigmenting effect가실제로 MITF와 E- box와의결합을저해하여나타나는지확인하기위해 EMSA를수행하였다. 그결과귀전우가 MITF와 E-box의결합을저해하며, MITF protein에특이적으로저해한다는것을확인할수있었다. 28

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46 감사의글 무작정화장품연구원이되겠다며뛰어들었던무모했던저에게많은것을가르쳐 주시고도움을주셨던분들께감사의말씀을전하고자합니다. 우선부모님께감사의말씀을먼저드리고싶습니다. 빵꾸난양말, 거칠어진손보며글썽이며걱정하던우리엄마너무고마워요^-^ 그리고항상따뜻하게이부자리지켜주던은근히자상한우리아빠나이많은늙은딸공부시키시느라고생많으셨습니다. 언넝효도할께요^-^ 그리고이름만들어도쨘 ~ 한우리아들추운데나라지키느라힘들지제대할때까지꼭건강히지내길 ~ 보고싶다 ~ 아들 진정한스승에대한존경이란말을느끼게해주신우리교수님^-^ 2 년전무작정무모하게찾아왔던저를기꺼이받아주시고조금은느리게성장하는저에게항상걱정하지말라며격려해주시고기다려주심에교수님너무감사드립니다. 교수님으로인해많이성장할수있었던것같습니다. 너무바쁘셔서건강걱정됩니다. 교수님 ~ 제가좋아하는거아시죠? 항상건강하시고좋은일만있으셔야해요^-^ 감사합니다. 사수김해종박사님^-^ 박사님저졸업해요 ~ 아무것도모르던저가르치시기힘드셨을텐데항상밝은미소로가르쳐주시고다른곳에가서도항상먼저안부연락주시고많이도와주셔서저졸업해요. 박사님감사합니다. 공부방향제시해주시는영범오빠, 실험실신경써주시는향복언니, 항상밝은미소로대해주시는바비다박사님모두감사드립니다. 2 년넘게보면서답답하실법도한데표정한번찡그리는법없는베트남프린스 용오빠 ~ 오빠테김령씨랑저는참많이도움받았던것같아요. 매번도움만 받다가웨스턴알려드릴기회가와서저기뻤어요. 오빠웨스턴대박나세요 ^^ 37

47 영원한선배 " 정현선배 " 선배나졸업해^^ 진짜고마운거알지? 선배도힘들었을텐데때론선배처럼때론친구처럼대해준선배가없었다면아마버티기힘들었을꺼야. 동갑이지만존경하는정현선배 ~ 은혜갚으려면빕스정도로안되는거알아... 돈마니벌어서맛난거마니사줄께^^ 정말고마워 ~ 무심하지만자상한치호오빠오빠 ~ 저부사수정도는해줄수있잖아요! 왜맨날저보면죽일라고해요! 저이제미친화학과아니고 ~ 미친생공해도되나요? ㅋㅋ오빠저마니답답했을텐데나름자상하게부사수처럼가르쳐주신거너무감사해요. 담에만나면좀친절하게대해주세요! ㅋㅋ 쉬운남자재동오빠^-^ 졸업하고진가발휘하고계신ㅋㅋ졸업하고도걱정해주시고이리저리알아봐주시고완전감동인거아시죠? 오빠가처음에이름도안불러주고여자얘야그래서사실서운했었는데 ~ 지내다보니오빠처럼정많고쉬운? ㅋㅋ사람없더라구요 ~ 오빠졸업후내기하는사람이없어서아이스크림못먹은지가ㅋㅋ덕분에실험실생활정말편하고즐거웠었습니다. 섹쉬한김령씨^^ 김령씨 ~ 언니 1 차고나빵차였을부터참힘들었지만같이여서참을수있었고좋았던거같아 ~ 그리고다행히도개그코드가맞아서우리참잼있었는데 ~ 언니정읍가고잘못보내 ~ 언니빨랑하이테크오빠랑결혼해 ~ 그래야내가얘들이랑가서축가부르고춤춰주지^^ ㅋㅋ혼자아니고언니랑둘이어서실험실생활즐거웠어요 ~ 귀염둥이윤! 엄!^--^ 사실너희는내첫후배라애착이가네 ~ 너희들이똘똘해서얼마나다행이었는지일을잘해줘서얼마나다행이라고생각했는지몰라ㅋㅋ이런걸내복이라고하지?^-^ 우리대장윤! 귀엽지만강한아이 ~ 일복이터져서탈이지만지켜보면서정현선배가왜널부사수로인정해주는지알겠더라구^^ 방장일힘들겠지만말 38

48 안들면얘기해다때려주러갈께^^ PCR 슬슬나오는것같던데 ~ 곧스페셜리스트가되실것이오^^ 자칭 2 호관죽순이엄양! 강해보이지만여린엄양^-^ 개그코드가맞는다며ㅋㅋ일처리가워낙깔끔해서역씨넌 ~ 치호오빠부사수가맞는것같아ㅋㅋ좋겠다 ~ 넌인정해주는거같던데 ~ ㅋㅋ글고 EMSA 때문에고생이많은거같은데 ~ 곧대박나길 ~ 고것을그냥!^^ 윤. 엄후배였지만너희들에게많이의지했었어 ~ 언넝취직해서돈마니벌어올께 ~ 빕스가자 오빠보단언니가더어울리는지혁오빠^^ 처음엔나이가있어서어렵진않을까걱정했는데특유의친화력으로잘맞춰주셔서개인적으로든든하고고맙게생각해요^^ 허리조심하시고오빠가아끼는아중이담비뺨치는분언넝만나시길바래요 ~ 내친구만기^^ 항상열심히인만기열정이느껴져서보기좋아너의따뜻한마음을조금더빨리알았더라면더빨리친구가되지않았을까싶네^^ 표현방식이달라도진심은통한다잖아언젠간알아줄꺼라여기시오 ~ 칩라인의스페셜리스트가되서 ~ 꼭인류의수명을 1 초연장하는꿈이뤄주길바래^^ 내친구우영이^^ ( 대가리라고하려고하다가속상할까봐큰맘먹고바꿈거임!) 튼튼하게생겼지만몸이약하고, 막자랐을것같지만은근귀하게자라고, 강할것같지만여린아이. 선배보다는친구가먼저여서힘들었겠지만항상따뜻히대해줘서고마웠어소원대로언넝웃음이예쁜여자친구만나서행복하게사시길 ~ 항상진지한상주^^ 어정쩡하게부사수가되서욕만먹고혼만나고힘들었을꺼야완전부사수였다면더혼났을텐데다행이라고생각해^^ㅋㅋ성실하게꾸준히인모습보기좋아공부하다보면자신감생길거야혹시실험하다가내도움이필요하면언제든지요청하시고^^ 39

49 대학원와서소중한인연맺게된혜란이와지윤양우리졸업하고도연락하자 ^^ 구박해서미안해요득주오빠, 귀염둥이슬기, 잘생긴동진이, 자상한다훈이, 귀여운일재모두모두고마웠어요 ^^ 40