微脂粒佐劑應用於雞點鼻免疫之研究

୯ҥѠ᡼εᏢ πᏢଣᄤᙴᏢଣ ᙴᏢπำᏢࣴز܌

റγፕЎ

Graduate Institute of Biomedical Engineering College of Medicine and College of Engineering

National Taiwan University Doctoral Dissertation

༾િಈ՘ᏊᔈҔܭᚊᗺሷխࣝϐࣴز

The intranasal delivery study of liposomal-vaccine to SPF chickens

මಹ๩

Li-Ping Tseng

ࡰᏤ௲௤Ǻ໳ကٺ റγ Advisor: Yi-You Huang, Ph.D.

ύ๮҇୯ 98 ԃ 6 Д

June, 2009

ᕴ

ᕴҞᒵ!

α၂ہ঩཮ቩۓਜ ... i

ᇞᖴ ... ii

ύЎᄔा ... iii

मЎᄔा ... iv

߄Ҟᒵ ...…vi

კҞᒵ ... vii

ಃ΋ക ᆣقϷЎ᝘ӣ៝ ...1

1.1 ᗹጢխࣝϐϟಏ ...1

1.2 ୏ނҔᗹጢௗᅿϐࣝभ...2

1.3 ՘Ꮚϐϟಏ ...3

1.4 ᗭಈ܄ࣝभ໺ᒡس಍ ...6

1.5 ༾િಈ՘Ꮚ ...6

1.6 િӭᗐᡏᆶᗹ๱܄ӭᗐᡏ ...10

1.7 ᚊଫᗹጢགࢉੰࢥ ...11

ಃΒക ፕЎࣴزҞޑ ...13

ಃΟക ׷਑ᆶБݤ ...14

3.1 ჴᡍ୏ނ ...14

3.2ੰࢥ ...14

3.3ჴᡍ၂ᛰϷϩ݋၂Ꮚ ...14

3.3.1၂Ꮚᛰࠔ...14

3.3.2לᡏ ...15

3.3.3༟ጤǵ࣒ዟᇙࠔ ...16

3. 4༾િಈхᙟੰࢥלচ ...16

3.4.1 ༾િಈଛБ...16

3.4.2 ᇙഢࢬำ ...17

3.4.3 ಈ৩ǵࣚय़ႝՏᆶхᙟ౗ ...17

3.4.4 ࣬ᙯ౽ྕࡋ...18

3.4.5 ᑻӀ༾િಈ ...18

3.4.6 ᚈ՘Ꮚ༾િಈ ...19

3.4.7 ሌԸӭᗐ༾િಈکΟиጤ༾િಈ ...19

3.5խࣝௗᅿǵ௦Ոکሷᆶ਻ᆅؑࢱన ...20

3.6ՈౚᏉ໣׭ڋ၂ᡍᆶሇનխࣝೱ่ϩ݋ݤ ...21

3.7װࢥჴᡍ ...22

3.8ᚊ๠᠌ಒझϩᚆ ...22

3.9Ӆ೫ขᡉ༾᜔ ...22

3.10 RT-PCRୀෳੰࢥ ...23

3.11׷਑ჹಒझϐࢥ܄ୀෳ ...24

3.12ୀෳಒझ୻ᎦనύNO ...24

3.13ՋБᗺᏀݤୀෳೈқ፦߄౜ ...25

ಃѤക ่݀ ...27

ಃϖക ૸ፕ ...40

ಃϤക ่ፕ ...48

ୖԵЎ᝘ ...49

߄ ...64

კ ...72

ว߄ፕЎӈ߄ ...93

ii

ᇞ ᇞᖴ!

!

!!!!!೭ҁፕЎޑֹԋ२ाགᖴࡰᏤԴৣޑࡰᏤаϷٮᔈкىޑ࿶຤Ǵᡣךჹܭࣴ

ز࿶຤ޑ٬ҔૈคኁคቾǶӆޣाགᖴα၂ہ঩ॺჹܭፕЎޑࡰ҅٠ගٮ೚ӭᝊ

຦ޑཀـǴ٬ҁፕЎ׳ᑪֹऍǶќѦǴाགᖴஒ߈Βίଫޑᚊޑ឴࣊Ǵ٬ךޑፕ

ЎԖى୼ޑኧᏵǴёаว߄ paperǶԜѦǴाགᖴჴᡍ࠻ᚊಔޑұՔॺԖǺऍذǵ

୯ሎǵ৖דǵۘᘶǵ੦ᆺǵ☰໥کշ౛ޚউǴаϷததЍජךޑᏢ׌ۂǺඵ๔ǵ

ηဃǵሎےǵ٫໥ǵ޲ӹک܃։Ǵך҉ᇻό཮ב૶ךॺ΋ӕոΚࣴزޑၸำǶ!

!

!!!!!ᖴᖴךޑৎΓۈಖӄΚЍ࡭ךǴ٬ךคࡕ៝ϐኁӦֹԋറγᏢՏǴགᖴךЈ

Ҟύޑ៓ΓǴᙖҗᚹ۟٬ךሦ৴ډࣴزޑᆒઓ൩ӵӕᚹ۟ޑᆒઓǶ!

iii

ύ

ύЎᄔा

!

ճҔ༾િಈхᙟࢲޑ(living)܈ѐࢲޑ(killing)ཥࠤੰࢥ(NDV)Ǵаᗺሷ(intranasal) БԄௗᅿᚊଫǴว౜ӧᚊଫሷ๚0਻ᆅؑࢱనᆶՈమύ೿Ԗౢғ੝౦܄ NDV לᡏǶ

๏ϒᚊଫௗᅿΟᅿόӕԋҽޑѮࠠӭቫ༾झ༾િಈǴځύ٬Ҕᕗિㄽᖌᡵ(PC)܈ᕗ

િㄽํữለ(PS)ಔԋޑ༾િಈǴӧ୏ނჴᡍύᗹጢϩݜࠠלᡏ(s-IgA)ᆶՈమלᡏ (IgG)Ϸфૈ܄לᡏΚሽ(HI titer)߄౜ໆӧٿԛௗᅿࡕלᡏ֡ගϲǴװࢥࡕߥៈ౗Ψ

ၲ 80-90%Ƕӧ༾િಈуΕિӭᗐᡏ(LPS)՘ᏊǴௗᅿܭᚊଫࡕǴӧճҔ PC ༾િಈ ಔ཮ቚуᗹጢלᡏᆶ PS ༾િಈಔቚу཮ՈమύלᡏΚሽǶ׳຾΋؁ǴబуሌԸӭ ᗐ(tremella)аቚу༾િಈޑᗹࡋǴۯߏ༾િಈӧᗹጢਔ໔Ǵᗺሷௗᅿࡕᗹጢϩݜ

ࠠלᡏᆶՈమύלᡏ֡ᡉ๱ගϲǴװࢥࡕԖ 100%ޑߥៈ౗Ƕ༾િಈ՘Ꮚჹܭխࣝ

ಒझϐᐒᙯҗӅ೫ขᡉ༾᜔ᢀჸډǴPC ಔԋҽ༾િಈ೏ᚊ๠᠌ѮᏘಒझझտໆၨ

ӭǶPC ༾િಈᆶ PS ༾િಈ֡ࢲϯᚊ๠᠌ѮᏘಒझౢғ NO ញܫǴځύ PC ༾િಈ NOޑញܫё೏ Bay 11-7085 ׭ڋᆶ U0126 ׭ڋǴ߄Ң PC ༾િಈӧԐයࢲϯᚊ๠

᠌ѮᏘಒझࢂа NF-κB ᕗለϯᆶ ERK ΢ෞޑ MEK ᕗለϯޑၡ৩ǶനࡕךॺճҔ ᕇளխࣝௗᅿ༾િಈ܈ሌԸӭᗐ༾િಈޑᚊଫǴว౜Πෞޑ p-ERK ᕗለϯޑ౜ຝ ӸӧǴځύ༾િಈ p-ERK ᕗለϯໆၨӭԶሌԸӭᗐ༾િಈ p-ERK ᕗለϯໆၨϿǴ p-ERKޑᕗለϯЬाߦ຾խࣝϸᔈوӛᡏనխࣝޑ Th2 ၡ৩ǴӢԜǴሌԸӭᗐ༾

િಈಔ೷ԋޑխࣝϸᔈࢂֹ᏾ޑಒझխࣝޑ Th1 ᆶᡏనխࣝޑ Th2 ၡ৩ޑғԋǶ!

!

!

ᜢᗖӷǺ༾િಈǵ՘Ꮚǵᗹጢǵཥࠤੰࢥǵ΋਼ϯේǵᗹጢᗹ๱ǵሌԸӭᗐ!

!

iv

Abstract

Liposomal-NDV vaccine intranasal administration to chicken can induce specific-NDV

antibody at mucosal and serum. The adjuvant effect of multi-lamellar vehicles (MLVs)

liposomes formulated with three phospholipids including

phosphatidylcholine-liposomes (PC-Lip), phosphatidylserine-liposomes (PS-Lip), and

stearylamine-liposomes (SA-Lip) was compared with that for virus alone using the

inactivated Newcastle disease virus (NDV) as model antigen. PC-Lip and PS-Lip

induced more NDV antibody titer after secondary immunizations. In response to

virulent viral challenge, all control animals died, whereas 80~90% of animals which

still survived. Added LPS with liposomal-NDV vaccine which enhanced chickens

produced antibody in serum and nasal/tracheal lavages. Furthermore, we combined

tremella with liposomal-NDV vaccine to enhance the muco-adhesive property and

prolong liposomes on the mucosal surface. Chickens received T-PC-Lip resulted in a

significant increase in HI titer also elicited a significant mucosal secretary

immunoglobulin A (s-IgA) response in tracheal lavages and a serum IgG response.

After virulent virus challenge, the protection rate of T-PC-Lip vaccine showed 100%

survival rate. The molecular mechanism of liposomal-adjuvant was study both in vitro

and in vivo. Confocal laser scan microscopy showed that PC-Lip were uptaken into

chicken spleen macrophages. PC-Lip and PS-Lip stimulated chickens spleen

v

macrophages produce NO. In response to PC-Lip stimulated, Bay 11-7085 and U0126

inhibited macrophages produced NO. PC-Lip was shown to be involved in both

activation of NF-κB!and!upstream ERK of MEK pathway. Moreover, PC-Lip and

T-PC-Lip were shown to be activated ERK pathway in vovo. In conclusion, T-PC-Lip is

an active adjuvant for chickens resulting in trigger Th1 pathway of cellular immunity

and Th2 pathway of humoral immunity.

Keyword: liposome, adjuvant, mucosal, NDV, nitric oxide, mucoadhesive, Tremella

vi

߄ ߄Ҟᒵ

߄΋

The particle size, zeta potential, phase transition temperature, and encapsulation efficiency of various compositions of liposomal-vaccine ... ….64 ߄Β

Serum hemagglutination-inhibition (HAI) antibody titers against NDV after the 1^st, 2^nd and 3^rd vaccinations ... ….65 ߄Ο

Nasal hemagglutination-inhibition (HAI) antibody titers against NDV after the 1^st, 2^nd and 3^rd vaccinations ... ….66 ߄Ѥ

Chickens against virulent NDV Sato strain challenge survival rate after the 1^st vaccination with different dose of vaccine……….67 ߄ϖ

Chickens against virulent NDV Sato strain challenge survival rate after the 1^st, 2^nd and 3^rd vaccinations ... ….68 ߄Ϥ

Viscosity of bioadhesive polysaccharide gels………..69 ߄Ύ

Serum hemagglutination-inhibition (HAI) antibody titers against NDV and survival rate after the 2^nd vaccinations ... ….70 ߄Ζ

Shedding of NDV challenge virus ... ….71

vii

კ კҞᒵ

კ΋ Antibody response ... ….72

კΒ Nasal specific-NDV antibody ... ….73

კΟ Serum specific-NDV antibody ... ….74

კѤ Antibody producining time ... ….75

კϖ Liposomal-NDV vaccine dose ... ….76

კϤ Anti-NDV s-IgA levels in tracheal lavages and sera ... ….77

კΎ LPS and Antibody producing ... ….79

კΖ Specific-NDV nasal antibody between primary and secondary immunization ... ….80

კΐ Specific-NDV serum antibody between primary and secondary immunization ... ….81

კΜ Nasal challenge virus dose ... ….82

კΜ΋ Nasal secreted antibody from immunized bioadhesive liposomal-vaccine. ….83 კΜΒ Serum antibody from immunized bioadhesive liposomal-vaccine ... ….84

კΜΟ CLSM of chicken macrophages………..85

კΜѤ Cell toxicity ... ….86

კΜϖ NO production from adjuvant stimulated ... ….87

კΜϤ NO production from liposomes stimulated ... ….88

კΜΎ NO production after treated with U0126 inhibitor ... ….89

კΜΖ NO production after treated with SB-203580 inhibitor ... ….90

კΜΐ NO production after treated with Bay 11-7085 inhibitor ... ….91

კΒΜ Western blot ... ….92

!

1

ಃ

ಃ΋കġ ᆣقϷЎ᝘ӣ៝

1.1 ᗹጢխࣝϐϟಏ

ठੰࠠ༾ғނӭҗஎЬᗹጢ೽ՏΕߟǴٯӵǺ΢ڥ֎ၰǵ੃ϯၰ܈ғ෗ၰ[1]Ƕ ځύаགࢉ΢ڥ֎ၰϐੰࢥӵଯচ܄࿥ᜪࢬՉ܄གߵੰࢥ (Highly pathogenic avian influenza, HPAI)کཥࠤੰࢥ(Newcastle disease virus, NDV)Ǵܰ٬ள࿥ᜪЇଆ ଯठੰ౗ࣗԿଯԝΫ౗[2,3]ǴӢԜᗹጢ೽Տޑխࣝϸᔈ׳ࣁխࣝᏢৎ܌ᜢݙǶ

࿥ᜪԖᜪ՟থ٢ᜪޑᗹጢխࣝس಍Ǵӧᗹጢ೽ՏڀԖܢᑇ༾ғނΕߟޑᗹጢ࣬

ᜢరЃಔᙃ(Mucosal-associated lymphatic tissue, MALT)ǴԜᗹጢ࣬ᜢరЃಔᙃх

ࡴ ڥ֎ၰ س಍ Ǻሷࠒോ ࣬ᜢర Ѓಔ ᙃ (Nasopharynx-associated lymphatic tissue, NALT)[4] ک Ѝ ਻ ᆅ ࣬ ᜢ ర Ѓ ಔ ᙃ (Brancheal-associated lymphatic tissue, BALT)[5,6]ǵဉगၰ࣬ᜢరЃಔᙃ(Gut-associated lymphatic tissue, GALT)[7-9]کᓐ

࣬ᜢరЃಔᙃ(Head-associated lymphoid tissue, HALT)[10]Ƕӧ೭٤ᗹጢ࣬ᜢరЃಔ ᙃ่ᄬҗ΢Ҝಒझᙟᇂܭᆫ໣ޑరЃಒझ(Lymphoid cell)[11]ǵరЃ΢Ҝಒझ (Lymphoepithelium) [12]܈ᘠݰ࣬ᜢ΢Ҝಒझ(Follicle-associated epithelium, FAE)

[13]Ǵӧᆅ๚ϣޑᗹጢᘠݰ࣬ᜢ΢Ҝಒझߕ߈Ԗ΋ᅿಒझڀԖόೕ߾ޑ༾้Л੝ϯ

ޑ M ಒझ(Microflod cell)ǴM ಒझᆙᎃܭరЃಒझǴځЬाфૈࣁឪΕלচǴ٠ ஒלচၮଌډరЃಔᙃǶלচ࿶җ M ಒझ۳ΠሀଌǴ຾Ε୻ओМඬ(Peyer’s patches, PP)ǴѬࢂ੝ϯޑԛભరЃಔᙃǴᆫ໣೚ӭלচև౜ಒझ(Antigen presenting cells, APCs)ǴӵǺѮᏘಒझ(Macrophages)ǵᐋँރಒझ(Dendritic cells, DCs)Ǵ೭٤לচ և౜ಒझஒלচឪΕࡕ཮ஒځ߄౜ܭಒझጢ΢Ǵ٬ T ಒझૈ୼ᒣ᛽לচԶᆶϐ่

ӝǴ຾ԶࢲϯరЃᘠݰύޑ B ಒझǴ೏ࢲϯޑ B ಒझזೲӦϩϯԋዀಒझ(Plasma cell)Ǵᚆ໒ᘠݰࡕϩݜ IgM לᡏǴӆ࿶לᡏᜪࠠᙯඤ(class switch)ԋ IgA ک IgG ฻

2

לᡏǴ೭٤לᡏаϩݜࠠԄ࿶΢Ҝಒझၮଌډᆅ๚ύکלচౢғբҔǴаၲډߥ

ៈஎЬܢᑇѦٰੰচ༾ғނϐӒ্[14-17]Ƕ

1.2 ୏ ୏ނҔᗹጢௗᅿϐࣝभ

୏ނௗᅿࣝभࢂԖਏ௓ڋ٠Ⴃٛ੯ੰޑБݤǴ࿥ᜪ٬Ҕᗹጢௗᅿޑࣝभ೿ࢂࢲ

ੰ ࢥ (live viral) ࠠ Ԅ ϐ ࣝ भ Ǵ Ҟ ߻ ᚊ ଫ ٬ Ҕ ޑ ࢲ ࢥ ࣝ भ ௗ ᅿ ी Ԗ ཥ ࠤ ੰ (Newcastle disease, ND)хࡴ Hichner type BǵHichner type LaSota strianǵHichner type Avinum strian฻Ǵа໯Нǵᗺሷǵᗺ౳܈ቔᜦ…฻฻БԄௗᅿ[18]ǹ໺ࢉ܄

Ѝ਻ᆅݹ(Infectious bronchitis, IB)ࢲࢥࣝभԖ H120 ک ND ޑ B1 ܈ LaSota షӝࣝ

भǴа໯Нǵᗺሷ܈ᗺ౳БԄௗᅿ[19,20]ǹ໺ࢉ܄๮М៶ੰ(Infectious bursal disease, IBD) Ǵ а ໯ Н ǵ ᗺ ሷ ܈ ᗺ ౳ Б Ԅ ௗ ᅿ [21] ǹ ໺ ࢉ ܄ ോ ᓐ ਻ ᆅ ݹ (infectious laryngotracheitis, ILT) Ǵа໯Нǵᗺሷ܈ᗺ౳БԄௗᅿ[22,23]Ƕᗹጢௗᅿࢲࢥࣝभ ޑᓬᗺࣁǺ1.խࣝߥៈਏ౗ၲ 90%а΢Ǵ2.ૈ୼Їଆᡏన܄խࣝϸᔈ(humoral immunity)ᆶಒझ܄խࣝϸᔈ(cell-mediated immunity) [18]ǹᚊଫௗᅿࢲࢥ࿶ኧঁД ࡕǴځᡏϣխࣝΚሽ཮࡭ុफ़եǴѻሡाӆԛаࢲࢥ܈όࢲϯੰࢥௗᅿᚊଫǴ଺

ࣁխࣝံமǴωளаԖֹ᏾ޑߥៈਏΚǶ୤٬ҔࢲࢥϝԖځӒᓀ܄ӵࢥΚ଑ᘜǴ ջ٬ځ܌٬Ҕޑלচࣁଯࡋีញޑੰࢥ܈ࢂ෧ࢥੰࢥǴϝόૈόԵቾ୷Ӣ႖жᒪ

໺଍ᙯޑёૈ܄ǶऩੰࢥวғँᡂǴ٬෧ࢥੰࢥਲ਼ᙯӣഁғࠠǴ܈کഁғࠠੰࢥ ਲ਼วғ୷Ӣख़ಔǴᏤठཥࢥਲ਼ޑౢғǴ೿Ԗёૈ೷ԋคݤႣයޑӒᐒ[24]Ƕཥࠤੰ

ࢥаᅽଭ݅܈ѹΒ⾺όࢲϯೀ౛ࡕջࣁԝࢥ(killed virus)Ǵ࿶җᗺሷک਻ᆅௗᅿБ ԄЇଆޑֽ೽܄խࣝϸᔈ࣬ჹӦКҔࢲࢥௗᅿޑ১[25]ǴҗᗹጢᆶՈమύϐלᡏό ىаၲډߥៈΚሽǶฅԶǴᓬذޑࣝभᔈڀഢԖΠӈ੝܄Ǻ1.ڀԖխࣝߥៈբҔϐ ਏ౗ၲ 80%а΢Ǵ2.ଯӼӄ܄Ǵ3. ௗᅿ٬Ҕ܄ߡճ[26]Ǵ4. ߡەǶࣁ٬ڀଯࡋӼ ӄ܄ՠեխࣝלচ܄ϐԝࢥૈ୼җᗹጢௗᅿǴ٬ளᚊଫౢғᓬؼϐֽ೽܄ᆶس಍

܄խࣝϸᔈǴ٠ܢלੰࢥϐװᔐаၲډԖਏӦߥៈǴ߾ሡा٬Ҕ՘Ꮚ(adjuvant)а

3

ၲԋҞޑǶ

1.3 ՘ ՘Ꮚϐϟಏ

՘Ꮚޑᐕў

ԝࢥלচǵԛൂՏלচ(subunit antigen)ǵᴏ两לচ(peptide antigen)ǵӭਡ㧿לচ (polynucleotide antigen)Ǵ ᗨ ฅځ Ӽӄ ܄ (safety) ε ൯ ග ϲ Ǵՠ ࢂ࣬ჹ Ӧ Ԗਏ ܄ (effective)ᆶխࣝΚਔਏࠅᒿϐफ़եǴӢԜǴԐයࣝभࣴزว৖ၸำ൩ޕၰ՘Ꮚӧ

ࣝभϐख़ा܄Ǵ܌аࣝभࣴزว৖ޑၸำύନᒧ᏷፾྽ϐלচѦǴΨว౜Ԗਏޑ

՘Ꮚё଺ࣁቚமࣝभਏΚϐނ፦Ƕ

Ȩ՘Ꮚȩ(Adjuvant)ǴྍԾܭ܎΍ЎȺadjuvareȻǴࢂȺᔅշȻϷȺуமȻϐཀ[27]Ƕ Υࢂӧࣝभύ೛ीҔٰቚၗխࣝਏ݀ޑނ፦܈ଛБջࣁ՘ᏊǶԐӧՋϡ1916ԃ MoignieکPinoyջ٬Ҕ᝜ނݨቚமխࣝϸᔈ[28]ǹՋϡ1924Կ1926ԃ໔ǴRamonว

౜ӧԖಒ๵ౢғޑқോᜪࢥન(diphtheria toxoid)٬ளֽ೽Ӧౢғวݹ౜ຝǴ཮Քᒿ

๱ჹᔈלচނ፦ޑלᡏεໆӦғԋ[28]ǹԿՋϡ1926ԃǴຯϞ80ӭԃ߻ǴGlenny et al. [17]ۈӧΓҔࣝभᇙᏊύబу᎑ጤ(ణ਻ϯ᎑)଺ࣁ՘ᏊǴԿϞ᎑ጤࢂ୤΋೏ऍ୯ १ࠔᛰނᆅ౛ֽ(US Food & Drug Administration)ਡ঑ёҔܭΓᜪϐ՘ᏊǶӧځд୯ ৎӵኻ࿉฻୯Ǵς࿶ӕཀёҔܭΓᜪ՘ᏊԖǺMF59(֖فᚢ౎ޑНхݨݨ፦՘Ꮚ) ଛӝࢬՉ܄གߵࣝभϐଛБǴౢࠔӜࣁFluadǹAS03(Нхݨݨ፦՘Ꮚ)ଛӝH5N1ࣝ

भϐଛБǴౢࠔӜࣁPrepandrixǹAS04՘ᏊǴ่ӝMPL(monophosphoryl lipid A)ک

᎑ጤǴଛӝHBV܈HPVࣝभଛБǴౢࠔӜϩձࣁFendrixکCervarix [29]Ƕ୏ނҔࣝ

भςਡ঑ϐ՘Ꮚ߾ࣁǺ࿥ᜪҔϐόࢲϯੰࢥౢࠔǺݨ፦՘ᏊӵSpecolکMontanide^® ISAسӈౢࠔǹങҔੰࢥԛൂՏࣝभౢࠔǺݨ፦՘ᏊӵNorvax-IPNVౢࠔǹ፜Ҕϐ όࢲϯԛൂՏࣝभǺݨ፦՘Ꮚӵ಻ើम੝࠶ϦљౢࠔǶ

4

౛

౛གྷޑ՘Ꮚ

ᓬؼޑࣝभڗ،ܭаΠޑӢનǺ1.לচǴ2.ௗᅿޑ୏ނނᅿǴ3.ௗᅿࣝभޑ೼৩Ǵ 4.՘ᏊǴ5.คୋբҔǴ6.ߡە[30]ǹ՘Ꮚࢂࣝभࠔ፦ϐᜢᗖǴ՘ᏊଛБޑᒧ᏷ૈ୼ቹ ៜלচޑ߄౜ૈΚа΢ࣝभౢࠔϐԖਏ܄Ǵՠόؼޑ՘ᏊуΕࣝभΨ཮ቚуࣝभ ϐୋբҔǴ຾Զቹៜډᡏన܄ᆶಒझ܄խࣝϸᔈϐ߄౜ǶԶ౛གྷޑ՘Ꮚሡ಄ӝа Π൳ঁाᗺǺ1.ӼӄคୋբҔϐ՘Ꮚ׷਑Ǵ2.ڀғނёफ़ှ܄ϐ׷਑Ǵ3.՘Ꮚғౢ

ԋҁե༹Ǵ4.ЇଆԖਏӦಒझ܄ᆶᡏన܄ޑխࣝϸᔈǴаၲډֹ᏾ӦߥምਏΚ[31]Ƕ

՘Ꮚᆶࣝभௗᅿ೼৩

ࣝभ٬Ҕ΢ϩᜪߚதӭኬ܄Ǵ٩ੰচᅿᜪǵੰচठੰ೼৩ǵלচࠠԄǵ՘ᏊᏊࠠ

܈ௗᅿБԄ฻ٰуа୔ϩ[31]Ƕࣝभ٬Ҕ΢ЬाϩߟΕԄ(invasion)ၟߚߟΕԄ(non- invasion)ٿᅿǴߟΕԄࣝभΞёϩࣁǺ1.ҜΠݙ৔(subcutaneous injection)Ǵ2.ԼԺ ݙ৔(intramuscular injection)Ǵ3.ऀڈ(puncture)ǹߚߟΕԄࣝभёϩࣁǺ1.αܺ(oral)Ǵ 2.ᗺ౳(intraocular)Ǵ3.ᗺሷ(intranasal)Ǵ4.Ҝጥ༡ܓǴ5.੆ݰǶቹៜࣝभਏΚനЬा

ޑӢનջࢂ՘ᏊǴ୷ܭࣝभխࣝௗᅿၡ৩Ǵᒧ᏷፾ӝޑ՘Ꮚ٬ҔǴ๱Ӝޑ MF59

՘Ꮚ଺ࣁݙ৔ᏊࠠӧΓᡏޑלᡏ߄౜ໆ೿ߚதڑ๱Ǵՠஒ MF59 ଛӝ H5N3 ࢬՉ܄

གߵੰࢥלচ࿶җᗹጢௗᅿܭΓᜪࡕǴӧՈమаϷᗹጢޑלᡏғԋໆᆶ҂ௗᅿޣ คৡ౦ǴMF59 ࣁᓬذޑݙ৔ࠠ՘ᏊǴՠࢂࢂӍ฻ޑᗹጢࠠ՘Ꮚ[32,33]Ƕ

՘Ꮚޑϩᜪᆶϟಏ

Ҟ߻ς࿶೏ว߄ܭЎ᝘ύޑ՘ᏊऊԖኧԭᅿϐӭ[34]Ǵόӕޑ՘ᏊԖόӕߦ຾խࣝ

ϸᔈޑᐒᙯǴҗܭӭख़Ϸ۶Ԝख़᠄ޑғނᏢ੝܄Ǵ՘ᏊޑϩᜪዴԖ֚ᜤǶ٩ྣȨௗ

ᅿ೼৩ȩϩᜪёϩࣁǺᗹጢࠠ՘Ꮚᆶߚᗹጢࠠ՘Ꮚǹ٩ྣȨᏊࠠȩϩᜪࣁǺНྋ నރǵݨ፦ރǵᗭಈރǵНጤރ…฻ǹ٩Ȩ੝܄ȩёϩࣁǺނ౛фૈ՘Ꮚҗ᝜ނ

5

ݨǵિ፦ǵࣚय़ࢲ܄Ꮚǵᆫ٢ለǵᆫҒᎇለǵፃᝯለǵ൳΍ᆫᗐ…฻฻ᇙԋ(ٯӵ

༾િಈǵ༾झǵ༾ಈکISCOM฻)ǴϯᏢфૈ՘ᏊҗϯᏢӝԋނ፦(ٯӵᆢғનCǵ ᆢғનE)Ǵғނфૈ՘Ꮚҗ༾ғނੰচ๵ނ፦܈୏ނǵ෌ނޑނ፦ᇙԋ (ٯӵ CT-BǵಒझᐟનǵғߏӢηǵિӭᗐǵmonophosphoryl lipid A (MPL)ǵCpG motifǵ ઇ໾॥ᜪࢥનTetanus toxoidǵқോᜪࢥનDiphetheria toxoidǵᆘᖋఎ๵Ѧࢥન Pseudomonas exotoxin)Ƕ׳຾΋؁ӦϩᜪǴ٩ྣЇวȨխࣝϸᔈȩϩᜪǺࢲϯӃϺ

܄խࣝ(innate immunity)ϸᔈ՘ᏊǴճҔੰচ࣬ᜢϩηࠠᄊ(Pathogen associated molecular patterns, PAMP)Ѧٰϐੰচϩη่ᄬǴ٬ኳԄᒣ᛽ڙᡏ(pattern recognition receptors)ৎ௼ύǴᜪ៕ڙᡏ(Toll-like receptor, TLR)ޑᒣᇡǴӵǺᆫҒ៛ᗐ(mannose, mannan)բҔܭToll-like receptor 2[35]Ǵpolyinosinic-polycytidylic acidᜪ՟༾ғނϐ ᚈިRNA(dsRNA)բҔܭTLR3[36]ǴॠᙔМ഍܄๵߄य़ϐિӭᗐ(lipopolysaccharide, LPS)բҔܭToll-like receptor 4[37]Ǵಒ๵ϐᚎЛ(flagellin)բҔܭToll-like receptor 5[38]Ǵ༾ғނϐൂިRNA(ssRNA)բҔܭTLR7܈TLR8[39,40]Ǵ༾ғނϐCpG DNA բҔܭTLR9[40]ǹբҔ٠ࢲϯࡕϺ܄խࣝ(adaptive immunity)ϸᔈ՘ᏊǴፓ௓ࡕϺ

܄խࣝϸᔈޑ௴୏ǴӵǺಒझᐟન(cytokines)܈ϯᏢᐟન(chemokines)ǴځύԖᗭ ಈ ౚ Ѯ Ꮨ ಒ झ ᆫ ပ ڈ ᐟ Ӣ η (granulocytemacrophage colony stimulating factor, GM-CSF)[41]܈ϟқ፦ન12(interlukin 12, IL-12) [42]฻฻Ƕ٩ྣȨ໺ሀس಍ȩ՘Ꮚ ёϩᜪࣁǺݨхН(Water-in-oil)ϐݨ፦՘Ꮚ[43,44]Ǵ᝜ނᡶᜪϐణ਼ϯ᎑܈້ᡶ [45]Ǵᗭಈၩᡏӵ༾ಈ(microparticles)[46]ǵ༾િಈ(liposomes)[47]ǵխࣝڈᐟፄӝ ނ(Immune stimulate complex, ISCOM)[48]ǵүੰࢥᗭಈ՘Ꮚ(Virus-like particles, virosome)[49]Ƕ

՘

՘ᏊޑфૈᆶբҔᐒڋ

Ꮲ౛΢Ǵόӕޑ՘ᏊԖߦ຾όӕխࣝϸᔈޑфૈکխࣝচ܄(immunogenicity)Ƕᘜ યрϖঁाᗺǺ΋ǵӦୱ܄ཷۺޑխࣝϸᔈ܄ǹΒǵঊ৤ਏᔈ(depot effect)౛ፕǴ

6

ӧௗᅿࡕלচѻೀܭֽ೽ϯ[50]ǹΟǵ՘Ꮚתᄽૻ৲ 0 (Signal 0)ǴЇᏤלচ،ۓՏ (epitope)ޑૻ৲ 1 (Signal 1)ک࿶ᒣ᛽ڙᡏϩ៏ੰচ࣬ᜢϩηࠠᄊޑૻ৲ 2 (Signal 2) [51] ǹѤǵૻ৲ 2 תᄽ՘Ꮚࢲϯ߃ۈ T ಒझǺϖǵ࿶Ѧጔ܄کϣጔ܄ޑӒᓀૻ৲

ࢲϯխࣝϸᔈ[52,53]Ƕ

1.4 ᗭ ᗭಈ܄ࣝभ໺ᒡس಍

ঊ৤ਏᔈࢂלচ໺ᒡس಍ૈ୼ԋфӦЇวխࣝϸᔈޑЬाচӢǶଷ೛՘Ꮚᅈىঊ

৤ਏᔈǴٗሶ՘ᏊޑϯᏢಔԋᆶ೛ी཮،ۓխࣝϸᔈޑࠠᄊǴٯӵǺҗӅӕڈᐟ ϩη܈לচ௓ڋǴ٬ B ಒझౢғόӕޑ isotype ޑלᡏǴ٬̐ಒझϩݜόӕޑಒझ

ᐟનǶԖ೚ӭ՘ᏊᜪࠠឦܭȬᗭಈ܄ࣝभ໺ᒡس಍ȭǴхࡴ༾િಈǵᆫӝޑ༾ಈǵ

ڼԯಈηǵүੰࢥᗭಈ՘Ꮚ….฻฻[54]Ƕҗܭёྋ܄לচ܈ԝࢥלচӧ࿶ᗹጢௗᅿ ਔ֡คݤၲډԖਏޑխࣝϸᔈǴӢԜǴஒ೭٤לচ࿶җᗭಈ܄ޑхᙟܭᗹጢ೽Տ

ௗᅿਔૈЇଆֽ೽Ӧᆶس಍܄ӦխࣝϸᔈǶ ᘜયΠӈӢન٬ᗭಈ܄՘ᏊԖਏޑ চӢǺ΋ǵᗭಈ܄՘Ꮚکלচ׎ԋޑѮϩηፄӝނ٬לচ׳ԖਏӦғӸܭᗹጢ ޑᕉნǴᗉխܭሇનНှ܈ઇᚯǹΒǵᗭಈ܄՘Ꮚᔅշלচ೯ၸᗹጢᠼЛቫǹ Οǵᗭಈ܄՘ᏊКёྋ܄ޑϩη׳ܰӧ Peyer’s patches ࿶җ M ಒझឪΕࡕЇᏤԿ

ౢғ IgA ୔ୱǹѤǵᗭಈ܄՘Ꮚёૈתᄽঊ৤ਏᔈ٬ளלচ጗ᄌញܫǶҗܭа΢

ӢનǴ٬ளֽ೽ᗹጢௗᅿࣝभޑௗᅿБԄளаჴ౜ǴӢԶϝԖ೚ӭࣴزი໗࡭ុ

ࣴزԖਏӦᗹጢࠠᗭಈ܄՘ᏊǶхࡴࣴز༾ಈޑ O’HaganǴࣴزխࣝڈᐟፄӝނ ޑ A.Osterhaus ک G.RimmelzwaanǴکࣴز༾િಈޑ S.Michalek ک N.ChildersǶ

1.5 ༾િಈ՘Ꮚ

༾િಈᙁϟ

༾િಈȐLiposomeȑΞᆀિ፦ᡏȐfat bodyȑǴҗિ፦܌ᄬԋޑүғނಒझϐၩᡏǶ

7

Ջϡ 1965 ԃम୯ࣽᏢৎ Bangham ฻Γ२ԛว౜Ǵஒᕗિ፦ᖓጢϩණܭН࣬ᕉნس

಍ύǴ཮׎ԋᜪ՟ࢩጳ୮ޑӭቫ่ᄬȐmultilamellar structureȑϐ೯೸܄િ፦λݰ [55]Ƕޔډ 1968 ԃࣽᏢৎ Sessa ک Weissman ҅ԄஒλݰڮӜࣁ LiposomeǴ٠ܴዴ ۓကǺ༾િಈࢂҗ΋ቫډኧቫᕗિ፦ᚈቫጢ܌ಔԋޑ༾λ៶ݰǴڀԖԾՉஏӝ (self-closing)ޑ੝܄[56]Ƕ༾િಈЬाࢂҗᕗિ፦܌ಔԋǴᕗિ፦ҁيڀԖ౧Н܄

ޑᅹ᜘ᆶᒃН܄ޑᓐ୷ǴӢԜёхᙟᒃН܄(hydrophilic)ނ፦ӧύޜౚᡏϣ೽ϐН

࣬୔ୱϷхᙟ౧Н܄(hydrophobic)ϐނ፦ᢃ൪ӧિᚈቫύǶќѦǴΨӢࣁ༾િಈࢂ

җᕗિ፦ϐϺฅނ፦܌ಔԋޑᜢ߯ǴځڀԖؼӳޑғނ࣬৒܄(biocompatibility)Ϸ ғނफ़ှ܄(biodegradable)ǴӢԜӧՋϡ 1972 ԃǴ໒ۈ೏Ҕٰբࣁᛰނᒡଌޑၩᡏ [57]Ƕ٠ӧՋϡ 1995 ԃǴਡ঑੝ਸଛБ༾િಈёࡼѺܭΓᜪϐᔈҔ[58] Ƕ

༾

༾િಈ܄፦ᆶϩᜪ

༾િಈ٩ಈηελǵિ፦ޑಔԋҽᆶႝ܄ϩᜪǶ΋૓٩ྣિᚈቫ่ᄬޑቫኧ Ȑnumber of lamellaeȑ܈ࢂಈηελ(size)ϩᜪǶεठ΢ёϩࣁΠӈѤᅿǺ΋ǵǵӭ ቫ༾झ༾િಈ( multilamellar vesicles, MLVs)೯தࢂҗϖঁа΢ӕЈቫ܌ಔԋǴځಈ ৩ऊࣁ 1μm а΢Ǵҗܭ؂ঁ༾િಈԖӭቫિᚈቫጢǴڀԖ࡭ុញܫޑфૈǴ೏х ᙟӧѦቫޑނ፦཮Ӄ೏ញܫрٰǴхᙟӧϣቫޑނ፦߾೏ᄌᄌញܫрٰǶΒǵൂ

ቫε༾झ༾િಈ(Large unilamellar vesicles, LUVs)җ΋ঁિᚈቫ܌хൎ࠾ഈ׎ԋǴ ಈ৩ऊࣁ 1000 nmǶΟǵൂቫύ༾झ༾િಈ( Intermediated unilamellar vesicles, IUVs) җ΋ঁિᚈቫᄬԋޑ༾િಈǴځಈ৩ऊӧ 100 nm ޑ฻ભǶѤǵൂቫλ༾झ༾િಈ ( Small unilamellar vesicles, SUVs)җ΋ঁિᚈቫ܌хൎ࠾ഈԶԋޑ༾િಈǴځಈ৩

ၨ MLVsǵLUVs ک IUVs λǴಈ৩ऊӧ 25~100 nmǶ༾િಈિ፦ޑಔԋҽǺ΋ǵ Ғݨᗐિ፦(Glyceroglycolipid)ǴٯӵǺъ٢ᑗࢴീᗐ(galactopyranoside)ǶΒǵܤӝ ᕗિ፦(Sphingophospholipid)ǴٯӵǺઓ࿶ᕗિ፦(sphingomyelin)ǶΟǵܤӝᗐિ፦

(Sphingoglycolipid)ǴٯӵǺઓ࿶ㄽữъ٢ࢴീᗐ(ceramide galactopyranoside)ǶѤǵ

8

Ғݨᕗિ፦ (Glycerophospholipid)ǴٯӵǺռᕗિ(Lecithin)Ƕ༾િಈႝ܄ϩᜪࢂ٩

ྣિ፦ޑᒧ᏷ಔԋǴᄬԋ஥ॄႝ಻ȐNegatively! chargedȑ༾િಈǴӵᕗિㄽํữ ለ( Phosphotidylserine, PS)ǵᕗિㄽҒݨ( Phosphotidylglycerol, PG)܈ᕗિㄽԼᎇ (Phosphotidylinositol, PI)Ƕ ஥ ҅ ႝ ಻ Ȑ Positively charged ȑ ༾ િ ಈ Ǵ ӵ ฯ િ ữ (Stearylamine, SA)Ƕ ٿ ܄ ႝ ಻ Ȑ Natural charge ȑ ༾ િ ಈ Ǵ ӵ ᕗ િ ㄽ ᖌ ᡵ (Phosphotidylcholine, PC)کᕗિㄽΌᎇữ(Phosphotidylethanolamine, PE)Ƕ༾િಈ଺

ࣁᛰނޑၩᡏਔǴё٩όӕિ፦ޑಔԋҽǵಈ৩ελǵࣚय़ႝՏޑଯեаၲډԖ ਏӦᛰނ܈לচᒡଌҞޑǶ

༾

༾િಈ՘Ꮚ

Ջϡ 1984 ԃଆ༾િಈۈ೏ᔈҔܭխࣝ՘Ꮚ[59,60]Ǵ༾િಈ೏ว౜ڀԖ௴୏խࣝϸ ᔈϐወΚǴࢂҗܭ༾િಈ଺ࣁᕎੱᛰނ໺ᒡس಍ਔǴ༾િಈനಖ೿೏ᆛރϣҜس

಍ȐReticuloendothlial System, RESȑύޑלচև౜ಒझȐAntigen Presenting Cell, APCȑϐ΋ޑѮᏘಒझȐMacrophageȑտᏘǶ༾િಈё೏ѮᏘಒझکځдӧՈనǵ రЃکಔᙃύ(ӵరЃ่ǵط᠌ک๠᠌)ޑտᏘಒझտᏘࡕԶញܫלচǶAlving Ꮲޣ ӧՋϡ 1991 ԃǵ Gregoriadis ᏢޣӧՋϡ 1990,1992 ک 1995 ԃว߄༾િಈ՘Ꮚ࿶

җݙ৔ௗᅿޑБԄǴૈ୼ԖਏӦЇวխࣝϸᔈ[59-62]Ƕ༾િಈ՘ᏊբҔޑᐒڋǴ ёૈࣁаΠӢનǺ΋ǵߥៈ٠ᛙۓלচǴኳүޑфૈ(ӵੰচ༾ғނޑ߄य़)ǹΒǵ ߦ຾ჹྗלচև౜ಒझЬ୏ᆶ೏୏ԄޑឪΕǹΟǵឪΕࡕቚу٠௓ڋלচೀ౛Ƕ

༾િಈᗹጢ՘Ꮚ

җܭ༾િಈ՘Ꮚ࿶җݙ৔ௗᅿǴᕇளؼӳޑխࣝϸᔈǶӢԜǴഌុԖᏢޣ஑ৎஒ

༾િಈࣝभௗᅿ೼৩ᔈҔܭᗹጢௗᅿБԄ[63]Ƕаቚуࣝभ٬Ҕޑ໩ᔈ܄ǵБߡ܄

ᆶԖਏ܄ǶᗹጢௗᅿБԄЬाԖαܺ(Oral)೼৩ᆶᗺሷ(Nasal)೼৩ǴനԐࣴز༾િ

9

ಈαܺࣝभܭεႵ୏ނኳԄس಍ǴૈԖਏ٬ൄనύౢғStreptococcus mutansלᡏё ႣٛೃУౢғ[64,65]ǶӕኬӦஒ༾િಈхᙟStreptococcus mutans೽ϩޑᗐೈқ่

ᄬǴܭΓᜪαܺௗᅿࡕǴ࿶җൄనᆶՈమᔠᡍ֡ૈගϲIgAלᡏϐғԋ[66,67]Ƕќ ஒᓅ໶۱๵ࢥનޑLipid Aхᙟܭӭቫ༾ݰ༾િಈ(MLV)ܭεႵαܺௗᅿࡕǴૈቚ уεႵဉၰϣIgAғԋ[68,69]Ƕᗹጢௗᅿ༾િಈࣝभ٠҂ڙज़ܭαܺ೼৩ޑނ౛܄

ࡀም(ٯӵǺगለઇᚯ೷ԋ༾િಈόᛙۓǴ٬ளלচ೏ሇનϩှ)ǶEl GuinkᏢޣӧ Ջϡ1989ԃ२ԛว߄Ў᝘Ǵᇥܴ༾િಈхᇗࢬՉ܄གߵੰࢥלচǴӕਔаᗺሷᆶ ݙ৔БԄኳԄௗᅿܭλႵǴ࿶ᗺሷௗᅿ༾િಈࣝभޑλႵӧሷؑࢱనύԖଯΚሽ ޑIgAלᡏǴаϷϿໆޑՈమύלᡏΚሽǴ࿶װࢥჴᡍ᛾ჴૈ୼ԖਏЪԭϩϐԭߥ

ៈλႵऐڙၸࢬՉ܄གߵੰࢥϐװᔐ[70]ǶՋϡ1995ԃde Haanکځࣴزი໗Ψว

౜Ǵ๏ϒλႵᗺሷௗᅿ༾િಈхᙟϐԛൂՏלচǴૈ࡭ុϩݜIgAלᡏߏၲϖঁД ޑਔ໔ܭλႵޤ೽ᆶሷ๚ǴىаߥៈλႵխܭੰࢥགࢉ[71]ǶќѦǴde Haanکځ

ࣴزი໗Ψว౜ǴᗺሷௗᅿλႵࡕǴלᡏϐғԋό໻໻ֽज़ӧሷ๚܈ޤ೽ғԋǴ ӧဉၰǵғ෗ၰکՈమ֡཮ϩݜלᡏғԋ[72]Ƕ

༾

༾િಈᗹጢ՘ᏊϐխࣝբҔᐒڋ

ӧ༾િಈ՘ᏊޑբҔϐΠǴלচև౜ಒझ(antigen-presenting cells, APCs)੝ձࢂѮ Ꮨಒझ(macrophages)ᆶᐋँރಒझ(dendritic cells, DCs)ࣁཱུख़ाޑفՅǴѬॺӢࣁ ӃϺ܄խࣝϸᔈ(innate immune response)ޑᐒڋᆶ༾િಈ՘Ꮚௗ᝻ࡕǴѮᏘಒझᆶ ᐋँރಒझ೏ࢲϯԶЇวࡕϺ܄խࣝϸᔈ(adaptive immune response)Ǵஒ༾િಈ՘

Ꮚझտǵೀ౛٠ஒ༾િಈхᙟϐלচև౜ܭTಒझǶѮᏘಒझࢲϯࡰ኱ϐ΋ࣁ΋਼

ϯේ(nitric oxide, NO)ϐғԋ[73,74]Ƕ྽NOҗѮᏘಒझғԋǴNO೷ԋಒझᐟનޑ ғԋǴ٠Їଆߕ߈రЃಒझϣޑૻ৲໺ሀǴхࡴ p38 mitogen-activated protein kinase (p38-MAPK)ǵextracellular signal-related kinase 1/2 (ERK1/2)ǵc-Jun N-terminal kinase (JNK)کphophatidylinositol 3-kinase (PI3K)ǶќѦǴૻ৲໺ሀނ፦ϐ΋ޑ nuclear

10

factor kappa B (NF-κB)Ψ ת ᄽ ख़ ा ޑ ف Յ Ǵ ࿶ ࢲ ϯ ࡕ ӧ ಒ झ ϣ ౢ ғ ᐟ ન ӵ co-stimulatory molecules typeΙinterferonǵchemokines܈cytokines[75]Ƕ

1.6 િ િӭᗐᡏᆶᗹ๱܄ӭᗐᡏ

િӭᗐᡏ

િӭᗐ (Lipopolysaccharide; LPS) ࣁϣࢥન (endotoxin) ΋ᅿǴࣁ਱ើМ഍܄๵ಒ झᏛޑЬाԋϩϐ΋ǶLPS നԐ೏଺ࣁ՘ᏊܭՋϡ 1956 ԃ[76]ǴLPS ޑനѦୁࣁ

΋ࢤڀԖלচ੝౦܄ޑӭᆫᗐǴᆀࣁ outer-polysaccharideǹҗܭځϣ֖ёᇨว஑΋

לᡏޑ O לচ(O-specific chain)ǴӢԜё೏ᔈҔܭᖏ׉ບᘐ଺ࣁᒣ᛽Ӛᅿόӕ਱ើ

М഍܄๵ޑ٩ᏵǶԿܭ LPS ύ໔΋ࢤၨอ೏ᆀࣁ core-polysaccharide (core region) ޑӭᆫᗐǴځаӅሽᗖޑ่ӝБԄೱௗ O-specific chain Ϸ lipid AǶLPS ࢂ΋ᅿٿ

܄ϩηǴ੝ቻԖ஥ॄႝǵԖၨଯޑऐ዗܄ᆶϩηᆫ໣ελ཮ᒿ pH کᡶᐚࡋόӕ Զόӕ฻ǶΨ҅Ӣځ౧Н܄Զᆶ༟ጤᜪᇙࠔԖமޑᒃکΚǶ྽ಒझڙډಒ๵ޑߟ ҍਔǴಒझ཮೏ϣࢥનڈᐟǴϣࢥનёࢲϯѮᏘಒझǴ٬ځϩݜಒझᐟનǴхࡴ

TNFǵIL-1ǵIL-6ǵIL-8 ฻ǶLPS ᏤठқՈౚȐMacrophagesǵNeutrophilsȑࢲϯ ϐၸำǴёᇥࢂಒझჹ਱ើМ഍܄๵ౢғߚ੝౦܄խࣝϸᔈϐ୷ҡǶѮᏘಒझ (macrophage) ᆶൂਡտᏘಒझ(mononuclear phagocyte)ࢂςޕӧϣࢥનڈᐟΠǴё

ౢғᆶϩݜӚᅿ cytokines (exǺTNF, IL)ޑಒझǴӧᡏϣϣࢥનᙖ cytokine ޑҸϟǴ

཮Їว endotoxemia хࡴ௳ՈੱᆶᏔ۔ཞ໾Ƕӧಒ๵ߟΕஎЬಒझਔҗಒझᏛ܌ញ ܫޑ LPS ཮ڈᐟᡏϣхࡴ T ಒझǵൂਡౚಒझǵѮᏘಒझǵ΢ҜಒझǵՈᆅϣ Ҝಒझӧϣӭᅿόӕಒझޑғ౛ϸᔈ (ٯӵ: ፓ࿯ނ፦ޑౢғǵझ໯բҔǵቚғϷ ϩϯ฻)ǴԶ೷ԋஎЬᝄख़ޑวݹǵНဍǵаϷ௳Ո܄ҶլǶҞ߻ςޕեᐚࡋޑ LPS

཮ࢲϯ Th ಒझǴ຾Զᐟϯ B ಒझౢғԖ஑΋܄ޑלᡏǶԶଯᐚࡋޑ LPS ߾ё аࢲϯߚ஑΋܄ޑ B ಒझᆶϐ่ӝǶ

11

ᗹ

ᗹ๱܄ӭᗐᡏ ሌԸӭᗐ

ȨқЕԸȩΞᆀࣁȨሌԸȩǵȨഓԸȩ܈ȨοԸȩǴځᏢӜࣁ Tremella fuciformis (TF)炻 ሌԸࢂሌԸࣽ෌ނሌԸޑηჴᡏǴӧύ୯೏ຎࣁ१ނޑ΋ᅿ܈ࢂӵံᛰޑȨύ

ᛰȩǶ߈ԃٰǴࣴزว౜ሌԸ๧ڗޑሌԸӭᗐڀԖ೚ӭғ౛ᛰ౛ޑࢲ܄[77]Ǵځύ ڀԖխࣝፓ࿯ϐфૈаቚமխࣝΚǴխࣝፓ࿯ࢲ܄җߚᐘણࠠԄޑӭᗐᡏǴ੝ձ

ࢂҗβ-glucanǴቚуಒझ܄ᆶᡏన܄խࣝфૈ[78,79]Ƕ

Οиጤ

Οиጤ(Xanthan gum (XG))җΜӷ޸ࣽ໵ᆭੰ๵Xanthomonas campestris܌ғԋޑ ӭᗐᡏǴҗҒ៛ᗐ(mannose)ǵဟ๻ᑗ(glucose)ǴϷဟᗐ⾺ለ(glucuronic acid)ᄬԋǶ ځЬ᜘җβ-1,4ᗖ่ޑဟ๻ᑗǶୁ᜘ჹЬ᜘ޑК౗ၨεǴୁ᜘܌֖Ԗޑ♐ለ୷ (carboxyl group)ᆶҗЧ✉ለ(pyruvic acid)Զٰޑॄႝ಻ǴᄬԋࣁᏱԖၨεॄႝ಻ޑ ለ܄ӭᗐᜪǶΟиጤ೏ቶݱ٬Ҕܭαܺᛰނޑ፟ࠠᏊǴ଺ࣁᛙۓᆶϩණᛰނҔ[80]Ƕ

1.7 ᚊଫᗹጢགࢉੰࢥ ཥࠤੰࢥޑੰࢥᏢ੝܄

ཥࠤੰࢥ(Newcastle disease virus, NDV)നԐ೏૶ၩܭ 1926 ԃӧӑࡋѭՋ٥ޑП ࠐۈᛈวࢬՉǴ٠ЪጭۯܭӄШࣚ[1]ǶҗചᜪϩᚆрٰǴࢂ΋ᅿୋᗹనੰࢥឦ (Paramyxovirus, PMV)Ǵ1956 ԃа߻ചᜪୋᗹనੰࢥؒԖܴᡉޑಃΒᅿՈమࠠǴ ՠ߈ԃςҗചᜪϩᚆډኧဂޑ PMV ੰࢥǴҔ໺಍ޑՈమᏢ၂ᡍǴஒϐϩᜪԋΐ ᅿόӕޑੰࢥဂǴջ PMV-1~PMV-9ǶཥࠤੰࢥځڀԖൂި RNAǴੰࢥᗭಈ΋૓

ࣁᐍ༝׎ǴԖਔࣁӭ׎ރǴځελऊ 150-300 nm [81]Ƕ

12

ཥ

ཥࠤੰࢥϐགࢉ೼৩ک໺ኞБԄ གࢉ೼৩Ǻ

ཥࠤੰࢥ࿶җ΢ڥ֎ډ຾ΕډᚊଫᡏϣǴੰࢥᗭಈ཮Ӄӧ਻ᆅᕷ෗ǴฅࡕញܫΕ ՈనൻᕉǴӆ຾Εϣ᠌Ꮤ۔ᕷ෗ௗ๱ΞញрੰࢥډՈనύ׎ԋಃΒঁൻᕉǴੰࢥ

຾Εઓ࿶س಍ࡕǴགࢉᚊଫ໒ۈวғཥࠤੰੱރǴ໒ۈ௨рੰࢥډൻᕉύǴੰࢥ ϐ௨р࿶ڥ֎਻ᆅϷᕨߡǶ

໺ኞБԄǺ

ཥࠤੰࢥᗭಈ࿶җڥ֎ၰޑϩݜނϷᕨߡ΋ଆ௨рᡏѦǴ٠ёᙖҗޜ਻໺ኞ๏ځ дᚊଫǶ

13

ಃ

ಃΒകġ ፕЎࣴزҞޑ

Ҟ߻ᇡࣁ༾િಈڀԖᗹጢ՘ᏊޑբҔǴՠډҞ߻ࣁЗǴᔈܭ࿥ᜪޑᗺሷௗᅿǴ ځխࣝਏ݀ϝ҂೏ࣴزǴҗܭ࿥ᜪ༾િಈᗺሷௗᅿࣝभኳԄޑલЮǴࣴزϝԖ ज़Ǵ੝ձࢂόӕ׷਑ϐ༾િಈ՘ᏊჹܭࡕϺ܄խࣝϸᔈޑቹៜǴ܌ޕ׳Ԗज़Ƕ Ԗ᠘ܭԜǴҁፕЎޑࣴزҞ኱ӵΠǺ

1. Кၨᗺሷௗᅿࢲཥࠤੰࢥᆶа༾િಈхᙟࢲཥࠤੰࢥǴӧᚊي΢Їวϐխࣝ

ϸᔈǶፕЎ२Ӄຑ՗༾િಈխࣝߦ຾לᡏғԋޑਏ݀Ǵᆶࡌҥ༾િಈᗺሷௗ

ᅿᚊଫϐ୏ނኳԄǶ

2. ௖૸όӕႝ܄׷਑ಔԋϐ༾િಈхᙟѐࢲϯϐཥࠤੰࢥܭᚊଫᗺሷௗᅿࡕϐ

խࣝϸᔈǴځύ௖૸༾િಈᗺሷௗᅿԛኧᆶխࣝϸᔈਏ݀Ƕ

3. а LPS ༡ᙟܭ༾િಈ߄य़Ǵᙖҗխࣝಒझ΢ TLR4 ϐӃϺ܄խࣝϸᔈϐᒣᇡǴ ԜᅿӃϺ܄խࣝಒझޑख़ाಒझ߄य़ௗڙᏔࣁჹຝǴКၨ LPS խࣝڈᐟᏊϐᇶ շ՘Ꮚਏ݀Ƕ

4. ቚу༾િಈᗹ๱ࡋǴаڀԖғނᗹ๱܄ϐΟиጤ܈ሌԸӭᗐ༡ᙟܭ༾િಈ߄

य़ǴКၨғނᗹጤ׷਑༾િಈௗᅿࡕܭᚊଫౢғխࣝϸᔈޑਏ݀Ƕ

5. ༾િಈ՘Ꮚ٬όࢲϯཥࠤੰࢥϐלᡏ߄ၲуமǴՠჹܭ༾િಈᆶᚊޑխࣝϸᔈ

فՅ٠όమཱǴӢԜፕЎޑനࡕ΋೽ϩࢂКၨ༾િಈჹܭᚊխࣝಒझϐᐒڋϐ

௖૸Ƕ

14

ಃ

ಃΟകġ ׷਑ᆶБݤ

3.1 ჴᡍ୏ނ

9-11Вសϐค੝ۓੰྍ Specific Pathogen Free (SPF)ᅿೈᆶ 4 ຼស SPF ٰՎМᚊ (white leghorn chickens)Ƕ୏ނٰྍࣁՉࡹଣၭہ཮ৎ੬ፁғ၂ᡍ܌୏ނҔᛰࠔᔠ ۓϩ܌ᖼວǶ୏ނჴᡍ٩Ᏽၭہ཮ϐ୏ނᅽճྣៈᆶਠϣჴᡍ୏ނჴࡼྗ߾Ƕ

3.2 ੰࢥ

(1) խࣝਲ਼

аᅽଭ݅όࢲϯཥࠤੰࢥ(La Sota strain)଺ࣁלচځੰࢥΚሽ 10^9.8 EID₅₀ /mLҗѠ

᡼ғނᇙᏊިҽԖज़ϦљගٮǶ (2) װࢥਲ਼

ੰࢥ՘ᛯਲ਼(Sato strain)ੰࢥΚሽ 1000 MLD җѠ᡼ғނᇙᏊިҽԖज़ϦљගٮǶ

3.3 ჴᡍ၂ᛰϷϩ݋၂Ꮚ 3.3.1 ၂Ꮚᛰࠔ

1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-palmitoyl-sn-glycero-3- phospho-L-serine (DPPS) ᖼ Ծ Avanti Polar Lipid

(USA), Epikuron 200SH Phosphophatidylcholine (PC) ᖼ Ծ Degussa BioActives (Hamburg, Germany)ˤStearylamine (SA)ˣ3β-Hydroxy-5-cholestene (Cholesterol)ˣ Carbonate-BicarbonateˣTween 20ǵChloroformǵTris-NaClˣTrisˣNaClˣH2SO4ˣ Bovine Serum Albumin (BSA) ǵ 1,1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate (DiI)ˣ PBS ˣ LPS (Escherichia coli

15

0127:B8)ˣ Xanthan gum ˣ DEPC water ˣ Isopropanol ک BCIP/NBT ᖼ Ծ Sigma-Alderich (St. Louis, MO)ˤ3,3’,5,5’’-tetramethyl benzidine (TMB)ˣProtease inhibitor cocktailک Phospotase inhibitor cocktailǵTaq enzymeˣdNTP ک SYBR green ᖼԾ Bionovas Biotechnology (USA)ˤp38 MAPK inhibitor (SB-203580)ˣMAPK kinase (MEK) inhibitor (U-0126) ک IκB phosphorylation inhibitor (Bay 11-7085) ᖼ Ծ Biomol (Plymouth, PA, USA)ǶBCA Kit ᖼԾ Pierce Biotechnology (Thermo Scientific, USA)ǶAgarose ک MTS ᖼԾ Promega (USA)ˤHigh Capacity cDNA Reverse Transcription kit ᖼ Ծ Applied Biosystems (USA) ˤ Ficoll-Paque ᖼ Ծ Amersham Pharmacia Biotech (USA)ˤRPMI-1640ˣRPMI-1640 W/O phenol redˣPSˣ Trypsin-EDTAˣFBSˣFCS ᖼԾ Gibco (USA)Ƕ

PrimerᖼԾ MB Mission Biotech (Taiwan)Ǻ Sato HNǺ

forwardǺ5’-GTA CCC TCC AAT CCC AAA CT-3’

reverseǺ5’-TCC GGA TCT GGT AAT CTT GC-3’

Chicken GAPDHǺ

forwardǺ5’-GGC CGC CTG GTC ACC AGG GCT GCC-3’

reverseǺ5’-GGA GGA GTG GGG GAG ACA GAA GGG-3’

3.3.2 ל לᡏ

(1) Goat anti-chicken IgG conjugated HRP ᖼԾ Bethyl Laboratories (USA)ˤ (2) Goat anti-chicken IgA conjugated HRP ᖼԾ Bethyl Laboratories (USA)ˤ (3) Goat anti-chicken IgM conjugated HRP ᖼԾ Bethyl Laboratories (USA)ˤ (4) Mouse anti-chicken antibody (CVI-ChNL-68.1) ᖼԾ Prionics AG (USA)

16

(5) Goat anti-mouse IgG-FITC ᖼԾ Invitrogen (USA)

(6) Beta-actin mouse monoclonal IgG1 ᖼԾ Santa Cruz Biotechnology (USA) (7) p-p-38 MAPK rabbit polyclonal IgG ᖼԾ Santa Cruz Biotechnology (USA) (8) p-ERK mouse monoclonal IgG2aᖼԾ Santa Cruz Biotechnology (USA)

(9) NF-kappa B p-50 rabbit polyclonal IgG ᖼԾ Santa Cruz Biotechnology (USA) (10) NF-kappa B p-65 rabbit polyclonal IgG ᖼԾ Santa Cruz Biotechnology (USA) (11) Goat anti-mouse IgG-AP ᖼԾ Santa Cruz Biotechnology (USA)

(12) Goat anti-rabbit IgG-AP ᖼԾ Santa Cruz Biotechnology (USA)

3.3.3 ༟ ༟ጤǵ࣒ዟᇙࠔᆶጢ

(1) ሇનխࣝೱ่ϩ݋烉Maxisorp ELISA Immuno plate 96 ༾ϾዬᖼԾ Nunc (USA) (2) ՈౚᏉ໣׭ڋ၂ᡍǺU ࠠ 96 ༾ϾዬᖼԾ Greiner (Germany)

(3) ಒझ୻ᎦǺ96 ༾Ͼǵ24 ༾Ͼک 6 ༾Ͼಒझ୻ᎦѳዬᖼԾ Nunc (USA) (4) ೈқ፦ᆶ΋਼ϯේୀෳǺ96 ༾ϾѳዬᖼԾ Greiner (Germany)

(5) PVDF Membrane (6) PC membrane

3.4 ༾િಈхᙟੰࢥלচ 3.4.1 ༾િಈଛБ

(1) િ፦ᐚࡋ 20mM, DPPCǺCholesterolɨ4Ǻ1

(2) િ፦ᐚࡋ 20mM, DPPCǺDPPSǺCholesterolɨ3Ǻ1Ǻ1 (3) િ፦ᐚࡋ 20mM, DPPCǺSAǺCholesterolɨ3Ǻ1Ǻ1

17

3.4.2 ᇙ ᇙഢࢬำ

(1) ஒિ፦ગख़ࡕуΕᑃα༝ۭᐨ౟ύǴуΕ፾ໆ chloroform ٬ځિ፦ֹӄྋှǶ (2) ᑃα༝ۭᐨ౟ௗ΢෧ᓸᐚᕭᐒǴН੎ྕࡋܭ 55ɗ࡭ុܜଳ 1 λਔԿ׎ԋֹ᏾ᖓ

ጢܭ౟ۭǶ

(3) уΕ NDV לচనܭ༝ۭᐨ౟

(4) ٬ҔН੎Ԅຬॣݢ᎜ᕏᐒǴ٬ᑃα༝ۭᐨ౟ϣϐᖓጢ᎜Πа׎ԋ༾િಈ

(5) ӆа௖ଞԄຬॣݢ᎜༾િಈа׎ԋൂቫεᗭ༾િಈ

3.4.3 ಈ৩ǵࣚय़ႝՏᆶхᙟ౗

(1)ಈ৩ϩ݋

I. ڗ༾િಈࣝभ࿼ܭଳృϐ 4mL Ѥय़೸ӀኬࠔኲύǴӆаΒԛНีញ II. ஒኬࠔኲ࿼ܭ BIC 90 PLUS ಈ৩ᄤࣚय़ႝՏϩ݋ሺ

III. ೛ۓୖኧ(хࡴᗹࡋǵשՅ౗฻)຾ՉෳໆǶ IV. ؂ঁኬࠔෳໆ 10 ԛǴڗளಈ৩ѳ֡ॶǶ

(2) ࣚय़ႝՏϩ݋

I. ኬࠔකΕႝཱུǴ࿼ܭ BIC 90 PLUS ಈ৩ᄤࣚय़ႝՏϩ݋ሺύௗ΢ႝྍǶ II. ೛ۓୖኧȐྕࡋǵྋᏊǵש৔౗…฻ȑ຾ՉෳໆǶ

III. ؂ঁኬࠔෳໆ 10 ԛǴڗளࣚय़ႝՏѳ֡ॶǶ

(3) хᙟ౗ϩ݋

I. ஒ༾િಈࣝभ࿼ܭᚆЈᆅǴᚆЈᙯೲ 100,000g ǴᚆЈਔ໔ 1 λਔǴྕࡋ 4ɗǶ II. ᚆЈϐࡕڗ΢మనǶ

III. ճҔϩӀӀࡋीᔠෳ֎Ӏॶ(OD 280nm)Ƕ

18

IV. ीᆉלচ೏хᙟϐᕴໆǶ

3.4.4 ࣬ ࣬ᙯ౽ྕࡋ

(1) ஒ༾િಈ࿼Ε၂ኬ৒Ꮤ(pan)ύǴаஏ࠾၂ኬ৒ᏔޑᐒᏔஒ၂ኬ৒Ꮤஏ࠾Ǵᗉխ నᡏѦᅅǶ

(2) Ѻ໒DSCႝྍǴ዗ᐒ20ϩដǶ

(3) ஒࡑෳኬࠔܫӧ᝗η΢ࡕஒ΢ᇂஏ࠾Ǵ೛ۓ௟ඔೲ౗(scanning rate)ǵ௟ඔྕࡋ ጄൎ(scanning range)฻ୖኧǶ

(4) ࡑس಍ѳᑽࡕ௴୏ჴᡍǶሺᏔ཮٩ྣ܌೛ۓޑჴᡍБݤ຾Չ࣬ᙯ౽ྕࡋޑໆ ෳǴ٠૶ᒵځ዗ໆᡂϯǶ

(5) ჴᡍ่״ࡕϩ݋კ׎ёள࣬ᙯ౽ྕࡋǶ

3.4.5 ᑻӀ༾િಈ

༾િಈଛБӵΠ

(1) િ፦ᐚࡋ 20mM, DPPCǺCholesterolǺDiIɨ4Ǻ1Ǻ0.04

(2) િ፦ᐚࡋ 20mM, DPPCǺDPPSǺCholesterolǺDiIɨ3Ǻ1Ǻ1Ǻ0.04 (3) િ፦ᐚࡋ 20mM, DPPCǺSAǺCholesterolǺDiIɨ3Ǻ1Ǻ1Ǻ0.04

ᇙഢࢬำ

(1) ஒᑻӀિ፦(DiI)ᆶિ፦ગख़ࡕуΕᑃα༝ۭᐨ౟ύǴуΕ፾ໆ chloroform ٬ځ

િ፦ֹӄྋှǶ

(2) ᑃα༝ۭᐨ౟ௗ΢෧ᓸᐚᕭᐒǴН੎ྕࡋܭ 55ɗ࡭ុܜଳ 1 λਔԿ׎ԋֹ᏾ᖓ ጢܭ౟ۭǶ

(3) уΕ PBS לচనܭ༝ۭᐨ౟

19

(4) ٬ҔН੎Ԅຬॣݢ᎜ᕏᐒǴ٬ᑃα༝ۭᐨ౟ϣϐᖓጢ᎜Πа׎ԋ༾િಈ

(5) ӆаᔒᓸᏔஒ༾િಈᔒᓸ׎ԋಈ৩֡΋ޑൂቫεᗭ༾િಈ

3.4.6 ᚈ ᚈ՘Ꮚ༾િಈ

ஒ 1 mL LPS (250 μg/mL) ᆶ΢ॊɦ3.4 ༾િಈхᙟੰࢥלচɧᇙഢளϐ༾િಈӚ 0.6 mL֡ϬషӝǶ

3.4.7 ሌԸӭᗐ༾િಈکΟиጤ༾િಈ

ஒ 1 mL 1% Tremella ܈ 1mL 0.2% Xanthan gum ᆶ΢ॊɦ3.4 ༾િಈхᙟੰࢥל চɧᇙഢளϐύ܄ႝ಻༾િಈ 1 mL ֡ϬషӝǶ

3.5 խࣝௗᅿǵ௦Ոᆶሷᆶ਻ᆅؑࢱన

(1)խࣝ߻௦Ո଺ࣁ഍܄ჹྣǺ

࿶Ոౚ׭ڋᏉ໣၂ᡍዴᇡ؂ଫᚊ NDV לᡏ HI Κሽɦ2Ǵۈ຾ՉխࣝௗᅿǶ ᚊଫᗺሷխࣝಔձᆶԛኧҗΠ߄߄ҢǺ

PBS ࢲཥࠤੰ

ࢥ༾િಈ

ࣝभௗᅿ Ꮚໆ

όࢲϯཥ ࠤੰࢥ༾

િಈࣝभ

ௗᅿᏊໆ

ӭᗐ٬Ҕ Ꮚໆ

΋ ԛ խ

ࣝ Β ԛ խ

ࣝ Ο ԛ խ

ࣝ

Con 30 μL — — — — — —

NDV — 30 μL — — — √ —

NDV — — 60 μL — √ √ √

PC-Lip — 30 μL — — — √ —

PC-Lip — — 30 μL — — √ —

PC-Lip — — 60 μL — √ √ √

PC-Lip — — 120 μL — √ — —

PS-Lip — 30 μL — — — √ —

20

PS-Lip — — 60 μL — √ √ √

SA-Lip — 30 μL — — — √ —

SA-Lip — — 60 μL — √ √ —

NDV-LPS — — 60 μL 100 μL √ √ √

PC-Lip-LPS — — 60 μL 100 μL √ √ √ PS-Lip-LPS — — 60 μL 100 μL √ √ √ SA-Lip-LPS — — 60 μL 100 μL √ √ —

NDV — — 100 μL — — √ —

PC-Lip — — 100 μL — — √ —

Tremella-PC-Lip — — 100 μL 100 μL — √ — Xanthan-gum-PC-Lip — — 100 μL 100 μL — √ —

(2)ᗺᗺሷௗᅿਔ໔

΋ԛխࣝຼស֡ࣁѤຼសǴΒԛխࣝਔຼស֡ࣁϖຼសǴΟԛխࣝਔຼស֡ࣁ Ϥຼស

(3)௦Ո

΋ԛխࣝಔ௦ՈВӧѤຼសǵϖຼសکϤຼសǹΒԛխࣝಔ௦ՈВӧѤຼសǵ ϖຼសǵϤຼសکΎຼសǹΟԛխࣝಔ௦ՈВӧѤຼសǵϖຼសǵϤຼសǵΎ

ຼសکΖຼសǹՈమڗள؁ᡯӵΠǺ

җᚊᓉેа 3.5 mL ੿ޜ௦Ոᆅ௦໣ՈనǴஒ੿ޜ௦Ոᆅа 2500 rpm ᚆЈ 30 ϩ ដǴڗளՈమа 56ɗН੎ 30 ϩដѐନံᡏǴϩးܭ༾ໆᚆЈᆅߥӸܭ-20ɗࡑל ᡏዴਏ٬ҔǶ

(4)ሷᆶ਻ᆅؑࢱనڗள

΋ԛխࣝಔࣁϤຼសਔڗளǴΒԛխࣝಔࣁΎຼសਔڗளǴΟԛխࣝಔࣁΖຼ

សਔڗளǹሷᆶ਻ᆅؑࢱనڗள؁ᡯӵΠǺ

21

ஒᚊаΒ਼ϯᅹӼ኷ԝǴِೲஒᚊଫᕵӛှওǴڗр਻ᆅஒ 4 mL ጗ፂనȐ1 mM PBS у 0.1% BSAȑݙΕǴҗᚊሷϾڗளሷᆶ਻ᆅؑࢱనǴӆϩးࡕߥӸܭ-20 ɗǶ

3.6 Ո Ոౚ׭ڋᏉ໣၂ᡍᆶሇનխࣝೱ่ϩ݋ݤ

(1)Ոౚ׭ڋᏉ໣၂ᡍ (HI test)

ஒ U ࠠ 96 ༾ϾዬݙΕ 50 μL 0.85%ǴՈమܭӇ΢ଏӇǴࡑՈమֹӄྋှࡕǴӚ ڗ 50 μL ՈమݙΕ U ࠠ 96 ༾Ͼዬޑಃ΋ӈǴೱុ 2 ७ีញՈమǴஒ 4HAU ϐ NDVੰࢥ 50 μL ݙΕ 96 Ͼܭ࠻ྕΠགբ 60 ϩដǴஒ 50 μL ϐ 1% RBC (red blood cell) ݙΕ 96 Ͼܭ࠻ྕΠགբ 60 ϩដǴаԺ౳ղ᠐ኧᏵǶ

(2)ሇનխࣝೱ่ϩ݋ݤ (ELISA)

ෳۓՈమǵሷᆶ਻ᆅؑࢱనϐלᡏ

I. ஒ NDV לচа coating buffer 1:1000 ีញǴݙΕ 50uL ܭ 96 ༾ϾዬύǴ࿼ܭ 4 ɗϸᔈ႖ڹ

II. ڗр 96 ༾Ͼዬ٠ஒྋనҕଳǴݙΕ wash buffer ܭ؂ϾύǴҕଳྋనǴख़ᙟ Ԝ୏բ 3 ԛǴҕଳ 96 ༾ϾዬύූᎩϐྋన

III. ؂ϾуΕ 50 μL blocking buffer ܭ࠻ྕΠբҔ 30 ϩដǹݙΕ wash buffer ܭ؂

ϾύǴҕଳྋనǴख़ᙟԜ୏բ 5 ԛǴҕଳ 96 ༾ϾዬύූᎩϐྋన IV. ӧ 96 ϾύуΕ 50 μL ϐלᡏᔠᡏܭ࠻ྕΠբҔ 1 λਔ

ՈమǺа sample conjugate buffer ีញϐՈమ ሷᆶ਻ᆅؑࢱనǺচ७ϐሷᆶ਻ᆅؑࢱన

V. ஒ 96 ༾ϾዬϣϐྋనҕଳǴݙΕ wash buffer ܭ؂ϾύǴҕଳྋనǴख़ᙟԜ

୏բ 4 ԛǴҕଳ 96 ༾ϾዬύූᎩϐྋన VI. ӧ 96 ϾύуΕ 50 μL לᡏܭ࠻ྕΠբҔ 1 λਔ

22

VII. ஒ 96 ༾ϾዬϣϐྋనҕଳǴݙΕ wash buffer ܭ؂ϾύǴҕଳྋనǴख़ᙟԜ

୏բ 4 ԛǴҕଳ 96 ༾ϾዬύූᎩϐྋన VIII. уΕ 50 μL TMB բҔ 30 ϩដ

IX. уΕ 50 μL 2M H2SO4

X. а OD 450 nm ሇનխࣝϩ݋ሺ/༾ໆዬղ᠐ሺୀෳ

3.7 װ װࢥჴᡍ

(1) ᗺሷװࢥᏊໆෳۓ

ஒᚊϩࣁ 7 ಔ؂ಔӚ 6 ଫǴ؂ಔϩձ๏ϒ 300 μL (1x10^1.0-7.0 EID₅₀/mL)ཥࠤੰࢥம ࢥਲ਼ Sato চనǴװࢥࡕᢀჸ 1 ຼǴۓက 1 MLD ϐੰࢥᏊໆǶ

(2) ᗺሷװࢥ၂ᡍ

࿶ௗᅿࣝभϐᚊଫܭխࣝࡕಃ 14 Ϻ๏ϒᗺሷװࢥ 1000 MLD ϐ Sato மࢥਲ਼Ǵᢀჸ 14Ϻ٠૶ᒵӚಔӸࢲଫኧᆶวੰੱރǶ

3.8 ᚊ๠᠌ಒझϩᚆ

(1) ஒ҂խࣝၸޑᚊаΒ਼ϯᅹ឴࣊ڗள๠᠌Ǵڗр๠᠌٠੆ݰܭӇ PBS ύ (2) ஒ๠᠌ᑃ࿗Ǵ๠᠌ಒझ጗጗ݙΕ 5 mL Ficoll-Paque ύǴܭ࠻ྕΠᚆЈ 30 ϩដ (3) ᇸᇸӦܜڗϩᚆрޑᜦރᕉ஥ቫϐಒझ

(4) а PBS ࢱಒझǴܭ࠻ྕΠᚆЈ 3 ϩដ

(5) ѐନ΢మనǴӆ጗጗уΕ RPMI-1640 ୻Ꭶన (6) а Trypen blue ܭᡉ༾᜔Πኧಒझ

3.9 Ӆ೫ขᡉ༾᜔

23

(1) ஒ҂խࣝၸޑᚊаΒ਼ϯᅹ឴࣊ڗள๠᠌Ǵа 10⁴cells/mL ᚊ๠᠌ಒझ୻Ꭶ ܭ 41ɗ 5% CO2ޑಒझ୻Ꭶጃ

(2) ѐନ୻ᎦనуΕό֖ FBS/FCS ϐ RPMI-1640 ୻ᎦనբҔၸڹܭ 41ɗ 5%

CO2ޑಒझ୻Ꭶጃ

(3) уа 10 μL Ӛಔԋҽϐɦ3.4.5 ᑻᑻӀ༾િಈɧǴӅӕ୻Ꭶ 24 λਔܭ 41ɗ 5%

CO2ޑಒझ୻Ꭶጃ (4) ѐନ୻ᎦనуΕӇ PBS

(5) ѐନ PBS уΕа PBS ଛᇙϐ 0.5% paraformaldehye 15 ϩដ (6) ѐନ 0.5% paraformaldehye ٠а PBS ᇸᇸࢱಒझ

(7) уΕа 3% BSA/PBS ଛᇙϐ CVI-ChNL-68.1 לᡏբҔ 1 λਔܭ 4ɗ (8) а PBS ᇸᇸࢱಒझ

(9) уΕа 3% BSA/PBS ଛᇙϐ Goat anti-mouse IgG-FITC לᡏբҔ 1 λਔܭ࠻ྕ

(10) аӅ೫ขᡉ༾᜔ Leica TCS SP5 Confocal Spectral Microscope Imaging System ᢀჸᑻӀ༾િಈ೏๠᠌ಒझϐൂਡ/ѮᏘಒझझտໆ

3.10 RT-PCR ୀෳੰࢥ

(1) ஒװࢥࡕӸࢲޑᚊଫаΒ਼ϯᅹ឴࣊Ǵڗр਻ᆅǵط᠌ǵ๠᠌کޤ᠌Ǵஒᔠᡏ

এܭ -80ɗ ࡑ٬Ҕ

(2) Ӛ೽Տϐಔᙃગख़ 50gǴуΕనᄊේࣴᑃԋણރǴуΕ 1mL TRIzol ᆶણ҃

ቃਗ਼షӝ

(3) уΕ 200 uL Chloroform ቃਗ਼షӝǴа 12000 rpm ᚆЈ 30 ϩដ

(4) ڗ΢మనऊ 500 uLǴуΕ฻ໆϐ isopropanol кϩషӝǴа 12000 rpm ᚆЈ 30 ϩដܭ 4ɗ

(5) ڗ΢మనǴуΕӇޑ 75%ଚᆒǴа 7500 rpm ᚆЈ 15 ϩដܭ 4ɗ (6) ѐନ΢మనǴࡑଚᆒචวࡕуΕ DEPC Н

24

(7) а 260/280 nm ෳ RNA પࡋᆶᐚࡋ

(8) ڗ2 μg/μLϐtotal RNAаABI reverse transcriptase kitܭ37 ɗբҔ2λਔ

(9) ڗ3 μL cDNAуΕSato primerǵTaqǵdNTPکΒԛНǴҗ95ɗଆۈ62ɗᚇӝ Ӆ30ঁൻᕉ

(10) ڗ10 μL PCRౢނǴуΕ2%ࢩ๼(֖SYBR green)ጤႝݚऊ30ϩដ (11) аUVጃྣጤКჹPCRౢނ

3.11 ׷ ׷਑ჹಒझϐࢥ܄ୀෳ

(1) ஒ҂խࣝၸޑᚊаΒ਼ϯᅹ឴࣊ڗள๠᠌Ǵа 10⁴cells/mL ᚊ๠᠌ಒझ୻Ꭶ 96 well ܭ 41ɗ 5% CO2ޑಒझ୻Ꭶጃ

(2) ѐନ୻ᎦనуΕ֖ 10%FBS/5%FCS ϐ RPMI-1640 ୻ᎦనբҔၸڹܭ 41ɗ 5% CO2ޑಒझ୻Ꭶጃ

(3) ѐନ 100 μL ୻Ꭶనуа 100 uL ୻Ꭶనᆶ 10 uL Ӛಔԋҽϐ༾િಈǴӅӕ୻

Ꭶ 24 λਔܭ 41ɗ 5% CO2ޑಒझ୻ጃ

(4) ؂ well уΕ 20 μL ϐ MTS ྋనǴբҔ 4 λਔܭ 41ɗ 5% CO2ޑಒझ୻Ꭶጃ (5) а 490 nm ሇનխࣝϩ݋ሺ/༾ໆዬղ᠐ሺୀෳ

3.12 ୀෳಒझ୻Ꭶనύ NO

(1) ஒ҂խࣝၸޑᚊаΒ਼ϯᅹ឴࣊ڗள๠᠌Ǵа 10⁶cells/mL ᚊ๠᠌ಒझ୻Ꭶ 24 well ܭ 41ɗ 5% CO2ޑಒझ୻Ꭶጃ

(2) ѐନ୻ᎦనуΕ֖ 10%FBS/5%FCS ϐ RPMI-1640 ୻ᎦనբҔၸڹܭ 41ɗ 5% CO₂ޑಒझ୻Ꭶጃ

(3) ѐ ନ 1.2 mL ୻ Ꭶ న у Ε 700 μL 10%FBS/5%FCS ϐ non phenol red RPMI-1640 ୻ᎦనǴуΕ p38 MAPK inhibitor (SB-203580) ˣMAPK kinase

25

(MEK) inhibitor (U-0126) ک IκB phosphorylation inhibitor (Bay 11-7085)ܭ 41ɗ 5% CO2୻Ꭶ 1 λਔ

(4) уΕ 1 μL,5 μL,10 μL Ӛಔԋҽϐ༾િಈǵӭᗐ׷਑ǴӅӕ୻Ꭶ 24 λਔܭ 41 ɗ 5% CO2ޑಒझ୻ጃ

(5) ஒಒझ୻Ꭶనу 50 μL ܭ 96 well ύуΕ Greiss reagent ྋన(1% sulfanilamide ک 50 μL ϐ 0.1% naphthylenediamine (ྋܭ 2.5% phosphoric acid solution)) (6) а 570 nm ሇનխࣝϩ݋ሺ/༾ໆዬղ᠐ሺୀෳ

3.13 Ջ ՋБᗺᏀݤୀෳೈқ፦߄౜

(1) ᚊଫ࿶җ༾િಈࣝभխࣝࡕ 2 ຼаΒ਼ϯᅹӼ኷ԝǴڗр๠᠌এܭ-80ɗࡑ

٬Ҕ

(2) ๠᠌ગख़٠୊࿗๠᠌ಔᙃǴуΕ RIPA buffer (ςуΕ protease inhibitor cocktail/phospotase inhibitor cocktail)ܭӇ΢բҔ 10 ϩដ

(3) ܭ 4ɗᚆЈ 30 ϩដࡕڗ΢మనǴߥӸܭ-20ɗ܈ջਔ٬Ҕ (4) ೈқ፦ᐚࡋа BCA Kit ୀෳ

(5) Ӛڗ 30 μg ೈқ፦າ SDS-PAGE

(6) ஒ SDS-PAGE ΢ޑೈқ፦ᙯ䭋ܭ PVDF Membrane

(7) ஒ PVDF Membrane ੆ܭ 3% BSA-PBST blocking solution 30 ϩដ (8) а PBST ࢱ PVDF Membrane 3 ԛ؂ԛ 10 ϩដ

(9) уΕа PBST ีញ 1000 ७ϐ Beta-actin לᡏǵp-p-38 MAPK לᡏǵp-ERK ל ᡏǵNF-kappa B p-50 לᡏک NF-kappa B p-65 לᡏǴܭ 4ɗբҔ႖ڹ

(10) а PBST ࢱ PVDF Membrane 3 ԛ؂ԛ 10 ϩដ

(11) уΕа PBST ีញ 5000 ७ϐ Goat anti-mouse IgG-AP ک Goat anti-rabbit IgG-APܭ࠻ྕբҔ 1 λਔ

(12) а PBST ࢱ PVDF Membrane 3 ԛ؂ԛ 10 ϩដ

26

(13) уΕ BCIP/NBT ևՅ

27

ಃ

ಃѤക ่݀

(΋)՘Ꮚ׷਑ϐނ౛܄፦ϩ݋

(1) ༾િಈಈ৩ᆶхᙟ౗ϩ݋

җΟᅿႝ܄ႝ಻ಔԋϐᕗિ፦ᄬԋޑӭቫ༾झ༾િಈ(MLV)Ǵхᙟϩණܭ጗ፂనύ ޑࢲཥࠤੰࢥ܈όࢲϐཥࠤੰࢥלচǴ࿶җႜ৔ಈ৩ϩ݋ሺϩ݋Ǵځѳ֡ಈ৩ࣁ 1030-1223 nmǴҗхᙟ౗၂ᡍϩ݋ӭቫ༾झ༾િಈޑੰࢥхᙟ౗ࣁύ܄ႝ಻༾િಈ PC-Lip ޑ 20.58% Ǵॄႝ಻༾િಈ PS-Lip ޑ 2.57% Ǵ҅ႝ಻༾િಈ SA-Lip ޑ 43.26%! (߄΋)Ƕ

(2) ༾િಈࣚय़ႝՏᆶ࣬ᙯ౽ྕࡋϩ݋

ࣁຑ՗༾િಈ՘Ꮚޑႝ಻੝܄Ǵஒᇙഢֹԋޑ༾િಈཥࠤੰࢥࣝभӚಔǴճҔࣚ

य़ႝՏϩ݋ሺᔠෳӚ༾િಈཥࠤੰࢥࣝभޑಈηႝ܄ǶவჴᡍኧᏵёـǴύ܄ႝ

಻༾િಈ PC-Lip ޑࣚय़ႝՏࣁ-17.70 mV Ǵॄႝ಻༾િಈ PS-Lip ޑࣚय़ႝՏࣁ -51.79 mVǴ҅ႝ಻༾િಈ SA-Lip ޑࣚय़ႝՏࣁ 33.52 mVǶ࿶җ዗ৡ༾ϩϩ݋ሺ (DSC)ϩ݋Ӛಔԋҽϐӭቫ༾झ༾િಈǴځ࣬ᙯ౽ྕࡋࣁύ܄ႝ಻༾િಈ PC-Lip ޑ 43.0ɗ Ǵॄႝ಻༾િಈ PS-Lip ޑ 62.0ɗǴ҅ႝ಻༾િಈ SA-Lip ޑ 58.0ɗ(߄΋)Ƕ

(Β)ࢲࢥᆶ༾િಈхᙟࢲࢥלᡏϩ݋

ѤຼសқՅٰՎМᚊϩԋΟಔǴ؂ಔӚΎଫᚊǶϩձ๏ϒᗺሷௗᅿࢲཥࠤੰࢥל চ30 μL(ࣁNDVಔ)Ǵаύ܄ႝ಻༾િಈхᙟࢲཥࠤੰࢥלচ30 μL(ࣁEPC-Lipಔ)Ǵ аϷௗڙPBS 30 μL(ࣁControlಔ)ǶӧխࣝࡕಃΎВǴҗᚊᓉે௦Ո٠ஒᚊଫ឴࣊

аڗளሷ๚ᆶ਻ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱ

28

నύϩݜࠠs-IgAלᡏᆶՈమύIgGלᡏ֡ࣁύ܄ႝ಻༾િಈхᙟࢲཥࠤੰࢥಔ (EPC-Lipಔ)ғԋלᡏΚሽၨଯǶᡉҢа༾િಈхᙟࢲੰࢥלচǴૈԖਏගϲᚊଫ ᡏϣלᡏғԋໆคፕӧֽ೽ޑᗹጢ೽Տ܈ࢂӄي܄ޑՈన೽Տ(კ΋)Ƕ

(Ο Ο)όӕႝ಻༾િಈхᙟࢲࢥלᡏϩ݋

ѤຼសқՅٰՎМᚊϩԋϖಔǴ؂ಔӚΜଫᚊǶϩձ๏ϒᗺሷௗᅿࢲཥࠤੰࢥל চ30 μL(ࣁNDVಔ)Ǵаύ܄ႝ಻༾િಈхᙟࢲཥࠤੰࢥלচ30 μL(ࣁEPC-Lipಔ)Ǵ

ॄႝ಻༾િಈхᙟࢲཥࠤੰࢥלচ30 μL(ࣁPS-Lipಔ)Ǵ҅ႝ಻༾િಈхᙟࢲཥࠤੰ

ࢥלচ30 μL(ࣁSA-Lipಔ)ǴаϷௗڙPBS 30 μL(ࣁControlಔ)Ƕٿຼࡕ຾ՉಃΒԛ ᗺሷխࣝௗᅿǴӧΒԛխࣝࡕٿຼǴҗᚊᓉે௦Ո٠ஒᚊଫ឴࣊аڗளሷ๚ᆶ਻

ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱనύϩݜࠠs-IgA לᡏаEPC-LipಔלᡏΚሽനଯ(p<0.05)ǴځԛࣁPS-Lipಔ(಍ी΢คᡉ๱ৡ౦) (კ Β)ǶՈమύIgGלᡏࣁEPC-LipಔלᡏΚሽനଯ(p<0.05)ǴځԛࣁPS-Lipಔ(಍ी΢

คᡉ๱ৡ౦) (კΟ)Ƕ

(Ѥ) לᡏғԋԔጕᆶ༾િಈхᙟόࢲϯੰࢥᏊໆКၨ

(1)לᡏғԋԔጕ

ѤຼសқՅٰՎМᚊϩԋΒಔǴ؂ಔӚΒΜ΋ଫᚊǶϩձܭ๏ϒᗺሷௗᅿࢲཥࠤ

ੰࢥלচ30 μL(ࣁLive NDVಔ)Ǵаύ܄ႝ಻༾િಈхᙟࢲཥࠤੰࢥלচ30 μL(ࣁ Liposomal-inactivated NDVಔ)Ƕܭᗺሷௗᅿࡕ؂ಔ؂ٿВ឴࣊ΟଫᚊǴаڗளሷ๚

ᆶ਻ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱనύϩݜࠠ

s-IgAלᡏǴӧಃΟВԿಃΖВᚊଫௗᅿLiposomal-inactivated NDVಔ֡Ԗၨӭໆϐ s-IgAלᡏғԋǴҗಃΐВଆᚊଫLive NDVಔޑϩݜࠠs-IgAלᡏ೴ᅌ෧Ͽՠࢂלᡏ ғ ԋ ໆ ֡ К ௗ ᅿ Liposomal-inactivated NDV ಔ ל ᡏ ғ ԋ ໆ ࣁ ӭ Ǵ Զ ௗ ᅿ

29

Liposomal-inactivated NDVಔޑᚊଫᗹጢ೽Տלᡏғԋໆ೴ᅌ෧ϿǶᡉҢа༾િಈ хᙟόࢲϯלচаᗺሷௗᅿᚊଫǴӧᗹጢ೽Տלᡏғԋวғਔ໔ၨࢲࢥِೲ(კ Ѥ)Ƕ

(2)༾ ༾િಈхᙟόࢲϯੰࢥᏊໆКၨ

ѤຼសқՅٰՎМᚊϩԋϖಔǴ؂ಔӚΎଫᚊǶϩձܭ๏ϒᗺሷௗᅿόӕᏊໆϐ όࢲϯཥࠤੰࢥלচǴϩձࣁ60 μL(ࣁVirus aloneಔ)Ǵ60 μL(ࣁLip PC-NDಔ)Ǵ30 uL(ࣁLow dose Lip PC-NDಔ)Ǵ120 μL(ࣁHigh dose Lip PC-NDಔ)аϷௗڙPBS 30 μL(ࣁControlಔ)ǶܭխࣝࡕٿຼǴஒᚊଫ឴࣊аڗளሷ๚ᆶ਻ᆅؑࢱనǶҗሇનխ

ࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱనύϩݜࠠs-IgAלᡏаௗᅿ60 uL(ࣁ Lip PC-NDಔ)לᡏΚሽനଯ(p<0.01)Ǵځԛࣁௗᅿ120 μL(ࣁHigh dose Lip PC-NDಔ) (p<0.05ᆶControlಔКၨ)ՠ໻КVirus aloneಔޑs-IgAלᡏғԋໆӭǴՠ಍ी΢คᡉ

๱ৡ౦(კϖ)ǶӢԜǴҁࣴزӧௗΠٰޑࣴزЬᚒǴКၨόӕႝ಻׷਑ಔԋ༾િಈ хᙟόࢲϯੰࢥ܈బуિӭᗐǵᗹ܄ӭᗐᡏϐࣴزύǴ֡а༾િಈхᙟ60 μLϐל চௗᅿᏊໆ଺КၨǶ

(ϖ)༾િಈхᙟόࢲϯཥࠤੰࢥלᡏϩ݋

(1)ᗹጢלᡏ߄౜ໆ

ѤຼសқՅٰՎМᚊϩԋϖಔǴ؂ಔӚΒΜଫᚊǶϩձܭ๏ϒᗺሷௗᅿӕᏊໆ(60 μL)Ǵՠόӕႝ಻༾િಈхᙟόࢲϯཥࠤੰࢥלচǴϩձࣁVirus aloneಔǵ PC-Lip ಔǵPS-LipಔǵSA-LipಔаϷௗڙPBS 30 μL(ࣁControlಔ)Ƕӧ΋ԛխࣝࡕ΋ຼǴ ᒿᐒڗΜଫᚊ຾Չᚊᓉે௦Ո٠ஒᚊଫ឴࣊аڗளሷ๚ᆶ਻ᆅؑࢱనǶӕਔǴஒ ഭᎩޑΜଫᚊ຾ՉಃΒԛᗺሷխࣝௗᅿǴӧΒԛխࣝࡕٿຼǴҗᚊᓉે௦Ո٠ஒ ᚊଫ឴࣊аڗளሷ๚ᆶ਻ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ

30

਻ᆅؑࢱనύϩݜࠠs-IgAלᡏКၨǴӧӚಔᚊଫௗڙΒԛխࣝௗᅿ༾િಈࣝभǴ

֡ёගϲᗹጢלᡏ߄౜ໆǴځύ΋ԛᆶΒԛխࣝࡕϩݜࠠs-IgAלᡏКၨԖᡉ๱ৡ ౦ಔձࣁௗᅿPC-LipಔǵPS-LipಔکSA-Lipಔ(p<0.05)Ƕ୤ௗᅿVirus aloneಔӧٿԛ לᡏКၨ֡คᡉ๱΢ϲǴ߄Ңӧᗹጢ೽Տѝௗᅿόࢲϯלচࢂคݤගϲלᡏ߄౜

ໆǶӧКၨόӕႝ܄༾િಈӚಔԋҽǴ࿶җ΋ԛᗺሷխࣝ܈ࢂΒԛᗺሷխࣝௗᅿ PC-LipಔלᡏΚሽനଯ(p<0.01) (კϤA)Ƕ

(2)Ո Ոమלᡏ߄౜ໆ

ᚊଫௗڙޑխࣝीฝӕ΢Ǵҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨՈమύanti-NDV IgGלᡏғԋໆǴӧӚಔᚊଫௗڙΒԛխࣝௗᅿ༾િಈࣝभǴ֡ёගϲӄي܄לᡏ ߄౜ໆǴځύ΋ԛᆶΒԛխࣝࡕIgGלᡏғԋໆКၨԖᡉ๱ৡ౦ಔձࣁௗᅿPC-Lip ಔ(p<0.05)Ƕၨ੝ձޑӧௗᅿVirus aloneಔǵPS-LipಔکSA-LipಔӧٿԛלᡏКၨ֡

คᡉ๱΢ϲǴ߄Ңӧᗹጢ೽ՏௗᅿόࢲϯלচǵPS-Lipхᙟ܈SA-LipࢂคݤගϲՈ మύלᡏ߄౜ໆǶӧКၨόӕႝ܄༾િಈӚಔԋҽǴ࿶җ΋ԛᗺሷխࣝ܈ࢂΒԛ ᗺሷխࣝௗᅿPC-LipಔלᡏΚሽനଯ(p<0.01) (კϤB)Ƕ

(Ϥ)િӭᗐᡏ༡ᙟ༾િಈхᙟόࢲϯཥࠤੰࢥלᡏϩ݋

(1) િӭᗐᡏ๏ϒਔ໔

ᚊଫᒿᐒϩԋٿಔǴ؂ಔӚϖଫᚊǴځύ΋ಔᚊଫӧխࣝௗᅿ༾િಈࣝभ߻ 24 λ ਔӃ๏ϒિӭᗐᡏ LPSǴќ΋ಔᚊଫխࣝௗᅿ༾િಈࣝभӕਔ๏ϒ༾િಈࣝभک

િӭᗐᡏ LPSǴܭխࣝࡕ΋ࢃයǴڗளሷ๚ᆶ਻ᆅؑࢱనǴว౜ӧӕਔ๏ϒ༾િ

ಈࣝभکિӭᗐᡏ LPS ಔձޑᚊଫόፕࢂ IgAǵIgG ک IgM ֡ౢғၨӭ஑΋܄ޑ לᡏ(კΖ)Ƕ

31

(2)ᗹ ᗹጢלᡏ߄౜ໆ

٩ྣխࣝௗᅿԛኧϩࣁ΋ԛխࣝ(Ύଫ)ǵΒԛխࣝ(Ύଫ)کΟԛխࣝ(Ύଫ)Ƕխࣝ

ය໔໔႖ࣁ΋ຼǴϩձܭ๏ϒᗺሷௗᅿӕᏊໆ(60 μL)Ǵՠόӕႝ಻༾િಈхᙟό ࢲϯཥࠤੰࢥלচǴϩձࣁ Virus alone ಔǵ PC-Lip ಔǵPS-Lip ಔǵSA-Lip ಔǴ

ௗᅿӕᏊໆ(160 μLǴځύ༾િಈࣝभ 60 μL ᆶિӭᗐᡏ LPS 100μL)όӕႝ಻༾િ

ಈхᙟόࢲϯཥࠤੰࢥלচӕਔ༡ᙟિӭᗐᡏ LPSǴϩձࣁ Virus-LPS ಔǵ PC-Lip-LPSಔǵPS-Lip-LPS ಔǵSA-Lip-LPS ಔǴаϷௗڙ PBS 30μL (ࣁ Control ಔ)ǶӧӚԛխࣝࡕٿຼǴᒿᐒڗΎଫᚊ຾Չᚊᓉે௦Ո٠ஒᚊଫ឴࣊аڗளሷ๚

ᆶ਻ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱనύϩݜࠠ

s-IgA לᡏКၨǴӧӚಔᚊଫௗڙΒԛխࣝௗᅿ༾િಈࣝभǴ֡ёගϲᗹጢ s-IgA לᡏ߄౜ໆǴ୤ᐱӧ PC-Lip-LPS ಔלᡏғԋໆϸԶफ़եǶ᏾ᡏКၨ࿶җ΋ԛᗺሷ խࣝ܈ࢂΒԛᗺሷխࣝௗᅿ PC-Lip ಔלᡏΚሽനଯ(p<0.01)ǴᡉҢિӭᗐᡏ LPS ༡ᙟ༾િಈӧᗺሷௗᅿࡕǴӧᗹጢғԋϐלᡏໆคܴᡉշ੻(კΐ)Ƕ

(3)Ոమלᡏ߄౜ໆ

ᚊଫௗڙޑխࣝीฝӕ΢Ǵҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨՈమύ Anti-NDV IgGלᡏғԋໆǴӧӚಔᚊଫௗڙٿԛխࣝௗᅿ༾િಈࣝभǴځύ໻ӧ PC-Lip ಔ (p<0.05)ǵPC-Lip-LPS(p<0.05)ಔک SA-LPS ಔࢂΒԛխࣝࡕלᡏғԋໆගϲ(კΐ)Ƕ

(Ύ)Ոౚ׭ڋᏉ໣၂ᡍ

(1)Ոమלᡏ߄౜ໆ

ѤຼសқՅٰՎМᚊϩԋΐಔǴ؂ಔӚΒΜ΋ଫᚊǶ٩ྣխࣝௗᅿԛኧϩࣁ΋ԛ խࣝ(Ύଫ)ǵΒԛխࣝ(Ύଫ)کΟԛխࣝ(Ύଫ)Ƕխࣝය໔໔႖ࣁ΋ຼǴϩձܭ๏

ϒᗺሷௗᅿӕᏊໆ(60 μL)Ǵՠόӕႝ಻༾િಈхᙟόࢲϯཥࠤੰࢥלচǴϩձࣁ

32

Virus aloneಔǵ PC-Lip ಔǵPS-Lip ಔǵSA-Lip ಔǴௗᅿӕᏊໆ(160 μLǴځύ༾

િಈࣝभ 60 μL ᆶિӭᗐᡏ LPS 100 μL)όӕႝ಻༾િಈхᙟόࢲϯཥࠤੰࢥלচ ӕਔ༡ᙟિӭᗐᡏ LPSǴϩձࣁ Virus-LPS ಔǵ PC-Lip-LPS ಔǵPS-Lip-LPS ಔǵ SA-Lip-LPSಔǴаϷௗڙ PBS 30 μL (ࣁ Control ಔ)Ƕܭ؂ຼᒿᐒڗΎଫᚊ຾Չᚊ ᓉે௦ՈǶаՈౚ׭ڋᏉ໣၂ᡍКၨǴӚಔᚊଫௗڙௗᅿ༾િಈࣝभܭ΋ԛխࣝǵ ΒԛխࣝکΟԛխࣝӧՈమύלᡏ߄౜ໆǶҗ߄ΒᡉҢӧ΋ԛխࣝࡕ PC-Lip ಔک PC-Lip-LPSಔޑՈమีញ 640 ७ࡕǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶPS-Lip ಔ Ոమ໻ૈีញԿ 40 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶPS-Lip-LPS ಔՈమ໻ૈ

ีញԿ 20 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶVirus alone ಔک Virus-LPS ಔՈ మ໻ૈีញԿ 10 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶSA-Lip ಔک SA-Lip-LPS ಔՈమ໻ૈีញԿ 8 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶӧௗڙΒԛխࣝௗᅿ ࡕǴӚಔՈౚ׭ڋᏉ໣Κሽ֡ගଯǴЀځӧ PS-Lip-LPS ಔՈమ໻ૈีញԿ 2560 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶЪӧ PC-Lip ಔǵPC-Lip-LPS ک PS-Lip ಔ ՈమϐՈౚ׭ڋᏉ໣ΚሽගଯԿ 1280 ७Ƕ୤ SA-Lip ಔک SA-Lip-LPS ಔՈమϐՈ ౚ׭ڋᏉ໣Κሽफ़եǶӧௗڙΟԛխࣝௗᅿࡕǴPC-Lip ಔǵPC-Lip-LPSǵPS-Lip ಔک PS-Lip-LPS ಔՈమ໻ૈีញԿ 10240 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣Ƕ Virus aloneಔک Virus-LPS ಔՈమ໻ૈีញԿ 10 ७ǴSA ಔک SA-Lip ಔ߾ؒԖෳ

ளኧᏵǶ

(2)ᗹ ᗹጢלᡏ߄౜ໆ

ᚊଫௗڙޑխࣝीฝӕ΢Ǵܭ؂ຼᒿᐒڗΎଫᚊଫ឴࣊аڗளሷ๚ᆶ਻ᆅؑࢱ నǶӕਔǴஒഭᎩޑᚊଫ຾ՉಃΒԛᆶಃΟԛᗺሷխࣝௗᅿǴӆ឴࣊ᚊଫаڗள ሷ๚ᆶ਻ᆅؑࢱనǶаՈౚ׭ڋᏉ໣၂ᡍКၨǴӚಔᚊଫௗڙௗᅿ༾િಈࣝभܭ

΋ԛխࣝǵΒԛխࣝکΟԛխࣝӧሷ๚ᆶ਻ᆅؑࢱనύלᡏ߄౜ໆǶҗ߄ΟᡉҢ ӧ΋ԛխࣝࡕ PC-Lip ಔᗹጢ೽Տϐלᡏีញ 32 ७ࡕǴϝฅૈԖਏӦ׭ڋआՈౚ

33

ޑᏉ໣ǶPC-Lip-LPS ಔᗹጢ೽Տϐלᡏีញ 16 ७ࡕǴϝฅૈԖਏӦ׭ڋआՈౚޑ Ꮙ໣ǶӧௗڙΒԛխࣝௗᅿࡕǴӚಔՈౚ׭ڋᏉ໣Κሽ֡ගଯǴЀځӧ PC-Lipǵ PC-Lip-LPSಔᗹጢ೽ՏϐלᡏีញԿ 64 ७ǴϝฅૈԖਏӦ׭ڋआՈౚޑᏉ໣ǶЪ ӧ PS-Lip ಔک PS-Lip-LPS ಔᗹጢ೽ՏϐՈౚ׭ڋᏉ໣ΚሽගଯԿ 32 ७Ƕ୤ SA-Lip ಔک SA-Lip-LPS ಔᗹጢ೽ՏϐՈౚ׭ڋᏉ໣Κሽफ़եǶӧௗڙΟԛխࣝௗᅿࡕǴ PC-Lipಔک PC-Lip-LPS ಔᗹጢ೽ՏϐלᡏีញԿ 256 ७ǴϝฅૈԖਏӦ׭ڋआՈ ౚޑᏉ໣ǶPS-Lip ಔǵPS-Lip-LPS ಔǵVirus alone ಔک Virus-LPS ಔᗹጢ೽ՏϐՈ ౚ׭ڋᏉ໣Κሽ֡ࣁ 32 ७ǶSA ಔک SA-Lip ಔ߾ؒԖෳளኧᏵǶ

(Ζ Ζ)װࢥ၂ᡍ (1)ᗺሷװࢥᏊໆ

٩୯ৎݤೕǴόࢲϯཥࠤੰࣝभਏΚ၂ᡍǴሡаཥࠤੰϐமࢥ(՘ᛯਲ਼)1,000MLD ԼԺݙ৔װᔐǴ࿶ΒຼᢀჸǴݙ৔ΒᏊໆޣ֡໪଼ӸǴௗᅿΒϩϐ΋Ꮚໆޣ໪Ԗ Ύϖʘа΢Ǵόևϸᔈ܈ևᇸ༾ϸᔈࡕऐၸԶ଼ӸǴჹྣᚊǴ໪ևڂࠠཥࠤੰੱ

ԶᔚԝǶՠҁࣴزයఈૈኳᔕཥࠤੰགࢉ೼৩ǴࡺஒװࢥБԄׯҗаᗺሷ๏ϒǴ ཥࠤੰமࢥਲ਼(՘ᛯਲ਼)1,000MLDǶ

२Ӄஒཥࠤੰமࢥ(՘ᛯਲ਼)଺ׇӈีញǴҗ 1×10^7.0 EID₅₀/mL ีញԿ 1×10^1.0 EID50/mLǴ؂ଫޜқಔ၂ᡍᚊଫ֡๏ϒ 300 μl ੰࢥనǶੰࢥనᐚࡋӧ 1×10^4.0 EID₅₀/mLਔǴԝΫ౗ၲډ 50 %(კΜ΋)ǶҗԜ่݀ளޕǴऩаᗺሷ๏ϒ 300 μl ཥ ࠤੰமࢥ(՘ᛯਲ਼)БԄװࢥǴځ 1 MLD ࣁ 1×10^4.0 EID50/mLǴ٠Ъჴᡍᚊଫ཮ӧװ ࢥࡕऊಃ 3-7 ϺϣԝΫǶࡺҁࣴزװࢥ၂ᡍஒ௦ҔǴᗺሷ๏ϒ 1,000 MLD ཥࠤੰ

மࢥ(՘ᛯਲ਼)Ǵջ๏ϒ؂ଫჴᡍᚊଫ 300 μL (1×10^7.0 EID50/mL)ཥࠤੰமࢥǶ

34

(2)༾ ༾િಈхᙟόࢲϯੰࢥᏊໆКၨᗺሷװࢥ่݀

ѤຼសқՅٰՎМᚊϩԋϖಔǴ؂ಔӚΜଫᚊǶϩձܭ๏ϒᗺሷௗᅿόӕᏊໆϐ όࢲϯཥࠤੰࢥלচǴϩձࣁ 60 μL (ࣁ Virus alone ಔ)Ǵ60 μL (ࣁ Lip PC-ND ಔ)Ǵ 30 μL (ࣁ Low dose Lip PC-ND ಔ)Ǵ120 μL (ࣁ High dose Lip PC-ND ಔ)аϷௗڙ PBS 30 μL (ࣁ Control ಔ)ǶܭխࣝࡕٿຼǴ຾Չװࢥ၂ᡍǴ؂ଫᚊᗺሷ๏ϒ 1,000 MLD ཥࠤமࢥ(՘ᛯਲ਼)Ǵջ๏ϒ؂ଫჴᡍᚊଫ 300 μL (1×10^7.0 EID50/mL)ཥࠤம ࢥǶӧ๏ϒ΋ԛௗᅿࡕޑჴᡍᚊଫύǴ60 μL (ࣁ Lip PC-ND ಔ)ёၲډ 80%ޑߥៈ

ΚǴ120 μL (ࣁ High dose Lip PC-ND ಔ) ёၲډ 50%ޑߥៈΚǴԶ 30 μL (ࣁ Low dose Lip PC-NDಔ)໻Ӹ 20%ᚊଫӸࢲǶ60 μL (ࣁ Virus alone ಔ)໻Ӹ 10%ᚊଫӸ ࢲǶௗڙ PBS 30μL (ࣁ Control ಔ)ᚊଫ߾ֹӄԝΫ(߄Ѥ)Ƕ

(3)િӭᗐᡏ༡ᙟ༾િಈхᙟόࢲϯཥࠤੰࢥКၨᗺሷװࢥ่݀

٩ྣխࣝௗᅿԛኧϩࣁ΋ԛխࣝ(Μଫ)ǵΒԛխࣝ(Μଫ)کΟԛխࣝ(Μଫ)Ƕխࣝ

ය໔໔႖ࣁ΋ຼǴϩձܭ๏ϒᗺሷௗᅿӕᏊໆ(60 μL)Ǵՠόӕႝ಻༾િಈхᙟό ࢲϯཥࠤੰࢥלচǴϩձࣁ Virus alone ಔǵ PC-Lip ಔǵPS-Lip ಔǵSA-Lip ಔǴ

ௗᅿӕᏊໆ(160 μLǴځύ༾િಈࣝभ 60 μL ᆶિӭᗐᡏ LPS 100 μL)όӕႝ಻༾િ

ಈхᙟόࢲϯཥࠤੰࢥלচӕਔ༡ᙟિӭᗐᡏ LPSǴϩձࣁ Virus-LPS ಔǵ PC-Lip-LPSಔǵPS-Lip-LPS ಔǵSA-Lip-LPS ಔǴаϷௗڙ PBS 30 μL (ࣁ Control ಔ)Ƕӧ΋ԛխࣝࡕٿຼǴᒿᐒڗΜଫᚊ຾Չᗺሷװࢥ၂ᡍǶӧΒԛխࣝࡕٿຼǴ ᒿᐒڗΜଫᚊ຾Չᗺሷװࢥ၂ᡍǶӧΟԛխࣝࡕٿຼǴᒿᐒڗΜଫᚊ຾Չᗺሷװ ࢥ၂ᡍǶᗺሷװࢥੰࢥᏊໆࣁ؂ଫჴᡍᚊଫ 300 μL (1×10^7.0 EID50/mL)ཥࠤੰம ࢥǶҗ߄ϖ܌ҢǴӧ๏ϒ΋ԛௗᅿࡕޑჴᡍᚊଫύǴPC-Lip ಔёၲډ 80 %ޑߥៈ

ΚǴPC-Lip-LPS ಔ߾ၲډ 40%ޑߥៈΚǴԶځᎩѤঁಔ߾໻Ԗեܭ 30 %ޑߥៈΚǶ ځύа Virus alone ಔനৡǴ໻Ԗ 10%ޑߥៈΚǶӧ๏ϒΒԛௗᅿࡕޑჴᡍᚊଫύǴ

35

PC-Lipಔёၲډ 90 %ޑߥៈΚǴPC-Lip-LPS ಔǵPS-Lip ಔǵPS-Lip-LPS ಔ֡ၲ

ډ 80%ޑߥៈΚǶVirus alone ಔǵVirus-LPS ಔک SA-Lip-LPS ಔ໻ၲډ 40%ޑߥ

ៈΚǶӧ๏ϒΟԛௗᅿࡕޑჴᡍᚊଫύǴPC-Lip ಔǵPC-Lip-LPS ಔک PS-Lip-LPS ಔёၲډ 100 %ޑߥៈΚ(߄ϖ)Ƕ

(ΐ ΐ)ᗹ๱ጤϐᗹࡋϩ݋

ஒ༾િಈᆶғނᗹ๱ጤ֡Ϭషӝࡕܭ 41ɗǴ୊ϪΚࣁ 0.09 s^-1 ளډϐᗹࡋॶ࣬К

ၨǴӵ߄Ϥ܌ҢǶӧԜךॺۓကပӧ 500~600 cp ࣁଯᗹࡋނ፦Ǵပӧ 200~300 cp ࣁύᗹࡋނ፦(߄Ϥ)Ƕ

(Μ)ᗹ๱ጤ༡ᙟ༾િಈхᙟόࢲϯཥࠤੰࢥϐלᡏϩ݋

(1)ᗹጢלᡏ߄౜ໆ

ѤຼសқՅٰՎМᚊϩԋϖಔǴ؂ಔӚΖଫᚊǶϩձܭ๏ϒᗺሷௗᅿӕᏊໆϐό ࢲϯཥࠤੰࢥלচǴϩձࣁ100 μL (ࣁNDV aloneಔ)Ǵ100 μL (ࣁLip-PC-NDVಔ)Ǵ 200 μL (ࣁT+Lip-PC-NDVಔ)Ǵ200 μL (ࣁXG+Lip-PC-NDVಔ)аϷௗڙPBS 30 μL (ࣁControlಔ)ǶӅ຾ՉٿԛᗺሷխࣝௗᅿǴܭΒԛխࣝࡕٿຼ຾Չᚊଫ឴࣊аڗள ሷ๚ᆶ਻ᆅؑࢱనǶҗሇનխࣝೱ่ϩ݋ݤ(ELISA)КၨǴሷ๚ᆶ਻ᆅؑࢱనύϩ ݜࠠs-IgAלᡏКၨǴT+Lip-PC-NDVಔӧᗹጢϩݜനӭໆϐAnti-NDV s-IgAלᡏ (p<0.01)Ǵ ځ ԛ ߾ ࣁ Lip-PC-NDV ಔ ϩ ݜ לᡏໆ ଯ ܭ XG+Lip-PC-NDV ಔ ᆶ NDV aloneಔ(p<0.05)(კΜ΋)Ƕ

(2)Ոమלᡏ߄౜ໆ

ᚊଫௗڙޑխࣝीฝӕ΢ǴܭΒԛխࣝࡕٿຼ຾Չᚊଫᓉે௦ՈǶҗሇનխࣝೱ

่ϩ݋ݤ(ELISA)КၨǴՈమύלᡏໆനଯࣁT+Lip-PC-NDVಔ(p<0.01)Ǵځԛ߾ࣁ

36

XG+Lip-PC-NDVಔ(კΜΒ)Ƕ

(3)Ո Ոౚ׭ڋᏉ໣၂ᡍϩ݋

ᚊଫௗڙޑխࣝीฝӕ΢ǴܭΒԛխࣝࡕٿຼ຾Չᚊଫᓉે௦ՈǶҗՈౚ׭ڋᏉ

໣၂ᡍϩ݋ՈమύלᡏύکੰࢥϐૈΚǶനଯޑՈౚ׭ڋᏉ໣၂ᡍלᡏΚሽ256ࣁ T+Lip-PC-NDVಔǴځԛ߾ࣁΚሽ64ឦXG+Lip-PC-NDVಔǵPC-Lip-NDVಔکVirus aloneಔ(߄Ύ)Ƕ

(4)װࢥࡕ่݀

ᚊଫௗڙޑխࣝीฝӕ΢ǴܭΒԛխࣝࡕٿຼ຾Չᚊଫᗺሷװࢥ၂ᡍǴᗺሷװࢥ

ੰࢥᏊໆࣁ؂ଫჴᡍᚊଫ300 μL (1×10^7.0 EID50/mL)ཥࠤੰமࢥǶҗ߄ΎᡉҢǴװࢥ ࡕᚊଫӧӚಔ໔วੰރᄊǴځύ௓ڋಔӄᡏӧಃ6.5Ϻౢғཥࠤੰޑᖏ׉ੱރǴӧ

ಃ8.5ϺԝΫຬၸъኧǶVirus aloneಔǵPC-LipಔکXG-PC-Lipಔӧಃ7ϺԖъኧᚊ ଫ ౢ ғ ᖏ ׉ ੱ ރ Ƕ T-PC-Lip ಔ ӧ װ ࢥ ࡕ Ԗ 100 % Ӹ ࢲ ౗ Ǵ Lip-PC-NDV ಔ ǵ XG+Lip-PC-NDVಔᆶNDV aloneಔ֡ѝᆢ࡭60 %Ӹࢲ౗(߄Ύ)Ƕ

(5)ᔠᡏϩ݋ੰࢥූӸໆ

ஒװࢥࡕӸࢲޑᚊଫаΒ਼ϯᅹ឴࣊Ǵڗр਻ᆅǵط᠌ǵ๠᠌کޤ᠌ᢀჸӧᚊᡏ ϣࢂցѻූ੮ Sato ੰࢥǶSato ੰࢥޑ RNA ࿶ RT-PCR ϩ݋ࡕ௓ڋಔᚊଫᡏϣූ੮ ޑੰࢥໆനӭЪӧӚঁϩ݋೽Տ೿ԖੰࢥޑූӸǶT-PC-Lip ಔӧ਻ᆅؒԖੰࢥ

ූ੮Ǵՠࢂӧޤ᠌ǵ๠᠌کط᠌೿ёаୀෳډϿໆޑੰࢥ RNAǶVirus alone ಔǵ PC-Lipಔک XG-PC-Lip ಔӧӚ೽Տ೿Ԗੰࢥޑ RNAǴ࿶ PCR ᔠۓϩ݋ډੰࢥ RNA ໆ߾֡คܴᡉৡ౦(߄Ζ)Ƕ

37

(Μ Μ΋)༾િಈܭᚊ๠᠌ѮᏘಒझࢲ܄ϐቹៜ (1)Ӆ೫ขᡉ༾᜔

ᚊ๠᠌ಒझ୻Ꭶܭ 41ɗЪ 5% CO2ޑಒझ୻ᎦጃǴуΕόӕႝ಻ಔԋϐᑻӀ༾

િ ಈ Ǵ Ӆ ӕ ୻ Ꭶ 24 λ ਔ Ƕ Ӆ೫ขᡉ༾᜔ Leica TCS SP5 Confocal Spectral Microscope Imaging System ᢀჸᑻӀ༾િಈ೏๠᠌ಒझϐൂਡ/ѮᏘಒझझտ ໆǴҗკΜΟᡉҢǴ༾િಈԋҽ PC-Lip ᆫ໣ၨӭໆܭᚊ๠᠌ಒझύǴځԛ߾ࣁ

༾િಈԋҽ SA-Lip炻Զ೏झտໆനϿޑࣁ༾િಈԋҽ PS-Lipˤ

(2)༾િಈಒझࢥ܄ϐϩ݋

ஒόӕ׷਑ϐ՘ᏊуΕᚊ๠᠌ѮᏘಒझӅӕ୻Ꭶ 24 λਔǴҗ MTS ၂ᡍϩ݋(კΜ Ѥ)ǴёޕӧჴᡍύᒧҔޑ׷਑೿όڀࢥ܄ǴᡉҢӧҁჴᡍύޑ༾િಈ׷਑ᆶғނ ᗹ܄׷਑ᆶ༾િಈ೿ёа٬ಒझ҅தғߏǶ

(3)ѮᏘಒझࢲϯ΋਼ϯේϐញܫ

՘Ꮚڈᐟխࣝಒझ٬ᚊޑѮᏘಒझౢғ NO ޑញрǶஒ՘Ꮚᆶᚊ๠᠌ѮᏘಒझӅ ӕ୻Ꭶ 24 λਔǴಒझ΢మనа Greiss reagent ୀෳ NO ޑ֖ໆǶ௓ڋಔޑᚊ๠᠌Ѯ ᏘಒझౢғϿໆޑ NOǶ๏ϒ༾િಈޑѮᏘಒझౢғޑ NO ࣁ 6.09±4.7 uM (p<0.05)Ǵ๏ϒΟиጤ܈િӭᗐᡏޑಔձ֡ౢғ NOǴЪᆶᏊໆԋ҅࣬ᜢ܄ǶฅԶǴ

๏ϒሌԸӭᗐޑಔձ߾คౢғ NOǶᡉҢ༾િಈǵΟиጤᆶિӭᗐᡏ֡཮೷ԋѮᏘ ಒझޑࢲϯ(კΜϖ)Ƕ

ӕኬӦǴךॺஒΟᅿόӕႝ಻׷਑ޑ༾િಈуΕ୻ᎦనύᆶѮᏘಒझӅӕ୻Ꭶ 24 λਔǴӚಔԋҽ༾િಈញܫрޑ NO ᆶ༾િಈᏊໆԋ҅࣬ᜢ܄Ǵځύ PS-Lip ౢғ നӭ NO(* p<0.05Ǵ**p<0.01 ᆶ௓ڋಔКၨ)ǶPC-Lip ڈᐟ NO ౢғޑໆԛϐǴӧ

38

٬ҔᏊໆࣁ 5 μL ޑ PC-Lip ਔ߾ܴᡉғԋ NO(# p<0.05)ǶSA-Lip ЇଆѮᏘಒझࢲ ϯޑ NO ໆ߾ࣁനϿ(კΜϤ)Ƕ

׳຾΋؁Ǵךॺӧಒझ୻ᎦనύуΕ׭ڋᏊǺϩձࣁ U0126 ׭ڋ MAPK kinase (MEK)ૻ৲໺ሀǵSB-203580 ׭ڋ p-38 MAPK ૻ৲໺ሀک Bay 11-7085 ׭ڋ IκB ᕗ ለϯૻ৲໺ሀǶU0126 ׭ڋᏊ׭ڋૻ৲໺ሀၡ৩ύ MAPK ৎ௼ԋ঩ϐ΋ޑ MEK1/2 ࣁ ERK ၡ৩ޑ΢ෞǴӧ٬Ҕ U0126 ׭ڋᏊКၨǴU0126 ׭ڋ LPS (1 μg/mL Ϸ 10 μg/mL)ڈᐟғԋޑ NOǴU0126 ׭ڋᏊᏊໆቹៜ׭ڋ NO ޑໆ(კΜΎ)ǶSB-203580 ࣁ pyridinyl imidazoles ϐ΋Ǵ׭ڋ p38-MAPK ૻ৲໺ሀၡ৩ǴSB-203580 (1 μM ܈ 5 μM)೿όૈ׭ڋ༾િಈڈᐟౢғ NOǴSB-203580 ዴૈ୼׭ڋ LPS ౢғ NOǴ׭ڋ Ꮚᐚࡋཇଯ׭ڋਏ݀ཇܴᡉ(კΜΖ)Ƕૉ෎୏ނಒझϣ NF-κB ৎ௼ᙯᒵӢηऩϝ

ೀܭᓉЗރᄊǴ߄Ң NF-κB ᆶ׭ڋࠠ IκB ೈқ፦׎ԋፄӝނǶ྽ಒझϣૻ৲໺ሀ Ӣη೏ࢲϯਔǴIκB ೈқ፦೏ᕗለϯЪҗ NF-κB ញܫǴௗ๱຾Չಒझਡޑᒪ໺ܰ

ՏǴௗ๱௴୏୷Ӣޑᙯ᝿ǶBay 11-7085 ᙖҗ׭ڋ IκB ᕗለϯаٛЗ NF-κB ޑࢲϯǶ Bay 11-7085ૈ୼׭ڋ༾િಈғԋ NOǴ٬Ҕޑ Bay 11-7085 ᐚࡋཇଯ׭ڋਏ݀ཇܴ

ᡉ炻٠Ъૈ୼ֹӄӦ׭ڋ LPS ౢғ NO(კΜΐ)Ƕ

ӧ༾િಈ՘ᏊᆶѮᏘಒझӅӕ୻Ꭶ 24 λਔǴҗ٬Ҕޑ׭ڋᏊёᇥܴ༾િಈڈᐟ٠ ࢲϯխࣝಒझޑૻ৲໺ሀၡ৩Ьाࣁࢲϯ NF-κB ၡ৩ᆶ MEK ၡ৩Ƕ

(4)ೈ ೈқ፦ᕗለϯૻ৲໺ሀ

༾િಈࣝभᗺሷࡕٿຼǴ༾િಈࣝभЇଆԖਏޑᗹጢלᡏᆶՈమלᡏǴӧ࿶җװ ࢥࡕว౜༾િಈࣝभ໻Ԗ60 %ޑߥៈ౗ǴӧT-PC-LipಔӧװࢥࡕԖ100 %Ӹࢲ౗Ƕ

༾િಈૈ୼ԖਏӦࢲϯѮᏘಒझ٠ڈᐟғԋNOǴՠࢂሌԸӭᗐคݤڈᐟѮᏘಒझ ғԋNOǴคݤᇥܴሌԸӭᗐޑխࣝᐒڋǶӢԜǴҁჴᡍ੝ӧᚊխࣝ༾િಈࡕڗள

39

๠᠌Ǵаᕕှ༾િಈӧᚊي΢ޑૻ৲໺ሀၡ৩ǴаՋБᗺᏀݤϩ݋ᚊ๠᠌ϣౢғ ޑૻ৲໺ሀೈқ፦Ƕךॺϩ݋p-38 MAPKǵp-ERKکp-NF-κB炻ว౜ӧխࣝٿຼࡕ

܌Ԗಔձޑᚊ೿คp-38 MAPKکp-NF-κBǴ୤ᐱӧp-ERKޑၡ৩Кၨ΢Ԗৡ౦Ǵځ ύT-PC-Lipಔೈқ፦ᕗለϯໆၨϿǴԶVirus aloneಔǵPC-LipಔکXG-PC-Lipಔ೿

Ԗܴᡉޑp-ERKӸӧ(კΒΜ)Ƕ