ĐẠI HỌC QUỐC GIA HÀ NỘI
TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN

Nguyễn Thị Tươi NGHIÊN CỨU KHẢ NĂNG CHỐNG VIÊM CỦA
CÂY NHỌ NỒI VÀ NGẢI CỨU THÔNG QUA THỤ
THỂ TLR4
LUẬN VĂN THẠC SĨ KHOA HỌC


TS. Trịnh Tất Cường Hà Nội - 2012
Luận văn tốt nghiệp Nguyễn Thị Tươi MỤC LỤC Trang
MỞ ĐẦU
1
Chƣơng 1. TỔNG QUAN
3
1. Khái niệm viêm
3
2. Tình hình nghiên cứu trong và ngoài nƣớc
3

2.1. Tình hình nghiên cứu ngoài nƣớc
3 2.1.1. Các chất kháng viêm
3


ng viêm
7 2.1.4.Tế ba
̀
o marcrophage
12 2.1.5. Các mô hình sàng lọc chất kháng viêm in vitro
13
2.1.5.1. 









  th 


NF-B (NF-B dependent luciferase reporter assay)
13







(TLRs)
17
2.1.5.4.












LPS
19

2.2. Tình hình nghiên cứu trong nƣớc
20
3. Cây nhọ nồi và cây ngải cứu

2.1.2. Hóa chất
27 2.1.3. Thiết bị nghiên cứu
27

2.2. Phƣơng pháp nghiên cứu
27 2.2.1.Tách chiết macrophage từ tủy xƣơng của chuột.
27 2.2.2.Đánh giá hoạt tính độc tố của Wedelolactone tới khả năng
sống của BMDM bằng kít CCK-8
27 2.2.3. Đánh giá khả năng sản xuất cytokine bằng ELISA Kit
28
2.2.3.1. Gii thiu v 
28

Luận văn tốt nghiệp Nguyễn Thị Tươi Chƣơng 3- KẾT QUẢ VÀ THẢO LUẬN
38

3. 1. Khả năng sống của tế bào không bị ảnh hƣởng bởi Wedelolactone
38

3.2. LPS kích thích tế bào BMDM sinh cytokine
39

3.3. Wedelolactone ức chế quá trình sinh cytokine thông qua thụ thể
TLR4 kích thích bằng LPS
40

3.4. LPS kích thích hoạt động của yếu tố tự phân bào (MAPK) thông
qua thụ thể TLR4
41

3.5. Wedelolactone ức chế quá trình phospho của yếu tố tự phân bào
bằng LPS kích thích thông qua thụ TLR4
42

3.6.Wedelolactone đã ức chế quá trình sinh ROS thông qua thụ thể
TLR4 kích thích bằng LPS
43

3.7. LPS kích thích hoạt động p47 phox trong quá trình tạo ra ROS
44


TT
Từ viết tắt
Nghĩa từ viết tắt
1.
BMDM
Bone Marrow-Derived Macrophage
2.
ELISA
Enzyme-Linked ImmunoSorbent Assay
3.
IL
Interleukin
4.
LPS
Lipopolysaccharides
5.
NF-
Nuclear factor kappa-light-chain-enhancer of activated B
cells
6.
ROS
Reactive oxygen species
7.
TLR
Toll-like receptor
8.
TNF
Tumor necrosis factor/ Tumor necrosis factor alpha
9.

Trang
h 1.










Glucocorticoid 


7

a Glucocorticoid
7

hong ca mt s TLR
10









(GR)
16















(TLRs)
18









in vivo 


trong BMDM
42
7.
Wedelolactone hoc Dex c ch      c

43
Luận văn tốt nghiệp Nguyễn Thị Tươi 18.
       a p47phox trong
BMDM.
44
19.
Wedelolactonc ch a p47phox
45
20.
Du ngi ci vi t 
46
21.
Tinh du ngi c     c ch   
cytokine ting LPS
47


g yc tng hi thu
ng    
ng ph n. S dng thun
gc thc v gii quyt nhng lo ngu qu
p.
n nay, vit ch mn gc t thc vt
 i nhng hiu qu c    m
Tr i hc Khoa hc T 
N s t ch
        n 
u khc kh 
 


Lut nghip Nguyn Th  2

                
nh
           

  


M 

 do b m bnh hay
ng t  hm bo v ci b hay sa
cha nhng v   li. 





 , 

(migrating cells) :  , ,  , 

, 
 











 an, 


 rostaglandins (PG), leukotrienes (LT), histamine, bradykinin, 
 (PAF) -1. 




  dn ti hiu
qu 

  
        chi        
huyn mch, thic b t 
hu qu tip sau dn tt ch
 vong [37].
Lut nghip Nguyn Th  4

2. c
c
2.1.1. 
3 :
2.1.1.1.  (NSAID)





 (COX), 








 

 (PG) 
thromboxane (TX). 

 





 (NSAID) 
 . 
















 prostaglandins (PG) 





 
 . 












[42].
2.1.1.2.



prostaglandins (PG), thromboxane leukotrienes. 
 i

 2 










 







 , lipomodulin hay renocortin
 15, 30  40 kDa. 





5

2.1.1.3. 












 . 





 ,











enzyme lysosom . 




















glycoprotein 8400 Da. 








nhn dng nh   mm bnh (pathogen-associated molecular patterns -
PAMPs)  vi khum. Sau khi nhn dng mm b th s
truy 

  (mediator)


n dng
u [24, 12] 
cng s (2009) 





  th 
  u t           Acid Deoxyribo
Nucleic (AND)  8].
Glucocorticoid (GC) t ng thn sn xut, gm hai
ch
t. Glucocorticoid  ocorticoid
(GR). T(GR)  th th 
t 8]. T  
nh  n cu    

, th     t Acid
Deoxyribo Nucleic (AND)  n cut vi phi
t    i t c hi  t t b










 Acid Deoxyribo Nucleic (AND)  - nuclear factor kappa-
light-chain-enhancer of activated B cells) (1).
Glucocorticoid thc hi
bng ni sinh c sn thi t nhiu ho
hc quan trng, chng hn trng ni sinh cn kinh v
h thng tim m   n ng min d     m.
Glucocorticoid 



 c ch min dc
 vi nhiu bm, mn cm min dch, d 
n ca t ,  p kh Mc
 Glucocorticoid tng h   i di    t v ch 
 di sinh ra nhng phn ng ph .






 , c c c   







  n
c nhiu hp ch u qu 



 Glucocorticoid 





Lut nghip Nguyn Th  7


 ng s 





 c ch ng c
yu t 

- AP-1 (activator protein -1) [34].
Lut nghip Nguyn Th  8 2: 

a t [33]






  t 













 .
Nhng NF- b i phc h  b t bt c
cn tr 

  .  







 




 ng phi t t tng h  thc
v



9

 in vitro 





 t
glucocorticoid  in vivo 










 t  
(pharmacokinetic)  , 
245186, 









2.1.3. toll-






ng bm sinh vm
b c hiu co v vt ch. 1996, 


 



 
ch i trong s n ci vi rung hiu qu chng
li nm [30]. T  c chc








 ng min dch bm sinh. 
chu











.  





 (LRR) 






 (-), 




(+), , pipopeptide, roi (flagellin)






t sn xu
      u t t    
(mitogen-activated protein kinases-MAPKs), hay phn  [31]. 




, 

 
 n min d
. 

 , TLR4 tham gia hong trong h thng min d




bng lipopolysccharides vi khun dc hiu nh
t ngoc t  ng min dch vi vi
khu ca TLR truy hii s hp
  ng ca m    u, bao gm MyD88 (myeloid
ng adaptor ca MyD88, chng
h    u ca TLR2 ph thu    (myeloid
differentiation primary response protein 88), hay TLR4 ph thun protein
a MyD88/TIR (TIRAP) hon th th toll/IL-1 




  gii h



 

  tr 
c x  mm bnh v mc.  
m khong 60% trong tng s ng h
t truy 
thng min dch. Ngo,  (2005) 
 th nhn d



 







 n t bin
c l hong vt thim
khung vi LPS [10]. 







4 MD-i din ch

 t trong danh
c ti. 



, 
















4  u tr nhi
dh chu

quan 

.
TLR4 

n dng minh chu


t C3H/

1

t
bi

 o v chng li nc t
t b t bin TLR4  m vi
khu th



 [43ng ca TLR4 




 n dch bo v chng l
m ngoc t h th















 nh
m t nm, nhim gram ho


vi khu



[20, 25, 41].
2.1.4.




i thng t bch cuth
ng trong h min dch c hi min dc
hiu  ng vn
ca t  nh. 







 .

























 
Lut nghip Nguyn Th  13

 in vitro 
8].

4. 







 [47]
2.1.5. in vitro



























 in vivo. 


 in vitro  10 














































  th 



 -B (NF-B
dependent luciferase reporter assay)
NF-







ng nhanh ("rapid-acting" primary transcription
factors) 
gian, 

































 :
Macrophage
Granulocyte
Lut nghip Nguyn Th  14






. AP-1(activation Protein-1) 










 

- IL-1, IL-6, IL-12, 


PG. -

 -B
  nh  . 
disulfiram, olmesartan dithiocarbamates 





 -



 [33]. Trong m














 -B, 







 
































 ng hunh quang k (luminator). 











8 









 Andrographis paniculata 
[14]. 

, 








, 
 (iNOS) 







 








  1/2 3








 -



. , 
















Lut nghip Nguyn Th  15
























-B
   y t  M   sung LPS,
chuyn gen luciferase, b   ch chit thc vt hoc ch  u.
u kh c ch biu hic dch chit tim
c cht ti
2.1.5.2. 







glucocorticoid


















 ,  .  ng
 t xut t t p ca
th th glucocorticoid 



 th th glucocorticoid [46]. Gn
,  (2009) 





 t ch

Ưc ch biu
hin gen luciferase
phụ thuc NF-κB
- Dch chit tim năng
- Cht tim năng
Marcrophage mang
gen luciferase
phụ thuc NF-κB
Lut nghip Nguyn Th  16

glucocorticoid in vitro  in vivo   u qu ca Compound A 


 y dexamethasone vn dn tng huy
    Compound A 

   
n Compound A 

 ra mi trong vic s dng
i t ca th th glucocorticoid t t xut thc v



nh
n ng ph [19]. 















 . 














(TNF-, IL-6, IL-
10)  th th

(6).

T bo
macrophage
LPS
- Dch chit thc vt
- Cht nghiên cu
Đo mc độ c chế cht
trung gian gii phng ra
C thể l phi tử ca GR
trong mu nghiên cu
c chế biu hin gene
GR-siRNA trong macrophage
Marcrophage c
GR b c chế
Cht c chế
GR RU486
Khng đnh l phi tử ca GR
trong mu nghiên cu
T bo
macrophage
LPS
- Dch chit thc vt
- Cht nghiên cu




(GR)
ng v   y t  , b sung cht c ch GR
 ch chit thc vt hoc chu
m c ch t trung gian giT kt qu  khng
i t GR trong mu. c ch biu hin gene GR-siRNA trong
Macrophage t nh phi t u.

2.1.5.3. 













(TLRs)


 khi TLR cng vc ch




 .  

 n thc sinh h
s t k nhng liu tr vii, mang li hiu qu trong
u tr bnh. Nhi  c ph      ng h




 nhng cht c ch hoi t c hiu ca TLR, chng
h7909, imiquimod, resiquimod hay chloroquine [10]  chng minh




 dc bit, 








th nghi

u b

i. 