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Seminario biomol
1. THE EFFECTS OF TWIST-2 ON LIVER
ENDOTOXIN TOLERANCE INDUCED
BY A LOW DOSE OF
LIPOPOLYSACCHARIDE
Peizhi Li, Min Li, Kun He, Kaichan Zhong, Jianping
Gong, and Haibo You.
MARÍA ANGÉLICA DÍAZ URIBE
DANIEL DUQUE
Molecular biology
2. LIPOPOLY(LPS) are polymers structures
SACCHARIDE
located in the outer membrane of
bacterial cell
IMPORTANCE in the pathogenesis of infectious
disease, as well as in the interaction
with host immune system.
4. SEPSIS
Response Host`s
mechanism
caused by the
release of certain
compounds of
the invasive
microorganisms
such as
endoxotins,
telcoico acid, etc.
Activate cellular
mediators:
macrophages,
neutrophils,
endothelial cells
which releases
uncontrollably
several humoral
mediators
(TNF- alfa, IL-1,
IL-6)
Endothelial
Damage
5. TWIST-2 PROTEIN
Is a basic helix–loop–
helix transcription
factors.
Act cooperatively to
regulate the expression
of other genes.
6. RELATION
There is a possible way to regulate the
expression of inflammatory genes
through twist-2 protein that can act
as negative regulator for cytokine
signaling by establishing a negative
feedback loop that repressed the NF–
κB-dependent cytokine pathway.
7. In this study, the main objective
was show the relationship
between Twist-2 and liver
endotoxin tolerance, developing
an animal model of ET to
observe the changes of Twist-2
expression in liver tissues.
8. Ratones machos
BABL
n= 24/grupo
Grupo ETT
Grupo NETT
Pre tratados con
inyección de LPS
Inyección salina
estéril
24hrs
Inyección de LPS
4 Subgrupos de 6
ratones
Sacrificar, recoger
muestras de
hígado y sangre
9. Es un
mecanismo de
protección
sepsis o una
inflamación
sistémica
(TGF)-β o (GC),
conducen al
huésped a un
estado de
refractariedad
temporal frente
a una nueva
exposición al
LPS
FINALIDAD
FUNDAMENTO
TOLERANCIA A LA ENDOTOXINA
desarrollo de
diferentes
pruebas con el
objetivo de
comprobar si La
Twist-2 pude
actuar como un
gen silenciador.
11. CAMBIOS
HEPATICOS
Los cortes en parafina con (HE) se realiza para
detectar los cambios morfológicos por
microscopía de luz.
FINALIDAD
marcador en la reacción inflamatoria, para
comprobar si la tolerancia a la endotoxina fue
efectiva.
12. demostrar una
variedad de
antígenos
Se utilizan
anticuerpos
marcados y con
la R(x) Ag –Ac
las estructuras
se colorean
con diferentes
reactivos.
FINALIDAD
Técnica de
inmunotinción
METODO
FUNDAMENTO
INMUNOCITOQUÍMICA
La tinción de
inmunocitoquimi
ca se realizó para
detectar la
expresión de
Twist-2
13. REACCIÓN EN CADENA DE LA POLIMERASA
CON TRANSCRIPCIÓN INVERSA (RT-PCR)
Técnica para
el estudio
de virus de
ARNm
FINALIDAD
RT-PCR investigar:
la relación entre Twist-2 y TNF-α en
ET
la expresión de Twist-2 Y TNF-α
ARNm
Amplificación
de Genes
Expresión génica
a partir de ARN
DNA
Polimerasa
En el tejido del hígado y KCS
DNAc
http://www.youtube.com/watch?v=HdK-Fe6wnm8
transcriptasa
inversa
14. WESTERN BLOT
Técnica analítica
localizar proteínas
capacidad de unión a anticuerpos
específicas
mediante
una
electroforesis en gel
15. ANALISIS
FINALIDAD
DE NF-kB
Factor nuclear
potenciador de las
cadenas ligeras
kappa de las
células B activadas
Control de
la transcripción del
ADN.
Explorar los
efectos de Twist-2
sobre NF-kB transactivación en los
KC.
17. FIGURA 1: Cambios histopatológicos del
tejido hepático 24 horas después de la
segunda estimulación con LPS.
Se demuestra que la tolerancia a
endotoxinas ayuda a que la lesión
hepática
no
progrese
tan
rápidamente.
Grupo
ETT
Grupo
NETT
19. Figura 3: Análisis de Western blot de la
expresión de la proteína Twist-2 en KC.
20. Figura 4: KC normales (control negativo) y
tinción inmunocitoquímica del Twist-2 en KC
aisladas de los grupos NETT y ETT.
Normal
ETT
NETT
21. Figura 5: Análisis de RT-PCR de los niveles
de mRNA del TNF- α
Tejido
hepático
Células de
Kupffer
22. Figura 6: Análisis de ELISA de los cambios en las
concentraciones del TNF- α en suero y medio de cultivo
de KC a 0, 1, 3 y 6h.
SUERO
MEDIO DE CULTIVO DE KC
• Los resultados coincidieron con la expresión del mRNA del TNF- α.
23. Figura 7: Cambios en la
activación del NF- KB en tejido
hepático y KC en NETT y ETT
24. Figura 8: Análisis de RT- PCR de los niveles del
mRNA de Twist-2 en KC, 24h después de
transfectada con shRNA Twist-2 y shRNA
control.
25. Figura 9: Efectos del shRNA del Twist- 2
en activación del KF- KB en KC del grupo
control, ETT y NETT 3h después del
tratamiento.
26. DISCUSSION
AUTHOR
Fairfax, B.P et
al.
Peng, Q. et al.
IDEA
Endotoxin tolerance is a significant protective mechanism to
prevent the LPS induced “cytokine storm” associated with onset
of sepsis and shock. Although a lot of molecular mechanisms
had been found, additional biological bases of this critical
immunological process were yet to be defined [18].
TLR4 is the major receptor for LPS; its function in LPS initiated
singling pathway and ET were investigated in many researches.
TLR4 engagement by LPS results in the recruitment of myeloid
differentiation primary response protein 88 (MYD88) and the
rapid assembly of a large multiprotein complex which include IL1 receptor-associated kinases, TNF receptor associated factor 6,
and cellular inhibitor of apoptosis proteins at the cytoplasmic
face of the plasma membrane [19, 20].
- X
27. DISCUSSION
AUTHOR
IDEA
This process contribute to the activation of two different pathways
Sharabi, A.B. et that involve the Rel family transcription factor NF–κB and c-Jun
al.
NH2-terminal kinase(JNK), p38 mitogen-activated protein kinase
family. Meanwhile, triggering of TLRs also induced Twist-2 proteins
expression in dendritic cells (DCs) [21].
Šošić, D. et al.
There were evidences linking Twist-2 to TLR signaling, since Twist-2
knockout exhibit increased LPS sensitivity, associated with elevated
TNF-α, IL-6, and IL-12 secretion and lack of ET in DCs. Furthermore,
Twist-2 can negatively regulate mammalian cytokine gene
expression through interaction with p65 and bind to the promoters
of the TNF-α and IL-1β genes in vivo and in vitro *10+.
- X
28. •
This topic must be investigate more, because today they don't know some of
action's mechanisms about how Twist- 2 operates, although much progress
has been made.
•
In future, Twist- 2 could be a part of treatment of sepsis another bacterial
infections that promote inflammation and shock, until now it has seen the
positive consequences in maintenance of liver function, an important organ
related with organism homeostasis.
•
laboratory tests are a useful tool to check gene regulation. using many
positive laboratory tests support the efficacy of the procedure.
•
These results are encouraging for the management of immunosuppression in
sepsis and/or noninfectious shock, and deserve further investigation in the
future, however the effects can make counterproductive in the human body.
31. Peizhi Li, Min Li, Kun He, Kaichan Zhong, Jianping Gong, and Haibo You. the
effects of twist-2 on liver endotoxin tolerance induced by a low dose of
lipopolysaccharide
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