seminario biomol
TRANSCRIPT
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Peizhi Li, Min Li, Kun He, Kaichan Zhong, JianpingGong, and Haibo You.
MARÍA ANGÉLICA DÍAZ URIBEDANIEL DUQUE
Molecular biology
THE EFFECTS OF TWIST-2 ON LIVER ENDOTOXIN TOLERANCE INDUCED BY A LOW DOSE OF LIPOPOLYSACCHARIDE
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(LPS) are polymers structures located in the outer membrane of bacterial cell
in the pathogenesis of infectious disease, as well as in the interaction with host immune system.
LIPOPOLY-SACCHARIDE
IMPORTANCE
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ENDOTOXIN
They are polysaccharidecomplex that is combined witha lipid and released from thecell walls of Gram-negativebacteria in the cell lysismoment, producing toxiceffects.
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SEPSIS
Response Host`smechanism
caused by therelease of certain
compounds of the invasive
microorganismssuch as
endoxotins, telcoico acid, etc.
Activate cellularmediators:
macrophages, neutrophils,
endothelial cells
which releasesuncontrollably
several humoral mediators
(TNF- alfa, IL-1, IL-6)
EndothelialDamage
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TWIST-2 PROTEIN
Is a basic helix–loop–helix transcription
factors.
Act cooperatively to regulate the expression
of other genes.
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RELATION
There is a possible way to regulate theexpression of inflammatory genesthrough twist-2 protein that can actas negative regulator for cytokinesignaling by establishing a negativefeedback loop that repressed the NF–κB-dependent cytokine pathway.
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In this study, the main objectivewas show the relationshipbetween Twist-2 and liverendotoxin tolerance, developingan animal model of ET toobserve the changes of Twist-2expression in liver tissues.
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Ratones machos BABL
n= 24/grupo
Grupo ETT
Pre tratados con inyección de LPS
4 Subgrupos de 6 ratones
Grupo NETT
Inyección salina estéril
Inyección de LPS
Sacrificar, recoger muestras de
hígado y sangre
24hrs
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TOLERANCIA A LA ENDOTOXINA
FUN
DA
MEN
TO
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
FIN
ALI
DA
D desarrollo de diferentes pruebas con el objetivo de comprobar si La Twist-2 pude actuar como un gen silenciador.
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TRANSFECCIÓNTWIST-2 SHRNA
FINALIDAD
Comprobar si Twist-2 shRNA eraeficiente para la inhibición de laexpresión de genes proinflamatorios
FUNDAMENTO
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Los cortes en parafina con (HE) se realiza para detectar los cambios morfológicos por microscopía de luz.
marcador en la reacción inflamatoria, para comprobar si la tolerancia a la endotoxina fue efectiva.
CAMBIOS HEPATICOS
FINALIDAD
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INMUNOCITOQUÍMICAFU
ND
AM
ENTO
Técnica de inmunotinción
demostrar una variedad de antígenos
MET
OD
O Se utilizan anticuerpos marcados y con la R(x) Ag –Ac las estructuras se colorean con diferentes reactivos.
FIN
ALI
DA
D
La tinción de inmunocitoquimica se realizó para detectar la expresión de Twist-2
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FINALIDAD
RT-PCR investigar:
la relación entre Twist-2 y TNF-α enET
la expresión de Twist-2 Y TNF-αARNm
En el tejido del hígado y KCS
http://www.youtube.com/watch?v=HdK-Fe6wnm8
REACCIÓN EN CADENA DE LA POLIMERASA CON TRANSCRIPCIÓN INVERSA (RT-PCR) Técnica para
el estudio de virus de
ARNm
Amplificación de Genes
Expresión génica a partir de ARN
DNA Polimerasa
transcriptasa inversa
DNAc
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WESTERN BLOT
Técnica analítica
localizar proteínas
capacidad de unión a anticuerposespecíficas mediante unaelectroforesis en gel
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FINALIDAD
Explorar los efectos de Twist-2 sobre NF-kB trans-activación en los
KC.
Control de la transcripción del
ADN.
ANALISIS
DE NF-kB
Factor nuclear potenciador de las
cadenas ligeras kappa de las
células B activadas
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Ensayo inmunoenzimático (ELISA)
FINALIDAD
Se utilizó ELISA paradeterminar los niveles de TNF-α en el plasma y elsobrenadante de las célulascultivadas de acuerdo con elprotocolo.
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FIGURA 1: Cambios histopatológicos deltejido hepático 24 horas después de lasegunda estimulación con LPS.
Se demuestra que la tolerancia aendotoxinas ayuda a que la lesiónhepática no progrese tanrápidamente.
Grupo ETT
Grupo NETT
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FIGURA 1: Análisis de RT-PCR de niveles relativos del mRNA de Twist-2
.
Tejido hepático
Células de Kupffer
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Figura 3: Análisis de Western blot de la
expresión de la proteína Twist-2 en KC.
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Figura 4: KC normales (control negativo) y tinción inmunocitoquímica del Twist-2 en KC aisladas de los grupos NETT y ETT.
ETT
NETT Normal
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Figura 5: Análisis de RT-PCR de los niveles de mRNA del TNF- α
Tejido hepático
Células de Kupffer
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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.
• Los resultados coincidieron con la expresión del mRNA del TNF- α.
MEDIO DE CULTIVO DE KCSUERO
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Figura 7: Cambios en la activación del NF- KB en tejido hepático y KC en NETT y ETT
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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.
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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.
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AUTHOR IDEA - X
Fairfax, B.P et al.
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].
Peng, Q. et al.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 IL-1 receptor-associated kinases, TNF receptor associated factor 6, and cellular inhibitor of apoptosis proteins at the cytoplasmic face of the plasma membrane [19, 20].
DISCUSSION
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AUTHOR IDEA - X
Sharabi, A.B. et al.
This process contribute to the activation of two different pathways that involve the Rel family transcription factor NF–κB and c-Jun NH2-terminal kinase(JNK), p38 mitogen-activated protein kinase family. Meanwhile, triggering of TLRs also induced Twist-2 proteinsexpression 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+.
DISCUSSION
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• This topic must be investigate more, because today they don't know some ofaction's mechanisms about how Twist- 2 operates, although much progresshas been made.
• In future, Twist- 2 could be a part of treatment of sepsis another bacterialinfections that promote inflammation and shock, until now it has seen thepositive consequences in maintenance of liver function, an important organrelated with organism homeostasis.
• laboratory tests are a useful tool to check gene regulation. using manypositive laboratory tests support the efficacy of the procedure.
• These results are encouraging for the management of immunosuppression insepsis and/or noninfectious shock, and deserve further investigation in thefuture, however the effects can make counterproductive in the human body.
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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
http://digital.bl.fcen.uba.ar/gsdl-282/cgi bin/library.cgi?a=d&c=tesis&d=Tesis_4874_Rearte