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Alpha-1 antitrypsin is a potential biomarker for hepatitis B
Xu-fei Tan, Shan-shan Wu, Shu-ping Li, Zhi Chen and Feng Chen
Alexander Aguilar AriasYulissa Victoria Bertel Serna
Biología Molecular
Universidad Pontificia BolivarianaFacultad Medicina
2011
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INTRODUCTION
HEPATITIS B
Liver disease caused by hepatitis virus
Belonging to the family Hepadnaviridae
Hepatocellular necrosis or inflammation
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It may be acute or chronic
Can result in: liver cirrhosis, liver cancer, liver failure and even death
One of the most common infections worldwide
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POSSIBLE CAUSES TO HEPATITIS B
Sex without condom use
Infected needlesToothbrush
or razor from an infected person
Blood transfusions
Spread the baby's mother at
the time of birth or breast-
milk
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ALPHA-1 ANTITRIPSIN
Serum protease inhibitor
Consists of 394 amino acids and three carbohydrate side chains.
Produced by hepatocytes
Principal defense in the lung against elastase
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Its concentration varies between 120 – 220 mg/dl measured by nephelometry
Is distributed 40% serum and 60% in the extravascular space
Electrophoresis is not very accurate so it is analyzed by electrofocusing
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AAT RELATIONSHIP WITH HEPATITIS
The liver secretes 34 mg/kg/24 hours
In inflammatory processes, tumor and infection can be increased from 3 to 5 times
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GENERAL AIM
To identify the major proteins in the progression of HBV infection from
mild stage to severe stage
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MATERIALES Y MÉTODOS
Información de los pacientes
31 pacientes con hepatitis B crónica.
10 convaleciente de hepatitis B aguda (AHB).
18 relacionados con el carcinoma hepato-celular (HCC).
12 donantes de sangre sanos (controles normales) se inscribieron en este estudio.
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Aprobado por el comité de ética local.
Todos los pacientes fueron HBA positivo.
Los pacientes con daños hepáticos crónicos y otras enfermedades fueron excluidos:
• Hepatitis C
• Hepatitis D
• VIH-1positivas y VIH-2 negativo
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2-DE
Análisis de mezclas complejas de proteínas extraídas:
• Células
• Tejidos
• Muestras biológicas.
Este análisis se da por dos etapas:
• La primera dimensión-es el isoelectroenfoque (IEF), que separa las proteínas según su punto isoeléctrico.
• La segunda dimensión-es de poliacrilamida-SDS electroforesis en gel (SDS- PAGE), que separa las proteínas de acuerdo a sus pesos moleculares.
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Figure 1 2-DE images of serum proteins from patients with mild (A) and severe (B) chronic hepatitis. The significantly higher expressed protein with 44kD (C) was further analysed by PMF
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MALDI-TOF-MS
Matrix-Assisted Laser Desorption/Ionization -Time-Of-Flight.
Técnica de ionización suave utilizada en espectrometría de masas.
Permite el análisis de biomoléculas y moléculas orgánicas grandes que tienden a hacerse frágiles y fragmentarse cuando son ionizadas por métodos más convencionales.
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Caracterización general implica:
• Análisis de la masa molecular utilizando cromatografía de permeabilidad en gel, dispersión de la luz y entre otros
• La secuencia de unidades de repetición utilizando espectroscopia de RMN
• Valoración de la utilización de análisis de endgroup, espectroscopia de RMN, o espectroscopia FT-IR
• Examen de pureza utilizando espectroscopia de RMN, análisis elemental y espectroscopia FT-IR.
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Figure 3 The high expression of ATT in severe hepatitis B patients (Ca: hepatic carcinoma; S: severe hepatitis B; M: mild hepatitis B; A: acute hepatitis; N: normal).
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Western Blotting
Variación de la transferencia Southern.
Técnica analítica usada para detectar proteínas específicas en una muestra determinada.
Los pasos a seguir para las proteínas:
• Electroforesis por la técnica de SDS-PAGE poliacrilamida gel electroforesis.
• Transfiere a un filtro, que se incuba con un anticuerpo.
• El anticuerpo es detectado de varias maneras.
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Immunohistoquímica
Identificación de un tejido (in situ) por medio de una interacción especifica entre un antígeno y un anticuerpo (marcado).
La célula es coloreada para demostrar la presencia y localización celular de una molécula.
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Figure 5 AAT expression in different cell lines. Six types of cells indicated were lysed and equal amount of proteins were subjected to SDS-PAGE. The fractionized proteins were blotted with anti-AAT and anti-Actin antibodies.
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RESULTADOS
Electroforesis de dos dimensiones (2-DE)
Huellas dactilares de péptidos de
masas
Técnicas de microarrays
Incremento significativo de la alfa 1 antitripsina
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FIGURA 1
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FIGURA 3
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FIGURA 5
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DISCUSSION
J. Kenonen et alFirstly introduced microarray technology in biomedical research.
He QY, ChiuMany studies focus on biomarker identification for HBV infection.
Poon TCHowever, the methodology used in these studies only detect proteins in the serum.
Rimm DLCombined 2-DE with high-throughput tissue microarray technology to study proteins that may be involved with HBV.
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CONCLUSIONS
ALEX AGUILAR ARIAS
1.Plasma proteins are important markers for many diseases, so it is very important to know a tests to determine the concentration of these proteins
2.Alpha-1-antitrypsin is an important biomarker for the diagnosis of hepatitis B
YULISSA BERTEL SERNA
1. Due to the different studies that exist for HBV can study various aspects to study it
2. If there is a good protein separation procedure can bring disadvantages and errors in the study of HBV
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MAPA CONCEPTUAL ALEX AGUILAR
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MAPA CONCEPTUALYULISSA BERTEL
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