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Lennis Bedoya Muñoz

ID: 000087035

Juan Carlos Agudelo Jiménez

ID:000149094

MOLECULAR BIOLOGY

UPB

Proteomic analysis of aqueous humor from patients with primary

open angle glaucoma.

Xiaoming Duan.

Anterior chamber cornea, iris, pupil and lens.

Filled with a watery fluid called the aqueous humor.

Increased pressure of aqueous humor in the eyes

Leading cause of irreversible visual

impairment and blindness on a global level.

Responsible for the supply of nutrients.

Removal of metabolic wastes from the avascular tissues of the eye.

Contains proteins secreted from anterior segment tissues.

Protein levels in AH change in many eye diseases.

PRIMARY OPEN-ANGLE TYPE

CLOSED-ANGLE GLAUCOMA

Age Especially for people over 60 years old

Family history

Race African or Hispanic descent

Myopia

Raised intraocular pressure (IOP).

No one knows exactly what causes glaucoma but some factors raise

PREVIOUS STUDIES REPORTED SOME PROTEINS ARE ELVATATED IN PATIENTS WITH POAG

First coined in 1995 Complement of a cell line, tissue, or organism.

Today Two definitions of proteomics are encountered.

PROTEOMIC USES

Identifying components of the proteome.

Comparing the expression of proteins between normal and diseased organs.

Determine how proteins interact with each other in vivo.

Identification and characterization of proteins post-translationally.

Study the structure and function of protein complexes.

Understand the organization of cells at the molecular level.

PROTEOMICS HAS BEEN USED TO STUDY EYE DISEASES

Understand Proteomic analysis of aqueous humor from patients with primary open

angle glaucoma to identify the possible roles of these AH proteins in POAG and

the respective relationships between POAG and elevated intraocular pressure

(IOP).

Estudiar proteínas diferenciales presentes en los pacientes con

POAG.

Distribución de las proteínas diferenciales presentes en el humor

acuoso.

Comprender de mejor manera la relación entre la biología

molecular y el desarrollo de los pacientes con POAG

Recolección de las muestras

BeijingTongren Eye Center

Capital médica universitaria de la China.

Antes de la cirugía

Aprobado por la declaración de Helsinki

Aprobación del consentimiento por escrito

El protocolo de estudio Aprobado por el comité de ética médica

Capital médica universitaria de Beijing

Comparar los resultados en los pacientes con POAG y en los pacientes control,

analizando la presencia o ausencia de factores que favorezcan el aumento de la PI

Paciente control Pacientes estudiados

Concentración total de proteínas presentes en el humor acuoso

Método de Bradford

ELECTROFORESIS EN GEL DE 2 DIMENSIONES

Separación de proteínas Movimiento en el campo eléctrico.

Determinar la cantidad de las proteínas presentes en los pacientes con

POAG y en los control de acuerdo al grado de tinción obtenido.

Determinar si hay incremento en el volumen de las manchas de los

pacientes con POAG y las pruebas control.

Identificación de proteínas

Aíslan proteínas obtenidas con la electroforesis 7

Proteólisis con tripsina modificada y bicarbonato de amonio

Obtener sobrenadante y extraer más extractos de péptidos

Realizar cromatografía de líquidos y espectrofotometría de masas

SDS-PAGE Separación del AH

TBS Lavado de la membrana

Determinar la presencia de anticuerpos anti PGDS

Confirmar la existencia de D- isomerasa de prostaglandina

H2(PGDS) en los pacientes con glaucoma

Pacientes con Glaucoma de ángulo abierto fue de 0.4382±0.1675 mg/ml (Rango=0.2796~0.6517 mg/ml).

Pacientes control fue de 3124±0.0567 mg/ml (rango=0.2484~0.3984 mg/ml).

Patrones en el Gel de Electroforesis de 2 Dimensiones

Paciente con Glaucoma Paciente Control

Pacientes con Glaucoma mostraron una mayor intensidad y número de manchas plateadas que los pacientes control.

Patrones en el Gel de Electroforesis de 2 Dimensiones

Se encontró diferencias significativas entre el volumen % entre los pacientes con Glaucoma y los pacientes Control.

Se aislaron 7 proteínas encontradas en las manchas producidas en el gel de electroforesis, mediante el kit nanoLC MS/MS para identificación.

Identificación de Proteinas

Identificación de Proteinas

Prostaglandina Humana H2 D-Isomerasa

Caspasa 14 transthyretin albumina transferrinaalbumina

Cistatina C

AQGFTEDTIVFLPQTDK

La Anti-Prostaglandina Humana H2 D-isomerasa estaba presente en todas las muestras de Humor Acuoso. Indicando niveles altos en pacientes con Glaucoma de Ángulo Abierto.

Analisis Western blott

Doctors Argument Agreed

Ferreira SM. “Although POAG has been widely considered a multifactorial disease, IOP is the most important known risk factor for the development of glaucomatous optic nerve damage.”

Grus FH, Joachim SC, Sandmann S, Thiel U, Bruns K, LacknerKJ, Pfeiffer N.

“Most recent insights regarding POAG have come from studies on animal models. Other studies of AH typically focus only on proteins. Grus et al. has studied transthyretin changes in AH of POAG using surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) ProteinChip arrays and two-dimensional electrophoresis.”

Author Argument Agreed

Choi J “Reducing IOP can still be considered the most beneficial treatment in terms of halting visual field damage progression, even in normal pressure glaucoma. However, the mechanisms of elevated IOP have not been fully elucidated. At present, it is well known that elevated IOP could result from a variety of cell deaths in the trabecular meshwork. AH, the circulated intraocular fluid, includes many kinds of cytokines, which could likely play significant roles in the trabecular meshwork.”

Doctors Argument Agreed

Denecker G “There were few reports about caspase 14 in eyes.Caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate-specific proteinases, which are mainly involved in inflammation and apoptosis. Previous studies demonstrated that caspase 8 and caspase 9 are the main kinds of caspase that play roles in the apoptosis of ocular tissues. According to this study, caspase14 might be involved in the apoptosis of ocular tissues by either directly mediating or inducing caspase 8 and caspase 9 activation.”

Molecular biology has allowed health personnel access to new

diagnostic tools that every day as close to knowing the true etiology of

diseases such as open-angle glaucoma and make decisions based on

facts and effective and timely treatments.

A wide variety of proteins, still unknown, are present in many

pathologies, that is why we must consider the molecular biology and

engineering as fundamental molecular diagnostic methods.

This study is very important because is a new opportunity to to show the

importance of proteomics in the diagnosis and treatment of deseases in which

traditional methods has been limited.

this article made a great contribution to molecular biology, medicine and

scientific world Because researchers not satisfied with talk of glaucoma but

also achieved to find the proteins that increased in POAG and analyze what is

their role in this pathology.

 

Juan Carlos Agudelo