GALNT11 as a new molecular marker in chronic lymphocytic leukemia
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Transcript of GALNT11 as a new molecular marker in chronic lymphocytic leukemia
Stefanía Bahamonde OlayaDiana Agudelo Jaramillo
Teacher Lina Martínez Medicine Faculty
III Semester
GENERALIDADES
LEUKEMIA is a neoplastic disease that occurs as a result of unregulated proliferation and incomplete modulation of cellular precursors of hematopoietic and lymphoid tissues.
In BONE MARROW tumor cells displace the remaining normal series and later spreads to other sides of the body:
Lymph-nodes Liver
Spleen
GRENERALIDADES
It is a type of cancer that begins in the white blood cells in the bone marrow.
Over time these cells tend to accumulate and many people with the disease do not show symptoms for at least a few years.
We have discovered two types: -one fast progress.- one slower progress.
GRENERALIDADES
B lymphocytes B lymphocytes (cells that protect the body against invading germs by maturing into plasma cells that produce antibodies) are the cells that are affected more frequently in CLL.
SYMPTOMS SYMPTOMS -Weakness -Sensation of fatigue -Weight-Loss -Fever -Enlarged lymph-nodes
INTRODUCTION
GALNT11 GALNT11 is a variety of the 14 GALNTs this overexpressed in patients with CLL, in B cells.
TN ANTIGEN TN ANTIGEN is the first product GalNAc transferase.
MOLECULAR MARKER MOLECULAR MARKER is a character or a gene may be used to indicate the presence of another gene.
INTRODUCTION
THIS ANALYSIS IS COSTLY AND DIFFICULT
THERE ARE TWO PROFILES AND A WIDE RANGE OF DIFFERENT MANIFESTATIONS. •(IGHV) MUTATED (M) ---> BETTER PROGNOSIS •(IGHV) UNMUTATED (UM) --> WORSE PROGNOSIS
THE ARTICLE IS BASED ON RECENT DEVELOPMENTS. SUCH US
INTRODUCTION
ALTERNATIVES WERE SEARCHED FOR AS MARKERS ZAP-70 CD38 LPL
ASSOCIATED WITH UM
BUT THE CORRELATION IS NOT ABSOLUTE.
INTRODUCTION
THEREFORE THE EXPRESSION OF THE ENZYMES OF THIS PROCESS IN CANCER CELLS (GALNTS) WAS INVESTIGATED.
SPECIFICALLY GALNT11
EXPRESION OF RESULTING OF AN INCOMPLETE O-GLYCOSYLATION EXPRESION OF RESULTING OF AN INCOMPLETE O-GLYCOSYLATION
ONE OF THE MOST IMPORTANT ALTERATIONS IN CANCER CELLS
TN ANTIGENTN ANTIGEN
GENERAL OBJETIVE
THE GOAL IS TO STUDY GALNT11 AS POSSIBLE NEW MOLECULAR
MARKER IN CHRONIC LYMPHOCYTIC LEUKEMIA.
TO USE IT AS A DIAGNOSTIC TEST FOR THIS SPECIFIC TYPE OF LEUKEMIA AND BUILD MORE APPROPRIATE AND SPECIFIC
MATERIALES Y MÉTODOSMATERIALES Y MÉTODOS
MATERIALES Y MÉTODOS
SANGRE PERIFÉRICACONTROLES
SANOS PACIENTES
CLL
CELULAS BCELULAS T
CENTRIFUGARONPURIFICARON
MATERIALES Y MÉTODOS
EXTRACCIÓN DE RNA
SE TRATÓ CON AMBION(TRATAMIENTO LIBRE DE DNA)
PARA IDENTIFICAR LA EXPRESIÓN DE DIFERENTES TEJIDOS
VARIOS CICLOS DE PCR CONVENCIONAL
MMLV(TRANSCRIPTASA REVERSA)
RT-PCR
LPL GALNT
MATERIALES Y MÉTODOS
QUANTITATIVE REAL TIME PCR (q-PCR)
SYBR GREEN (SUSTITUTO DE BROMURO DE ETIDIO) TINCIÓN
CONTROL DE CONTAMINACION( 6 DONANTES SANOS)PCR POR DUPLICADO(TODO) FUSION DE CURVAS
PRIMERS
AMPLIFICACIÓN GAPDH (GEN DE REFERENCIA)
CD3 (MARCA DE LINFOCITOS T)
GALNT11
AMBION Y RETROTRASNCRIPCIÓN
FLUORESCENCIA EN RELACIÓN A LA CANTIDAD DE ÁCIDOS NUCLEICOS
MATERIALES Y MÉTODOS
ANTICUERPOS Y LECTINAS
SE FORMO
DETECTAN PRESENCIA DE TN ANTIGENO
ANTIGALNAC-ANTIBODY DE
CONEJO
MAB B72.3 (AC)
MAB 83D4 (AC) IGM
DETECTA PROTEÍNA RECOMBINADA DE GALNAC-T11
PROTEINAS DE UNION ESPECIFICA Y REVERSIBLE A CARBOHIDRATOS
VVB4 LECTIN (SIGMA)
MATERIALES Y MÉTODOS
EXTRACCION DE PROTEINAS,SDS PAGE, WESTERN BLOT
SEPARACION DE PROTEINAS SEGUN TAMAÑO POR EF EN PAGE
FILTRO
EN ESTA EF SON DISUELTAS EN SDS(DETERGENTE DUODENIL SULFATO ) CARGA
NEGATIVA
GAPDH AC FUE USADA COMO CONTROL
INCUBACION CON AC
CUANTIFICACION DE PROTEINAS POR BCA-ENSAYO
CUANTIFICACION RELATIVA POR GENTOOLS
QUIMIOLUMINISCENCIA
MATERIALES Y MÉTODOS
CITOMETRÍA DE FLUJO
IDENTIFICA INCLUSO PEQUEÑAS POBLACIONES CELULARES
UNION AC CON MOLECULA FLUORESCENTE ( ABSORBE LUZ UV)
CON ALTA ESPECIFICIDAD
A PARTIR DEL CD Y MIDE VARIOS PARAMETROS
INMUNOFLUORESCENCIA
REACCION ANTIGENO-AC
TRANSCRIPTASA INVERSA
PBS( BUFFER FOSFATO SALINO)
DETECCIÓN DE HORMONAS Y PROTEÍNAS
RESULTS
Control
VVB4VVB4
Daudi
Anticuerpo secundario
RESULTS
Jurkat Tn (VVB4) 20 Tn (VVB4)
5/6 Muestra de pacientes con CLL, se encuentran en el rango de 22-51% con marcador de VVB4.
RESULTS
Jurkat Tn (83D4) 20 Tn (83D4)
Muestra de pacientes con CLL, se encuentran en el rango de 0.1–0.9% con marcador de mAB 83 D4.
RESULT
A549 sialyl-Tn 41 sialyl-Tn
6 CLL PACIENTS
Control Positivo
ANTI SIALYL-TN ANTICUERPO B72.3
ANTI SIALYL-TN ANTICUERPO B72.3
RESULTS
CONTROL NEGATIVO
Intensidad depende de la concentración
de la PCR
Intensidad depende de la concentración
de la PCR
POSITIVO- CLL- CÉLULAS T- JURKAT
NEGATIVO- CÉLULAS B- DAUDI
RESULTS
?
CD3MARCADOR CÉLULAS T
CD3MARCADOR CÉLULAS T
Buena expresiónde GalNAc-Tn
RESULTS
Intensidad según expresión de proteína.
Proteínas GalNAc-Tn se expresan bien en muestras
de CLL
RESULTS
ANTI-T11
ANTI-CD19CD19
MARCADOR CÉLULAS B
CD19MARCADOR CÉLULAS B
RESULTS
PBMC from healthy donors
T11 CD19 T11 + CD19
ANTI-T11ANTI-CD19
RESULTS
ANTI-T11
ANTI RABBIT TOTAL Ig
ANTI-T11 + PEPTIDO
MUY ESPECÍFICOMUY ESPECÍFICO
RESULTS
MUTADOS = MEJOR PRONÓSTICO
NO MUTADOS = MAL PRONÓSTICO
RESULTS
DISCUSSION
DISCUSSION
CONCLUSION
CONCLUSION
MAPA
Stefanía Bahamonde Olaya
MAPA
Diana Agudelo Jaramillo
BIBLIOGRAPHY
- MARTINEZ SÁNCHEZ, Lina María. (2012) . Class book , Molecular Biology , 7 ed. Colombia, Medellín: UPB Medical Faculty, Editorial Universidad Pontificia Bolivariana pp.7-8.
- M.G. Libisch, M. Casás, ML. Chiribao, P. Moreno, A. Cayota. (2013 September 17). GALNT11 as a new molecular marker in chronic lymphocytic leukemia. Elsevier B.V. Retieved from: http://www.sciencedirect.com/science/article/pii/S0378111913012572