Tesis profesional presentada por Vania Elvira Bonifaz Peña [vania.bonifaz@gmail.com]

Maestría en Biotecnología. Departamento de Ciencias Químico-Biológicas. Escuela de Ingeniería y Ciencias, Universidad de las Américas Puebla.

Jurado Calificador

Presidente: Dr. Solon Javier Garcés Eisele
Vocal y Director: Dr. José Luis Sánchez Salas
Secretario: Dr. José Daniel Lozada Ramírez

Cholula, Puebla, México a 13 de julio de 2007.

Resumen

Background: Hepatitis C virus (HCV) infection is a global medical problem. The current standard of care for chronic hepatitis C (CHC) is pegylated interferon plus ribavirin therapy, but this treatment is expensive, has significant side effects and, at best, is only 50% effective. Silymarin, the main active ingredient of milk thistle, is a natural antioxidant that is used by patients with CHC, although its efficacy for decreasing HCV levels or ameliorating CHC remains uncertain. HCV infection is associated with increased...

Palabras clave: Hepatitis C Virus, Silymarin, Heme Oxygeanse-1.

Resumen (archivo pdf, 91 kb).

Índice de contenido

Índices (archivo pdf, 82 kb)

Glosario (archivo pdf, 78 kb)

Capítulo 1. Abstract (archivo pdf, 91 kb)

Capítulo 2. Introduction (archivo pdf, 424 kb)

  • 2.1 a. Viruses
  • 2.2 b. Hepatitis Viruses
  • 2.3 c. Hepatitis C Virus
  • 2.4 d. Epidemiology
  • 2.5 e. Clinical Features
  • 2.6 f. HCV Replication
  • 2.7 g. HCV Therapy
  • 2.8 h. Complementary and Alternative Medicine (CAM)
  • 2.9 i. Silymarin
  • 2.10 j. Heme Oxygenase-1 (HO-1)
  • 2.11 k. Importance of the Replicons

Capítulo 3. Overall Goal (archivo pdf, 90 kb)

Capítulo 4. Specific Aims (archivo pdf, 90 kb)

Capítulo 5. Hypothesis (archivo pdf, 90 kb)

Capítulo 6. Justification (archivo pdf, 90 kb)

Capítulo 7. Materials and Methods (archivo pdf, 144 kb)

  • 7.1 a. Chemicals and Antibodies
  • 7.2 b. Routine Cell Culture
  • 7.3 c. Treatment and RNA Isolation
  • 7.4 d. Reverse Transcription
  • 7.5 e. Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)
  • 7.6 f. Treatment and Western Blot
  • 7.7 g. MTT Assay
  • 7.8 h. Statistical Analysis of Data

Capítulo 8. Results (archivo pdf, 1 mb)

  • 8.1 a. Determination of optimum Fetal Bovine Serum concentrations for induction of HCV core, HCV NS5A and HO-1 mRNAs in CNS3 and 9-13 cells
  • 8.2 b. Dose response and time course studies of mRNA levels in CNS3 cells treated with Silymarin
  • 8.3 c. Silymarin down-regulates HCV core protein in CNS3 cells
  • 8.4 d. Dose response and time course studies of selected mRNA levels in 9-13 cells treated with Silymarin
  • 8.5 e. Silymarin up-regulates HCV NS5A protein levels in 9-13 cells 42
  • 8.6 f. HO-1 mRNA induction by Silymarin in wild-type Huh7-cells 44
  • 8.7 g. Dose response experiments of Silymarin and interferon on HCV core and HO-1 mRNA levels in CNS3 cells
  • 8.8 h. Effects of the combined treatment of Silymarin and Interferon on HCV core mRNA in CNS3 cells
  • 8.9 i. Silymarin alone or in combination with interferon has similar effects on HCV core protein levels in CNS3 cells
  • 8.10 j. Effects of increasing concentrations of Silymarin and interferon on HCV NS5A and HO-1 mRNA levels in 9-13 cells at 24 and 48 h
  • 8.11 k. The combination of Silymarin and interferon have the same effects on HCV NS5A mRNA levels as treatment with Silymarin alone in 9-13 cells at 24 h
  • 8.12 l. Silymarin alone or in combination with interferon has similar effects on HCV NS5A protein levels in 9-13 cells
  • 8.13 m. Silymarin alone or in combination with interferon up-regulates HO-1 mRNA levels in Huh-7 cells
  • 8.14 n. Silymarin does not affect expression of Bach1 or Nrf2 transcription factors in wild type Huh-7, CNS3 and 9-13 cells
  • 8.15 o. HCV regulation by Silymarin is not related by the Jak-Stat Pathway
  • 8.16 p. Cytotoxicity of Silymarin in CNS3, 9-13 and Huh-7 cells

Capítulo 9. Discussion (archivo pdf, 145 kb)

Capítulo 10. Conclusions (archivo pdf, 94 kb)

Referencias (archivo pdf, 146 kb)

Apéndice A. I- Standard Curves for Quantitative RT-PCR Assays (archivo pdf, 99 kb)

Apéndice B. II- BCA Method to measure protein concentrations (archivo pdf, 93 kb)

Apéndice C. III- Prepare a 1mM stock solution of MTT formazan solution: 3.35mg of MTT formazan in 10mL of MTT solvent (0.1N HCl in isopropanol) (archivo pdf, 86 kb)

Bonifaz Peña, V. E. 2007. Effects of Silymarin on Hepatitis C Virus and Heme Oxygenase-1 Gene Expression in Human Liver Cells. Tesis Maestría. Biotecnología. Departamento de Ciencias Químico-Biológicas, Escuela de Ingeniería y Ciencias, Universidad de las Américas Puebla. Julio. Derechos Reservados © 2007.