Using Microrna C.Elegans Mutants to Analyze the Reciprocity Between Heathspan and Longevity

摘要

In an attempt to develop ultimate measures of extending lifespan in humans, age research using rniRNAs in the organisms like Caenorhabditis elegans, drosophila and the mice in reference to development, aging, cancer and neurodegenerative disease has been significantly sprouted over the recent years.Researchers in this field have always identified signaling pathways and conserved genes which boost longevity with an assumption that healthspan of the long-lived species will be enhanced as well.[1-3] The major challenge underlying these researches is whether extending lifespan would automatically subject an organism to living a robust life.In this project, we made use of the Caenorhabditis elegans a transcendent organism among others used for aging research to scrutinize the contradistinction between healthspan and longevity.We hypothesized that decline in functionality capacities was independent of organism's maximum lifespan and carried out a number of assays related to different signaling pathways that modulate lifespan together with some other parameters for measuring the degree of health in the C.elegans, and these were executed on the wild type as well as four other microRNA mutant including, mir-51, mir-67, mir-83, mir-240and mir-260.The survey was conducted in such a way to determine the health of the organisms as they aged using different tests i.e.feeding rate test (pharyngeal pumping rate), fertility/reproductive health test and response to external stimuli (heat stress resistance test) together with lifespan test.Compared to N2, mir-240 outlived the rest of the mutant types with approx 37％ increase in mean lifespan, mir-51 and mir-67 also significantly lived longer than N2 while mir-260 lived shorter than N2 with approx.22％decline in lifespan, and mir-51 had no significant change in mean lifespan compared to N2.By analyzing, chronologically (actual number of days) and the physiologically (in terms of percentage of maximal lifespan), our data revealed that some of the long lived mutants could surpass the wild type in a given age-related test but failed in another.Also by taking a comparison between healthspan and lifespan, we realized that just like the wild type N2, mutants mir-51 and the longevity mir-83, mir-67 spent above 64％ of their total life time in an emasculated state unlike their counterpart short living mir-260mutants which spent only approximately 48％ of the total lifespan in a frailly state.The analysis from the data was that healthspan and lifespan were distinct and independent entities that did not be correlated.Understanding the mechanisms by which lifespan and healthspan correlates using age-degenerative changes would help to clearly explain the mechanism of ageing and to understand interventions of boosting longevity incorporated with improved health living by finding possible solutions to neurodegenerative disorders, cancer, heart diseases and other age-related disease hence suppressing frailly conditions at elderly stage.

目录

声明

Abstract

TABLE OF CONTENTS

Chapter 1：Introduction

1．1 AIM AND SIGNIFICANCE OF THE STUDY

1．2 THE BIOLOGY OF AGEING

1．2．1 Programmed theories of ageing

1．2．2 Damage-based theory of ageing

1．2．3 Potential interventions into the ageing process

1．3 THE C．ELEGANS AMODEL ORGANISM IN RESEARCH

1．3．1 An overview and scientific classification of the C．elegans

1．3．2 Reproduction and life cycle of the C．elegans

1．3．3 Economic importance and C．elegans as prime model organism

1．3．4 Age-related changes that occur in the C．elegans

1．3．5 Pathways involved in aging of the C．elegans

1．4 THE MICRORNA(miRNA)

1．4．1 The miRNA biogenesis pathway

1．4．2 The molecular mechanism of miRNA

Chapter 2：Materials and Methods

2．1 EQUIPMENT，COMMON REAGENTS USED AND PREPARATIONS

2．1．1 List of equipment and reagents

2．1．2 Materials and their preparation

2．2 CULTURING AND MAINTENANCE OF ORGANISMS

2．2．1 Bacterial growth and seeding plates for worm culturing

2．2．2 Worm culturing and maintenance

2．3 LIFESPAN ANALYSIS PROTOCOL

2．4 HEALTHSAPAN ANALYSIS ASSAYS

2．4．1 Reproductive Health／Fertility assay

2．4．2 Pharyngeal Pumping rate(feeding rate)

2．4．3 Heat stress resistance

Chapter 3：Results and Discussion

3．1 LIFESPAN ANALYSIS

3．2 HEALTHSPAN ASSAY

3．2．1 Reproductive health test

3．2．2 Pharyngeal Pumping rate(feeding rate)

3．2．3 Heat stress resistance test

3．3 COLLATION OF HEALTHSPAN AND GEROSPAN

Chapter 4：Summary and Future directions

4．1 Summary and conclusion

4．2 Future direction

References

Acknowledgments

Appendix