Lecture 6 - Endocrine methodologies Flashcards
Using an in vivo model, how would you identify or prove the existence of an endocrine tissue?
- remove organ - surgically, chemically, deafferentation
- note effects on whole animal function and specific systems
- always use sham-operated animals as controls
What are the advantages and limitations of in vivo models?
Advantages
- most physiologically relevant
- biological variability can be minimized with the use of inbred lines
Disadvantages
- organ might be vital
- must ensure that organ is completely destroyed/removed, otherwise lack of feedback will induce hypertrophy/hyperplasia
- parameters and complexity of integration hard to control and account for
What are the advantages and limitations of in vitro models?
Advantages
- easier to control for environment and interactions
Disadvantages
- less physiologically relevant
- not good for long term action
What model (technique) would you use to analyze the effect of hormones on activation of an intracellular enzyme?
In vitro models are ideal for the study of specific hormone action on a specific target = more defined of a system
- with the use of cell cultures - could use immortalized cells (easily accessible) and then move to primary cell culture to confirm
A new molecule secreted by the funny gland is suspected of being a hormone acting on feeding behaviour. What would you do to show it is a hormone?
In vivo:
- to identify endocrine tissue –> remove = surgically, chemically, deafferentation
- note effects on whole animal function and specific system
- use sham operated animals as controls
In vitro:
- to identify endocrine tissue - when you’re looking at one thing with a direct effect –> isolated organ/tissue; splices/pieces of organs; isolated cells; sub cellular fraction
What is a cel line?
Immortalized cells - derived from carcinomas –> rapid growth
Advantages and disadvantages of using cell lines as opposed to primary tissue culture?
Advantages:
- no need to collect tissue from animals
- easy to grow and manipulate
Disadvantages:
- since they are modified cells –> limited reliability
- is this they way normal cells would respond?
- change characteristics over time
Describe the perfusion system of cell culture. Advantages/disadvantages?
Cell culture = static environment
-accumulation of metabolites and secreted factors (possible artificial paracrine/autocrine effects)
Perfusion system can offer advantage of cell culture with more of a physiological “twist”
What is an agonist?
Substance able to mimic the effect of a hormone (at receptor level)
- can be natural/artificial
- acts like a hormone
- binds to hormone receptor
What is an antagonist?
Substance which blocks or inhibits the effect of a hormone (at receptor level)
Why are agonists/antagonists useful?
work at a receptor level to either artificially enhance/inhibit hormone effect (controlling mechanism)
How are antibodies raised and how can you make a hormone more antigenic?
Antibodies produced as a defence mechanism against foreign compounds = antigens
Two types of immune responses:
1. Cell mediated response = destruction of self cells gone wrong (infected/cancerous) - cytotoxic T lymphocytes
- Humoral response = generation of soluble antibodies against foreign antigens - B lymphocytes which are pre-committed to respond to a limited exposure to an antigen –> primary immune response –> secondary immune response
Protein hormones are typically antigenic because their structures differ between species
Steroid hormones have low molecular weight and their structures are the same in different species - need to be linked to a large protein in order to be antigenic
Using an adjuvant + antigen = enhances immune response (e.g. Freund’s complete adjuvant = emulsion of water in oil containing killed bacteria)
Describe the process of producing monoclonal antibodies
Monoclonal antibodies = large proteins (antigens) which possess several epitopes
Steps:
- immunize animal with antigen
- remove spleen, isolate cells and screen for cells producing desired Ab
- fuse Ab producing cells with myeloma cell line to immortalize cells
- purify hybridize cell line and grow up to produce large amounts of Ab
