6: ECM, Histopathology (Tissues) Flashcards
Extracellular matrix (ECM)
Everything that surrounds cell
Not mobile
Cell deposit proteins and carbohydrates around themselves
Communicates with nearby cells via surface receptors
Helps keep structural support of tissue
Helps cells function and influences what they do
Connective tissue is composed of (2)
ECM
Component cells
4 components of ECM
Collagen - fibril forming, non-fibril forming
Multiadhesive glycoprotiens - laminins, fibronectins
Elastic fibres - elastin, fibrillin
proteoglycans
4 connective tissues in ECM
Bone
Cartilage
Tendon
Vitreous humour
Collagen
Fibrous
28 types
Multiple genes
Most abundant protein
Fibre layering affects strength
Collagen structure
Triple helix
three a chains
gly-x-y
stiff helix
glycine - H side chain =pack together
Composure of collagen fibre
One alpha chain –> 3 alpha chains–> collagen fibril –>collagen fibre
Collagen synthesis
1)one alpha chain
2) hydroxylation (add OH) - Vit C is a cofactor{scurvy}, glycosylation (add carbohydrate chain) x3
3) Become triple helix, bonds form between chains
[4) IF not secreted: keep non collagenous domains- unless fibrillar collagens]
4) IF secreted: cleavage of non collagenous domains
5) Fibrils form, crosslinking occurs
Basement membranes
Non fibrillar collagens - type IV
flexible / bendy
join together laterally & head to tail
rotary shadowing = self assembly
form a sheet
Where are basement membranes found
Flexible ECM around tubes/under epithelium
Essential to kidney for GBM
Diabetic nephropathy
thick/too much ECM in membrane, Filtrate unable to enter nephron
results in renal failure
Alport syndrome
Collagen IV disorder
GBM becomes split and laminated
Elastic tissues are required in
Arteries/vessels
in skin
lungs
Elastic fibres
In absence of stretching force = elastin proteins in compact conformation
When subject to stretching force - elastin proteins elongate, but remain attached by cross links
Elastic fibres consist of
elastin core
microfibrils (fibrillin)
Marfans syndrome
Fibrillin disorder- affects elastin production, loss of elastic tissue
Tall, slender people-tissues collapse, e.g eye lens dislocate, aneurisms burst, vessels not elastic due to lack of elastin
Two types of glycoproteins
Laminins
Fibronectins
Laminins
3 different chains
large proteins
cross shaped
bind to many other ECM components
Fibronectins
In fluids
soluble and insoluble
U shaped
Interact with other ECM components
Fibronectins are involved in
Involved in embryogenesis, tissue repair and clotting
Laminin infection causes
muscular dystrophy
epidermolysis bullosa
Proteoglycans
core protein + GAG chain
Often negative
depending on repeating unit there are 4 categories of GAGs
GAG chains are
two amino sugars joined together = disaccharide unit
types of GAGs
Hyaluronan - vitreous humour and synovial fluid
Chondroitin sulphate and Dermatan sulphate
Heparan sulphate
Keratan sulphate
- dependant on varying repeating unit
Too much ECM can lead to
Fibrotic disorders
- liver fibrosis - cirrhosis
- kidney fibrosis - diabetic nephropathy
- lung fibrosis - idiopathic pulmonary fibrosis (IPF)
To little/loss of ECM can lead to
Lack of support
e.g osteoarthritis
Taking a biopsy
remove tissue - scalpel, needle
preserve with formalin - to keep integrity
put in wax
cut into tiny sections
mount on a glass slide for microscopy
What is histopathology used for
Cancer diagnosis
observing if treatment works
What stain is used to see leucocytes in a biopsy
H&E stain (Hematoxylin and eosin)
- makes nuclei and cytoplasmic granules visible
What is the Ziehl-Neelsen stain used for in biopsies
Staining acid-fast bacteria red
- aids diagnosis of tuberculosis infection
Resection specimens
Specimens used to observe efficiency of treatment. more treatment needed?
allowing definitive diagnosis of disease from tissues surgically removed
5-7 days for results
Frozen specimens
Remove tissue in surgery
Freeze with cryostat machine
Cut & place on microscope
stain
rapid diagnosis of mass during surgery
30min until results
needs to be free of any chemicals
Cytopathology
Observing individual cells
Histopathology
Observing tissues as a whole
Procedure of Cytopathology
- Collect cells
- Smear them on slide
- Stain
- Examine under microscope
Fine needle aspiration
least invasive - doesn’t purse
can access relatively inaccessible tissues
N: only singular cells removed, cannot comment on architecture of tissue and cell interactions
Antibody structure
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bottom - heavy chain Fc- attaches to self cells
top -antigen binding site, top half of Y is variable, bottom half is constant
top outside - light chain
Histochemistry
Using antibodies to detect specific molecules
attach Fc part - called: conjugations
4 Types of conjugations
Enzymes (ELISA)
Fluorescent probes (flow cytometry)
Magnetic beads
Drugs
Direct ELISA
Primary antibody conjugate - directly binds to antigen
1. Antibodies attached in bottom of well
2. Addition of patient sample
3. Sample washed, Enzyme-labeled antibody added
3. Sample washed, Reaction observed
Indirect ELISA
Secondary antibody conjugate - Antibody binds to antigen which has detectable molecule attached
ELISA is used to
determine levels/presence of a certain molecule
Flow cytometry
Antibodies are fluorescent
1. Add antibodies
2. Run sample through laser
3. Flourophores excited
4. Colour (what is in sample) and scattering (molecular surface) measured
Bone consists of 70% water and 30% dry mass, up to 80% of which is collagen. what architectural property of collagen fibres suits it best for this role?
Tough due to perpendicular fibre arrangement
Withstand pressure during stretching and impact
10% of tendon is made up by elastin fibres, what property does this lend to tendons to make it useful for their role?
Tendon flexibility in order to contract and relax muscles
Why is a fine needle aspiration used instead of a biopsy?
Can penetrate relatively inaccessible tissues
Obtain a sample without need for surgery
What are epithelial cells classified according to
layering and shape
layers of epithelial cells
simple, stratified, pseudostratified
shape of epithelial cells
squamous, cuboidal, columnar
type of epithelial cells in mouth, oesophagus and vaginal tissue
non-keratinised stratified squamous
type of epithelial cells in the stomach
Simple columnar
type of epithelial cells in the small intestsine
ciliated simple columnar
type of epithelial cells in large intestine
simple columnar
type of epithelial cells in the anal canal
upper 2/3: simple columnar
lower 1/3: non-keratinised stratified squamous
type of epithelial cells in the epidermis of the skin
keratinised stratified squamous
type of epithelial cells in the trachea and main bronchi of lungs
ciliated pseudostratified columnar
type of epithelial cells in the alveoli of lungs
simple squamous
Biopsy
procedure removing tissue from a living body to be examined under a microscope
time taken for results 2-3days
Post-mortem
examining cell type on a body after death
smear
microscopically studying a specimen which is thinly spread across the slide with a swab
H&E stain identifies
Haematoxylin stains nuclei blue
Eosin stains cytoplasm pink
Gram stain identifies
Bacteria
Ziehl-Neelson stain identifies
Acid-fast bacteria (mainly myobacteria) - TB
Leishman stain identifies
Blood smears (contains eosin and methylene blue)
Antibody stain identifies
Detection of proteins in a sample
Osteoarthritis is an erosive disease caused by ECM degradation. What is the mechanism of degradation
Aggrecanases cleave aggrecan
How is aggrecan cleaved
Aggrecanases and metalloproteinases cleave aggrecan
Fragments released into synovial fluid
Hyaline cartilage is rich in
aggrecan
GAG (Glycosaminoglycans) of aggrecan are
highly sulphated leading to multiple negative charges
these charges attract cations such as Na+
Large quantities of water are attracted to the osmotically active cations
Retained water creates a cushioning effect
under compressive load, water is given up, but retained as load is reduced
What happens to aggrecan in osteoarthritis
Aggrecanases and metalloproteinases cleave aggrecan with age leading to loss of aggrecan fragments to synovial fluid
lack of cushioning at the end of long bones leads to joint pain and swelling
What would happen if the secretory machinery in epithelial cells wasn’t polarised
secretion would occur through both apical and basolateral compartments
- without polarisation , cell is unable to distinguish between the apical and basolateral surfaces for secretion, leading to secretion through both compartments
How do integrins link the ECM
Collagen fibril > fibronectin > integrin dimers> adaptor proteins > actin filament
Primary function of cuboidal epithelial cells
secretion
absorption
barrier functions
commonly found lining ducts of glands and small tubule organs e.g kidneys, thyroid
