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Wound healing vs. regeneration
Regeneration of epithelium is slower; Regeneration of epithelium is faster
within an hour, they start to divide and migrateLays down and remains there; Lays down and is digested soon after; lack of basal lamina allows for expansion
Wound Healing vs. Regeneration
- Regular amount
- Increased protease activity, as well as inhibitors and activators due to the fact that room needs to be made for all of the cells moving into the area to regenerate the tissue
- There are a variety of cells
- There are a variety of undifferentiated, as well as de-differentiated cells
- Scar tissue created (large amounts of collagen are secreted by fibroblasts)
- No scar tissue created
- Less functional than undamaged tissue
- Same functionality
What are the steps of wound healing?
- blood clot (hemostasis)
- inflammation (2-5 days)
- proliferative phase (2 days-3 weeks)
- --> granulation, contraction (pull things together), epithelialization (fill the whol ething then put epithelium)
tissue is still not as strong as uninjured tissue
What is the key to restoration?
- new growth by an organism of organs, tissues, etc. that have been lost, removed, or injured
- Key: when they grow back, no evidence of injury; no scar tissue, right place and size, completely functional
it is limited in vertebrates; ex: salamanders (defense mechanism)
Amphibians that regenerate are?
urodele: any of an order (Caudata syn. Urodela) of amphibians (as newts and salamanders) that have a tail throughout life
What can salamanders do?
they can grow back tail, limbs, lower jaw, and ocular tissue, including lens and retina
epimorphic growth: key to process of regeneration in amphibians is a structure known as a blastema
regeneration involves dedifferentiation of adult structuers in order to form an undifferentiated mass of cells, called the blastema
A blastema is a mass of un/de-differentiated cells capable of growth and regeneration into organs or body parts; characteristic and essential feature of regeneration; respond to signals in the environment
Blastema in detail
When limb is cut off, a layer of epidermis covers site of cut. it transforms into a layer of signaling cells called the apical epithelial cap (AEC)
Fibroblasts also migrate across the amputation surface to meet at the center of the wound. These fibroblasts multiply to form a blastema
Characteristics of Regeneration in terms of WBC
similarities with wound healing and regeneration
WBC: macrophages and leukocytes come in , but signlaing molecules are heard differently than in regeneration
What happens with the newt in their eye?
with the newt, there are cells that make up the iris called pigmented epithelial cells (PEC). In the absence of the lens, they de-differentiate and initally become epithelial layer int eh front of lens and trans-diferentiate into the lens and is perfectly functional
What was the experiment done using blastema cells?
Blastema cells obtained by cutting limb, letting them grow, and feeding them under various conditions
Some dishes= myotubes (beginning of formation of muscle cells), express mature muscle-like proteins
- Labeled cells, reinserted them into the blastema and followed label
- 1) In some places, the muscles de-differentiated
- 2) destabilizing environment of blastema can get them to go backwards
Some proteins that do this are retinoblastoma proteins
tumor-suppressor protein discovered because in young children, with both mutants, they got tumors in the retinas
Rb stops cell proliferation
Explain how retinoblastoma protein works.
The gene for it is widely expressed. In normal cells, the protein is inactivated by phosphorylation and activated by dephosphorylation.
Active (dephosphorylated) pRb binds and inactivates the cellular transcription factor E2F1, the function of which is required for cell cycle progression
In a G0 cell, active RB protien binds to E2F to inactivate the regulatory protien and thus keep the cell from transcribing
In a proliferating, cell, Rb is phosphorylated; and, it releases the protein, allowing it to activate transcription
What experiment was done with Rb?
They raised the serum concentration and put a signal molecule in, causing phosphorylation of Rb, and thus cell proliferation
Something about blastema allows this to happen
On a gel, the Rb-P in a low serum concentration has only one band, signifying teh activated Rb. In higher serum, there is both activated and inactivated, the latter of which is going to have a higher molecular weight because of the phosphate group
What did they do to determine what forms with arm amputation of blastemas?
They took two newts and cut off their arm at different locations, creating a distal and a proximal amputation.
When the distal blastema was inserted into the proximal blastema, it caused both the whole arm to form, as well as the portion of the hand corresponding to the distal cut.
When the distal blastema is placed on top of the proximal blastema, a correct arm forms
They allowed the blastema to form and cut off the blastemas.
What signals allow the cells to know where they lie to form into the proper structure?
one signaling molecule identified as key is retanoic acid.
They took a ceramic bead and coated it with retanoic acid==> ampuations and implantation of bead
- the closer to the body the bead was placed, the more parts were made
- As retinoic acid diffuses out, it becomes more distal and the signal strength weakens. With a weaker signal, less structures form.
What happens a few days after the wound is made in a B6 and MRL mouse?
there is no real difference after the wound is made
Day 5—A scab forms in the B6 mouse
MRL mouse: stem-like cells begin to become evident in the MRL mouse; the scab is shed and new epithelium is present. Because this epithelium is forming much more rapidly, a blastema forms.
B6: B6 mouse has re-epithelialized, and the presence of a hole is still evident; it is smaller but still present
MRL: a blastemal formed in the MRL mouse and much more cell growth is evident hafter ten days
not much proliferation is seen in the B6 mouse; in the MRL mouse, two blastemal meet and fuse to one another, causing a continuous tissue layer
Overall, explain the B6 vs. MRL mouse
the B6 mouse has stopped growing and dense collagen is present in the area; in the MRL mouse, islands of cartilage form where there used to be a hole but there is no sign of a hole
What are other characteristics that were discovered?
Staining for the basal lamina in B6 and MRL mice:
- B6: basal lamina was maintained
- MRL: lays down basal lamina but then is digested; communication between underlying epidermal tissue and cells--> growth
What did a microarray show?
The blastema was cut off with the epidermis and dermis, etc.
Take a healing hole, cut it out, extract mRNA from it and perform a primitive microarray (similar to Northern blot with probes and membrane)
Cells associated with regeneration process expressing Pref-1; stem-like cell maintaining stem cell characteristic b expressing Pref-1; array is spotted twice
The RNA obtained revealed the expression of Pref-1, barely on days zero and five, and significantly on day 20.
What was difference in Pref-1 expression in MRL and B6?
In comparison to the B6 mouse, using real-time PCR, the expression of Pref-1 was determined. Pref-1 becomes up-regulated much more quickly and to a larger extent in the MRL mouse than in the B6 mouse.
What was determined using laser capture microdissection (LCM)
Pref-1 expression, via LCM, was determined to be expressed in the dermis. Localization of Pref-1 was tehn carried out via in situ hybridization. Blastema cells adjacent to the cartilage hybridized.
Explain use of the antibody probe for Pref-1
Pref-1 was detected in the intact cartilage immediately adjacent to the wound site in the B6 ear. Staining appeared immediately adjacent to the wound site in MRL as well, but also within the blastema
Aside from an antibody probe, what other probe was used?
- Nucleotide probe
- cells tucked into cartilage associated with cartilage express Pref-1
dots are places where cells express Pref-1 and where a band of cartilage will grow
Essentially, what is Pref-1 needed for?
it is needed for part of the pathway needed for formation of the bllastema
What is one more characteristic besides the basal lamina?
increased expression of protease activity, as well as protease inhibitors and activators in MRL mice
How did they look for expression of the proteases?
MMP: secreted by cells in pro-enzyme form; has one MW and is activated by cleavage
Proteins were separated by size, incubated in a buffer, and placed in a gel made of gelatin; proteases digest the protein in gel, leaving a blank open spot
This showed increased protease activity in MRL mice