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5. Frontiers of Biomedical Engineering: Cell Culture Engineering
Poziom:
Temat: Edukacja
Professor Mark Saltzman:
There were a few things I
didn't get to finish last time
in talking about particularly
gene transfer in mammals.
I want to finish with that and
then start on the topic for this
week which is an introduction to
cellular physiology.
In particular,
cell culture technology or how
you culture cells outside the
body.
This is the slide that I
left on last time and the idea
here was to use plasmids,
which I talked about last
Thursday, to introduce genes
into animals.
Here, the plasmid is directly
injected into a cell.
The cell is a fertilized egg,
so you take a fertilized egg
and you directly inject plasma
DNA into the pronucleus,
into one of the
pronucleus--pronuclei.
This solves a major problem
which I'm going to talk about
just at the beginning of class
here in a slide or two,
is 'how to get DNA into cells?
We talked last week,
or the week before,
about the plasma membrane and
something about the chemistry of
the plasma membrane:
a lipid bilayer that's stable
in water because it's arranged
in such a way that the
hydrophilic parts of the lipid
molecules are exposed to both
the outer and the inner surface
of the cell.
There's lipid chains in between
so cell membranes are lipid rich
layers that can exist in water.
Because of that they're
barriers, they separate the
inside of the cell from the
outside of a cell,
and only certain kinds of
molecules can pass through a
cell membrane.
It has to be small,
and it has to be lipid soluble
in order for it to pass through
a cell membrane.
Well, plasmid DNA is
neither of those.
It's very water soluble and
it's a big molecule,
a very big molecule;
it can't naturally get inside
cells on its own.
We talked about this a little
bit in section last week,
when you eat vegetables or
meat,
you're eating a lot of DNA but
that doesn't enter the cells of
your body because of the barrier
properties of cell membranes.
To get genes and gene transfer
vectors into cells is a problem.
Here, the problem is solved by
injecting it directly into the
cell, shown here,
and then that's one issue with
gene delivery.
The second issue is that the
gene vector, in this case the
plasmid, has to be compatible
with the cells that you're
trying to express the gene in.
that means that the cells have
to be able to replicate the DNA
on the plasmids,
in order to make many more
copies of it.
It has to have an origin of
replication, which we talked
about last time,
which is compatible with the
cells.
It also has to have a
promoter for the gene that the
cells can recognize.
In this case the promoter,
which is a sequence of DNA that
is positioned right in front of
the gene of interest,
the promoter is the
betagalactoglobulin promoter in
this case, and
betagalactoglobulin is a milk
protein.
You're taking advantage of a
promoter system,
or a gene activation system,
that this species knows about
because adult animals can make
milk.
When they make milk they have
signals for turning on the
betagalactoglobulin promoter and
expressing the gene of interest.
Both of those things have to be
correct.
You have to be able to get the
DNA into the cell,
it has to be incorporated into
a vector of some sort,
and the vector has to be
compatible with the species and
the cells that you've provided
the DNA to.
In this case,
if everything goes right,
you inject the DNA into the
pronucleus,
you implant this fertilized egg
into a foster mother,
the mother gives birth to
offspring,
it develops and when it
develops this foreign DNA is in
the offspring.
When this animal makes milk,
it makes all the normal milk
proteins, but it also activates
your gene of interest as well.
So you could collect your gene
of interest in the milk.
Does that make sense?
This concept of introducing
foreign genes,
genes that are made of
recombinant DNA into animals
using this kind of technique is
widely used in biomedical
research.
One of the ways that it's used
is to take the fertilized egg
from an animal,
a mouse, usually it's a mouse
and inject in a vector that
contains a gene that is involved
in human disease of some sort.
One of the big problems
with studying human diseases is
that you can study how they
occur in people but there's only
a limited amount that you can
learn from people.
If animals get a similar
disease then you can learn about
that disease progression in much
more detail in the animal.
But, unfortunately,
there aren't good animal models
for many of the diseases that
we'd like to study.
Alzheimer's disease is a good
one.
Millions of humans affected by
Alzheimer's disease,
other species don't get
Alzheimer's disease.
You can produce animals that
have a disease that's similar in
some ways to Alzheimer's disease
by taking the genes that are
involved in Alzheimer's and
introducing them into mice,
for example.
You do that by expressing the
genes in the mice in the way
that I described on the last
slide and it's shown in a little
bit more detail on this slide.
Those animals that you produce
are called transgenic animals
because they're expressing,
usually at high levels,
a transgene or a gene that's
not normally present in their
species.
How would you accomplish
this kind of - how would you
accomplish gene transfer in
adult humans?
Those two examples I just gave
you, one had to directly inject
a gene containing vector into
the pronucleus of a fertilized
egg and you don't have that
opportunity in adults.
You already have an adult
organism and you'd like to get
the gene transferred.
Before we talk about how to do
it, and we don't have perfect
ways to do it yet but I'll
describe some of the ways that
are used,
I want to talk just briefly on
this slide about what the goals
for gene therapy might be.
There's a variety of
different ways to think about
using gene therapy and they're
illustrated in this picture.
One way is to replace a gene
that's defective in a disease.
Usually this is a disease
that's caused by a defect in a
single gene.
We talked about sickle cell
anemia last time,
that's an example.
Another example is cystic
fibrosis.
Cystic fibrosis is a disease
that affects many organ systems,
but particularly the lung.
You get cystic fibrosis because
one protein that's made by your
lung epithelium is not expressed
properly.
You're able to make the protein
but the protein doesn't function
properly.
You could potentially treat
cystic fibrosis by providing the
correct gene and providing it to
all the cells of the lung
epithelium.
Because of this,
because cystic fibrosis is a
disease that's caused by a
single gene defect,
there's been great interest in
trying to treat cystic fibrosis
with gene therapy.
The idea would be to try to
give, usually in the
lungs--people have thought about
this because that's where the
main manifestations of the
disease occur--and introduce a
gene vector into the lungs,
perhaps by having a patient
inhale it or by somehow
instilling into the lungs,
in such a way that these gene
vectors get taken up by lung
cells and the gene gets
expressed throughout the lung.
This might be a plasmid.
It might be a plasmid that
contains a promoter that works
in lung cells and that has the
cystic fibrosis gene.
Well, now,
how would you get the gene into
the cells?
Well, one way you can get it is
by mixing the gene with lipids,
with lipid molecules.
Special lipids that do this are
called cationic lipids.
They're lipids but they also
have a charged portion,
a positively charged portion
which interacts with DNA.
So you form DNA lipid complexes
and because the DNA is complexed
with lipids it's more soluble in
membranes and more likely to
enter cells.
That's one way to do it and
I'll talk about it in other ways
in just a minute.
You get the idea here,
is to deliver the gene that
you're interested in directly to
the tissue where it's being used
and in this case,
the idea is to introduce a gene
that's missing.
Now, in other cases you
might want to introduce a gene
that's not even normally present
in people.
You're doing that because you
want to treat disease in a
different way.
One way that that's done is by
introducing genes into cancer
cells, genes that aren't
normally expressed in any
mammalian cell,
but that will cause the cancer
cell to die.
This example here,
the idea is to introduce a gene
into tumor cells of a tumor.
It's a gene that allows you to
deliver a drug that's not active
but the gene causes that active
- that inactive drug to be
converted into an active form
that kills the cell.
This is called a suicide gene.
Basically, you're introducing
this gene into cells that when
you give the right inactive drug
that will cause the cell to make
chemicals that kill it.
Does that make sense?
This is a way of
introducing cytotoxicity or cell
killing ability into a cell,
and that's been used to treat
many kinds of cancers,
particularly cancers of the
brain.
The problem is the same,
how do I get the gene that I
want into only the cells that I
want?
You can see that it would be
problem here if the gene that
you were trying to give to the
cancer cells also went to normal
cells--that would be a problem.
This is a common problem in
gene therapy.
You would like to get the gene
that you're interested in
expressed in some population of
cells within the body but not in
other cells.
So, making gene therapy
specific is a key problem in
making it work.
These are two examples I
just gave you of introducing
genes that affect the life of a
cell,
either by making it express a
protein that it's not making
properly that's important to its
life,
or by intentionally killing a
cell by having it express a
fatal gene.
The other examples on the
bottom of the slide here are
introducing genes that affect
neighboring tissues.
So one way to do this might be
to - if you're trying to treat a
heart that has disease and
usually it's disease in one of
the coronary vessels or the
blood vessels that serve the
heart,
that provide blood to the heart.
Coronary artery disease is one
of the major causes of death in
western countries.
It occurs because blood flow
gets reduced in that blood
vessel and so the heart muscle,
which is actively beating all
the time, needs large quantities
of oxygen provided by blood
can't get the blood through the
vessel.
Here, a concept is to
introduce a gene that causes the
process of new blood vessel
growth.
If you could introduce that
gene you'd make the chemical,
in this case it's a protein
called vascular endothelial
growth factor.
The name of the molecule isn't
important, but it's a molecule
that stimulates the heart to
produce new blood vessels around
it.
This is introducing a gene,
it's a natural gene but it's
not - it's a natural gene that's
found in humans and is actually
expressed all over your body in
different levels,
but you're concentrating it or
over expressing it in one
particular region of tissue in
order to have a particular
effect.
You're taking a normal gene and
you're over expressing it or
expressing it more abundantly
than normal in one particular
spot of the body to have an
effect.
One last example here is
that you might want to put a
gene in that makes a protein in
a tissue that doesn't ordinarily
make it so that that tissue can
make large quantities of the
protein and serve sort of as a
source of that protein as a
drug.
The example might be insulin.
Your pancreas normally makes
insulin.
Diabetics whose pancreas does
not function properly,
so don't make enough insulin,
don't have enough insulin
present for normal glucose
metabolism so they can't handle
sugars properly.
What if you injected the gene
for insulin into their muscle so
that their muscle cells in one
particular location started
making insulin?
Then that insulin would
accumulate in the muscle,
enter the blood,
and circulate all over the body
and so you'd turn the muscle
into an insulin making tissue.
Those are some example of
different strategies for using
gene therapy in people.
Well, I talked about the
problem of using plasmids in
gene therapy,
that it's very difficult to get
them into cells.
There are other vectors that
are very efficient at getting
DNA into cells.
Viruses, in particular,
are very effective at
delivering their viral DNA into
cells.
If you catch a cold you get
exposed to someone who has a
virus that's causing an upper
respiratory infection,
they cough near you that virus
enters your respiratory system,
it infects cells of your
respiratory system and those
viruses replicate.
They basically make many,
many copies of their DNA and
they make all of their viral
proteins and they assemble into
new viral units.
Well, what if you could
hijack that virus to carry genes
that you want?
You could do that by using the
techniques we described with
restriction enzymes and ligases
to cut and paste the viral
genome so that it contains the
gene of interest to you.
You make a recombinant virus
that has all the normal
components of the virus but also
has a gene that you want.
Now, all you have to do is
introduce that virus into an
organism and the virus will do
what it naturally does,
which is infect some cells.
Now, the other nice thing about
viruses is not only are they
very efficient but most viruses
are specific for certain kinds
of cells.
The kinds of viruses that we
get when we have colds are very
good at infecting the
respiratory system.
If you get a flu that affects
your intestinal system then
those viruses are infecting gut
cells.
There's other viruses that
infect cells of the brain,
of the kidney,
of the liver.
Hepatitis is a virus that
specifically affects the liver,
so viruses are often specific
to certain kinds of cells.
That is also an advantage of
using them as gene therapy
vehicles because they will only
infect cells that they are -
that they have an affinity for
or that they're prone to affect.
This picture here,
we're going to come back to
this later in two weeks when we
start talking about vaccines,
and we're going to talk about
how you make vaccines for
viruses and so in doing that
we're going to think about the
life cycle of a virus.
This is a life cycle of a
particular class of viruses
called retroviruses.
You know about retroviruses,
the most famous and important
one at this time is HIV,
which is a retrovirus.
This shows the greatly
magnified compared to the size
of a cell because viruses are
much, much smaller than cells.
They might be 100 nanometers or
so in diameter,
whereas, a cell is ten microns
or so in diameter.
These very small particles are
able to specifically recognize
certain kinds of cells.
They enter the cell after
recognizing, they fall apart
inside the cell,
they reproduce their DNA,
or their genetic material using
host mechanisms.
They, in some cases,
they integrate their DNA into
the host chromosome,
that's what retroviruses do and
they make viral proteins.
Because they've made many
copies of their genome and
they've made all the proteins
that are necessary to - for
assembly of a new virus,
then this cell can make many
new viruses.
That's another advantage of
using viruses as a gene therapy
vector.
They can reproduce and so you
only need to give a few of them
in order to make many,
many inside the body.
Why isn't that the solution
to gene therapy?
If you can genetically engineer
viruses like this and viruses
have all those good properties
as gene therapy vehicles that I
described.
Why isn't this a solution?
Well, there are problems with
viruses as well.
Retroviruses,
for example,
can only insert - can only
transduce and express their DNA
in cells that are dividing.
So if it's not a cell that's
going through division you can't
use a retrovirus to express the
protein.
They integrate their DNA into
the host genome and you might
not want to make that kind of a
change in the cell.
These are some problems with
retro viruses.
Because of this people have
sought other kinds of viruses to
use as vectors and one that's
commonly used is an adenovirus.
Adenoviruses are viruses that
give upper respiratory
infections, they cause colds.
They can transduce non-dividing
cells so that's a good part.
They can insert genes into
non-dividing cells and cause
those genes to be expressed.
A problem with adenoviruses is
that your immune system
recognizes them,
and if your immune system
recognizes the virus then it can
attack the virus and eliminate
it from the body.
Now, normally that's a good
thing because if you're out in
the world and you get infected
with adenoviruses you recover
from it,
your immune system can get rid
of it.
But if you're trying to use
that virus as a gene delivery
vector and your body gets rid of
it then the therapy has failed,
it's ended.
People have tried to produce
viruses that are different,
adenoviruses for example,
that your immune system can't
recognize.
Because of this,
because of the natural
properties of viruses which
sometimes are unwanted like in
retroviruses,
and the natural properties of
your immune system which can
cause you to reject viruses,
we've had great problems in
making viruses that are both
safe and effective for gene
therapy in people.
I talked about just not
using viruses but using plasmid
DNA together with lipids and
that's another strategy for gene
therapy.
We'll talk later about other
kinds of vehicles when we talk
about new methods for delivery
of drugs.
That's what I wanted to say in
finishing up last week's topic
of DNA technology.
What I want to do for the rest
of the time today is talk about
- a little bit about cell
physiology but I'm not going to
say too much for a couple of
reasons.
I think most of you know
something about the sort of
architecture and normal
structure of a cell and the
components within it and the
chapter,
Chapter 5, has a good
description of that for those of
you want a refresher.
We'll talk a little bit about
the principles of cellular
physiology and then I'll move
next time to talking about how
you culture cells outside of the
body and what that's useful for.
I said this sentence in one
of the first two classes,
'all cells are the same',
and you know that's true.
We talk about cells as being
kind of the fundamental unit of
humans and other multi-cellular
organisms.
Why do we talk about them as
the fundamental units?
Because cells on their own are
alive, they can do the things
that we associate with life,
they replicate,
they reproduce,
they metabolize,
they can move,
they can grow,
and they can use nutrients and
make wastes, and they work
cooperatively in order to
perform some function.
This is a picture that I
showed you before,
but basically tells you
something that you already know,
that humans are made up of
organ systems,
organs and organ systems,
those organs are composed of
tissues.
What I didn't mention before
was the four major types of
tissues which make up the organs
of our body: muscle tissues,
and the function of muscle
tissues is to do work,
to contract in order to do
work,
nervous tissue and the function
of nervous tissue is to
communicate using electrical
impulses primarily to
communicate,
epithelial tissues which form
linings in the body so your skin
is an epithelial tissue,
the lining of your gut is an
epithelial tissue,
the linings of all the glands
in your body like the pancreas
are epithelial tissues,
and connective tissues which
are responsible for holding all
these things together into one
unit.
All cells are the same and
I say that because any cell
within you or within me is the
same in many respects.
It has a cell membrane.
If I looked inside it has the
same kinds of components inside,
it has the same DNA inside the
nucleus.
Let's take a step back and ask
are all kinds of cells really
the same and start by talking
about two cells that I know you
know are quite different.
Cells that I took from a human
and cells that are in a
bacterium.
What are the differences
between a bacterial cell and a
human cell?
What are some of the
differences between a bacterial
cell and a human cell?
Student:
[inaudible]Professor
Mark Saltzman:
The bacterium doesn't have
a nucleus,
and in fact,
they don't have very well
formed organelles in general.
Human cells have well-described
organelles inside of them.
You took biology in high
school, you learned the names of
these organelles and their
principle functions,
the nucleus,
the endoplasmic reticulum,
the Golgi apparatus,
mitochondria,
lysosomes.
Those things are all reviewed
in the chapter in the book.
I'm not going to talk about
them in detail but please look
at that to remind yourself of
those kinds of specialized
sub-cellular units which are
present in cells in humans but
not present in bacteria.
Why aren't they present in
bacteria?
Why don't bacteria need a
distinct nucleus and that's
necessary in a human cell?
What else is different about
human cells and bacteria other
than these formed organelles?
Student:
[inaudible]Professor
Mark Saltzman:
Human cells don't have a
cell wall but most bacteria do.
There are different kinds of
cell walls in bacteria but they
have a rigid cell wall and our
cells don't have a rigid cell
wall,
we have a fluid lipid like cell
wall.
Why is that the case?
Well, you could think about the
cell wall of a bacterium as its
skeleton, it's what protects it
from mechanical forces that are
out in the world.
We have other ways of
protecting;
many of our cells are inside
our body protected by
specialized cells of our
skeletal system.
They don't need that kind of
mechanical strength.
Another important
difference between human cells
and bacterial cells is their
size.
Bacterial cells are small,
one to two microns in diameter
and this shows a picture of a
common bacterium called E.
coli. This is a bacterium
that's normally present in
humans;
it colonizes all of our
intestinal systems.
It's a common bacterium and
most strains of E.
coli are not harmful to
people, in fact,
they provide us with some
beneficial properties.
A human cell shown here on the
opposite side has these formed
organelles that I talked about a
minute ago,
mitochondria,
Golgi, endoplasmic reticulum,
nucleus.
It's encased within a membrane,
a cell membrane which we talked
about the structure of.
It's much larger than a
bacterium, it's ten to 30
microns let's say instead of one
to two microns,
so almost ten times bigger.
One of the reasons why
human cells have compartments is
because their functions are
segregated into regions.
The nucleus contains all the
DNA, and so the early stages of
gene expression,
transcription,
happened inside the nucleus
where all the DNA is.
One of the reasons why you
would want to segregate things
into certain regions is that
molecules have to move in order
to accomplish these things;
molecules have to move from one
place to another.
In general,
inside of a cell molecules move
by a process called diffusion.
They move from regions of high
concentration to regions of low
concentration by this natural
flow down a concentration
gradient.
Movement down a concentration
gradient occurs fairly rapidly
over short distances like a
micron or so,
like the size of a bacterium.
Diffusion mixes things very
well, but as the size gets
bigger, diffusion becomes very
slow.
You're familiar with this,
if I had a jar of acetone or
some kind of a volatile liquid,
let's say I have perfume.
I have a jar of perfume and I
sit it here, if the room was
perfectly still the molecules of
perfume would diffuse out of
this jar and they'd start moving
through the room and those of
you in the first row would smell
it within a few minutes,
sometime later you'd smell it
one row back,
sometime later - and the
farther away you get the longer
it takes.
Because of the physics of
diffusion it doesn't go linearly
with distance.
That is, it doesn't take one
second to go the first,
and then two seconds to go to
the second, and three seconds to
go to third.
It varies with the square of
distance so it goes - in order
to go twice as far it takes four
times as long.
These details aren't important,
it is described in your book,
but the thing that is important
to know is that diffusion occurs
fast over short distances,
but because of the physics of
diffusion it occurs slowly over
longer distances.
Because of that you can't count
on molecules moving rapidly over
the size of a cell that's ten to
30 microns in diameter.
Because of that,
in order to efficient function,
then you group all the like
activities together.
That's one way of thinking why
animal cells have cells from
animals and humans have
sub-cellular compartments.
Another thing that's
different about human cells and
animal - human cells and
bacterial cells is that many
bacterial cells can swim and can
exist in suspension,
that means just suspended in a
fluid.
In fact, that's their preferred
state is suspended in a fluid
where nutrients are widely
available to them,
and where they can swim from
one spot to another in order to
get the nutrients they need,
or in order to run away from
toxic compounds.
If you're a bacterium your
whole life is about swimming to
nutrients and running away from
things that would kill you.
Human cells,
most human cells,
don't function very well in
suspension.
If you take cells from my skin
and disperse them and we'll talk
about ways to do that next time,
and you try to maintain them in
a beaker.
Even if that beaker had all the
nutrients that skin cells needed
they wouldn't survive very well
in solution,
they tend to need to be stuck
to a surface in order to
survive.
That's what this picture on
the bottom shows you here.
It's a picture of cells
actually growing and moving in
cell culture.
This is a cell from connective
tissue called a fibroblast.
Instead of being round like
this cartoon of a cell when you
have the cell in culture,
in a plastic or a glass dish,
it tends to stick to the dish
and spread out so it looks much
more like a pancake,
a funny shaped pancake,
but a pancake than a sphere.
This property of cells in
culture is called anchorage
dependence - that the cell needs
to be anchored to a surface in
order to function properly.
That's another difference
between human cells and
bacteria.
Now, what does that have to
do with the life of a cell
inside the body?
It's - this picture on the
bottom shows the cell stuck to
plastic.
Certainly that's not what it's
like when it's inside the body.
Well, it's true that it's not
stuck to plastic but it is stuck
to other materials The other
materials that it's stuck to
include other cells that
surround it and this complex
matrix which surrounds all of
the cells in our body called
extracellular matrix and I'll
get to that in just a few
moments.
In some senses most of the
cells in our body are stuck to a
surface.
They're stuck to a surface
that's provided by the
environment that's around them,
they're not swimming free in
solution.
Well, you know that cells
in our body can reproduce.
Most of you have probably at
some point or another learned
about the cell cycle,
or the process of a cell going
from being a single cell to
being two cells.
That's shown schematically
here, let me talk about the
bottom diagram first,
which shows the process of
mitosis.
Mitosis is an orderly sequence
of events by which one parent
cell becomes two daughter cells.
Several things happen during
mitosis, first,
when a cell enters the process
of mitosis it already has twice
the DNA that it needs.
So before a cell enters mitosis
it has to have synthesized its
DNA so it has two copies of all
of its chromosomes.
What happens during mitosis
is that the cell is able to
separate this double set of
chromosomes into two sets and
that happens in a way that's
shown in this cartoon here.
It separates those duplicated
chromosomes to two sides of the
cell, physically separates them,
and the cell itself pinches off
to form two new cells.
If this happens correctly,
each of the new cells has
exactly the same composition as
the parent cell.
Mitosis is one event that
happens during this larger event
called the cell cycle.
It's usually shown like a clock
is shown here,
where time 0,
which is at the top here is
right at the end of mitosis
where you have two new cells
that are formed.
If I followed one of them it is
in a phase that's called G1
first and this is a resting
phase before it enters the
process of synthesizing new DNA
in preparation for another round
of mitosis.
G2 is a phase that indicates
the gap between when DNA
synthesis occurs and when the
next mitotic event occurs.
If cells were actively
proliferating,
that is you had a population of
cells that are actively
proliferating,
they would be going through
continuous rounds of the cell
cycle.
New cells are formed,
they wait a bit,
they synthesize DNA,
they wait a bit,
they divide,
and on, and on,
and on.
Well, not all of the cells
in your body are dividing at any
one time.
Not all the cells are dividing;
in fact, cells in your brain
don't proliferate at all.
Some small subsets do but most
of the cells in your brain don't
proliferate and that's because
they get trapped at one portion
of the cell cycle.
They get arrested in the G1
phase, for example,
and they never progress through
to the S phase where they
synthesize DNA.
Cells can stop in their
lifetime at some point in the
cell cycle and persist for long
periods of time.
This process of cell
proliferation or cell division
is, of course,
very important during
development of an embryo.
During development of the
embryo it goes from being a
single cell, the fertilized egg,
to being a multi-cellular
organism with billions of cells
in it.
Those billions of cells come
from successive rounds of cell
division.
Now, I said before that all
cells are the same.
When I talked about mitosis,
I talked about cells making
perfect copies of each other,
so each parent cell becoming
two daughter cells that are the
same.
So if that was all that was
happening then an embryo would
just be many,
many cells that are all
identical and you know that's
not the case.
Cells start to change and they
change in complex ways so that
eventually when a fetus is born
it has some cells that make up
its brain,
some that make up the heart,
some that make up the liver and
the skin, and all those cells
are different in important ways.
I'm going to talk next
week, or next time on Thursday,
about the processes that lead
to differences between cells.
The general word for that
process of making differences
between cells is
differentiation.
When cells become more like
cells of the mature brain
they're said to differentiate
into brain cells.
When they become more like the
liver, they differentiate into
liver cells.
So we're going to talk about
that process more next time.
What I want you to remember now
is that abundant cell
proliferation takes place during
development.
All of the cells that are
reproduced during development
are the same in certain ways,
they contain the same DNA,
they're all progeny of the same
fertilized egg,
but differences are acquired
within specific cell populations
during the process of
development.
Some of those differences
in cells are obvious if you look
at the cells in an adult
organism.
In fact, some cells are very
different from the cells that
are proliferating early in
development.
One example of a very different
cell is a red cell or red blood
cell, these are the cells that
give blood it's red color.
They make up about 45% to 50%
of the volume of blood is red
cells.
They're red because they
contain a special protein called
hemoglobin which is very
concentrated inside the cell.
Hemoglobin, as you know,
is an oxygen carrier and so red
blood cells concentrate in bags
of hemoglobin which circulate in
your blood and they carry
oxygen.
Well, it was not very hard for
me to draw this red blood cell
here, it's just an oval,
and one reason I draw it like
an oval, is that that's
basically what it looks like if
you look at it.
One of the most obvious things
about a red blood cell is it
doesn't have nucleus,
it has no nucleus.
Red blood cells are terminally
differentiated cells.
They have become as different
as they can be.
In fact, they're no longer
capable of reproducing;
all they can do is exist as red
blood cells and then die.
So, they've lost one of the
essential characteristics of
these cells that are the same
that I talked about earlier.
That's just an example of a
cell that in its very mature or
differentiated form is quite
different from other cells
inside the body.
On the other side of this
diagram I show a picture of a
neuron.
This is just an example of what
a neuron might look like.
Here's the nucleus in the
middle of the cell body and what
- if you looked at a picture of
a variety of different cells
that were isolated from adult
organisms you would recognize
this immediately as a neuron or
something that's like a neuron
because of the many small thin
projections that come out of the
body of the cell.
Neurons have these projections
because one important property,
or one important function of
neurons, is to communicate with
each other.
The way that they communicate
with each other is by physically
touching each other,
and the way that they connect
across distances is by having
these process that extend from
one place to another.
Like many cells in neurons,
the shape of the cell is
intimately related to its
function within the organism.
That's obvious when you look at
neurons and it will be obvious
when we look at other kinds of
cells as well.
Because of this,
often a biologist can
discriminate between different
kinds of cells that they see in
section of a tissue for example,
merely by its shape and the
characteristics that it has.
One could easily,
for example,
differentiate between a red
cell and a neuron,
but a skilled biologist who's
looked at many,
many different cells could
differentiate between different
types of neurons.
Maybe by the different
branching patterns that these
processes had,
or how many processes were on a
different kind of neuron,
you'd be able to tell something
about its function and probably
something about where it came
from.
You might be able to tell just
by looking at it what was - if
there was a disease what was
wrong with it.
In between these two
pictures I show a picture of a
fibroblast.
Fibroblasts are specialized
cells that exist in connective
tissues.
The connective tissue is one of
those four main types of tissue,
they're supporting material -
supporting tissues that surround
other cells in the body and give
the cell - and give the body
much of its mechanical property.
One of the important functions
of a fibroblast is to allow for
healing of wounds.
This shows a fibroblast that is
very stretched out.
Remember when I showed you that
picture of cells attached to
plastic a few minutes ago;
I said those were fibroblasts
that were growing on a culture
dish.
If I looked within the
skin, one of the lower layers of
your skin, the connective tissue
layer,
you'd find many fibroblasts
there and they're just waiting
in a more rounded shape than
this,
just waiting to do their job.
Their job begins if you happen
to cut yourself,
for example,
or you get a wound.
When you get a wound several
things start to happen.
First, hopefully your blood
clots and so that stops you from
dying from loss of blood there,
but your body tries to heal
this wound that's created.
One of the first steps in that
healing is that fibroblasts like
this cell crawl into the space
that's created by the wound and
they grab a hold of both sides
of the wound and contract and
try to pull it together.
To do that they have to be able
to stretch out and they have to
be able to pull.
If you look at this picture of
this fibroblast you can imagine
that it's firmly stuck up here.
It's firmly adherent to the
surface here,
it's firmly adherent at the
back here,
and in between its trying to
pull these ends closer together,
you can imagine that that's
what it's doing,
and in fact,
that's one of the functions of
fibroblasts.
This is another example of how
the shape of a cell can tell you
something about its function,
how you can discriminate
between different cells by
looking at how they look in
their native environment.
Humans are collections of
cells, all cells are the same in
important ways,
but cells acquire differences
during development,
and so we are collections of
billions of cells.
What holds us together so that
when I leave the room most of my
cells go with me?
What holds all these cells that
are doing different things
together?
Well, one thing that holds
cells together is that they're
able to adhere to each other.
That cells that form tissues
often have junctions that hold
them together so that they don't
fall apart and they exist as a
solid piece of tissue.
This diagram is supposed to
make you think about the
intestinal tract,
this tube that runs through our
bodies and allows us to acquire
nutrients from the environment.
This tube, this hollow tube,
which food passes through is
made up of many,
many epithelial cells which
directly abut one another and
would be a flat sheet except
that it's rolled up into a tube.
It's this tube-like structure,
which is made of many,
many epithelial cells all
directly adjacent to each other,
is what separates the outside
world from our internal bodies.
We take bits of the outside
world like food and we swallow
them.
One can imagine that when
they're inside this tube here,
they're still really outside
the body.
If it passed directly through
the tube and nothing happened to
it, it would come out to the
outside world again.
There's a continuous path from
the outside world right through
the intestine.
That food only becomes part of
us if it gets absorbed through
the intestinal wall.
That is, if it can be absorbed
through this layer of
epithelium.
What happens in the intestine
is that food gets broken down
into constituent molecules,
some of those molecules are
absorbed into our bodies.
They become part of us,
and the rest get - we get rid
of and they remain part of the
outside world.
It's these cells that are
sitting at the surface here that
are responsible for determining
what becomes part of us and what
stays outside.
One of the ways they can do
this is if there's no space in
between these cells.
This cell can only be an
effective barrier if there's no
way to get past it.
Cells of the epithelium are
actually welded to one another
through these junctions called
tight junctions.
These junctions,
these red dots here are
actually made up of proteins,
some are synthesized by one
cell,
some are synthesized by each
other, and they lock together to
form a very tight barrier,
to form a tight junction
between the two cells.
Because of that,
if I eat something that's rich
in glucose, so it has lots of
sugar molecules,
the sugar molecules can't pass
between the cells.
That's how tight the junctions
are, not even a small molecule
like glucose can pass between
them.
In order for glucose to
enter our body it has to go
through the cells.
In order for it to get inside
the cells and go through them
there have to be transporters
which specifically allow glucose
to pass into the cell and then
out into our body.
There's a couple of features of
cellular physiology which are
shown here.
One is that cells form tissues
that are mechanically intact,
they have mechanical integrity
because they can adhere to one
another.
The other is that this kind of
adherence gives the tissue a
property that's useful,
in this case the property is it
can serve as a barrier to
nutrients from entering our
body.
The other reason why cells stay
together is that they're
surrounded by a matrix that's
called extracellular matrix.
That matrix has properties that
vary in different locations in
the body, but basically it's a
highly hydrated or water-rich
gel.
You know what Jell-o is?
Everybody knows what Jell-o is,
right?
It's usually colored because
there's food dye in it,
but it wouldn't be colored in
its natural state.
Jell-o is just a hydrated
solution of collagen and it
contains a lot of water.
A piece of Jell-o has probably
99% water in it,
but it has a shape.
If you make Jell-o and you make
it right, then once it's set or
gelled, it has mechanical
properties.
If you made it in a cup and you
tried to pour it out it would
still have the shape of the cup
when it came out.
Because of this my mother used
to make Jell-o that had fruit
suspended in it,
and the fruit doesn't sink to
the bottom like you'd expect it
to sink through water,
but it stays suspended inside.
Anybody's mother make Jell-o
with fruit in it?
That's how cells exist in
extracellular matrix.
Extracellular matrix in most
tissues is abundant in collagen
and other proteins like
collagen.
This collagen is highly
hydrated, it forms a gel that
has water-like properties but it
also has solid properties.
We'll talk about exactly
what a gel is later when we talk
about biomechanics and cells are
suspended within it.
The extracellular matrix or the
collagen gel gives the tissue
integrity, and in part,
determines its mechanical
properties.
Student:
[inaudible]
Professor Mark Saltzman:
Where does the collagen
come from?
The collagen is excreted by
cells themselves,
so the matrix is made by cells.
In fact, fibroblasts,
which I talked about before,
are very good producers of
collagen, they produce the
collagen matrix within which
they live.
Some cells digest collagen.
What extracellular matrix you
have in any particular tissue is
there because there's a balance
between it being produced by one
kind of cell and digested by
another,
and you're in this sort of
state of dynamic equilibrium.
Okay, I'm going to stop there
and we'll continue on
Thursday.
