First project!
My Project:
Enzymatic Exposure of Collagen Fibers in Cultured Chondrocyte Extra Cellular Matrix
Aim:
The extra cellular matrix is a complex network of macromolecules including glycoprotein, polysaccharides and proteoglycans. The extra cellular matrix of tissue is composed of water, proteoglycan, hyaluronic acid and collagen. Within this matrix, numerous growth factors and enzymes carry out their functions as cells respond to their local environment. Cartilage consists of about 5% cells and 95% extracellular matrix by volume (1), and is therefore a good model system in which to study extracellular matrix. The goal of this study is to visually characterize the extracellular matrix structure of RCS cells which are exposed to a range of enzymes over a range of culture ages.
Introduction:
Collagen fibers embedded in POLYSACHRIDE needs to degrade.
Hyaluraanin acid is. POLYSACHRIDE.
Aggrecan: Aggrecan is the shortened name of the large aggregating chondroitin sulphate proteoglycan. Aggrecan, which is one of the most widely studied proteoglycans, is abundant; it represents up to 10% of the dry weight of cartilage (articular cartilage is up to 75% water).
There can be some confusion about the use of the term aggrecan, and to what it refers. Many individual monomers of aggrecan bind to hyaluronic acid to form an aggregate; it is the monomer which is termed aggrecan. These aggregates are comprised of up to 100 monomers attached to a single chain of hyaluronic acid (HA).
The primary role of aggrecan appears to be a physical one, as it brings about an osmotic swelling and maintains the high levels of hydration in the cartilage extracellular matrix. In this way aggrecan plays a crucial role in the normal function of articular cartilage, which is found at the ends of long bones.
The extracellular matrix of articular cartilage is comprised of fibril forming collagens, aggrecan and many other important molecules. The fibrillar collagens form a network which has a very high tensile strength, and which entraps the aggrecan molecules. The presence on aggrecan of a very large numbers of chondroitin sulphate chains generates an osmotic swelling pressure. It is this which results in the wet weight of articular cartilage being 75% water. http://bssv01.lancs.ac.uk/gig/pages/pg/aggrecan.htm
Aggrecan has core protein that is attached to series of polysaccharide those include chondroytin sulfate and heparin sulfate.
We will be plating our RCS cells into six well plates and growing them on cover slips.The cells will be treated with three different types of enzymes.
1. Hyaluronidases: Sigma. H-4272
Hyaluronidase is an enzyme that is widely distributed in nature. It is derived from bovine (cow) testicular tissue extracts, although it is also present in the testes of other mammals. This enzyme breaks down the hyaluronic acid in connective tissue, increasing tissue permeability. It also enhances the diffusion of subcutaneously injected agents. Hyaluronidase is used for many different pharmacological purposes and also in surgery. This is a quite interesting enzyme. If interested in knowing the difference between hyaluronidase and hyaluronic acid and their pharmacological purpose... visit: http://www.ashp.org/shortage/hyaluronidase.cfm?CFID=4364102&CFTOKEN=17716244
2. Heparinase III: Sigma. H-8891:
There are three different types of Heparinase: I, II and III. Source of all three Heparinase is Flavobacterium heparinum (recombinant).
Two of them are saturated. I am still not sure what the main difference between all of them is. Heparinase III cleaves heparin sulfate exclusively, and does not cleave unfractionated heparin or low molecular weight heprains. It has clinical and non-clinical application. Most important: Heparinase III releases growth factors and mitogenic substances from extracellular matrices, thereby stimulating wound repair in an animal model of impaired wound healing. Therefore is useful in acceleration of wound healing in patients with venous ulcers such as seen in diabetic patients or decubitus ulcers. If interested in knowing the details about Heparinase III visit: http://www.ibex.ca/Assets/PDF/ds-hepiii.pdf
3. Chondroitinase ABC: Sigma. C-2905:
If interested in knowing the differences between Chondrotine A, B and C: http://bssv01.lancs.ac.uk/gig/pages/gag/cspages.htm#cs_abc_ase
Digestion buffer 0.1M TRIS/HCl pH 8.0; [1mM NaF (to inhibit sulphatases),- NOT NORMALLY ADDED]
For digesting GAGs to completion use 0.3units/mg GAG, 37 C.This enzyme produces unsaturated disaccharides that can be monitored at 232nm. To assay the enzyme, monitor the increase in A232 over the first 5 minutes of reaction under controlled conditions. Dividing the increase in absorbance in 1 minute by the molar extinction coefficient (5.5) gives the (ยต/m?)mole released per aliquot.
Method and Materials:
Cell culture: Rat chondrosarcoma (RCS) cells are stable differentiated chondrocytes that express type II, IX, and XI collagens but not type I and type X collagens (2). RCS cell lines will be grown in Opti-MeM media, (containing10% FBS supplemented with 100IU/ml penicillin, and 0.5 mM sodium pyruvate). These cell lines will be cultured at standard cell culture conditions. (4 to 5% of Co2 )
Enzymatic treatment: Cells will be grown in six well plates and on cover slips. Then these cells will be treated with three different enzymes: Hyaluronidases, Heparinase III and Chondroitinase ABC. (Working on Protocol!!)
Staining: After the enzymatic treatment, cells will be stained with different stains. After the successful staining, we will make the slides permanent. Stains:
Saffron, aniline blue, potassium iodine
Discussion:
This experiment will be just the beginning of our project to understand the structure and compostion of the pericellular matrix, which will have broad relevance to regulate extracellular matrix assembly and organization. Extra cellular matrix is very important and complex subject. Our aim is to break this complexity into simplicity and understand it with much confidence in order to perform many experiments and get results. From here, we will do different experiments in order to see what happens when one component of the ECM is absent verses present and many more. I am very excited to learn and get this project done as soon as possible so that I can move to next level. If any of you are interested in the topic and want to have your name on poster for this spring. Please join me. The more the merrier!!
Results: No Conclusion yet!! (In one month)
Literature cited:
1.Poole, C.A. (1993) Joint Cartilage Degradation; Basic and Clinical Aspects, (eds.J.F. Woessner, Jr. and D.S. Howell) Marcell Dekker, Inc, New York.
2.Mukhopadhyay, K., Lefebvre, V., Zhou, G., Garofalo, G., Kimora, J. H., & de Crombrugghe, B. (1995). J. Biol. Chem. 270, 27711 27719 . [PubMed][Free Full Text]
Related Readings:
http://www.ijdb.ehu.es/fullaccess/fulltext.0009/ft707.pdf
http://www.ucdmc.ucdavis.edu/ctrr/Chen-HaudenschildMolecBiolCell1995.pdf
http://dityatev.gmxhome.de/Dityatev_CSPG.pdf
http://www.aber.ac.uk/~ecmwww/journal/smi/pdf/cm98-11.pdf
Enzymatic Exposure of Collagen Fibers in Cultured Chondrocyte Extra Cellular Matrix
Aim:
The extra cellular matrix is a complex network of macromolecules including glycoprotein, polysaccharides and proteoglycans. The extra cellular matrix of tissue is composed of water, proteoglycan, hyaluronic acid and collagen. Within this matrix, numerous growth factors and enzymes carry out their functions as cells respond to their local environment. Cartilage consists of about 5% cells and 95% extracellular matrix by volume (1), and is therefore a good model system in which to study extracellular matrix. The goal of this study is to visually characterize the extracellular matrix structure of RCS cells which are exposed to a range of enzymes over a range of culture ages.
Introduction:
Collagen fibers embedded in POLYSACHRIDE needs to degrade.
Hyaluraanin acid is. POLYSACHRIDE.
Aggrecan: Aggrecan is the shortened name of the large aggregating chondroitin sulphate proteoglycan. Aggrecan, which is one of the most widely studied proteoglycans, is abundant; it represents up to 10% of the dry weight of cartilage (articular cartilage is up to 75% water).
There can be some confusion about the use of the term aggrecan, and to what it refers. Many individual monomers of aggrecan bind to hyaluronic acid to form an aggregate; it is the monomer which is termed aggrecan. These aggregates are comprised of up to 100 monomers attached to a single chain of hyaluronic acid (HA).
The primary role of aggrecan appears to be a physical one, as it brings about an osmotic swelling and maintains the high levels of hydration in the cartilage extracellular matrix. In this way aggrecan plays a crucial role in the normal function of articular cartilage, which is found at the ends of long bones.
The extracellular matrix of articular cartilage is comprised of fibril forming collagens, aggrecan and many other important molecules. The fibrillar collagens form a network which has a very high tensile strength, and which entraps the aggrecan molecules. The presence on aggrecan of a very large numbers of chondroitin sulphate chains generates an osmotic swelling pressure. It is this which results in the wet weight of articular cartilage being 75% water. http://bssv01.lancs.ac.uk/gig/pages/pg/aggrecan.htm
Aggrecan has core protein that is attached to series of polysaccharide those include chondroytin sulfate and heparin sulfate.
We will be plating our RCS cells into six well plates and growing them on cover slips.The cells will be treated with three different types of enzymes.
1. Hyaluronidases: Sigma. H-4272
Hyaluronidase is an enzyme that is widely distributed in nature. It is derived from bovine (cow) testicular tissue extracts, although it is also present in the testes of other mammals. This enzyme breaks down the hyaluronic acid in connective tissue, increasing tissue permeability. It also enhances the diffusion of subcutaneously injected agents. Hyaluronidase is used for many different pharmacological purposes and also in surgery. This is a quite interesting enzyme. If interested in knowing the difference between hyaluronidase and hyaluronic acid and their pharmacological purpose... visit: http://www.ashp.org/shortage/hyaluronidase.cfm?CFID=4364102&CFTOKEN=17716244
2. Heparinase III: Sigma. H-8891:
There are three different types of Heparinase: I, II and III. Source of all three Heparinase is Flavobacterium heparinum (recombinant).
Two of them are saturated. I am still not sure what the main difference between all of them is. Heparinase III cleaves heparin sulfate exclusively, and does not cleave unfractionated heparin or low molecular weight heprains. It has clinical and non-clinical application. Most important: Heparinase III releases growth factors and mitogenic substances from extracellular matrices, thereby stimulating wound repair in an animal model of impaired wound healing. Therefore is useful in acceleration of wound healing in patients with venous ulcers such as seen in diabetic patients or decubitus ulcers. If interested in knowing the details about Heparinase III visit: http://www.ibex.ca/Assets/PDF/ds-hepiii.pdf
3. Chondroitinase ABC: Sigma. C-2905:
If interested in knowing the differences between Chondrotine A, B and C: http://bssv01.lancs.ac.uk/gig/pages/gag/cspages.htm#cs_abc_ase
Digestion buffer 0.1M TRIS/HCl pH 8.0; [1mM NaF (to inhibit sulphatases),- NOT NORMALLY ADDED]
For digesting GAGs to completion use 0.3units/mg GAG, 37 C.This enzyme produces unsaturated disaccharides that can be monitored at 232nm. To assay the enzyme, monitor the increase in A232 over the first 5 minutes of reaction under controlled conditions. Dividing the increase in absorbance in 1 minute by the molar extinction coefficient (5.5) gives the (ยต/m?)mole released per aliquot.
Method and Materials:
Cell culture: Rat chondrosarcoma (RCS) cells are stable differentiated chondrocytes that express type II, IX, and XI collagens but not type I and type X collagens (2). RCS cell lines will be grown in Opti-MeM media, (containing10% FBS supplemented with 100IU/ml penicillin, and 0.5 mM sodium pyruvate). These cell lines will be cultured at standard cell culture conditions. (4 to 5% of Co2 )
Enzymatic treatment: Cells will be grown in six well plates and on cover slips. Then these cells will be treated with three different enzymes: Hyaluronidases, Heparinase III and Chondroitinase ABC. (Working on Protocol!!)
Staining: After the enzymatic treatment, cells will be stained with different stains. After the successful staining, we will make the slides permanent. Stains:
Saffron, aniline blue, potassium iodine
Discussion:
This experiment will be just the beginning of our project to understand the structure and compostion of the pericellular matrix, which will have broad relevance to regulate extracellular matrix assembly and organization. Extra cellular matrix is very important and complex subject. Our aim is to break this complexity into simplicity and understand it with much confidence in order to perform many experiments and get results. From here, we will do different experiments in order to see what happens when one component of the ECM is absent verses present and many more. I am very excited to learn and get this project done as soon as possible so that I can move to next level. If any of you are interested in the topic and want to have your name on poster for this spring. Please join me. The more the merrier!!
Results: No Conclusion yet!! (In one month)
Literature cited:
1.Poole, C.A. (1993) Joint Cartilage Degradation; Basic and Clinical Aspects, (eds.J.F. Woessner, Jr. and D.S. Howell) Marcell Dekker, Inc, New York.
2.Mukhopadhyay, K., Lefebvre, V., Zhou, G., Garofalo, G., Kimora, J. H., & de Crombrugghe, B. (1995). J. Biol. Chem. 270, 27711 27719 . [PubMed][Free Full Text]
Related Readings:
http://www.ijdb.ehu.es/fullaccess/fulltext.0009/ft707.pdf
http://www.ucdmc.ucdavis.edu/ctrr/Chen-HaudenschildMolecBiolCell1995.pdf
http://dityatev.gmxhome.de/Dityatev_CSPG.pdf
http://www.aber.ac.uk/~ecmwww/journal/smi/pdf/cm98-11.pdf
posted by T at 7:13 PM
0 Comments:
Post a Comment
<< Home