INVESTIGATING EFFECTS OF IGF-1 CISSUS QUADRANGULARIS AND FAGUS CRENATA ON THE PRODUCTION OF COLLAGEN IN MOUSE FIBROBLAST CELLS

Done by:Allison MeadowsGabi Guzman Cheng Yong Jian Er Yuan Zhi

CONTENTS Introduction Rational Objectives Hypothesis Materials Methods Timeline References

INTRODUCTIONTendinosis is a chronic form of tendinitis which occurs when mostly type-1 collagen that composes the tendon degenerates through repetitive motion and ageing (Summers, 2003)

Current treatments for tendon repair often contain compounds that stimulate collagen production (Haukipuro, 1991)

INTRODUCTIONCurrent treatments for tendon repair IGF-1 is a growth hormone that has been shown to be an in-vitro stimulant of type-1 collagen synthesis (Ivarsson, 1998)

Studies by J Indian Medical Association have shown that the daily use of C.quadrangularis increases collagen production in the body

INTRODUCTIONFagus Crenata is known to exhibits a superior effect of prevention of aging by promoting the production of collagen in the dermal fibroblast cells. (Y., Nakayama, N., Kojima, 1998)

Studies have also shown that F.Crenata have the effect of strongly promoting dermal fibroblast collagen biosynthesis (Takada et al, 2007)

RATIONAL We aim to investigate the effectiveness of

Cissus quadrangularis, IGF-I and Fagus crenata to stimulate the synthesis of collagen in mouse embryo fibroblast cells to develop new treatments for tendonitis.

OBJECTIVES Determine the effectiveness of Cissus

quadrangularis and Fagus Crenata, and to determine the effectiveness of C.quadrangularis and F.crenata when combined with IGF-1 on the stimulation of synthesis of collagen in mouse embryo fibroblast cells

HYPOTHESIS Our hypothesis is that:

Cissus quadrangularis and Fagus crenata increase production of collagen synthesis in mouse embryo fibroblast cells when used both separately and together with IGF-1

VARIABLE

•Amount and type of fibroblast cells treated•Environmental conditionsControlled

Variable•Treatment of fibroblasts cells•Type of herbs used (For AOS and HCI)•Concentration of substances added to

fibroblast cells

Independent Variable

•Amount of collagen produced by fibroblast cellsDependent

Variable

MATERIALS 3T6 Swiss Albino Nuclear Lystate Cissus quandrangularis Fagus Crenata IGF-1 Tissue Culture flask – 25cm2

CO2 incubator Dulbecco’s Modified Eagle Medium Variable volume micropipettes and pipette tips Vortex mixer Centrifuge machine Spectrophotometer Masson Trichrome staining kit Sircol Assay Kit

METHODSTests will be carried out in vitro with IGF-1 and C.quadrangularis (F.crenata for HCI) in DME medium at different concentrations

Fibroblast cells would be separately treated with IGF-1, C.quadrangularis (F.crenata for HCI) and a combination of both

Mouse fibroblast cells will be cultured in a CO2 chamber at 37°C at a volume of 0.25ml/cm2 for a period of 5 days

METHODS

2 controls will be created for comparison of the results obtained in the experiment

One set of control would be IGF-1 being added into the culture medium while being cultured in the exact same environment as the ones before

The second set of control would be the fibroblast cells being left to grow normally without anything being added

METHODS

Masson Trichrome staining will be done on the cells and ImageJ will be used measure the stained collagen

Sircol collagen assay will be done to determine the exact concentration of the amount of collagen produced by the mouse fibroblast cells

Triplicates will be carried out and the results obtained would be compared with the control

TIMELINEDate AOS HCINov-Dec Determine the

concentration of substances to be experimented on

Read up more articles regarding

project

Jan-Feb

Experiment

Obtaining of materials for experiment

Feb-March

ExperimentApril-May

June-July Summer Break

21st August Preparation for Project’s Day Finals presentation

REFERENCES Cissus Quadrangularis—The Best Kept Secret in

Bodybuilding. (2007). Retrieved September 28, 2009, from USPLabs Web site: http://www.usplabsdirect.com/supercissus.html

Erickson, L. (2002). Future treatments. Retrieved May 21, 2009, from http://www.tendinosis.org/future.html

Erickson, L. (2002). The Tendinosis Injury. Retrieved September 24, 2009, from http://www.tendinosis.org/injury.html

Takda, K., Suzuki, R., Guentert, M., Inomata, S., Hamada, C., and Sakiguchi, T., (June 14 2007) Anti-Aging composition and collagen production promoting composition

REFERENCES Borg, T. K., Ivarsson, M., McWhirter, A., Rubin, K.

(1998). Type I collagen synthesis in cultured human fibroblasts regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers. Matrix Biology. 16, 409-425.

Daha, M. R., Geest, R. N., Lam, S., VanKooten, C., Verhagen, N. A. M. (2004). Secretion of collagen type IV by human renal fibroblasts is increased by high glucose via a TGF-B-independent pathway. Nephrol Dial Transplant, 19, 1694-1701.

Leeson, C. R., Leeson, T. S., & Paparo, A. A. (1985). Connective tissue proper. Textbook of Histology. Philidelphia: W. B. Saunders Company.

REFERENCES Ghahary, A. , Houle , Kilani, T. , Scott, G. , Shen

and Tredget, E. , (2000), Vol. 17, No. 3, Pages 167-176. Mannose-6-Phosphate/IGF-II Receptors Mediate the Effects of IGF-1-Induced Latent Transforming Growth Factor β1 on Expression of Type I Collagen and Collagenase in Dermal Fibroblasts

Baroni, G., Benedetti, A., Casini, A., Folli, F., Gaglotti, G., Macarri, G., Marucci, L., Orlandoni, P., Perego, L., Ridolfi, F. and Sario, A. (1999) Insulin and Insulin-Like Growth Factor-1 Stimulate Proliferation and Type I Collagen Accumulation by Human Hepatic Stellate Cells: Differential Effects on Signal Transduction Pathways

Thank you for your kind attention!

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