Author

Shelby Hott

Defense Date

5-24-2006

Graduation Date

2006

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Biological Sciences

School

Bayer School of Natural and Environmental Sciences

Committee Chair

John S. Doctor

Committee Member

Ellen S. Gawalt

Committee Member

Kyle W. Selcer

Committee Member

Phil G. Campbell

Committee Member

Richard P. Elinson

Keywords

differentiation, Insulin-like growth factor, PAPP-A, pregnancy associated plasma protein-A, proliferation

Abstract

Pregnancy associated plasma protein-A (PAPP-A) plays an important role in the activity of the Insulin-like Growth Factor (IGF) family by proteolytically cleaving IGF-Binding Protein-4 (IGFBP-4) causing it to dissociate from IGF-I in vivo. This increases the bioavailability of IGF-I, allowing IGF-I to participate in growth and development. My goal was to evaluate the ability of PAPP-A to directly stimulate proliferation and differentiation of human adult mesenchymal stem cells (hAMSC) cells in vitro.

Commercially available PAPP-A, comprised of a heterotetrameric complex consisting of two PAPP-A subunits and two pro-MBP (proform of eosinophil major basic protein) subunits, was used in the following studies. Proliferation of hAMSC in response to PAPP-A was evaluated via CyQUANT and BrdU incorporation. Cells were arrested at G0 prior to treatment with 200 ng/mL PAPP-A. CyQUANT and BrdU incorporation results did not show statistically significant evidence of proliferation of hAMSC when treated with PAPP-A.

To test for differentiation, hAMSC were treated with mesenchymal stem cell growth medium -- MSCGM (negative control), an osteogenic supplement (positive control) or 200 ng/mL PAPP-A in MSCGM for multiple time points. hAMSC were harvested for RNA extraction followed by qPCR analysis to detect osteoblastic differentiation via multiple marker genes. qPCR results showed a statistically significant increase in Runx2 and alkaline phosphatase gene expression in hAMSC when treated with PAPP-A for 12 and 18 hours respectively.

My results indicate that addition of PAPP-A to cell culture medium does not affect proliferation of hAMSC. However, treatment with PAPP-A for 12 and 18 hours increases Runx2 and alkaline phosphatase gene expression respectively in hAMSC suggesting that PAPP-A plays a role in osteoblast differentiation in an in vitro environment. Further elucidation of this role is necessary to determine PAPP-A's involvement in osteoblast development and its potential therapeutic role in bone degenerative diseases such as osteoporosis.

Format

PDF

Language

English

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