科研之友
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摘要
Background and Objectives TGF-beta 1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-beta 1 from virally inactivated human platelets.
Study Design and Methods Apheresis platelet concentrates (N = 12) were solvent /detergent (S/D) treated (1% TnBP/1% Triton X-45; 31 degrees C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7.5. The column was washed to eliminate unbound proteins and the S /D agents. Bound proteins were eluted using a 1 M NaCl-PBS buffer pH 7.5 (DEAE-eluate). The content in TGF-beta 1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S /D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions.
Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-beta 1 at a mean concentration of about 102 ng/ml, whereas EGF, beta-FGF were at about 0.72 and 0.18 ng/ml, respectively. The content in TnBP and Triton X-45 was <2 ppm.
Conclusion A fraction enriched in TGF-beta 1 can be prepared from virally inactivated human platelet lysates using an easily scale process. Its interest in regenerative medicine and cell therapy will be evaluated in further studies.
- 出版日期2011-10
