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Experiment Study of Placental Factor Purification and Its Activity on PC12 Cells

Author: XuYangZuo
Tutor: ZhangXueRong
School: Guangxi Medical University
Course: Biochemistry and Molecular Biology
Keywords: Placenta immunoregulatory factor Gel chromatography Biological activity PC12 cells Proliferation Differentiation Placenta immunoregulatory factor purification fourth peak
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 40
Quote: 1
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Abstract


The first part of the separation of the placenta immunoregulatory factor purification and activity detection purposes purified placental immunomodulatory factor (placenta factor PF), most significant and screening for active component. Methods fresh human placenta to the fascia, saline rinse, Shredded homogenate, after repeated freezing and thawing, dialysis, lyophilization take outer dialysate was loaded on a Sephadex G-25 gel chromatography column, eluted, collecting each component. By cultured lymphocytes identification activity of PF purified product using the MTT assay, reverse phase high performance liquid chromatography of analytically pure product purity, matrix-assisted laser desorption ionization serial time-of-flight mass spectrometry molecular weight of the product. Results PF by Sephadex G-25 gel column purification to obtain a fourth peak having to significantly promote the activity of lymphocyte proliferation, is the main active ingredient of the placenta immunoregulatory factor is the molecular weight for 6737.813Da the polypeptide, and its purity was 82%, The Conclusion placenta immunoregulatory factor obtained by Sephadex G-25 gel column purification fourth peak is the main active ingredient of placenta immunoregulatory factor, a purity of 82%, the molecular weight of 6737.813Da. Objective: To observe the second part of the placenta immunomodulatory growth factor on PC12 cells placental immunomodulatory factor (placenta factor PF), the impact of the growth of PC12 cells. Methods: PC12 cells observed the PF survival of PC12 cells cultured in vitro serum-free and PF cultured PC12 cell proliferation, low serum ordinary optical microscope observation of morphological changes of PC12 cells using the MTT assay. Results: After to cultivate 44 hours of PC12 cells in serum-free medium with different concentrations of PF, PF concentration of 50μg/ml, 25μg/ml, 12.5μg/ml and 6.25μg/ml can significantly improve the serum-free culture of PC12 cells survival (P lt; 0.05). The PC12 cells cultured in low serum culture 68 hours after adding different concentrations of PF, PF at a concentration of 12.5μg/ml and 6.25μg/ml can significantly improve the amount of PC12 cells (P LT; 0.01). Cultured for 10 days low serum cultured PC12 cells with different concentrations of PF, PF concentration 3.13μg/ml and 1.56μg/ml role 6th day, cells grow protrusions. Conclusion: PF can improve the viability of PC12 cells serum-free culture, promote PC12 cell proliferation and induce differentiation of PC12 cells. The third part of the PF purification fourth peak (PFIV) the impact of the growth of PC12 cells Objective: To observe the placenta immunoregulatory factor purification fourth peak (PFIV) Growth of PC12 cells. METHODS: Cultured PC12 cells using MTT assay the observation PFIV vitro serum low serum cultured the survival and PFIV of PC12 cells cultured PC12 cell proliferation, ordinary optical microscope observation of morphological changes of PC12 cells. Results: After 44 hours of serum-free culture of PC12 cells with different concentrations of PFIV culture, at a concentration of 15μg/ml, 7.5μg/ml 3.75μg/ml and 1.88μg/ml can significantly improve PC12 cells in serum-free culture The survival rate (P lt; 0.05). The PC12 cells cultured in low serum culture 68 hours after adding different concentrations of PFIV can significantly improve the amount of PC12 cells (P LT; 0.01), at a concentration of 0.47μg/ml and 0.235μg/ml. Low serum cultured PC12 cells with different concentrations of PFIV cultured for 10 days, PFIV in concentration for 0.118μg/ml, and 0.059μg/ml the role of the sixth day, the cells grow protrusions. Conclusion: placenta immunoregulatory factor fourth peak (PFIV) can improve the survival rate of PC12 cells in serum-free culture, promote PC12 cell proliferation, induces differentiation of PC12 cells.

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