Huang Qian, Wang Yun-dan, Wu Tao, Jiang Shan, Hu Yan-ling, Pei Guo-xian
Department of Orthopedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangdong, China.
Chin Med J (Engl). 2009 Jan 5;122(1):83-7.
Platelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma.
The gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
PRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-beta1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P < 0.05), and the effects of fraction B and C showed no significant difference compared to the PRP group (P > 0.05). Fraction A and D showed no significant difference to the negative control group (P > 0.05).
The growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.
富含血小板血浆（PRP）作为生长因子的储存载体已成功应用于临床，但大多数情况下血小板为自体来源。然而，大量采血对贫血或全身健康状况不佳的患者有不利影响。为克服这些局限性，本研究旨在分离同源富含血小板血浆中的生长因子。
应用Superdex - 75柱凝胶色谱法将PRP上清液分离为4个主要组分。然后将这4个组分进行真空冷冻干燥，并重新溶解于磷酸盐缓冲盐水中。采用二辛可宁酸蛋白测定法检测PRP及4个组分中的蛋白质浓度；采用夹心酶联免疫吸附测定法测定血小板衍生生长因子 - AB（PDGF - AB）和转化生长因子β1（TGF - β1）水平。通过3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法测定各组分对人骨髓间充质干细胞（MSCs）增殖的影响。
通过凝胶色谱法将PRP上清液分离为4个主要组分。蛋白质回收率为96.72%。在这4个组分中，组分B含有最高的TGF - β1和PDGF - AB水平以及最高的蛋白质浓度。MSCs的细胞增殖曲线表明，与未处理的培养物相比，组分B和C诱导MTT值显著增加（P < 0.05），并且组分B和C的作用与PRP组相比无显著差异（P > 0.05）。组分A和D与阴性对照组无显著差异（P > 0.05）。
PRP上清液中的生长因子可通过凝胶色谱法初步分离为4个组分，并冻干组分保留了生长因子的生物活性。生长因子主要存在于组分B和C中，并能有效促进细胞增殖。
