Effects of different volume fractions of platelet-rich plasma on the proliferation and migration of vascular endothelial cells

doi:10.3969/j.issn.2095-4344.0331

Abstract

Abstract:

BACKGROUND: Platelet-rich plasma (PRP) plays an important role in promoting angiogenesis of vascular endothelial cells. Different volume fractions of PRP have definite influence on vascular endothelial cells, but it is not clear PRP at which volume fraction is optimal.
OBJECTIVE: To investigate the effect of PRP at different volume fractions on the proliferation and migration of vascular endothelial cells in vitro.
METHODS: Hearts of Sprague-Dawley rats were mechanicaIly minced and enzymatically digested with collagenase. Vascular endothelial cells were counted and cultured in the DMEM culture medium containing various volume fractions of autologous PRP (10%, 20%, 50%) respectively. The cell morphology was observed under an inverted microscope, and cellular proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Afterwards, scratch test was utilized to detect the cell migration ability and the optimal volume fraction was determined.
RESULTS AND CONCLUSION: After addition of PRP, the well-grown endothelial cells presented with regular shape, changed into polygon-like and became large with time. Different volume fractions of PRP in particular 20% PRP cultured for 72 hours could promote cell proliferation. According to scratch test, 20% and 50% PRP significantly promoted the cell migration, especially 20% PRP. These results indicate that PRP can promote the proliferation and migration of vascular endothelial cells in vitro, and 20% is the optimal volume fraction.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Endothelial Cells, Platelet-Rich Plasma, Cell Proliferation, Cell Movement, Tissue Engineering

CLC Number:

R318

Zhang Ming-yu, Ren Bo, Yang Zhen, Zhang Xian, Zheng Jiang. Effects of different volume fractions of platelet-rich plasma on the proliferation and migration of vascular endothelial cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(28): 4469-4474.

Figures/Tables 2

2.2 细胞形态学观察结果倒置相差显微镜观察，原代血管内皮细胞呈球形或椭圆形，折光性较强，接种24 h后细胞开始贴壁，呈不规则排列，最初细胞形态呈短梭形或三角形(图1A)，随着培养时间延长，细胞开始分裂增殖、迁移并逐渐呈规律性、方向性排列(图1B)；培养5 d后细胞可长满培养瓶底面积80%-90%。此时细胞形成紧密排列的铺路卵石样结构(图1C)。传代后细胞形态和原代相似，随着传代次数增多细胞生长速度较原代明显增快。 2.3 血管内皮细胞鉴定结果第3代血管内皮细胞进行血管Ⅷ因子免疫细胞化学染色。细胞浆呈棕黄色沉淀，其中在胞核周围更为明显，细胞核不显色，细胞的边界清晰(图2)。随机选择3张细胞爬片，在每张片子中任意选择3个视野进行计数，Ⅷ因子染色细胞的阳性百分率为(95.23±3.72)%，提示血管内皮细胞纯度较高。 2.4 MTT法测定各组吸光度值各组加入富血小板血浆干预后细胞形态规则，生长旺盛，随培养时间延长大部分细胞变成多角形，细胞较大。随着富血小板血浆体积分数梯度升高，镜下观察到细胞生长旺盛、密集，并且胞体丰满；与对照组相比细胞增殖明显增快，胞浆颗粒较少，细胞间连接紧凑。不同体积分数的富血小板血浆均对血管内皮细胞增殖具有促进作用(表2)。培养48 h后，体积分数为20%和50%富血小板血浆组中吸光度值高于空白对照组 (P < 0.05)，表明富血小板血浆可明显促进血管内皮细胞增殖，其中以培养72 h体积分数为20%富血小板血浆组对细胞的增殖促进作用最为明显(图3)。 2.5 划痕实验结果各组划痕愈合的相对距离比较结果见表3。体积分数为10%富血小板血浆组与空白对照组比较差异不明显。当体积分数为20%富血小板血浆和体积分数为50%富血小板血浆作用时，细胞相对迁移距离较空白对照组增强(图4)，统计结果显示体积分数为20%富血小板血浆组细胞相对迁移距离最远(74.23±3.78)%，与空白对照组比较差异有显著性意义。"

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