自体血小板血浆对角膜基质细胞生物学功能的影响

Effect of autologous platelet plasma on biological function of corneal stromal cells

目的观察自体血小板血浆对体外培养的角膜基质细胞生物学功能的影响。方法采用二次离心法分离兔全血获得富血小板血浆（platelet—richplasma，PRP）和贫血小板血浆（platelet—poorplasma，PPP）；原代培养兔角膜基质细胞，取第3代细胞用于实验。在角膜基质细胞中分别加入体积分数5．0％的PRP和PPP，倒置显微镜下观察细胞生长情况，对比PRP和PPP对角膜基质细胞的促生长情况，以确定自体血小板血浆中促增殖作用的最有效成分。采用不同浓度的PRP（体积分数分别为2．5％、5．0％、10．0％）作用于角膜基质细胞，以及采用不同浓度PRP（体积分数分别为2．5％、5．0％、20．0％）联合FBS作用于角膜基质细胞，CCK-8法检测PRP、PRP联合FBS促进角膜基质细胞增殖的作用。对不同浓度（体积分数分别为2．5％、5．0％）PRP作用的角膜基质细胞进行免疫荧光法检测其表达的平滑肌型肌动蛋白（a-smooth muscle actin，a-SMA）．结果加入体积分数5．0％PRP组促细胞增殖作用强于加入体积分数5．0％PPP组。PRP在体积分数为2．5％～10．0％时，均能促进角膜基质细胞的增殖，其中体积分数为5．0％时作用最强；体积分数5．0％PRP与FBS联合作用时的吸光度值随时间的变化均明显高于两者单独作用时的吸光度值（均为P〈0．05），而体积分数20．0％的PRP与FBS联合作用时的吸光度值随时间的变化低于FBS单独作用时的吸光度值。不同浓度PRP处理的角膜基质细胞均可见a-SMA的阳性表达。结论低浓度的PRP能有效促进角膜基质细胞的增殖及活化，有利于角膜基质损伤的快速修复，促进伤口愈合，防止严重并发症的发生。

Objective To investigate the effect of autologous platelet plasma on the biological function of corneal stromal cells cultured in vitro. Methods Plateletrich plasma （PRP） and platelet-poor plasma （PPP） were isolated from the rabbit blood by two-step centrifugation method. Corneal stromal cells of rabbits were primarily cultured, and cells at the third generation were used in the experiment. Corneal stromal cells were cultured with volmnn fraction 5.0% PRP or volunm fraction 5.0% PPP, respectively. Inverted microscope was performed to observe the cell growth. Effects of PRP and PPP on promoting growth of corneal stromal cells were compared to determine the most effective component in autologous platelet plasma for promoting proliferation. CCK-8 assay was used to analyze effects of PRP with various concentrations （ volumn fraction 2.5% ,5.0% , 10.0% ） and PRP with various concentrations （volumn fraction 2.5% ,5.0% , 20. 0% ） combining with FBS on promoting the proliferation of corneal stromal cells. Immunofluorescence was used to detect the expression of a-smooth muscle actin （a-SMA） in corneal stromal cells after being treated with various concentrations of PRP（volumn fraction 2.5% and 5.0% ）. Results The effect of volumn fraction 5.0% PRP on promoting cell proliferation was better than that of volunm fraction 5.0% PPP, PRP with concentration from 2.5% to 10.0% could all promote the proliferation of corneal stromal cells, and 5.0% PRP was the best. The absorbance value of corneal stromal cells with 5.0% PRP combining with FBS was significantly higher than that with 5.0% PRP or FBS alone with the changes of time （ all P 〈 0.05 ）. The absorbance value of corneal stromal cells with 20.0% PRP combining with FBS was lower than that with FBS alone with the changes of time. Positive expression of a-SMA was found in corneal stromal cells after being treated with various concentrations of PRP. Conclusion PRP with lower concentration can promote the proliferation and activation of corneal stromal cells,is good for the rapid healing of corneal stroma injury and wound healing. It can also prevent serious complications from happening.