| 摘要: |
| 目的 通过对比分析富血小板血浆(platelet rich plasma, PRP)与血小板裂解液(platelet lysate, PL)对体外培养的大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)生长增殖、细胞周期的作用，探讨二者对大鼠BMSCs生长增殖的影响。方法 取20只4周龄SD大鼠，采用全骨髓分离培养法扩增传代BMSCs，并采用流式细胞仪鉴定；另取30只12周龄SD大鼠心内采血，梯度离心法获得PRP，采用3次离心结合反复冻融法制备PL。取生长状态良好的第3代BMSCs进行实验，根据培养基成分不同将实验分为7组，普通完全培养基组(A组)、1%PRP条件培养基组(B组)、1%PL条件培养基组(C组)、5%PRP条件培养基组(D组)、5%PL条件培养基组(E组)、10%PRP条件培养基组(F组)及10%PL条件培养基组(G组)。MTT检测BMSCs增殖情况；免疫荧光检测BMSCs中增殖细胞核抗原(PCNA)的表达情况；流式细胞仪检测BMSCs细胞周期的变化；蛋白质印迹检测CyclinD1和p27 Kip1蛋白表达。 结果 培养24、48、72 h时，1%、5%、10% PRP与1%、5%、10% PL条件培养基组均可以促进BMSCs的增殖，并具有时间、剂量依赖性(P<0.05)；相同浓度的PRP与PL对BMSCs增殖作用的差异均无统计学意义(P>0.05)。免疫荧光检测显示，PRP与PL均可以促进PCNA蛋白表达(P<0.05)，且呈剂量依赖性(P<0.01)。流式细胞仪检测显示，不同浓度PRP及PL条件培养基组与普通完全培养基组比较，G0/G1期细胞比例逐渐减少，S、G2/M期细胞比例逐渐增多，增殖指数(PI)升高，差异具有统计学意义(P<0.05)，并呈剂量依赖性(P<0.05)；然而，相同浓度的PRP及PL对BMSCs细胞周期的影响相似，差异均无统计学意义(P>0.05)。蛋白质印迹检测结果显示，5%PRP与5%PL能促进CyclinD1表达上调和p27 Kip1表达下调。结论 不同浓度PRP与PL能够通过上调CyclinD1以及抑制p27 Kip1蛋白的表达，加快体外培养的大鼠BMSCs的细胞周期进程，促进BMSCs增殖，并呈现显著的时间、剂量依赖性；BMSCs增殖过程中，相同浓度PL与PRP的促增殖效果相同，PL可能代替PRP促进骨缺损修复。 |
| 关键词: 骨髓间充质干细胞 富血小板血浆 血小板裂解液 组织工程 细胞周期 细胞增殖 |
| DOI： |
| 投稿时间：2013-07-02修订日期：2013-07-22 |
| 基金项目:唐山市科技局重点实验室建设项目(10140201A-12). |
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| Effects of platelet rich plasma and platelet lysate on proliferation of rat bone marrow mesenchymal stem cells: a comparative study |
| GUO Jing1,GOU Xiang-bo2,ZHANG Wen-li1,LI Qi-jia1,WANG Zhi-qiang3* |
(1. Medical Experimental and Research Center, Hebei United University, Tangshan 063000, Hebei, China
2. Department of Pharmacology, College of Basic Medical Sciences, Hebei United University, Tangshan 063000, Hebei, China
3. Department of Orthopaedics, the Affiliated Hospital of Hebei United University, Tangshan 063000, Hebei, China
*Corresponding author.) |
| Abstract: |
| Objective To compare the effects of platelet rich plasma (PRP) and platelet lysate (PL) on proliferation and cell cycle of cultured rat bone marrow stem cells (BMSCs). Methods BMSCs were obtained from twenty 4-week-old SD rats using the whole bone marrow isolation and cultivation method and were identified with flow cytometry. Blood samples were taken from the hearts of thirty 12-week-old SD rats and gradient centrifugation was used to prepare PRP, and PL was obtained after three times of centrifugation and repeated freezing and thawing. The third generation of BMSCs with good growth state were divided into seven groups according to different culture media: ordinary complete medium (A group), 1% PRP-conditioned medium (B group), 1% PL-conditioned medium (C group), 5% PRP-conditioned medium (D group), 5% PL-conditioned medium (E group), 10% PRP-conditioned medium (F group), and 10% PL-conditioned medium (G group). The proliferation of BMSCs was assessed by MTT assay. The PCNA protein expression was assessed by immunofluorescence method. Cell cycle of BMSCs was tested by flow cytometry. Western blotting analysis was used to analyze CyclinD1 and p27 Kip1 protein expression of BMSCs. Results The proliferation of BMSCs was significantly promoted by 1%, 5%, 10% PRP- and 1%, 5%, 10% PL-conditioned media after cultured for 24 h, 48 h, and 72 h in a time- and dose-dependent manner (P<0.05), with the effect of PRP and PL being similar at the same concentration (P>0.05). Immunofluorescence assay showed that PRP and PL both significantly promoted PCNA protein expression in a dose-dependent manner (P<0.01). Flow cytometry showed that different concentrations of PRP- and PL-conditioned media, compared with the ordinary complete medium, resulted in gradually reduced cells in G0/G1 phase, gradually increased cells in S, G2/M phase, and significantly increased PI (P<0.05), with the changes in a dose-dependent manner. However, the effects of the same concentration of PRP and PL on cell cycle were not significantly different (P>0.05). Western blotting analysis showed that 5% PRP and 5% PL significantly up-regulated CyclinD1 and down-regulated the expression of p27 Kip1. Conclusion Different concentrations of PL and PRP can accelerate cell cycle progression of cultured rat BMSCs by up-regulation CyclinD1 and inhibiting p27 Kip1, promoting the proliferation of BMSCs in a time- and dose-dependent manner. PL and PRP at the same concentration have the same proliferation-promoting effect, indicating that PL may be used as an alternative of PRP to promote the repair of bone defects. |
| Key words: bone marrow mesenchymal stem cells platelet-rich plasma platelet lysate tissue engineering cell cycle cell proliferation |
