摘要
目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。
AIM： There are some theories that suspect the effect of platelet-rich plasma （PRP） on the osteogenic differentiation of bone precursor cells. In this paper, we explore the inhibitory effect of PRP on the osteogenic differentiation of human bone marrow mesenchymal stem cells （MSCs） cultured in vitro. METHODS： The experiment was conducted at the tissue-engineering laboratory of Southern Medical University from May to November 2005. ①5 mL bone marrow was harvested from the anterior superior iliac spine of 6 healthy volunteers, respectively. Human MSCs were cultured and amplified. PRP was obtained by centrifuging venous bloods （10 mL） for twice to induce MSCs. The human MSCs and PRP were obtained from the same volunteers. ②Alkaline phosphate （ALP） staining： The fourth passage MSCs were divided into two groups： PRP group was added PRP at final concentration of 100 g/L; fetal bovine serum （FBS） group was only added FBS of the same volume. At the 7^th day, ALP staining was performed. The black particle or massive sediment in periplast was of ALP positive MSCs. ③Calcium depositions staining： Another fourth passage MSCs were also divided into the same two groups. At the 19^th day of culture, calcium deposition staining was performed with 0.1% alizarin red-Tris Hcl （pH 8.3） at 37℃ for 30 minutes. The red calcium deposition was positive.④Gene expression of Cbfal： The fourth passage MSCs were grouped with the previous method. On days 3, 7, 12, and 16, RT-PCR was used to assay mRNA expression of Cbfal. ⑤Morphology of cells： The growth condition and morphology of cells were observed by inverted phase contrast microscope during the experiment. RESULTS： ①ALP staining results showed that at the 7^th day, compared with the FBS group, the ALP positive number of cells in PRP group was significantly decreased, furthermore, the black particle in periplast of ALP positive MSCs was also decreased. ALP positive MSCs of PRP was poor positive. ②Calcium depositions staining results suggested that at the 19^th day, some calcium depositions were found on the surface of cells in FBS group, but no obvious calcium nodus was observed. In the PRP group, there was little calcium deposition. ③Gene expression of Cbfal： At the 3^rd, 7^th, 12^th, and 16^th days, the gene expression of Cbfa1 in two groups was increased gradually. And there was no obvious difference at the same time point. ④Morphology of cells： MSCs of PRP group grew rapidly. MSC confluence was earlier than FBS group as observed under inverted phase contrast microscope. During culture, MSCs of FBS group began to aggregate at day 14 to 16, but not become intensive cluster. However, MSCs of PRP grew rapidly without appearance of aggregation. In the initial stage of culture, most MSCs of two groups presented shuttle shape, but at day 14 to 16, polygon shape MSCs in FBS group were more than those in PRP group. CONCLUSION： PRP can inhibit ALP secretion and calcium depositions of human MSCs, and directly inhibit the osteogenic differentiation of human MSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第28期5494-5498,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家高科技研究发展计划(八六三)重点项目(2003AA205010)
国家自然科学基金(30300367)
广州市科技攻关重大专项子课题(200481-E0031)~~
