富血小板纤维蛋白对人成骨样细胞分泌VEGF、PDGF及表达VEGFR-2的影响

发布时间：2018-02-23 23:41

本文关键词： Choukroun’s富血小板纤维蛋白人成骨样细胞血管内皮生长因子血小板衍生生长因子 VEGFR-2 血管化　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的与背景: 以增加骨量为目的的自体来源、异体来源、异种来源和人工合成的骨替代材料现在正广泛应用于牙科学及颌面外科学。血管新生被认为在骨替代物移植的成功愈合和成骨中起重要作用。骨生成与血管新生的相互作用是由成骨细胞、内皮细胞及它们的前体细胞通过自分泌-旁分泌网络产生的各种因子调控的。促血管生长因子不仅可以刺激血管新生，而且可以直接间接地促进成骨[3]。本实验选取促血管生成因子中特征性的两种为研究对象——VEGF和PDGF。研究Choukroun's PRF自身分泌二者的效应和其对人成骨细胞生物学的影响。探讨PRF作为局部给予促血管生长因子来促进骨血管化的可行依据。方法: 体外培养人成骨细胞，严格根据制备流程制备人Choukroun's PRF。实验一分为三组：1、空白对照组：培养的MG-63不做任何处理。2、实验组：培养MG-63时加入1块PRF膜。3、PRF对照组：培养1块PRF膜。实验点选取第二天、第四天、第六天、第八天、第十天，分别计数空白对照组与实验组MG-63的细胞数，并绘制生长曲线；分别收集三组培养液，采用ELISA方法测量培养液中VEGF、PDGF含量。实验二分为三组：1、空白对照组：培养的MG-63不做任何处理。2、实验组（1/4PRF）：培养MG-63时加入四分之一块PRF膜。3、实验组（1PRF）：培养MG-63时加入一块PRF膜。实验点选择第6天，采用Western Blot方法检测各组MG-63细胞VEGFR2表达。结果: 1、细胞计数结果及细胞生长曲线绘制加入一块PRF膜的实验组，MG-63细胞数在五个实验点（D2\D4\D6\D8\D10）均明显高于空白对照组（P0.05）。 2、ELISA试剂盒检测VEGF浓度实验组在每个实验点所测得的培养液中VEGF浓度均较空白对照组和PRF对照组高（P0.05）。 3、ELISA试剂盒检测PDGF浓度实验组在各个实验点所测得的培养液中PDGF浓度均较空白对照组和PRF对照组高（P0.05）。 4、Western Blot检测MG-63细胞VEGFR-2表达使用ImageJ软件灰度分析，加入PRF膜的实验组VEGFR-2蛋白表达明显高于对照组，实验组（1PRF） VEGFR2蛋白表达高于实验组（1/4PRF）。结论: 本实验通过在培养液内添加Choukroun’s PRF及阴性空白对照，对比研究Choukroun’s PRF对人成骨样细胞MG-63细胞株增殖和分泌VEGF、PDGF影响，表面表达VEGFR-2的影响。得出以下结论： 1、Choukroun’s PRF明显促进人成骨样细胞增殖，并缩短细胞生长的潜伏期，加速细胞进入指数生长期。 2、Choukroun’s PRF膜可以至少在10天内缓慢稳定的释放生长因子。 3、Choukroun’s PRF有效增加了环境中VEGF浓度，并促进人成骨样细胞分泌VEGF，提示其可以通过由VEGF介导的血管新生途径促进血管化。 4、Choukroun’s PRF有效增加环境中的PDGF浓度，且维持其较高水平；Choukroun’s PRF可以促进成骨样细胞分泌PDGF；提示其可以通过PDGF强烈促有丝分裂能力促进血管生成和促进成骨。 5、Choukroun’s PRF刺激人成骨样细胞表面表达VEGFR-2量增多，，且呈剂量依赖性。提示人成骨样细胞对VEGF利用度更高，反应更灵敏，反馈更强烈。 6、Choukroun’s PRF可以作为局部给予生长因子的材料应用于临床。
[Abstract]:The purpose and background of the study:
In order to increase bone mass for autologous, allogeneic objective, heterogeneous sources and synthetic bone substitute materials are now widely used in dentistry and maxillofacial surgery. Angiogenesis is considered in bone replacement graft healing and play an important role in the interaction of bone. The new bone formation and blood vessels by osteoblast precursor cells, endothelial cells and their through autocrine paracrine factors generated by the network. The regulation of vascular growth factor can not only stimulate angiogenesis, and can directly and indirectly promote the effect of bone [3]. in this experiment, two kinds of characteristic angiogenic factor as the research object, and VEGF PDGF. study of Choukroun's PRF two and its secretion of the human osteoblast biology. To investigate the PRF as the local administration of angiogenic factors to promote bone vascularization in feasible According to.
Method:
Human osteoblasts were cultured in vitro, and human Choukroun's PRF. was prepared strictly according to the preparation process
Experiments were divided into three groups: 1 control group: Cultured MG-63.2 without any treatment, the experimental group: MG-63 culture with 1 piece of PRF film.3, PRF control group: the culture of 1 pieces of PRF film. The second day, selecting experimental points for fourth days, sixth days, eighth days, tenth days, respectively. Count the number of cells in blank control group and experimental group MG-63, and the growth curve were collected; three groups cultured in the culture medium of VEGF, measured by ELISA method, the content of PDGF.
In experiment two, divided into three groups: 1 control group: Cultured MG-63.2 without any treatment, the experimental group (1/4PRF): MG-63 culture added a 1/4 PRF film.3, experimental group (1PRF): MG-63 culture into a piece of PRF film. The choice of sixth days, the expression of cells was detected by MG-63 VEGFR2 using Western Blot method.
Result:
1, cell count results and cell growth curve drawing
The number of MG-63 cells at five experimental sites (D2D4D6D8D10) was significantly higher than that in the blank control group (P0.05) in the experimental group adding a PRF film.
2, ELISA kit was used to detect the concentration of VEGF
The concentration of VEGF in the experimental group was higher than that in the blank control group and the PRF control group at each test point (P0.05).
3, ELISA kit was used to detect the concentration of PDGF
The concentration of PDGF in the experimental group was higher than that in the blank control group and the PRF control group (P0.05).
4, Western Blot detection of VEGFR-2 expression in MG-63 cells
The expression of VEGFR-2 protein in the experimental group was significantly higher than that in the control group by ImageJ software gray analysis. The expression of VEGFR2 protein in the experimental group (1PRF) was higher than that in the experimental group (1/4PRF). The expression of VEGFR-2 in the experimental group was higher than that in the experimental group (1/4PRF).
Conclusion:
In this experiment, we added Choukroun 's PRF and negative blank control in the culture medium, and compared the effects of Choukroun' s PRF on the proliferation and secretion of VEGF, PDGF and the expression of VEGFR-2 on the human osteoblast like cell MG-63 cell line.
1, Choukroun 's PRF obviously promotes the proliferation of human osteoblast like cells, and shortens the incubation period of cell growth and accelerates the cells to enter the exponential growth period.
2, Choukroun 's PRF membrane can slowly and steadily release growth factor for at least 10 days.
3, Choukroun 's PRF effectively increased the VEGF concentration in the environment, and promoted the secretion of VEGF from human osteoblast like cells, suggesting that it can promote vascularization through VEGF mediated angiogenesis.
4, Choukroun 's PRF can effectively increase the PDGF concentration in the environment, and maintain its high level; Choukroun' s PRF can promote the osteoblast like cells to secrete PDGF, suggesting that it can promote the angiogenesis and promote osteogenesis through the strong mitosis ability of PDGF.
5, Choukroun 's PRF stimulated the increase of VEGFR-2 expression on human osteoblast like cells in a dose-dependent manner, suggesting that human osteoblast like cells have higher VEGF utilization, more responsive and more intense feedback.
6, Choukroun 's PRF can be used as a local growth factor for clinical application.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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