發(fā)布時(shí)間：2018-02-23 23:41

  本文關(guān)鍵詞： Choukroun’s富血小板纖維蛋白 人成骨樣細(xì)胞 血管內(nèi)皮生長因子 血小板衍生生長因子 VEGFR-2 血管化　出處：《吉林大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的與背景: 以增加骨量為目的的自體來源、異體來源、異種來源和人工合成的骨替代材料現(xiàn)在正廣泛應(yīng)用于牙科學(xué)及頜面外科學(xué)。血管新生被認(rèn)為在骨替代物移植的成功愈合和成骨中起重要作用。骨生成與血管新生的相互作用是由成骨細(xì)胞、內(nèi)皮細(xì)胞及它們的前體細(xì)胞通過自分泌-旁分泌網(wǎng)絡(luò)產(chǎn)生的各種因子調(diào)控的。促血管生長因子不僅可以刺激血管新生，而且可以直接間接地促進(jìn)成骨[3]。本實(shí)驗(yàn)選取促血管生成因子中特征性的兩種為研究對象——VEGF和PDGF。研究Choukroun's PRF自身分泌二者的效應(yīng)和其對人成骨細(xì)胞生物學(xué)的影響。探討PRF作為局部給予促血管生長因子來促進(jìn)骨血管化的可行依據(jù)。 方法: 體外培養(yǎng)人成骨細(xì)胞，嚴(yán)格根據(jù)制備流程制備人Choukroun's PRF。 實(shí)驗(yàn)一分為三組：1、空白對照組：培養(yǎng)的MG-63不做任何處理。2、實(shí)驗(yàn)組：培養(yǎng)MG-63時(shí)加入1塊PRF膜。3、PRF對照組：培養(yǎng)1塊PRF膜。實(shí)驗(yàn)點(diǎn)選取第二天、第四天、第六天、第八天、第十天，分別計(jì)數(shù)空白對照組與實(shí)驗(yàn)組MG-63的細(xì)胞數(shù)，并繪制生長曲線；分別收集三組培養(yǎng)液，采用ELISA方法測量培養(yǎng)液中VEGF、PDGF含量。 實(shí)驗(yàn)二分為三組：1、空白對照組：培養(yǎng)的MG-63不做任何處理。2、實(shí)驗(yàn)組（1/4PRF）：培養(yǎng)MG-63時(shí)加入四分之一塊PRF膜。3、實(shí)驗(yàn)組（1PRF）：培養(yǎng)MG-63時(shí)加入一塊PRF膜。實(shí)驗(yàn)點(diǎn)選擇第6天，采用Western Blot方法檢測各組MG-63細(xì)胞VEGFR2表達(dá)。 結(jié)果: 1、細(xì)胞計(jì)數(shù)結(jié)果及細(xì)胞生長曲線繪制 加入一塊PRF膜的實(shí)驗(yàn)組，MG-63細(xì)胞數(shù)在五個(gè)實(shí)驗(yàn)點(diǎn)（D2\D4\D6\D8\D10）均明顯高于空白對照組（P0.05）。 2、ELISA試劑盒檢測VEGF濃度 實(shí)驗(yàn)組在每個(gè)實(shí)驗(yàn)點(diǎn)所測得的培養(yǎng)液中VEGF濃度均較空白對照組和PRF對照組高（P0.05）。 3、ELISA試劑盒檢測PDGF濃度 實(shí)驗(yàn)組在各個(gè)實(shí)驗(yàn)點(diǎn)所測得的培養(yǎng)液中PDGF濃度均較空白對照組和PRF對照組高（P0.05）。 4、Western Blot檢測MG-63細(xì)胞VEGFR-2表達(dá) 使用ImageJ軟件灰度分析，加入PRF膜的實(shí)驗(yàn)組VEGFR-2蛋白表達(dá)明顯高于對照組，實(shí)驗(yàn)組（1PRF） VEGFR2蛋白表達(dá)高于實(shí)驗(yàn)組（1/4PRF）。 結(jié)論: 本實(shí)驗(yàn)通過在培養(yǎng)液內(nèi)添加Choukroun’s PRF及陰性空白對照，對比研究Choukroun’s PRF對人成骨樣細(xì)胞MG-63細(xì)胞株增殖和分泌VEGF、PDGF影響，表面表達(dá)VEGFR-2的影響。得出以下結(jié)論： 1、Choukroun’s PRF明顯促進(jìn)人成骨樣細(xì)胞增殖，并縮短細(xì)胞生長的潛伏期，加速細(xì)胞進(jìn)入指數(shù)生長期。 2、Choukroun’s PRF膜可以至少在10天內(nèi)緩慢穩(wěn)定的釋放生長因子。 3、Choukroun’s PRF有效增加了環(huán)境中VEGF濃度，并促進(jìn)人成骨樣細(xì)胞分泌VEGF，提示其可以通過由VEGF介導(dǎo)的血管新生途徑促進(jìn)血管化。 4、Choukroun’s PRF有效增加環(huán)境中的PDGF濃度，且維持其較高水平；Choukroun’s PRF可以促進(jìn)成骨樣細(xì)胞分泌PDGF；提示其可以通過PDGF強(qiáng)烈促有絲分裂能力促進(jìn)血管生成和促進(jìn)成骨。 5、Choukroun’s PRF刺激人成骨樣細(xì)胞表面表達(dá)VEGFR-2量增多，，且呈劑量依賴性。提示人成骨樣細(xì)胞對VEGF利用度更高，反應(yīng)更靈敏，反饋更強(qiáng)烈。 6、Choukroun’s PRF可以作為局部給予生長因子的材料應(yīng)用于臨床。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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