巴曲酶对体外培养下内皮祖细胞的数量和功能的影响

发布时间：2018-06-16 21:15

  本文选题：巴曲酶 + 内皮祖细胞　； 参考：《安徽医科大学》2008年硕士论文

【摘要】： 目的: 血管内皮祖细胞(Endothelial progenitor cells,EPCs)是血管内皮的前体细胞,它们不仅参与胚胎期的血管发育,也存在于成年机体的骨髓及外周血,在成体血管新生中起重要作用。近年来的研究表明EPCs在下肢缺血性疾病的发生、发展中扮演了重要角色,血管内皮祖细胞数量减少预示血管内皮修复能力降低,血管疾病发生率增高。巴曲酶(Batroxobin,DF-521)是一种由蝮蛇毒液中提纯,精制而出的丝氨酸蛋白,目前临床上广泛用于包括脑梗塞、慢性下肢动脉硬化性闭塞症(ASO)在内的各种缺血性疾病的治疗,体现出其可能对于血管内皮的功能障碍有保护作用。内皮的损伤与修复之间的动态平衡是维持其正常功能的关键,EPCs是参与修复的重要因素。考虑DF-521可能通过影响EPCs促进内皮功能的改善,本文经试验观察其对于外周血EPCs数量、增殖、迁移和粘附能力的影响,旨在进一步探讨DF-521的作用机制,并为体外扩增EPCs寻找可能的诱导剂,从而提供更充足并良好的血管组织工程细胞。 方法: 实验血液样本来自健康志愿者,用于对照组和药物干预组的EPCs数量及功能检测。采用密度梯度离心法从外周血获得单个核细胞,将其接种在人纤维连接蛋白包被的培养板上。DMEM培养基培养4d,用PBS洗掉非贴壁细胞,换培养液继续培养至7d,用PBS洗掉非贴壁细胞,贴壁细胞供实验用。激光共聚焦显微镜鉴定FITC—UEA—Ⅰ和Dil—acLDL双染色阳性细胞为正在分化的EPCs;贴壁细胞随机分成4组,培养24h后干预组分别加入不同浓度的巴曲酶(0.05BU·ml~(-1)、0.1BU·ml~(-1)、0.2BU·ml~(-1))培养24h,再将其在倒置荧光显微镜下计数。分别采用MTT比色法、改良的Boyden小室和黏附能力测定实验来观察EPCs的增殖能力、迁移能力和黏附能力,再根据结果选择差异较显著的剂量再进行DF-521对EPCs数量及功能影响的时效关系实验。 结果: 1.EPCs的鉴定 分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。用Dil-acLDL和FITC-UEA-Ⅰ对细胞染色后,通过激光共聚焦显微镜鉴定,FITC-UEA-Ⅰ和Dil-acLDL双染色阳性细胞为正在分化的EPCs。 2.巴曲酶对外周血EPCs数量的影响 不同浓度的巴曲酶与EPCs培养24h均能增加其数量,其中以0.1BU·ml~(-1)在培养24h时最为显著。 3.巴曲酶对外周血EPCs增殖功能的影响 采用MTT比色法检测巴曲酶对EPCS增殖能力的影响,结果显示:不同浓度的巴曲酶均显著增加EPCs的增殖能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。 4.巴曲酶对外周血EPCs迁移功能的影响 采用改良的Boydne小室检测巴曲酶对EPCs迁移能力的影响,在200倍显微镜下计数迁移的细胞。巴曲酶明显提高EPCs的迁移能力,其中以0.1BU·ml~(-1)在培养24h时最为显著。 5.巴曲酶对外周血EPCs黏附功能的影响 为了观察巴曲酶对外周血EPCs豁附能力的影响,先将不同浓度的巴曲酶与EPCs培养24h,然后将相同数量的EPCs重新接种到包被有人纤维连接蛋白的培养板上培养30h,结果显示0.05 BU·ml~(-1)的巴曲酶明显提高EPCs的黏附能力,且在培养24h时最为显著。 结论: 1.巴曲酶可以增加外周血EPCs数量,提高EPCs的增殖能力、迁移能力和黏附能力。 2.巴曲酶对EPCs数量和功能的影响呈一定的时间依赖关系; 3.可以从外周血中的单个核细胞分离和培养血管内皮祖细胞。
[Abstract]:Objective:
Endothelial progenitor cells (EPCs) is a precursor cell of vascular endothelial cells. They not only participate in the development of blood vessels in the embryonic stage, but also exist in the adult's bone marrow and peripheral blood, which play an important role in the formation of adult angiogenesis. In recent years, research has shown that EPCs plays an important role in the development of ischemic disease of the lower limbs. The decrease in the number of vascular endothelial progenitor cells indicates a decrease in vascular endothelial progenitor cells and an increase in the incidence of vascular diseases. Batroxobin (DF-521) is a kind of serine protein purified from the venom of Agkistrodon ackkistrodon venom, and is currently widely used in clinical applications including cerebral infarction, chronic arteriosclerotic occlusive disease of the lower extremities (ASO). The treatment of ischemic disease shows that it may have protective effects on vascular endothelial dysfunction. The dynamic balance between injury and repair of endothelium is the key to maintain its normal function. EPCs is an important factor involved in the repair. Considering that DF-521 may promote the improvement of endothelial function by affecting the EPCs, this article is observed by the experiment. The effect of EPCs quantity, proliferation, migration and adhesion ability in peripheral blood is designed to further explore the mechanism of DF-521, and to find the possible inducer for the expansion of EPCs in vitro, thus providing more sufficient and good vascular tissue engineering cells.
Method:
The experimental blood samples from the healthy volunteers were used to detect the number and function of EPCs in the control group and the drug intervention group. The density gradient centrifugation was used to obtain the mononuclear cells from the peripheral blood. The 4D was cultured on the.DMEM medium of the human fibronectin package, and the non adherent cells were washed out with PBS, and the culture medium was continued to be cultured to 7. D, the non adherent cells were washed out with PBS, and the adherent cells were used for the experiment. The FITC UEA - I and Dil - acLDL positive cells were identified as the differentiated EPCs. The adherent cells were randomly divided into 4 groups. After the culture, the intervention group was cultured with different concentrations of batroxobin (0.05BU ml~ (-1), 0.1BU. 4h was counted under the inverted fluorescence microscope. The MTT colorimetry, the improved Boyden chamber and adhesion ability test were used to observe the proliferation, migration and adhesion of EPCs, and then the effect of DF-521 on the number of EPCs and the effect of function on the function of DF-521 was then selected.
Result:
Identification of 1.EPCs
The isolated cells were cultured for 7d and formed spindle shaped endothelia cells. After staining with Dil-acLDL and FITC-UEA- I, the cells were identified by laser confocal microscopy, and FITC-UEA- I and Dil-acLDL double staining positive cells were differentiated EPCs..
The effect of 2. Batroxobin on the number of EPCs in peripheral blood
Different concentrations of Batroxobin and EPCs could increase the number of 24h cultured, and 0.1BU (ml~) (-1) was the most significant when cultured 24h.
The effect of 3. Batroxobin on the proliferation of EPCs in peripheral blood
MTT colorimetric assay was used to detect the effect of Batroxobin on the proliferation of EPCS. The results showed that the proliferation ability of EPCs was significantly increased by different concentrations of batroxobin, and 0.1BU. Ml~ (-1) was the most significant in the culture of 24h.
The effect of 4. Batroxobin on the migration of EPCs in peripheral blood
The modified Boydne chamber was used to detect the effect of Batroxobin on the mobility of EPCs, and the migrating cells were counted under 200 times microscopes. The migration ability of EPCs was obviously improved by Batroxobin, and 0.1BU. Ml~ (-1) was the most significant in the culture of 24h.
The effect of 5. Batroxobin on the adhesion of EPCs in peripheral blood
In order to observe the effect of Batroxobin on the immunity of EPCs in peripheral blood, 24h was cultured with different concentrations of Batroxobin and EPCs, and then the same number of EPCs was reinoculated on the culture plate of the coated fibronectin. The results showed that the 0.05 BU. Ml~ (-1) batroxobin increased the adhesion ability of EPCs, and was the best for 24h. It is significant.
Conclusion:
1. batroxobin can increase the number of EPCs in peripheral blood and increase the proliferation, migration and adhesion of EPCs.
2. batroxobin had a certain time dependence on the quantity and function of EPCs.
3., endothelial progenitor cells can be isolated and cultured from peripheral blood mononuclear cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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