本文選題：巴曲酶 + 內(nèi)皮祖細(xì)胞��； 參考：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的: 血管內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs)是血管內(nèi)皮的前體細(xì)胞,它們不僅參與胚胎期的血管發(fā)育,也存在于成年機(jī)體的骨髓及外周血,在成體血管新生中起重要作用。近年來的研究表明EPCs在下肢缺血性疾病的發(fā)生、發(fā)展中扮演了重要角色,血管內(nèi)皮祖細(xì)胞數(shù)量減少預(yù)示血管內(nèi)皮修復(fù)能力降低,血管疾病發(fā)生率增高。巴曲酶(Batroxobin,DF-521)是一種由蝮蛇毒液中提純,精制而出的絲氨酸蛋白,目前臨床上廣泛用于包括腦梗塞、慢性下肢動(dòng)脈硬化性閉塞癥(ASO)在內(nèi)的各種缺血性疾病的治療,體現(xiàn)出其可能對(duì)于血管內(nèi)皮的功能障礙有保護(hù)作用。內(nèi)皮的損傷與修復(fù)之間的動(dòng)態(tài)平衡是維持其正常功能的關(guān)鍵,EPCs是參與修復(fù)的重要因素�？紤]DF-521可能通過影響EPCs促進(jìn)內(nèi)皮功能的改善,本文經(jīng)試驗(yàn)觀察其對(duì)于外周血EPCs數(shù)量、增殖、遷移和粘附能力的影響,旨在進(jìn)一步探討DF-521的作用機(jī)制,并為體外擴(kuò)增EPCs尋找可能的誘導(dǎo)劑,從而提供更充足并良好的血管組織工程細(xì)胞。 方法: 實(shí)驗(yàn)血液樣本來自健康志愿者,用于對(duì)照組和藥物干預(yù)組的EPCs數(shù)量及功能檢測。采用密度梯度離心法從外周血獲得單個(gè)核細(xì)胞,將其接種在人纖維連接蛋白包被的培養(yǎng)板上。DMEM培養(yǎng)基培養(yǎng)4d,用PBS洗掉非貼壁細(xì)胞,換培養(yǎng)液繼續(xù)培養(yǎng)至7d,用PBS洗掉非貼壁細(xì)胞,貼壁細(xì)胞供實(shí)驗(yàn)用。激光共聚焦顯微鏡鑒定FITC—UEA—Ⅰ和Dil—acLDL雙染色陽性細(xì)胞為正在分化的EPCs;貼壁細(xì)胞隨機(jī)分成4組,培養(yǎng)24h后干預(yù)組分別加入不同濃度的巴曲酶(0.05BU·ml~(-1)、0.1BU·ml~(-1)、0.2BU·ml~(-1))培養(yǎng)24h,再將其在倒置熒光顯微鏡下計(jì)數(shù)。分別采用MTT比色法、改良的Boyden小室和黏附能力測定實(shí)驗(yàn)來觀察EPCs的增殖能力、遷移能力和黏附能力,再根據(jù)結(jié)果選擇差異較顯著的劑量再進(jìn)行DF-521對(duì)EPCs數(shù)量及功能影響的時(shí)效關(guān)系實(shí)驗(yàn)。 結(jié)果: 1.EPCs的鑒定 分離獲得的單個(gè)核細(xì)胞培養(yǎng)7d后形成了梭形的內(nèi)皮樣細(xì)胞。用Dil-acLDL和FITC-UEA-Ⅰ對(duì)細(xì)胞染色后,通過激光共聚焦顯微鏡鑒定,FITC-UEA-Ⅰ和Dil-acLDL雙染色陽性細(xì)胞為正在分化的EPCs。 2.巴曲酶對(duì)外周血EPCs數(shù)量的影響 不同濃度的巴曲酶與EPCs培養(yǎng)24h均能增加其數(shù)量,其中以0.1BU·ml~(-1)在培養(yǎng)24h時(shí)最為顯著。 3.巴曲酶對(duì)外周血EPCs增殖功能的影響 采用MTT比色法檢測巴曲酶對(duì)EPCS增殖能力的影響,結(jié)果顯示:不同濃度的巴曲酶均顯著增加EPCs的增殖能力,其中以0.1BU·ml~(-1)在培養(yǎng)24h時(shí)最為顯著。 4.巴曲酶對(duì)外周血EPCs遷移功能的影響 采用改良的Boydne小室檢測巴曲酶對(duì)EPCs遷移能力的影響,在200倍顯微鏡下計(jì)數(shù)遷移的細(xì)胞。巴曲酶明顯提高EPCs的遷移能力,其中以0.1BU·ml~(-1)在培養(yǎng)24h時(shí)最為顯著。 5.巴曲酶對(duì)外周血EPCs黏附功能的影響 為了觀察巴曲酶對(duì)外周血EPCs豁附能力的影響,先將不同濃度的巴曲酶與EPCs培養(yǎng)24h,然后將相同數(shù)量的EPCs重新接種到包被有人纖維連接蛋白的培養(yǎng)板上培養(yǎng)30h,結(jié)果顯示0.05 BU·ml~(-1)的巴曲酶明顯提高EPCs的黏附能力,且在培養(yǎng)24h時(shí)最為顯著。 結(jié)論: 1.巴曲酶可以增加外周血EPCs數(shù)量,提高EPCs的增殖能力、遷移能力和黏附能力。 2.巴曲酶對(duì)EPCs數(shù)量和功能的影響呈一定的時(shí)間依賴關(guān)系; 3.可以從外周血中的單個(gè)核細(xì)胞分離和培養(yǎng)血管內(nèi)皮祖細(xì)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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