本文選題：人臍靜脈內(nèi)皮細(xì)胞 + 菲牛蛭素��； 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 目前，動(dòng)脈血管狹窄、梗塞、血栓形成等引起的組織器官缺血性疾病成為危害人類健康的常見疾病之一。本實(shí)驗(yàn)擬對(duì)菲牛蛭素進(jìn)行如下研究：通過凝血酶誘導(dǎo)體外培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞(HUVEC)，建立凝血酶損傷模型；以肝素（heparin sodium，hep.）為陽(yáng)性對(duì)照藥，觀察菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC活性、對(duì)vWF、PAI-1、t-PA分泌及其mRNA表達(dá)活性的影響，這些研究結(jié)果有助于我們探討菲牛蛭素在溶栓、抗凝及纖溶過程中的作用，為其應(yīng)用于臨床防治血栓性疾病提供新的理論依據(jù)。 方法 1.HUVEC的培養(yǎng)及鑒定 用預(yù)先配制的RPMI-1640完全培養(yǎng)基體外培養(yǎng)HUVEC，用0.25%胰蛋白酶消化后制備懸液，將細(xì)胞懸液按比例移入新的培養(yǎng)瓶?jī)?nèi)進(jìn)行傳代培養(yǎng)。倒置顯微鏡下觀察HUVEC的細(xì)胞形態(tài)，用兔抗人Ⅷ因子相關(guān)抗原鑒定方法對(duì)HUVEC進(jìn)行內(nèi)皮細(xì)胞鑒定。 2.菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC細(xì)胞活性的影響 凝血酶損傷模型的建立選取生長(zhǎng)狀態(tài)良好的HUVEC進(jìn)行實(shí)驗(yàn)。選擇凝血酶終濃度為0、5U/ml、10U/ml、15U/ml、20U/ml、30U/ml、40U/ml分別作用于HUVEC，采用MTT法測(cè)定492nm處光密度值(OD492)，以其反應(yīng)細(xì)胞活力；依照試劑盒操作方法，檢測(cè)凝血酶作用于HUVEC后培養(yǎng)上清液中乳酸脫氫酶(LDH)活性，以反映細(xì)胞損傷程度。 細(xì)胞活性的測(cè)定(1)菲牛蛭素(0.1U/ml~5U/ml)和肝素(1mg/ml~4mg/ml)分別作用于HUVEC，采用MTT比色法測(cè)定細(xì)胞活力，確定菲牛蛭素和肝素的實(shí)驗(yàn)濃度。(2)實(shí)驗(yàn)將分為六組進(jìn)行，即空白對(duì)照組、凝血酶模型組、肝素組和菲牛蛭素小、中、大濃度組，加樣培養(yǎng)24h后，收集細(xì)胞培養(yǎng)上清液-20℃凍存；MTT比色法測(cè)定細(xì)胞活力，以其反映細(xì)胞增殖活性；依照LDH試劑盒測(cè)定方法，檢測(cè)培養(yǎng)上清液中LDH活性，以其反映細(xì)胞受損傷的程度。 3.菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC對(duì)vWF、PAI-1、t-PA mRNA表達(dá)活性的影響 依照RNA提取方法提取RNA，利用瓊脂糖凝膠電泳方法檢測(cè)RNA的質(zhì)量及其完整性，根據(jù)逆轉(zhuǎn)錄試劑盒逆轉(zhuǎn)錄方法合成cDNA。通過實(shí)時(shí)熒光定量PCR法，測(cè)定HUVEC對(duì)vWF、PAI-1、t-PAmRNA的相對(duì)表達(dá)量,從而反映菲牛蛭素對(duì)以上因子mRNA表達(dá)活性的影響。 4.菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC培養(yǎng)上清液中vWF、PAI-1、t-PA分泌量的影響 解凍HUVEC培養(yǎng)上清液，以1000r/min的速度離心,棄沉淀物，酶聯(lián)免疫吸附試驗(yàn)法（ELISA）測(cè)定vWF、PAI-1、t-PA的釋放量。 結(jié)果 1.HUVEC鑒定結(jié)果 生長(zhǎng)狀態(tài)良好的HUVEC在顯微鏡下呈典型的鋪路石樣鑲嵌排列；兔抗人Ⅷ因子相關(guān)抗原鑒定結(jié)果顯示:HUVEC胞漿內(nèi)可見棕黃色顆粒，核周密集、胞核藍(lán)染，說(shuō)明細(xì)胞內(nèi)含有內(nèi)皮細(xì)胞所特有的第Ⅷ因子相關(guān)抗原的存在，因此證實(shí)培養(yǎng)的細(xì)胞為內(nèi)皮細(xì)胞。 2.菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC細(xì)胞活性的測(cè)定結(jié)果 凝血酶最佳誘導(dǎo)濃度確定與空白組比較，凝血酶各組（5U/ml~40U/ml）細(xì)胞活力明顯降低，隨著凝血酶濃度增加培養(yǎng)上清液中LDH活性顯著升高(P0.01)，當(dāng)凝血酶"g20U/ml時(shí)培養(yǎng)上清液中LDH活性趨于穩(wěn)定。根據(jù)凝血酶對(duì)HUVEC的細(xì)胞活力和培養(yǎng)上清液中LDH活性的測(cè)試結(jié)果，選擇凝血酶終濃度15U/ml作為最佳誘導(dǎo)濃度。 細(xì)胞活性的測(cè)定結(jié)果(1)在一定濃度范圍內(nèi)，隨著菲牛蛭素濃度增加，細(xì)胞增殖活性升高，0.25U/ml時(shí)細(xì)胞增殖活性最高，細(xì)胞增殖率達(dá)28.4%；繼續(xù)增加菲牛蛭素濃度("g0.3U/ml)細(xì)胞增殖活性降低，菲牛蛭素"g0.4U/ml時(shí)，與空白對(duì)照組比較細(xì)胞增殖活性差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；當(dāng)菲牛蛭素"g2U/ml時(shí)，與空白對(duì)照組比較，菲牛蛭素對(duì)HUVEC細(xì)胞活力呈現(xiàn)明顯的抑制作用(P0.01)，當(dāng)菲牛蛭素終濃度為5U/ml時(shí)，細(xì)胞毒活性達(dá)到24.61%。(2)與凝血酶組比較，Hep.組和菲牛蛭素小、中、大濃度組細(xì)胞活力均升高,差異有顯著的統(tǒng)計(jì)學(xué)意義(P0.01)；Hep.組與菲牛蛭素小、中、大劑量組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與空白對(duì)照組（393.3±5.8）U/ml比較，凝血酶組和Hep.組培養(yǎng)上清液中LDH活性升高明顯(P0.01)，凝血酶組達(dá)(575.4±5.1) U/ml；與凝血酶組比較，菲牛蛭素中、大劑量組培養(yǎng)上清液中LDH活性顯著降低(P0.01)，大劑量組LDH活性最低，為(375.0±5.2)U/ml。 3.RT-PCR檢測(cè)菲牛蛭素對(duì)凝血酶誘導(dǎo)HUVEC對(duì)vWF、PAI-1、t-PAmRNA表達(dá)活性測(cè)定結(jié)果 與空白對(duì)照組比較：凝血酶組vWF、PAI-1mRNA表達(dá)活性顯著增加(P0.01)、t-PA mRNA表達(dá)活性明顯降低(P0.01)，vWF mRNA相對(duì)表達(dá)量為空白對(duì)照組的1.67倍、PAI-1mRNA相對(duì)表達(dá)量為空白對(duì)照組9.28倍、t-PA mRNA相對(duì)表達(dá)量為空白對(duì)照組0.31倍；菲牛蛭素中、大濃度組t-PAmRNA表達(dá)活性明顯增加(P0.01)，最高可為空白對(duì)照組的2.58倍；與凝血酶組比較：Hep.組、菲牛蛭素小、中、大劑量組vWF、PAI-1mRNA表達(dá)活性顯著降低(P0.01)而t-PAmRNA表達(dá)活性明顯增加(P0.01)。 4.HUVEC培養(yǎng)上清液中vWF、PAI-1、t-PA含量測(cè)定結(jié)果 與空白對(duì)照組比較：凝血酶模型組和菲牛蛭素小劑量組vWF、PAI-1含量明顯增加(P0.01)、t-PA含量明顯減少(P0.01)，其中模型組vWF活性為(2122.4±231.3)U/ml、PAI-1含量為(59.90±7.80ng、t-PA含量為(6.68±0.66)ng；菲牛蛭素小、中、大劑量組t-PA含量明顯增加(P0.01)，在（16.20±4.37~17.27±4.73）ng范圍之間。與凝血酶組比較：Hep.組、菲牛蛭素中、大劑量組vWF、PAI-1含量明顯減少(P0.01)，其中菲牛蛭素大劑量組vWF活性為（1361.2±198.1）U/ml，PAI-1含量為（42.30±6.58）ng；菲牛蛭素小、中、大劑量組t-PA含量明顯增加(P0.01),最高可達(dá)(17.27±4.73）ng，為模型組的2.6倍。 結(jié)論 1.凝血酶對(duì)HUVEC具有損害作用，能夠破壞內(nèi)皮細(xì)胞的完整性，誘導(dǎo)內(nèi)皮細(xì)胞在抗血栓性能方面發(fā)生改變。 2.菲牛蛭素對(duì)HUVEC細(xì)胞活力具有雙向作用：低濃度菲牛蛭素對(duì)細(xì)胞增殖活力具有促進(jìn)作用；高濃度菲牛蛭素對(duì)細(xì)胞增殖具有抑制作用，隨著菲牛蛭素濃度增加，細(xì)胞毒活性增加。 3.高濃度菲牛蛭素具有保護(hù)VEC免受凝血酶損傷作用，調(diào)節(jié)內(nèi)皮細(xì)胞在抗凝、纖溶和血小板活化方面的作用，使內(nèi)皮細(xì)胞的生理功能恢復(fù)正常。其作用可能主要與菲牛蛭素對(duì)抗凝血酶誘導(dǎo)內(nèi)皮細(xì)胞的損傷，抑制vWF和PAI-1、增加t-PA的分泌及其mRNA的表達(dá)，增加t-PA/PAI-1比值相關(guān)。 目的 通過體外培養(yǎng)HUVEC，以人血管內(nèi)皮細(xì)胞生長(zhǎng)因子(VEGF)為陽(yáng)性對(duì)照藥，觀察低濃度菲牛蛭素對(duì)HUVEC細(xì)胞活力及其分泌釋放VEGF量的影響；以雞受精卵為研究對(duì)象，制備雞胚絨毛尿囊膜(CAM)模型，觀察菲牛蛭素對(duì)CAM血管新生作用的影響，為菲牛蛭素在治療冠心病、心肌梗死、腦梗死等缺血性疾病方面提供新的觀點(diǎn)。 方法 1.ELISA法測(cè)定菲牛蛭素對(duì)HUVEC培養(yǎng)上清液中VEGF分泌量的影響 體外培養(yǎng)HUVEC,選擇生長(zhǎng)狀態(tài)良好的細(xì)胞進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分為空白對(duì)照組、陽(yáng)性對(duì)照組(VEGF)及菲牛蛭素組�？瞻讓�(duì)照組不加任何處理因素，陽(yáng)性對(duì)照組含20ng/ml的VEGF，根據(jù)實(shí)驗(yàn)1.第二部分低濃度菲牛蛭素對(duì)HUVEC細(xì)胞活力測(cè)定結(jié)果，選擇菲牛蛭素終濃度為0.15U/ml、0.20U/ml、0.25U/ml、0.30U/ml、0.40U/ml五個(gè)濃度進(jìn)行實(shí)驗(yàn)。將培養(yǎng)上清液稀釋5倍后，ELISA法檢測(cè)低濃度菲牛蛭素對(duì)HUVEC培養(yǎng)上清液中VGEF含量的影響。 2.菲牛蛭素對(duì)雞胚絨毛尿囊膜(CAM)血管新生作用的影響 選擇新鮮雞受精卵置于全自動(dòng)孵化箱內(nèi)孵育至第8天，用手術(shù)刀片磨切標(biāo)記面中上1/3畫定開窗位置處，暴漏CAM制成假氣室，制備CAM模型；孵化箱內(nèi)穩(wěn)定12小時(shí)后選擇存活健康雞胚隨機(jī)分為無(wú)菌生理鹽水組，菲牛蛭素終濃度5U/ml、11U/ml、22U/ml3個(gè)劑量組；每胚0.02ml加樣于載體上，每天一次，共三天；雞胚孵育第12天，假氣室內(nèi)加入甲醇和甲醛的等量混合液2.5ml，，室溫靜置30min，去掉蛋殼及卵殼膜，最大面積暴露CAM，用數(shù)碼相機(jī)拍照；將CAM置于10×解剖顯微鏡下觀察并計(jì)數(shù)血管數(shù)目。 結(jié)果 1.HUVEC培養(yǎng)上清液中VEGF含量檢測(cè)結(jié)果 與空白對(duì)照組比較：菲牛蛭素0.15U/ml、0.2U/ml、0.25U/ml、0.3U/ml劑量組培養(yǎng)上清液中VEGF含量明顯增加(P0.01)，其中0.25U/ml時(shí)含量最高，達(dá)（1.808±0.046）ng/ml,比空白對(duì)照組高80.7%。 2.菲牛蛭素對(duì)CAM血管生成作用結(jié)果 與生理鹽水(NS)對(duì)照組比較：菲牛蛭素各組血管總數(shù)均明顯增加(P0.01)，其中大血管數(shù)、中血管數(shù)增加明顯(P0.01)，終濃度22U/ml組大血管數(shù)是生理鹽水對(duì)照組的12倍，中血管數(shù)為對(duì)照組3.2倍；小血管的數(shù)目增加則不顯著(P0.05)。 結(jié)論 1.低濃度菲牛蛭素具有促進(jìn)血管內(nèi)皮細(xì)胞分泌釋放VEGF的功能； 2.菲牛蛭素對(duì)CAM血管新生具有促進(jìn)作用。
[Abstract]:objective
At present, ischemic diseases of tissues and organs caused by arterial stenosis, infarction and thrombosis have become one of the common diseases which are harmful to human health. This experiment is to study the pheirin in human umbilical vein endothelial cells (HUVEC) cultured in vitro by thrombin, and to establish a thrombin damage model with heparin (heparin sodiu). M, hep.) as a positive control drug, the effects of pheidin on HUVEC activity induced by thrombin and on the secretion of vWF, PAI-1, t-PA and the expression of mRNA are observed. These results are helpful for us to explore the role of pheidin in thrombolytic, anticoagulant and fibrinolysis, and provide a new theoretical basis for its application in clinical prevention and treatment of thrombotic diseases.
Method
Culture and identification of 1.HUVEC
The preprepared RPMI-1640 complete culture medium was used to culture HUVEC in vitro, and the suspension was prepared by 0.25% trypsin digestion. The cell suspension was transferred into the new culture bottle and carried out in the new culture bottle. The cell morphology of HUVEC was observed under inverted microscope. The endothelial cells of HUVEC were identified by Rabbit anti human factor VIII related antigen identification method.
Effect of 2. pheniranin on thrombin induced HUVEC cell activity
The thrombin damage model was established to select the HUVEC with a good growth state. The final concentration of thrombin was selected as 0,5U/ml, 10U/ml, 15U/ml, 20U/ml, 30U/ml, 40U/ml respectively on HUVEC, and the MTT method was used to determine the light density value (OD492) at 492nm (OD492) to reflect the activity of the cells. The thrombin was tested after the action of the thrombin on HUVEC. The activity of lactate dehydrogenase (LDH) in supernatant was cultured to reflect the extent of cell damage.
Determination of cell activity (1) phenanthrin (0.1U/ml~5U/ml) and heparin (1mg/ml~4mg/ml) respectively on HUVEC, determination of cell vitality by MTT colorimetry, determination of the experimental concentration of phenanthrene and heparin. (2) the experiment will be divided into six groups, namely, blank control group, coagulase model group, heparin group and phenanthrin small, medium, large concentration group, and sample culture After 24h, the cell culture supernatant was collected at -20 centigrade, and the cell viability was measured by MTT colorimetric assay to reflect cell proliferation activity, and the activity of LDH in the culture supernatant was detected in accordance with the LDH kit determination method, in order to reflect the extent of cell damage.
Effects of 3. pheniranin on thrombin induced expression of vWF, PAI-1 and t-PA mRNA in HUVEC
RNA was extracted by RNA extraction method, and the quality and integrity of RNA were detected by agarose gel electrophoresis. The relative expression of HUVEC on vWF, PAI-1 and t-PAmRNA was measured by RT RT, and the effect of phenanthrene on the expression of mRNA was reflected by HUVEC.
Effects of 4. pheniranin on vWF, PAI-1 and t-PA secretion in HUVEC supernatant induced by thrombin
Thawing HUVEC was used to culture supernatant, centrifugated at 1000r/min speed, discarded sediment and enzyme linked immunosorbent assay (ELISA) was used to measure the release of vWF, PAI-1 and t-PA.
Result
1.HUVEC identification results
The HUVEC with a good growth state was arranged in a typical paved stone mosaic under the microscope, and the identification results of Rabbit anti human factor VIII related antigen showed that brown yellow granules were found in the cytoplasm of HUVEC, dense perinuclear and blue dye, indicating the existence of the VIII related antigen specific to endothelial cells in the cells, thus confirming the cultured cells. It is an endothelial cell.
Determination of thrombin induced HUVEC cell activity by 2. pheniranin
The optimum induction concentration of thrombin was compared with that of the blank group. The activity of 5U/ml~40U/ml cells decreased significantly, and the activity of LDH in the supernatant increased significantly (P0.01) with the increase of the concentration of thrombin in the culture supernatant (P0.01). When thrombin "g20U/ml", the activity of LDH in the culture supernatant tended to stabilize. The activity of the thrombin to HUVEC and the culture of the supernatant were in accordance with the thrombin. In the LDH activity test, the thrombin end concentration 15U/ml was chosen as the best induction concentration.
The results of cell activity determination (1) in a certain concentration range, with the increase of the concentration of pheirin, the proliferation activity of cells increased, the proliferation activity of 0.25U/ml was the highest and the cell proliferation rate reached 28.4%, and the proliferation activity of pheirin ("g0.3U/ml) cells decreased, and phenanthrin hirudin" g0.4U/ml, compared with the blank control group, the cell proliferation was increased. There was no significant difference in colonization activity (P0.05); when phenanthrin "g2U/ml", phenanthrene showed a significant inhibitory effect on HUVEC cell viability compared with the blank control group (P0.01). When the final concentration of phioxin was 5U/ml, the cytotoxic activity reached 24.61%. (2) compared with the thrombin group, the Hep. group and the phenanthrin group were small, medium, and large. The difference had significant statistical significance (P0.01), and there was no significant difference between group Hep. and pheirin in small, medium and large dose groups (P0.05). Compared with the blank control group (393.3 + 5.8) U/ml, the activity of LDH in the thrombin group and the culture supernatant of Hep. group was elevated to Gao Mingxian (P0.01), the thrombin group was (575.4 + 5.1) U/ml; and thrombin was used as a thrombin. Compared with the control group, the activity of LDH in the supernatant of the high dose group was significantly reduced (P0.01), and the LDH activity in the high dose group was the lowest (375 + 5.2) U/ml..
3.RT-PCR determination of the expression of HUVEC, vWF, PAI-1 and t-PAmRNA in mice induced by thrombin by phenanthrin
Compared with the blank control group, the expression of vWF and PAI-1mRNA in the thrombin group increased significantly (P0.01), the expression of t-PA mRNA was significantly decreased (P0.01), the relative expression of vWF mRNA was 1.67 times that of the blank control group, the relative expression of PAI-1mRNA was 9.28 times that of the blank control group, and the relative expression of t-PA mRNA was 0.31 times that of the blank control group, and the phenanthrene hirudin was large. The expression activity of t-PAmRNA in the concentration group was significantly increased (P0.01), which was 2.58 times higher than that in the blank control group. Compared with the thrombin group, the Hep. group, phenanthrene hirudin was small, medium, large dose group vWF, PAI-1mRNA expression activity decreased significantly (P0.01), and the expression of t-PAmRNA was significantly increased (P0.01).
Determination of vWF, PAI-1 and t-PA contents in 4.HUVEC supernatant
Compared with the blank control group, the content of vWF in the thrombin model group and the small dose group of phenanthrin was significantly increased (P0.01), and the content of t-PA decreased significantly (P0.01). The vWF activity of the model group was (2122.4 + 231.3) U/ml, PAI-1 content was (59.90 + 7.80ng, t-PA content was (6.68 + 0.66) ng, and phenanthrene hirudin was small, medium, and large dose group increased significantly. 0.01) in the range of (16.20 + 4.37~17.27 + 4.73) ng. Compared with thrombin group: group Hep., phenanthrin, large dose group vWF, PAI-1 content decreased significantly (P0.01), vWF activity of high dose group of pheirin was (1361.2 + 198.1) U/ml, PAI-1 content was (42.30 + 6.58) ng, phenanthrin was small, medium, large dose group t-PA content obviously increased (P0.01) The highest (17.27 + 4.73) ng was 2.6 times that of the model group.
conclusion
1. thrombin has a damaging effect on HUVEC, which can destroy the integrity of endothelial cells and induce changes in the antithrombotic properties of endothelial cells.
2. phenanthrene hirudin has a two-way effect on the activity of HUVEC cells: low concentration phenanthrin has a promoting effect on cell proliferation, and high concentration of phenanthrin has a inhibitory effect on cell proliferation. With the increase of pheirin concentration, cytotoxic activity is increased.
3. the high concentration of pheidin, which protects VEC from thrombin damage, regulates the role of endothelial cells in anticoagulant, fibrinolysis and platelet activation to restore the physiological function of endothelial cells. The effect may be mainly with pheidin against the injury of thrombin induced endothelial cells, the inhibition of vWF and PAI-1, and the secretion of t-PA and the secretion of the endothelial cells. The expression of mRNA was associated with the increase of the t-PA/PAI-1 ratio.
objective
The effect of low concentration of pheirin on the activity of HUVEC cells and the release of VEGF in HUVEC cells was observed by cultured human vascular endothelial growth factor (VEGF) in vitro. The chick embryo chorioallantoic membrane (CAM) model was prepared from the fertilized egg of chicken, and the effect of phenanthrene on the angiogenesis of CAM was observed. The effect of phenanthrene on the angiogenesis of HUVEC was observed. Bovine vermiculin provides new perspectives in the treatment of ischemic diseases such as coronary heart disease, myocardial infarction and cerebral infarction.
Method
Effect of phenanthrin on the secretion of VEGF in HUVEC supernatant by 1.ELISA
HUVEC was cultured in vitro, and the cells with good growth state were selected. The experiment was divided into blank control group, positive control group (VEGF) and phenanthrene hirudin group. The blank control group had no treatment factors, and the positive control group contained 20ng/ml VEGF. According to the results of the assay of HUVEC cell viability with the low concentration phenanthrin in the 1. second parts of the experiment, the phenanthrene was selected. The final concentration of vermiculin was five concentrations of 0.15U/ml, 0.20U/ml, 0.25U/ml, 0.30U/ml and 0.40U/ml. After 5 times the dilution of the supernatant, the ELISA method was used to detect the effect of the low concentration of pheirin on the content of VGEF in the culture supernatant of HUVEC.
Effect of 2. pheniranin on angiogenesis of chick embryo chorioallantoic membrane (CAM)
The fertilized egg of fresh chicken was incubated in a fully automatic incubator for eighth days, and the upper 1/3 of the surgical blade was used to paint the window position at the top of the window. A false gas chamber was made out of CAM, and the CAM model was prepared. After 12 hours in the incubator, the viable healthy chicken embryos were randomly divided into aseptic saline group, and the final concentration of phenanthrene birudin was 5U/ml, 11U/ml, 22U/ml. 3 dose groups, each embryo 0.02ml was added to the carrier, once a day for three days; chicken embryo was incubated for Twelfth days, the mixture of methanol and formaldehyde was added to the pseudo gas room for 2.5ml, 30min was inserted at room temperature, the eggshell and eggshell were removed, the maximum area was exposed to CAM, and the digital camera was taken. The CAM was placed under the 10 x microscope to observe the number of blood vessels and count the number of blood vessels. Eyes.
Result
Detection results of VEGF content in 1.HUVEC culture supernatant
Compared with the blank control group, the content of VEGF in the culture supernatant of phenanthrene 0.15U/ml, 0.2U/ml, 0.25U/ml and 0.3U/ml increased significantly (P0.01), and the content of 0.25U/ml was the highest, reaching (1.808 + 0.046) ng/ml, which was higher than that in the blank control group.
Effect of 2. phenanthrene hirudin on the angiogenesis of CAM
Compared with the normal saline (NS) control group, the total number of blood vessels in each group was significantly increased (P0.01), the number of large vessels and the number of blood vessels increased significantly (P0.01). The number of large blood vessels in the final concentration 22U/ml group was 12 times that of the normal saline control group, the number of the middle blood vessels was 3.2 times that of the control group, and the increase of the small blood vessels was not significant (P0.05).
conclusion
1. low concentration of phenanthrin can promote the secretion and release of VEGF from vascular endothelial cells.
2. phenanthrin has a promoting effect on angiogenesis of CAM.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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