菲牛蛭素对凝血酶诱导内皮细胞活性、vWF、PAI-1、t-PA分泌、mRNA表达及对血管新生作用的研究

发布时间：2018-04-20 18:01

本文选题：人脐静脉内皮细胞 + 菲牛蛭素　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的目前，动脉血管狭窄、梗塞、血栓形成等引起的组织器官缺血性疾病成为危害人类健康的常见疾病之一。本实验拟对菲牛蛭素进行如下研究：通过凝血酶诱导体外培养的人脐静脉内皮细胞(HUVEC)，建立凝血酶损伤模型；以肝素（heparin sodium，hep.）为阳性对照药，观察菲牛蛭素对凝血酶诱导HUVEC活性、对vWF、PAI-1、t-PA分泌及其mRNA表达活性的影响，这些研究结果有助于我们探讨菲牛蛭素在溶栓、抗凝及纤溶过程中的作用，为其应用于临床防治血栓性疾病提供新的理论依据。方法 1.HUVEC的培养及鉴定用预先配制的RPMI-1640完全培养基体外培养HUVEC，用0.25%胰蛋白酶消化后制备悬液，将细胞悬液按比例移入新的培养瓶内进行传代培养。倒置显微镜下观察HUVEC的细胞形态，用兔抗人Ⅷ因子相关抗原鉴定方法对HUVEC进行内皮细胞鉴定。 2.菲牛蛭素对凝血酶诱导HUVEC细胞活性的影响凝血酶损伤模型的建立选取生长状态良好的HUVEC进行实验。选择凝血酶终浓度为0、5U/ml、10U/ml、15U/ml、20U/ml、30U/ml、40U/ml分别作用于HUVEC，采用MTT法测定492nm处光密度值(OD492)，以其反应细胞活力；依照试剂盒操作方法，检测凝血酶作用于HUVEC后培养上清液中乳酸脱氢酶(LDH)活性，以反映细胞损伤程度。细胞活性的测定(1)菲牛蛭素(0.1U/ml~5U/ml)和肝素(1mg/ml~4mg/ml)分别作用于HUVEC，采用MTT比色法测定细胞活力，确定菲牛蛭素和肝素的实验浓度。(2)实验将分为六组进行，即空白对照组、凝血酶模型组、肝素组和菲牛蛭素小、中、大浓度组，加样培养24h后，收集细胞培养上清液-20℃冻存；MTT比色法测定细胞活力，以其反映细胞增殖活性；依照LDH试剂盒测定方法，检测培养上清液中LDH活性，以其反映细胞受损伤的程度。 3.菲牛蛭素对凝血酶诱导HUVEC对vWF、PAI-1、t-PA mRNA表达活性的影响依照RNA提取方法提取RNA，利用琼脂糖凝胶电泳方法检测RNA的质量及其完整性，根据逆转录试剂盒逆转录方法合成cDNA。通过实时荧光定量PCR法，测定HUVEC对vWF、PAI-1、t-PAmRNA的相对表达量,从而反映菲牛蛭素对以上因子mRNA表达活性的影响。 4.菲牛蛭素对凝血酶诱导HUVEC培养上清液中vWF、PAI-1、t-PA分泌量的影响解冻HUVEC培养上清液，以1000r/min的速度离心,弃沉淀物，酶联免疫吸附试验法（ELISA）测定vWF、PAI-1、t-PA的释放量。结果 1.HUVEC鉴定结果生长状态良好的HUVEC在显微镜下呈典型的铺路石样镶嵌排列；兔抗人Ⅷ因子相关抗原鉴定结果显示:HUVEC胞浆内可见棕黄色颗粒，核周密集、胞核蓝染，说明细胞内含有内皮细胞所特有的第Ⅷ因子相关抗原的存在，因此证实培养的细胞为内皮细胞。 2.菲牛蛭素对凝血酶诱导HUVEC细胞活性的测定结果凝血酶最佳诱导浓度确定与空白组比较，凝血酶各组（5U/ml~40U/ml）细胞活力明显降低，随着凝血酶浓度增加培养上清液中LDH活性显著升高(P0.01)，当凝血酶"g20U/ml时培养上清液中LDH活性趋于稳定。根据凝血酶对HUVEC的细胞活力和培养上清液中LDH活性的测试结果，选择凝血酶终浓度15U/ml作为最佳诱导浓度。细胞活性的测定结果(1)在一定浓度范围内，随着菲牛蛭素浓度增加，细胞增殖活性升高，0.25U/ml时细胞增殖活性最高，细胞增殖率达28.4%；继续增加菲牛蛭素浓度("g0.3U/ml)细胞增殖活性降低，菲牛蛭素"g0.4U/ml时，与空白对照组比较细胞增殖活性差异无统计学意义(P0.05)；当菲牛蛭素"g2U/ml时，与空白对照组比较，菲牛蛭素对HUVEC细胞活力呈现明显的抑制作用(P0.01)，当菲牛蛭素终浓度为5U/ml时，细胞毒活性达到24.61%。(2)与凝血酶组比较，Hep.组和菲牛蛭素小、中、大浓度组细胞活力均升高,差异有显著的统计学意义(P0.01)；Hep.组与菲牛蛭素小、中、大剂量组间比较差异无统计学意义(P0.05)。与空白对照组（393.3±5.8）U/ml比较，凝血酶组和Hep.组培养上清液中LDH活性升高明显(P0.01)，凝血酶组达(575.4±5.1) U/ml；与凝血酶组比较，菲牛蛭素中、大剂量组培养上清液中LDH活性显著降低(P0.01)，大剂量组LDH活性最低，为(375.0±5.2)U/ml。 3.RT-PCR检测菲牛蛭素对凝血酶诱导HUVEC对vWF、PAI-1、t-PAmRNA表达活性测定结果与空白对照组比较：凝血酶组vWF、PAI-1mRNA表达活性显著增加(P0.01)、t-PA mRNA表达活性明显降低(P0.01)，vWF mRNA相对表达量为空白对照组的1.67倍、PAI-1mRNA相对表达量为空白对照组9.28倍、t-PA mRNA相对表达量为空白对照组0.31倍；菲牛蛭素中、大浓度组t-PAmRNA表达活性明显增加(P0.01)，最高可为空白对照组的2.58倍；与凝血酶组比较：Hep.组、菲牛蛭素小、中、大剂量组vWF、PAI-1mRNA表达活性显著降低(P0.01)而t-PAmRNA表达活性明显增加(P0.01)。 4.HUVEC培养上清液中vWF、PAI-1、t-PA含量测定结果与空白对照组比较：凝血酶模型组和菲牛蛭素小剂量组vWF、PAI-1含量明显增加(P0.01)、t-PA含量明显减少(P0.01)，其中模型组vWF活性为(2122.4±231.3)U/ml、PAI-1含量为(59.90±7.80ng、t-PA含量为(6.68±0.66)ng；菲牛蛭素小、中、大剂量组t-PA含量明显增加(P0.01)，在（16.20±4.37~17.27±4.73）ng范围之间。与凝血酶组比较：Hep.组、菲牛蛭素中、大剂量组vWF、PAI-1含量明显减少(P0.01)，其中菲牛蛭素大剂量组vWF活性为（1361.2±198.1）U/ml，PAI-1含量为（42.30±6.58）ng；菲牛蛭素小、中、大剂量组t-PA含量明显增加(P0.01),最高可达(17.27±4.73）ng，为模型组的2.6倍。结论 1.凝血酶对HUVEC具有损害作用，能够破坏内皮细胞的完整性，诱导内皮细胞在抗血栓性能方面发生改变。 2.菲牛蛭素对HUVEC细胞活力具有双向作用：低浓度菲牛蛭素对细胞增殖活力具有促进作用；高浓度菲牛蛭素对细胞增殖具有抑制作用，随着菲牛蛭素浓度增加，细胞毒活性增加。 3.高浓度菲牛蛭素具有保护VEC免受凝血酶损伤作用，调节内皮细胞在抗凝、纤溶和血小板活化方面的作用，使内皮细胞的生理功能恢复正常。其作用可能主要与菲牛蛭素对抗凝血酶诱导内皮细胞的损伤，抑制vWF和PAI-1、增加t-PA的分泌及其mRNA的表达，增加t-PA/PAI-1比值相关。目的通过体外培养HUVEC，以人血管内皮细胞生长因子(VEGF)为阳性对照药，观察低浓度菲牛蛭素对HUVEC细胞活力及其分泌释放VEGF量的影响；以鸡受精卵为研究对象，制备鸡胚绒毛尿囊膜(CAM)模型，观察菲牛蛭素对CAM血管新生作用的影响，为菲牛蛭素在治疗冠心病、心肌梗死、脑梗死等缺血性疾病方面提供新的观点。方法 1.ELISA法测定菲牛蛭素对HUVEC培养上清液中VEGF分泌量的影响体外培养HUVEC,选择生长状态良好的细胞进行实验。实验分为空白对照组、阳性对照组(VEGF)及菲牛蛭素组。空白对照组不加任何处理因素，阳性对照组含20ng/ml的VEGF，根据实验1.第二部分低浓度菲牛蛭素对HUVEC细胞活力测定结果，选择菲牛蛭素终浓度为0.15U/ml、0.20U/ml、0.25U/ml、0.30U/ml、0.40U/ml五个浓度进行实验。将培养上清液稀释5倍后，ELISA法检测低浓度菲牛蛭素对HUVEC培养上清液中VGEF含量的影响。 2.菲牛蛭素对鸡胚绒毛尿囊膜(CAM)血管新生作用的影响选择新鲜鸡受精卵置于全自动孵化箱内孵育至第8天，用手术刀片磨切标记面中上1/3画定开窗位置处，暴漏CAM制成假气室，制备CAM模型；孵化箱内稳定12小时后选择存活健康鸡胚随机分为无菌生理盐水组，菲牛蛭素终浓度5U/ml、11U/ml、22U/ml3个剂量组；每胚0.02ml加样于载体上，每天一次，共三天；鸡胚孵育第12天，假气室内加入甲醇和甲醛的等量混合液2.5ml，，室温静置30min，去掉蛋壳及卵壳膜，最大面积暴露CAM，用数码相机拍照；将CAM置于10×解剖显微镜下观察并计数血管数目。结果 1.HUVEC培养上清液中VEGF含量检测结果与空白对照组比较：菲牛蛭素0.15U/ml、0.2U/ml、0.25U/ml、0.3U/ml剂量组培养上清液中VEGF含量明显增加(P0.01)，其中0.25U/ml时含量最高，达（1.808±0.046）ng/ml,比空白对照组高80.7%。 2.菲牛蛭素对CAM血管生成作用结果与生理盐水(NS)对照组比较：菲牛蛭素各组血管总数均明显增加(P0.01)，其中大血管数、中血管数增加明显(P0.01)，终浓度22U/ml组大血管数是生理盐水对照组的12倍，中血管数为对照组3.2倍；小血管的数目增加则不显著(P0.05)。结论 1.低浓度菲牛蛭素具有促进血管内皮细胞分泌释放VEGF的功能； 2.菲牛蛭素对CAM血管新生具有促进作用。
[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.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【参考文献】