可溶性环氧化合物水解酶抑制剂对内皮祖细胞功能的影响

发布时间：2018-03-09 21:56

本文选题：内皮祖细胞　切入点：培养　出处：《中南大学》2012年博士论文　论文类型：学位论文

【摘要】：第一部分小鼠骨髓来源内皮祖细胞的分离、培养及鉴定目的研究小鼠骨髓来源内皮祖细胞的分离培养方法并对其进行鉴定。方法 1.小鼠骨髓来源内皮祖细胞培养：用密度梯度离心法从小鼠股骨及胫骨骨髓中分离单个核细胞,按5×105/cm2密度置于包被有人纤维连接蛋白的细胞培养皿中,加入含5%FBS和生长因子的EBM-2培养基培养。显微镜下观察不同时间点种植的内皮祖细胞形态变化。 2.培养的内皮祖细胞双荧光染色：荧光显微镜下观察细胞摄取Dil-AcLDL及结合FITC-UEA-1特性,染色双阳性细胞为EPCs。 3.小鼠骨髓来源内皮祖细胞培养的鉴定：培养七天后用流式细胞仪检测CD34、CD133、CD31、Flk-1等内皮祖细胞表面标志的表达。结果 1.分离单个核细胞培养4天后,显微镜下可见集落形成,集落中央为圆形细胞,周围为梭形细胞围绕；7天后见培养的细胞集落明显扩大,集落中央圆形细胞向梭形细胞转化；第8天可见培养细胞形成条索状；第10天培养的内皮祖细胞可达80%-90%融合,以类圆形和多角形细胞为主,也可见梭形细胞。 2.显微镜下计数Dil-AcLDL及结合FITC-UEA-1染色双阳性细胞为95%以上。 3.流式细胞仪检测结果示细胞表面特异性抗原含量分别为CD34(53.89±0.34)%;CD133(52.79±0.67)%;CD31(36.67±0.93)%; Flk-1(43.88±0.48)%。结论通过形态学、(DiI-ac-LDL/FITC-UEA-1)荧光双染色和流式细胞检测证明培养的细胞为内皮祖细胞。第二部分可溶性环氧化物水解酶抑制剂t-AUCB体外对小鼠EPCs功能的影响及机制目的检测不同浓度可溶性环氧化物水解酶抑制剂t-AUCB (trans-4-[4-(3-adamantan-1-ylureido-cyclohexyloxy]-benzoic acid))及PPARγ(peroxisome proliferator activated receptory)受体阻滞剂(GW9662)干预后对内皮祖细胞增殖、粘附、迁移、归巢、血管生成、分泌VEGF (vascular endothelial growth factor)及HIF1-α(hypoxia inducible factor1alph)的影响及机制。方法内皮祖细胞接种7天后用不同浓度(0、1、10、50、100μmol/Lt-AUCB、GW9662+100μmol/L t-AUCB)干预细胞,后取相同数量1×105个细胞分别进行如下实验： 1.将细胞消化至96孔板,按不同浓度t-AUCB干预24小时后孔内加MTT,在酶联免疫监测仪上测定各孔光吸收值,计算其对增殖的影响。 2.细胞接种于纤连蛋白包被的12孔培养板,37℃孵育1h,除去未贴壁细胞,倒置显微镜下计数粘附细胞数,计算其对粘附的影响。 3.细胞添加至transwell板的上层,下层添加含有不同浓度t-AUCB的迁移缓冲液,37℃孵育4h后,测定迁移至下层的细胞数,计算t-AUCB对迁移的影响。 4.细胞与2μg/mL Dil-ac-LDL共同培养1h,心肌梗死手术后立即给小鼠尾静脉注射,术后24h,心脏切小粒,循序在酶中消化,用胎牛血清中和,离心后在荧光显微镜下记数Dil标记的阳性细胞数,计算t-AUCB对内皮祖细胞归巢的影响。 5.细胞接种于预先孵育的Matrigel板上,加入不同浓度t-AUCB孵育24h后倒置显微镜下观察血管生成情况。 6.细胞进行消化、提取蛋白,经western blot检测其分泌VEGF及HIF1-α能力。结果 1.从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs的增殖能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs增殖能力(P0.05)；PPARγ受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。 2.从0μmol/L-100μmol/L,随着浓度增加,t.AUCB可呈浓度依赖性增强EPCs的粘附能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs粘附能力(P0.05)；PPARγ受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。 3.从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs的迁移能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs迁移能力(P0.05)；PPAR7受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。 4.从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs的归巢能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs归巢能力(P0.05)；PPARγ受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。 5.从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs的血管生成能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs血管生成能力(P0.05)；PPARγ受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。 6.从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs合成VEGF及HIF1-α能力,与对照组(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB显著增强EPCs合成VEGF及HIF1-α能力(P0.05)；PPARγ受体阻滞剂(GW9662)可抑制其上述功能(P0.05)。结论从0μmol/L-100μmol/L,随着浓度增加,t-AUCB可呈浓度依赖性增强EPCs的EPCs增殖、粘附、迁移、归巢、血管生成及分泌VEGF及HIF-α能力,该作用可能通过PPARγ受体发挥作用。第三部分t-AUCB处理后的EPCs体内注射对小鼠缺血心肌的影响及机制目的研究不同浓度t-AUCB及PPARy受体阻滞剂(GW9662)处理后的EPCs体内注射对小鼠心肌梗死的影响及机制方法 1.通过结扎小鼠左前降支制作心肌梗死模型,并记录梗死心电图。 2.梗死后1小时分别通过尾静脉注射200μ1不同浓度(0、1、10、50、100μmol/L、100μmol/L t-AUCB+GW9662)干预的小鼠内皮祖细胞。 3.在心肌梗死后24小时、3天、7天、14天、28天处死小鼠,取出心脏,行福尔马林固定,测定梗死心肌面积占结扎点横切面以下心脏面积百分比。 4.通过免疫组化检测VEGF及Ⅷ (factor Ⅷ)测定梗死心肌边缘区血管生成数。 5.对相同实践不同浓度干预组梗死心肌面积比及血管生成数量进行比较。 6.对相同浓度组干预后不同时间点梗死心肌而积比及血管生成数量进行比较。结果 1.制作心肌梗死模型时,结扎瞬间可见结扎线远端心肌活动度减弱,左室前壁苍白后紫绀,行心电图检查表现为前壁导联ST段弓背向上抬高,表明模型制作成功。 2.相同时间点不同浓度移植组心梗面积百分比示：心梗移植后24小时不同浓度干预组间面积比无显著差异(P=0.481)；移植后3天、7天、14天、28天,随着浓度增加,各十预组面积比逐渐减小,100μmol/L t-AUCB+GW9662组面积比明显大于100μ/L组,不同浓度t-AUCB各干预组间存在显著差异(P0.05)。 3.相同浓度t-AUCB干预后不同时间点心梗面积百分比示： (1)0μmol/L组示：从24小时至14d,随着时间延长,心梗面积比逐渐增大；从14d至28d,面积比逐渐缩小,各时间点间具有显著差异(P0.05)。 (2)1μmol/L组示：从24h至3d,面积比较前逐渐缩小；3天至14天,面积比较前逐渐增大；14天至28天,面积比较前逐渐缩小,各时间点间具有显著差异(P0.05)。 (3)10、50、100μmol/L组示：从24小时至7天,面积比逐渐缩小；7天至14天,面积比较前逐渐增大,但仍小于24小时面积比；从14天至28天,面积比较前逐渐缩小,各时间点间具有显著差异(P0.05)。 (4)100μmol/L t-AUCB+gw9662组示：从24小时至3天,面积比逐渐增大；3天至7天,面积比较前逐渐减小；7天至14天,面积比较前增大；从14天至28天,心梗面积比较前明显缩小。各时间点间具有显著差异(P0.05)。 4.相同时间点不同浓度干预组血管数结果示：随着浓度增加,各干预组血管生成数明显增多,t-AUCB+gw9662组结果明显低于100μmol/L组。各组间均具有显著差异(P0.05)。 5.相同浓度t-AUCB干预组不同时间点血管数比较示：随着干预后时间延长,各干预组血管生成数明显增多,100μmol/L t-AUCB+gw9662组结果明显低于100μmol/L组。各组间均具有显著差异(P0.05)。结论sEH抑制剂t-AUCB参与正向调控EPCs修复梗死心肌,促使心肌缺血区血管重建；t-AUCB正向调控EPCs与激活EPCs上PPARγ有关。
[Abstract]:Isolation, culture and identification of bone marrow derived endothelial progenitor cells from the first part of mice
Objective to study the isolation and culture of endothelial progenitor cells from bone marrow of mice and to identify them.
Method
The 1. mouse bone marrow derived endothelial progenitor cells: mononuclear cells were isolated from femur and tibia of mice bone marrow by density gradient centrifugation, according to 5 * 105/cm2 density coated with fibronectin in the cell culture dish, containing 5%FBS and growth factor in cultured in EBM-2 medium supplemented. Morphological changes of endothelial progenitor cells grown in different time observed under the microscope.
2. cultured endothelial progenitor cell double fluorescence staining: fluorescence microscope observation cell uptake of Dil-AcLDL and FITC-UEA-1 characteristics, dyed double positive cells are EPCs.
3. identification of culture of mouse bone marrow derived endothelial progenitor cells: after seven days of culture, the expression of CD34, CD133, CD31, Flk-1 and other endothelial progenitor cells surface markers were detected by flow cytometry.
Result
1. mononuclear cells were isolated after 4 days of culture, microscope, colony formation, colony central circular cells around the spindle cells around; after 7 days cultured cell colony was significantly expanded, the central colony round cells to spindle cell transformation; cell culture for eighth days to form a visible streak; tenth up to the day of cultivation of endothelial progenitor cells 80%-90% fusion, with round and polygonal cells, also visible fusiform cells.
The number of double positive cells with Dil-AcLDL and FITC-UEA-1 staining under 2. microscopes was more than 95%.
3. flow cytometry results showed that the content of cell surface specific antigen was CD34 (53.89 + 0.34)%, CD133 (52.79 + 0.67)%, CD31 (36.67 + 0.93)%, Flk-1 (43.88 + 0.48)%, respectively.
Conclusion by morphology, (DiI-ac-LDL/FITC-UEA-1) fluorescence double staining and flow cytometry, the cultured cells are endothelial progenitor cells.
The effect and mechanism of the second part of soluble epoxide hydrolase inhibitor t-AUCB in vitro on EPCs function in mice
Objective to detect different concentrations of soluble epoxide hydrolase inhibitor t-AUCB (trans-4-[4- (3-adamantan-1-ylureido-cyclohexyloxy]-benzoic acid)) and PPAR (peroxisome proliferator activated receptory) gamma receptor blocker (GW9662) intervention on the proliferation of endothelial progenitor cells, adhesion, migration, homing, angiogenesis, secretion of VEGF (vascular endothelial growth factor) and HIF1- (hypoxia inducible factor1alph) of the alpha effect and mechanism.
Methods endothelial progenitor cells were inoculated for 7 days, then the cells were treated with different concentrations (0,1,10,50100, mol/Lt-AUCB, GW9662+100, mol/L and t-AUCB). The same number of 1 * 105 cells was taken as follows:
1., the cells were digested to 96 hole plates. After intervening for 24 hours at different concentrations of t-AUCB, MTT was added into the hole. The optical absorption value of each hole was measured by enzyme linked immunosorbent monitor, and the effect on proliferation was calculated.
2. cells were seeded on fibronectin coated 12 hole culture plate, incubated at 37 degrees 1H, removed the non adherent cells, counted the number of adherent cells under inverted microscope, and calculated the effect on adhesion.
3. cells were added to the upper layer of Transwell board, the lower layer was added with different concentration of t-AUCB migration buffer, and incubated 4H at 37 C, the number of cells migrated to the lower layer was measured, and the effect of t-AUCB on migration was calculated.
4. cells and 2 g/mL Dil-ac-LDL 1H co culture, by injecting the mice with myocardial infarction immediately after surgery, postoperative 24h, cardiac chop, digestive enzyme in sequence, and with fetal bovine serum, positive cells labeled Dil under fluorescent microscope counting number after centrifugation, calculating the effects of t-AUCB on endothelial progenitor cell homing.
5. cells were inoculated on the pre incubated Matrigel board, and the angiogenesis was observed under the inverted microscope after adding different concentrations of t-AUCB to incubate 24h.
6. cells were digested and extracted, and the ability to secrete VEGF and HIF1- alpha was detected by Western blot.
Result
1., from 0 mu mol/L-100 to mol/L, with increasing concentration, t-AUCB could enhance EPCs proliferation in a concentration dependent manner. Compared with the control group (0 mol/L), 1,10,50100 mol/Lt-AUCB significantly enhanced EPCs proliferation ability (P0.05), and PPAR gamma receptor blocker (GW9662) could inhibit its above functions (P0.05).
2., from 0 mu mol/L-100 to mol/L, t.AUCB could enhance EPCs adhesion in a concentration dependent manner. Compared with the control group (0 mol/L), 1,10,50100 mol/Lt-AUCB significantly enhanced EPCs adhesion ability (P0.05), and PPAR gamma receptor blocker (GW9662) could inhibit the above functions.
3., from 0 mu mol/L-100 to mol/L, with increasing concentration, t-AUCB could enhance EPCs migration in a concentration dependent manner. Compared with the control group (0 mol/L), 1,10,50100 mol/Lt-AUCB significantly enhanced EPCs migration ability (P0.05), and PPAR7 receptor blocker (GW9662) could inhibit the above functions.
4., from 0 mu mol/L-100 to mol/L, with increasing concentration, t-AUCB could enhance EPCs homing ability in a concentration dependent manner. Compared with the control group (0 mol/L), 1,10,50100 mol/Lt-AUCB significantly enhanced EPCs homing ability (P0.05), and PPAR gamma receptor blocker (GW9662) could inhibit its above functions (P0.05).
From 5. to 0 mu mol/L-100 mu mol/L, along with the increase of concentration, t-AUCB showed a concentration dependent enhancement of angiogenesis ability of EPCs, and the control group (0 mol/L) compared to 1,10,50100 mol/Lt-AUCB EPCs significantly enhanced the ability of angiogenesis (P0.05); PPAR gamma receptor blocker (GW9662) can inhibit the function (P0.05).
From 6. to 0 mu mol/L-100 mu mol/L, along with the increase of concentration, t-AUCB showed a concentration dependent enhancement of EPCs synthesis of VEGF and HIF1- alpha, and the control group (0 mol/L) compared to 1,10,50100 mol/Lt-AUCB significantly enhanced EPCs synthesis of VEGF and HIF1- alpha (P0.05); ability of PPAR gamma receptor blocker (GW9662) can inhibit the the function (P0.05).
Conclusion from 0 micron mol/L-100 mol/L, t-AUCB can enhance EPCs proliferation, adhesion, migration, homing, angiogenesis and secretion of VEGF and HIF- alpha in a concentration dependent manner, which may play a role through PPAR gamma receptor. T-AUCB can enhance EPCs proliferation, adhesion, migration, homing, angiogenesis and secretion of VEGF and HIF- alpha.
The effect and mechanism of the third part of t-AUCB treated EPCs injection on the ischemic myocardium of mice
Objective to study the effect and mechanism of EPCs injection of different concentrations of t-AUCB and PPARy receptor blockers (GW9662) on myocardial infarction in mice
Method
1. the myocardial infarction model was made by ligation of the left anterior descending branch of the mice, and the infarct electrocardiogram was recorded.
2. the mouse endothelial progenitor cells were injected with different concentrations (0,1,10,50100 mu mol/L, 100 mu mol/L t-AUCB+GW9662) through the tail vein for 1 hours after the infarction.
3. after 24 hours, 3 days, 7 days, 14 days, 28 days after myocardial infarction, the mice were killed, and the heart was removed. Faure Marin's fixation was used to measure infarcted myocardium and the percentage of heart area below the transverse section of ligature.
4. by immunohistochemical detection of VEGF and VFFL (factor VIII) determination of myocardial infarction vascular fringe generation number.
5. compared the infarcted myocardial area ratio and the amount of angiogenesis in the intervention group with the same practice.
6. a comparison was made between the cumulative ratio of the myocardial infarction and the amount of angiogenesis at different time points after the same concentration group.
Result
The model of myocardial infarction 1. production, ligation of instant ligature distal myocardial visible decreased activity, left ventricular anterior wall pale cyanosis, electrocardiogram examination showed anterior ST segment arched elevation, indicating that the model was established successfully.
At the same time in 2. different concentrations of transplantation group myocardial infarction area percentage showed myocardial infarction 24 hours after transplantation in different concentrations of the intervention group had no significant difference between the area ratio (P=0.481); 3 days after transplantation, 7 days, 14 days, 28 days, along with the increase of concentration, the ten pre group area ratio decreased, 100 mol/L in group t-AUCB+GW9662 the area ratio is greater than 100 /L group, there were significant differences between the intervention group and different concentrations of t-AUCB (P0.05).
3. the percentage of the infarct size at different times after the intervention of the same concentration of t-AUCB was shown as follows:
(1) 0 mu mol/L group showed that the area ratio of myocardial infarction increased gradually from 24 hours to 14d, and the area ratio decreased from 14d to 28d. There was a significant difference between each time point (P0.05).
(2) 1 mu mol/L group showed that the area from 24h to 3D decreased gradually before the comparison. 3 days to 14 days, the area increased gradually. From 14 days to 28 days, the area decreased gradually, and there was a significant difference between each time point (P0.05).
(3) 10,50100 mol/L group showed that the area ratio decreased from 24 hours to 7 days, and the area increased from 7 days to 14 days, but it was still less than 24 hour area ratio. From 14 days to 28 days, the area decreased gradually, and there was a significant difference between each time point (P0.05).
(4) 100 mol/L t-AUCB+gw9662 group showed: from 24 hours to 3 days, the area ratio increases gradually; 7 days to 3 days, area ratio decreased before; 7 to 14 days, area increased; from 14 days to 28 days, the infarct size was reduced. Compared with significant differences between different time points (P0.05).
4. at the same time point and different concentration, the number of vessels in intervention group showed that with the increase of concentration, the number of angiogenesis in each intervention group increased significantly, and the results in group t-AUCB+gw9662 were significantly lower than those in 100 mol/L group. There was a significant difference between all groups (P0.05).
5. at the same concentration of t-AUCB, the number of vessels at different time points in the intervention group showed that the number of angiogenesis increased significantly in each intervention group as compared with that in the intervention group. The results in 100 mol/L t-AUCB+gw9662 group were significantly lower than those in the 100 mol/L group. There was a significant difference between the groups (P0.05).
Conclusion sEH inhibitor t-AUCB participates in the positive regulation of EPCs to repair infarcted myocardium, and promotes myocardial ischemia area revascularization. T-AUCB positively regulates EPCs and EPCs PPAR activation.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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