本文選題：內(nèi)皮祖細(xì)胞　切入點(diǎn)：培養(yǎng)　出處：《中南大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分小鼠骨髓來源內(nèi)皮祖細(xì)胞的分離、培養(yǎng)及鑒定 目的研究小鼠骨髓來源內(nèi)皮祖細(xì)胞的分離培養(yǎng)方法并對其進(jìn)行鑒定。 方法 1.小鼠骨髓來源內(nèi)皮祖細(xì)胞培養(yǎng)：用密度梯度離心法從小鼠股骨及脛骨骨髓中分離單個(gè)核細(xì)胞,按5×105/cm2密度置于包被有人纖維連接蛋白的細(xì)胞培養(yǎng)皿中,加入含5%FBS和生長因子的EBM-2培養(yǎng)基培養(yǎng)。顯微鏡下觀察不同時(shí)間點(diǎn)種植的內(nèi)皮祖細(xì)胞形態(tài)變化。 2.培養(yǎng)的內(nèi)皮祖細(xì)胞雙熒光染色：熒光顯微鏡下觀察細(xì)胞攝取Dil-AcLDL及結(jié)合FITC-UEA-1特性,染色雙陽性細(xì)胞為EPCs。 3.小鼠骨髓來源內(nèi)皮祖細(xì)胞培養(yǎng)的鑒定：培養(yǎng)七天后用流式細(xì)胞儀檢測CD34、CD133、CD31、Flk-1等內(nèi)皮祖細(xì)胞表面標(biāo)志的表達(dá)。 結(jié)果 1.分離單個(gè)核細(xì)胞培養(yǎng)4天后,顯微鏡下可見集落形成,集落中央為圓形細(xì)胞,周圍為梭形細(xì)胞圍繞；7天后見培養(yǎng)的細(xì)胞集落明顯擴(kuò)大,集落中央圓形細(xì)胞向梭形細(xì)胞轉(zhuǎn)化；第8天可見培養(yǎng)細(xì)胞形成條索狀；第10天培養(yǎng)的內(nèi)皮祖細(xì)胞可達(dá)80%-90%融合,以類圓形和多角形細(xì)胞為主,也可見梭形細(xì)胞。 2.顯微鏡下計(jì)數(shù)Dil-AcLDL及結(jié)合FITC-UEA-1染色雙陽性細(xì)胞為95%以上。 3.流式細(xì)胞儀檢測結(jié)果示細(xì)胞表面特異性抗原含量分別為CD34(53.89±0.34)%;CD133(52.79±0.67)%;CD31(36.67±0.93)%; Flk-1(43.88±0.48)%。 結(jié)論通過形態(tài)學(xué)、(DiI-ac-LDL/FITC-UEA-1)熒光雙染色和流式細(xì)胞檢測證明培養(yǎng)的細(xì)胞為內(nèi)皮祖細(xì)胞。 第二部分可溶性環(huán)氧化物水解酶抑制劑t-AUCB體外對小鼠EPCs功能的影響及機(jī)制 目的檢測不同濃度可溶性環(huán)氧化物水解酶抑制劑t-AUCB (trans-4-[4-(3-adamantan-1-ylureido-cyclohexyloxy]-benzoic acid))及PPARγ(peroxisome proliferator activated receptory)受體阻滯劑(GW9662)干預(yù)后對內(nèi)皮祖細(xì)胞增殖、粘附、遷移、歸巢、血管生成、分泌VEGF (vascular endothelial growth factor)及HIF1-α(hypoxia inducible factor1alph)的影響及機(jī)制。 方法內(nèi)皮祖細(xì)胞接種7天后用不同濃度(0、1、10、50、100μmol/Lt-AUCB、GW9662+100μmol/L t-AUCB)干預(yù)細(xì)胞,后取相同數(shù)量1×105個(gè)細(xì)胞分別進(jìn)行如下實(shí)驗(yàn)： 1.將細(xì)胞消化至96孔板,按不同濃度t-AUCB干預(yù)24小時(shí)后孔內(nèi)加MTT,在酶聯(lián)免疫監(jiān)測儀上測定各孔光吸收值,計(jì)算其對增殖的影響。 2.細(xì)胞接種于纖連蛋白包被的12孔培養(yǎng)板,37℃孵育1h,除去未貼壁細(xì)胞,倒置顯微鏡下計(jì)數(shù)粘附細(xì)胞數(shù),計(jì)算其對粘附的影響。 3.細(xì)胞添加至transwell板的上層,下層添加含有不同濃度t-AUCB的遷移緩沖液,37℃孵育4h后,測定遷移至下層的細(xì)胞數(shù),計(jì)算t-AUCB對遷移的影響。 4.細(xì)胞與2μg/mL Dil-ac-LDL共同培養(yǎng)1h,心肌梗死手術(shù)后立即給小鼠尾靜脈注射,術(shù)后24h,心臟切小粒,循序在酶中消化,用胎牛血清中和,離心后在熒光顯微鏡下記數(shù)Dil標(biāo)記的陽性細(xì)胞數(shù),計(jì)算t-AUCB對內(nèi)皮祖細(xì)胞歸巢的影響。 5.細(xì)胞接種于預(yù)先孵育的Matrigel板上,加入不同濃度t-AUCB孵育24h后倒置顯微鏡下觀察血管生成情況。 6.細(xì)胞進(jìn)行消化、提取蛋白,經(jīng)western blot檢測其分泌VEGF及HIF1-α能力。 結(jié)果 1.從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs的增殖能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs增殖能力(P0.05)；PPARγ受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 2.從0μmol/L-100μmol/L,隨著濃度增加,t.AUCB可呈濃度依賴性增強(qiáng)EPCs的粘附能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs粘附能力(P0.05)；PPARγ受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 3.從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs的遷移能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs遷移能力(P0.05)；PPAR7受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 4.從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs的歸巢能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs歸巢能力(P0.05)；PPARγ受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 5.從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs的血管生成能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs血管生成能力(P0.05)；PPARγ受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 6.從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs合成VEGF及HIF1-α能力,與對照組(0μmol/L)相比,1,10,50,100μmol/Lt-AUCB顯著增強(qiáng)EPCs合成VEGF及HIF1-α能力(P0.05)；PPARγ受體阻滯劑(GW9662)可抑制其上述功能(P0.05)。 結(jié)論從0μmol/L-100μmol/L,隨著濃度增加,t-AUCB可呈濃度依賴性增強(qiáng)EPCs的EPCs增殖、粘附、遷移、歸巢、血管生成及分泌VEGF及HIF-α能力,該作用可能通過PPARγ受體發(fā)揮作用。 第三部分t-AUCB處理后的EPCs體內(nèi)注射對小鼠缺血心肌的影響及機(jī)制 目的研究不同濃度t-AUCB及PPARy受體阻滯劑(GW9662)處理后的EPCs體內(nèi)注射對小鼠心肌梗死的影響及機(jī)制 方法 1.通過結(jié)扎小鼠左前降支制作心肌梗死模型,并記錄梗死心電圖。 2.梗死后1小時(shí)分別通過尾靜脈注射200μ1不同濃度(0、1、10、50、100μmol/L、100μmol/L t-AUCB+GW9662)干預(yù)的小鼠內(nèi)皮祖細(xì)胞。 3.在心肌梗死后24小時(shí)、3天、7天、14天、28天處死小鼠,取出心臟,行福爾馬林固定,測定梗死心肌面積占結(jié)扎點(diǎn)橫切面以下心臟面積百分比。 4.通過免疫組化檢測VEGF及Ⅷ (factor Ⅷ)測定梗死心肌邊緣區(qū)血管生成數(shù)。 5.對相同實(shí)踐不同濃度干預(yù)組梗死心肌面積比及血管生成數(shù)量進(jìn)行比較。 6.對相同濃度組干預(yù)后不同時(shí)間點(diǎn)梗死心肌而積比及血管生成數(shù)量進(jìn)行比較。 結(jié)果 1.制作心肌梗死模型時(shí),結(jié)扎瞬間可見結(jié)扎線遠(yuǎn)端心肌活動(dòng)度減弱,左室前壁蒼白后紫紺,行心電圖檢查表現(xiàn)為前壁導(dǎo)聯(lián)ST段弓背向上抬高,表明模型制作成功。 2.相同時(shí)間點(diǎn)不同濃度移植組心梗面積百分比示：心梗移植后24小時(shí)不同濃度干預(yù)組間面積比無顯著差異(P=0.481)；移植后3天、7天、14天、28天,隨著濃度增加,各十預(yù)組面積比逐漸減小,100μmol/L t-AUCB+GW9662組面積比明顯大于100μ/L組,不同濃度t-AUCB各干預(yù)組間存在顯著差異(P0.05)。 3.相同濃度t-AUCB干預(yù)后不同時(shí)間點(diǎn)心梗面積百分比示： (1)0μmol/L組示：從24小時(shí)至14d,隨著時(shí)間延長,心梗面積比逐漸增大；從14d至28d,面積比逐漸縮小,各時(shí)間點(diǎn)間具有顯著差異(P0.05)。 (2)1μmol/L組示：從24h至3d,面積比較前逐漸縮��；3天至14天,面積比較前逐漸增大；14天至28天,面積比較前逐漸縮小,各時(shí)間點(diǎn)間具有顯著差異(P0.05)。 (3)10、50、100μmol/L組示：從24小時(shí)至7天,面積比逐漸縮小；7天至14天,面積比較前逐漸增大,但仍小于24小時(shí)面積比；從14天至28天,面積比較前逐漸縮小,各時(shí)間點(diǎn)間具有顯著差異(P0.05)。 (4)100μmol/L t-AUCB+gw9662組示：從24小時(shí)至3天,面積比逐漸增大；3天至7天,面積比較前逐漸減��；7天至14天,面積比較前增大；從14天至28天,心梗面積比較前明顯縮小。各時(shí)間點(diǎn)間具有顯著差異(P0.05)。 4.相同時(shí)間點(diǎn)不同濃度干預(yù)組血管數(shù)結(jié)果示：隨著濃度增加,各干預(yù)組血管生成數(shù)明顯增多,t-AUCB+gw9662組結(jié)果明顯低于100μmol/L組。各組間均具有顯著差異(P0.05)。 5.相同濃度t-AUCB干預(yù)組不同時(shí)間點(diǎn)血管數(shù)比較示：隨著干預(yù)后時(shí)間延長,各干預(yù)組血管生成數(shù)明顯增多,100μmol/L t-AUCB+gw9662組結(jié)果明顯低于100μmol/L組。各組間均具有顯著差異(P0.05)。 結(jié)論sEH抑制劑t-AUCB參與正向調(diào)控EPCs修復(fù)梗死心肌,促使心肌缺血區(qū)血管重建；t-AUCB正向調(diào)控EPCs與激活EPCs上PPARγ有關(guān)。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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