缺氧条件下脑微血管内皮细胞与间充质干细胞的相互影响

发布时间：2018-04-19 13:18

本文选题：间充质干细胞 + 脑微血管内皮细胞　；参考：《第三军医大学》2008年硕士论文

【摘要】： 研究背景: 骨髓间充质干细胞(mesenchymal stem cells, MSC)是来源于骨髓的成体干细胞,具有多潜能分化性,已证明MSC修复和分化为多种组织的能力,尤其在修复和重建血管方面成为缺血性疾病治疗研究的热点。脑微血管内皮细胞(brain microvascular endothelial cells, BMEC)是构成血脑屏障的最主要组成部分,具有细胞间紧密连接、极少的胞饮囊泡和维持神经组织离子和代谢稳定的特殊跨膜转运系统等独有生理特点。在缺血性脑血管疾病中,BMEC的损伤是导致血脑屏障开放、脑水肿发生,从而加重神经元细胞损伤的重要因素,同时缺血半暗区脑微血管的修复与新生也是挽救缺血受损神经元的关键。当前,在干细胞治疗缺血性脑血管病的研究方面,多数研究聚焦于干细胞修复受损的神经组织,然而在干细胞对脑缺血部位受损的血脑屏障和微血管内皮细胞影响的研究并未深入。本实验以缺氧条件下培养BMEC模拟缺血性脑血管疾病中血脑屏障和脑微血管所处的病理生理环境,观察BMEC对共培养的MSC分化的影响,以及MSC以旁分泌方式对BMEC增殖、迁移和血脑屏障模型通透性的影响,为MSC应用于缺血性脑血管疾病的治疗提供基础实验依据。研究目的: 1.分离、培养及鉴定大鼠BMEC和人骨髓MSC,建立缺氧条件下BMEC与MSC直接与间接共培养的模型,观察缺氧条件下BMEC对直接与间接共培养的MSC分化的影响。 2.测定MSC与BMEC条件培养液中血管内皮生长因子(vascular endothelial growth factor,VEGF)和基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)含量,观察缺氧条件下MSC以旁分泌方式对BMEC增殖、迁移及其单层通透性的影响。研究方法: 1.分离、培养及鉴定大鼠BMEC和人骨髓MSC:采用两次酶消化法(0.1%Ⅱ型胶原酶,0.1%胶原酶/分散酶)和密度梯度离心法得到纯化的脑微血管片段,加入含10ng/ml bFGF、20%胎牛血清和100μg/ml肝素钠的DMEM高糖完全培养液,接种于涂布Ⅳ型胶原和纤连蛋白的塑料培养瓶,免疫荧光细胞化学法鉴定BMEC的vWF表达;用密度梯度离心法分离人骨髓MSC,采用流式细胞术鉴定MSC的CD29、CD34、CD44、CD105和Flk-1表达。建立缺氧条件下BMEC与MSC直接与间接共培养的模型:缺氧实验在37℃、93%N2、5%CO_2、2%O_2的缺氧培养箱内进行;间接共培养使用孔径0.4μm的Millicell Culture Plate Inserts,BMEC以10%胎牛血清的DMEM低糖(DMEM-10)接种于上层(5×10~3/well),将MSC以DMEM-10接种于下层24孔板内(5×10~3/well);直接共培养将两种细胞以5000:5000cells/ml的比率混合于DMEM-10后接种于培养瓶或盖玻片上。观察缺氧条件下BMEC对共培养的MSC分化的影响:分别将直接和间接共培养的细胞在正常和缺氧条件下培养5天,采用流式细胞术(定量检测Flk-1)和免疫荧光细胞化学法(定性检测Flk-1和vWF)对MSC的分化程度进行分析。2.①测定MSC与BMEC条件培养液中VEGF和MMP-9含量:收集正常和缺氧条件下两种细胞的条件培养基得到正常的脑微血管内皮细胞条件培养基(BMEC~(CM N))、间充质干细胞条件培养基(MSC~(CM N)),以及缺氧条件下脑微血管内皮细胞条件培养基(BMEC~(CM H))、间充质干细胞条件培养基(MSC~(CM H)),用ELISA检测各条件培养基中VEGF和MMP-9的含量;②观察各条件培养基对BMEC增殖和迁移的影响:使用六种不同培养基(DMEM-10、MSC~(CM N)、MSC~(CM H)、煮沸的MSC~(CM H)培养基、添加抗VEGF抗体和MMP -9抑制剂I的MSC~(CM H)对BMEC进行培养,采用Cell Counting Kit-8方法检测各条件培养基对BMEC增殖的影响,采用Transwell培养体系检测各条件培养基对BMEC迁移的影响;③观察各条件培养基对BMEC单层通透性的影响:使用MilliCell-ERS Voltohmmeter测量不同条件培养基(DMEM-10、MSC~(CM N)、MSC~(CM H)、含VEGF抗体的MSC~(CM H)和含MMP-9抑制剂I的MSC~(CM H))对BMEC单层通透性的影响。结果: 1.流式细胞仪检测结果表明MSC呈CD29、CD44、CD105阳性表达,CD34和Flk-1阴性表达;免疫荧光细胞化学法检测BMEC呈vWF阳性表达。常氧和缺氧条件下的间接和直接共培养的细胞均能良好生长。常氧条件下间接共培养MSC的Flk-1和vWF蛋白均为阴性表达。缺氧条件下间接共培养的(7.58±0.58)% (n=6,P=0.034) MSC开始表达Flk-1蛋白,激光共聚焦显微镜也显示少量细胞开始出现红色荧光,但未见绿色荧光的vWF蛋白表达。正常和缺氧条件下直接共培养5 d时,开始表达Flk-1蛋白的MSC分别占共培养混合细胞数的(13.76±1.67)% (n=6,P0.001)和(23.64±2.50)% (n=6,P0.001),两者相比有显著性差异(n=6,P0.001);激光共聚焦也显示部分MSC开始表达Flk-1,而在常氧共培养细胞中未发现Flk-1阳性的细胞同时表达vWF,值得关注的是,缺氧直接共培养的混合细胞中,部分Flk-1阳性细胞开始同时表达vWF的绿色荧光。 2.①ELISA检测条件培养基中VEGF、MMP-9的含量:缺氧导致BMEC和MSC条件培养基中VEGF含量均明显增高;MSC~(CM H)中VEGF含量明显高于BMEC~(CM H);BMEC~(~(CM N))中未检测到MMP-9,MSC~(CM H)中MMP-9含量明显高于MSC~(CM N)和BMECCM H。②条件培养基对BMEC增殖和迁移的影响:与DMEM-10相比,MSC~(CM N)明显增强了BMEC的增殖,而MSC~(CM H)对BMEC的增殖作用又明显强于MSCCM N(0.947±0.103与0.532±0.028,P0.001,n=6);MSC~(CM H)促增殖作用因煮沸而完全丧失,同时VEGF的阻断抗体可明显抑制MSC~(CM H)对BMEC的增殖作用(0.947±0.103与0.419±0.034,P0.001,n=6),而MMP-9抑制剂对BMEC的增殖影响不明显(0.947±0.103与0. 902±0.065,P=0.963,n=6)。与DMEM-10对照相比,MSC~(CM N)明显增多了BMEC迁移细胞的数量,而MSC~(CM H)对BMEC的迁移作用又明显强于MSC~(CM N)(238±27与154±24,P0.01,n=6);VEGF的阻断抗体可明显抑制MSC~(CM H)对BMEC的迁移作用(238±27与150±20,P0.001,n=6),而MMP-9抑制剂对BMEC的迁移的抑制则更为明显(238±27与106±18,P0.001,n=6);与对增殖的影响类似,MSC~(CM H)促迁移作用经煮沸处理而完全丧失。③检测24h内不同条件培养基对内皮单层通透性的影响:常氧条件下DMEM-10培养的BMEC电阻值在24 h内保持在较稳定范围内,缺氧状态DMEM-10培养BMEC的电阻值在缺氧6~18 h内出现明显下降,约在18 h达到最低为处理前的(77.2±1.8)%;缺氧状态MSC~(CM H)培养的BMEC的电阻值在2 h内出现急剧增大,约在2 h达到最低,为处理前的(50.5±2.6)%,在随后的2～24 h电阻值略有回升但仍处于较低水平;抗VEGF抗体和MMP-9抑制剂I使MSC~(CM H)培养的BMEC电阻值下降明显趋缓,最低电阻值分别为处理前的(60.3±3.6)%和(76.0±2.4)%。结论: 1.常氧条件下BMEC仅通过旁分泌细胞因子不足以诱导MSC分化,BMEC能够通过细胞直接接触诱导共培养的MSC向内皮分化,缺氧在诱导MSC向内皮分化的过程中发挥重要作用,缺氧条件下,直接共培养的BMEC能诱导更多的MSC更彻底地向内皮分化。 2.①MSC条件培养基中MMP-9和VEGF的含量均明显高于BMEC条件培养基,缺氧可介导BMEC和MSC条件培养基中MMP-9和VEGF的含量明显升高。②MSC可通过旁分泌方式促进内皮细胞的增殖和迁移,MMP-9在MSC以旁分泌方式促进BMEC迁移过程中发挥了重要作用,而VEGF则同时在促进BMEC增殖和迁移过程中发挥重要作用。③MSC可通过旁分泌方式导致BMEC单层通透性的急剧增大,缺氧状态下MSC所分泌的大量MMP-9和VEGF是其导致BMEC单层通透性的急剧增大的原因,这是MSC应用于缺血性脑血管疾病的治疗中值得慎重考虑的问题。
[Abstract]:Research background:
Bone marrow mesenchymal stem cells (mesenchymal stem cells (MSC)) are adult stem cells derived from bone marrow and have multiple potential differentiation. It has proved the ability of MSC to repair and differentiate into a variety of tissues, especially in the repair and reconstruction of blood vessels as a hot point for the treatment of ischemic disease. Brain microvascular endothelial cells (brain microvascular endothel). Ial cells, BMEC) is the most important component of the blood brain barrier, which has the unique physiological characteristics such as close intercellular connection, very few vesicles, and special transmembrane transport system to maintain the ion and metabolic stability of the nerve tissue. In ischemic cerebrovascular disease, the damage of BMEC is caused by the opening of the blood brain barrier and the occurrence of brain edema. The key factors for cell injury in heavy neurons, and the repair and regeneration of the cerebral microvessels in the ischemic penumbra are also the key to save the ischemic neurons. Most studies focus on the repair of damaged nerve tissue in stem cells in the stem cell treatment of ischemic cerebrovascular disease. However, it is damaged by stem cells in the cerebral ischemic areas. The effects of blood brain barrier and microvascular endothelial cells were not deeply studied. In this experiment, the pathological and physiological environment of blood brain barrier and cerebral microvascular in ischemic cerebrovascular disease was simulated under hypoxia conditions, and the effect of BMEC on the differentiation of co cultured MSC, and the proliferation, migration and blood brain barrier model of BMEC by MSC by parathellate secreting methods were observed. The effect of type permeability provides a basic experimental basis for the application of MSC in the treatment of ischemic cerebrovascular diseases.
The purpose of the study:
1. isolation, culture and identification of rat BMEC and human bone marrow MSC, and establish a direct and indirect co culture model of BMEC and MSC under the condition of hypoxia, and observe the effect of BMEC on the direct and indirect co culture of MSC under the condition of hypoxia.
2. the contents of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and matrix metalloproteinase -9 (matrix metalloproteinases-9, MMP-9) in MSC and BMEC conditioned medium were measured, and the effects of paracrine on proliferation, migration and monolayer permeability under hypoxia were observed.
Research methods:
1. isolation, culture and identification of rat BMEC and human bone marrow MSC: using two enzyme digestion (0.1% type II collagenase, 0.1% collagenase / dispersing enzyme) and density gradient centrifugation to obtain the purified cerebral microvascular fragments, adding DMEM high sugar complete culture solution containing 10ng/ml bFGF, 20% fetal bovine serum and 100 g/ml heparin sodium, inoculated with type IV collagen and fiber. The vWF expression of BMEC was identified by immunofluorescence cytochemical method, and human bone marrow MSC was separated by density gradient centrifugation. The expression of CD29, CD34, CD44, CD105 and Flk-1 in MSC was identified by flow cytometry. The model of BMEC and MSC directly and indirectly cultured under the condition of hypoxia was established: the hypoxia experiment was at 37, and 93%N2,5%CO_2,2%O_2 was missing. In the oxygen incubator, Millicell Culture Plate Inserts with 0.4 m pore diameter was indirectly co cultured, BMEC was inoculated on the upper layer (5 x 10~3/well) with the DMEM low sugar (DMEM-10) of the 10% fetal bovine serum, and MSC was inoculated in the lower layer (5 * 10~3/well) in the lower layer (5 x 10~3/well), and the two kinds of cells were mixed at the ratio after mixing directly. The effects of BMEC on the differentiation of co cultured MSC were observed under the condition of hypoxia: direct and indirect co cultured cells were cultured under normal and anoxic conditions for 5 days respectively. Flow cytometry (quantitative detection of Flk-1) and immunofluorescent cytochemistry (qualitative detection of Flk-1 and vWF) were used to differentiate the degree of differentiation of MSC. Analysis of the content of VEGF and MMP-9 in the conditioned medium of MSC and BMEC: to collect the conditioned medium of normal cerebral microvascular endothelial cells (BMEC~ (CM N)), the conditioned medium of mesenchymal stem cells (MSC~ (CM N)) and the conditioned medium of the cerebral microvascular endothelial cells (BM) under hypoxia conditions (BM), and the conditioned medium of the cerebral microvascular endothelial cells (BM) under hypoxia conditions (BM), and the conditioned medium (BM) (BM) under the condition of hypoxia (BM), and the conditioned medium (BM) (BM) in the condition of hypoxia conditions (BM), and the conditioned medium of the cerebral microvascular endothelial cells (BM) under hypoxia conditions (BM), and the conditioned medium of the cerebral microvascular endothelial cells (BM) under hypoxia conditions (BM), and the condition medium of the cerebral microvascular endothelial cells (BM) under hypoxia conditions (BM), and the condition medium of the cerebral microvascular endothelial cells under the condition of hypoxia (BM) (BM),.2. EC~ (CM H)), the conditioned medium of mesenchymal stem cells (MSC~ (CM H)), the content of VEGF and MMP-9 in the conditioned medium was detected by ELISA. (2) the effects of the culture medium on the proliferation and migration of BMEC were observed. Six different medium (DMEM-10, MSC~), boiling water culture medium were used. The MSC~ (CM H) was used to culture the BMEC, and the Cell Counting Kit-8 method was used to detect the effect of the culture medium on the proliferation of BMEC, and the effect of the culture medium on the BMEC migration was detected by the Transwell culture system. Thirdly, the influence of the medium on the permeability of BMEC monolayer was observed. The effects of DMEM-10 (MSC~, CM N), MSC~ (CM H), MSC~ (CM H) containing VEGF antibody and the inhibitor of the inhibitor containing the inhibitor of VEGF on the permeability of monolayer were studied.
Result:
The results of 1. flow cytometry showed that MSC was CD29, CD44, CD105 positive, CD34 and Flk-1 negative expression, and immunofluorescent cytochemical method was used to detect the positive expression of vWF in BMEC. The indirect and direct co cultured cells under the condition of normoxic and hypoxia could grow well. The Flk-1 and vWF protein of CO cultured MSC under normal oxygen condition were all negative tables. The indirect co culture (7.58 + 0.58)% (n=6, P=0.034) MSC began to express Flk-1 protein in the hypoxic condition. The laser confocal microscope also showed that a small number of cells began to appear red fluorescence, but no green fluorescent vWF protein was expressed. When the normal and hypoxia conditions were directly co cultured 5 D, the MSC that began to express the Flk-1 protein accounted for the co culture mixture respectively. The cell number (13.76 + 1.67)% (n=6, P0.001) and (23.64 + 2.50)% (n=6, P0.001) were significantly different (n=6, P0.001). Laser confocal microscopy also showed that some MSC began to express Flk-1, while no Flk-1 positive cells were found to express vWF in normal oxygen co culture cells. It is worth paying attention to the direct co culture of mixed cells in hypoxia. Some Flk-1 positive cells began to express vWF green fluorescence simultaneously.
2. ELISA detected the content of VEGF, MMP-9 in the conditioned medium: the content of VEGF in the medium of BMEC and MSC was significantly higher than that in the condition medium of BMEC and MSC; the VEGF content in MSC~ (CM H) was obviously higher than BMEC~ (CM). Effect of migration: compared with DMEM-10, MSC~ (CM N) significantly enhanced the proliferation of BMEC, while MSC~ (CM H) significantly increased the proliferation of BMEC than MSCCM N (0.947 + 0.103 and 0.532 + 0.028, P0.001,). 103 and 0.419 + 0.034, P0.001, n=6), and the effect of MMP-9 inhibitors on the proliferation of BMEC was not obvious (0.947 + 0.103 and 0.902 + 0.065, P=0.963, n=6). Compared with DMEM-10, MSC~ (CM N) significantly increased the number of BMEC migration cells, and the migration of MSC~ (238 + 27 and 154 + 0.947); The antibody could obviously inhibit the migration of MSC~ (CM H) to BMEC (238 + 27 and 150 + 20, P0.001, n=6), while the inhibition of MMP-9 inhibitor to BMEC migration was more obvious (238 + 27 and 106 + 18, P0.001, n=6), and similar to the effect on proliferation. MSC~ (CM) promoted the loss of migration through boiling. The effect of the permeability of the skin monolayer: the BMEC resistance value of DMEM-10 culture under the condition of atmospheric oxygen is kept in a relatively stable range within 24 h. The resistance value of the BMEC in the hypoxia state DMEM-10 culture decreases obviously in the anoxic 6~18 h, and the lowest is before the treatment (77.2 + 1.8)%, and the resistance value of the BMEC is 2 within the oxygen deficiency state MSC~ (CM H). There is a sharp increase, at the minimum of about 2 h, for (50.5 + 2.6)% before processing, and a slight increase in the subsequent 2~24 h resistance, but still at a lower level; the resistance to VEGF and MMP-9 inhibitors I makes the BMEC resistance value of MSC~ (CM H) decreased obviously, and the minimum resistance value is divided into (60.3 + 3.6)% and (76 + 2.4)% before treatment.
Conclusion:
1. BMEC can not induce MSC differentiation only through paracrine cytokine, BMEC can induce co cultured MSC to differentiate into endothelium through direct cell contact, and hypoxia plays an important role in inducing MSC to endothelial differentiation, and the direct co cultured BMEC can induce more MSC to differentiate into endothelium more thoroughly under hypoxia conditions.
2. (1) the content of MMP-9 and VEGF in the conditioned medium of MSC was significantly higher than that in the BMEC conditioned medium, and the content of MMP-9 and VEGF in BMEC and MSC conditioned medium was significantly increased by hypoxia. (2) MSC can promote the proliferation and migration of endothelial cells by paracrine mode. MMP-9 plays an important role in promoting BMEC migration in MSC by paracrine mode. In addition, VEGF plays an important role in promoting the proliferation and migration of BMEC. (3) MSC can lead to a sharp increase in the permeability of BMEC monolayer through paracrine mode. A large number of MMP-9 and VEGF secreted by MSC in anoxic state are the cause of the rapid increase in the permeability of BMEC monolayer, which is the treatment of MSC in ischemic cerebrovascular disease. A matter of prudence in the treatment.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R743;R361.2

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