低氧预处理人脂肪间充质干细胞联合脐血单核细胞治疗大鼠急性心肌梗死

发布时间：2018-07-03 11:21

本文选题：低氧预处理 + 脂肪间充质干细胞　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：[背景]心肌梗死是严重威胁人类健康的疾病。我国每年约有2.9亿人罹患心血管疾病,并有350万人因此而丧生。心血管疾病死亡率位居我国城乡居民总死亡原因的首位,且呈逐年攀升趋势。目前心肌梗死的主要治疗手段还是药物和手术,虽然它们能够促进血管再通,减轻心肌的持续性损伤,但二者对梗死的心肌组织并无再生作用,且不能有效逆转心肌梗死后的纤维化。干细胞移植是一种新兴的治疗方法,其目的是促进缺血区心肌和血管生成,减少心肌细胞凋亡,减轻心室重塑。脂肪间充质干细胞(Adipose-derived mesenchymal stem cells,ASC)以其独特的优势,逐渐成为当前研究的热点。虽然ASC的安全性和有效性已得到初步认识,但其在体内的存活和显效还受到多个环节的影响。为了提高干细胞在局部的存活效率,研究者们开始对干细胞进行体外预处理或者对组织微环境进行改善,以利于细胞的长期存活和发挥修复功能。低氧预处理(Hypoxia precondition,HP)是增加干细胞存活率的简单有效的方法。为了适应移植后在梗死区局部的低氧环境,在体外对ASC进行低氧培养,以此增强细胞对低氧的耐受性和抗凋亡能力,同时促进其分泌多种生长因子,有利于细胞进入体内后发挥修复和再生功能。脐血单核细胞(Cord blood mononuclear cells,CBMNCs)能够促进血管新生和血管内皮化,改善梗死局部的微循环。内皮细胞的再血管化可以为移植的干细胞带来营养和氧气,有利于干细胞在局部的存活。因此,将体外预处理与体内微环境的改善结合起来,可能更有利于细胞在局部的长期存活。[目的]1.研究ASC的培养,鉴定和体外诱导分化能力；以及CBMNCs分离和冻存备用；2.检测低氧预处理前后ASC的基因表达变化；并体外模拟心梗环境培养HP-ASC和ASC,比较二者基因表达水平的变化；3.评价HP-ASC治疗大鼠心肌梗死是否优于ASC；4.评价HP-ASC联合CBMNCs治疗大鼠心肌梗死是否优于单一细胞移植效果。[方法]1.以健康成人脂肪抽吸术后的脂肪组织为实验对象,用胶原酶消化分解后,离心取下层SVF细胞,接种培养。用MTT法测量细胞密度并绘制ASC生长曲线；流式细胞术检测ASC细胞表面标记；分别以成骨、成脂、成软骨诱导液诱导ASC分化,利用组织化学染色和免疫蛆化染色检测其多向分化潜能。来自健康婴儿的脐血离心沉降法分离出CBMNCs,检测细胞数量及活性细胞比例,冻存备用。2.ASC在低氧(2%O2)条件下培养24h,提取细胞RNA,比较HP-ASC与未处理ASC基因表达情况；模拟体内心梗局部微环境,取大鼠心梗后3d的心脏组织,研磨成浆,加入血清及必须氨基酸等配制成心梗混合液。将HP-ASC和ASC在此混合液中培养5d,检测细胞在微环境诱导下基因表达的变化；3.两月龄Wistar雌性大鼠30只,按照随机数法将实验动物随机分为3组,HP-ASC组,ASC组和PBS组,每组各10只大鼠。结扎冠状动脉左前降支建立心肌梗死损伤模型,动物心梗后即刻心肌内注射干细胞(总量：2×106)或PBS。细胞移植后7d和15d每组分别处死5只大鼠,收集心脏组织,制作冰冻组织切片和石蜡组织切片。比较各组之间心梗部位的大体形态学变化,心梗区占左心室比例,细胞移植效率,细胞凋亡数量和新生血管密度。4. 两月龄Wistar雌性大鼠60只,按照随机数法将实验动物随机分为4组,HP-ASC组,HP-ASC+CBMNCs组,CBMNCs组和PBS组,每组各15只大鼠。模型成功后即刻进行细胞移植,细胞总量为2×106(联合组细胞比例1：1)。每组动物分别在手术后7d,15d和30d各取5只处死。比较各组之间心梗部位的大体形态学变化,心梗区占左心室比例和室壁厚度,细胞移植存活率和新生血管密度。[结果]1.原代ASC接种6h后,部分细胞开始贴壁生长,24-48h后细胞开始出现集落样增长(CFU)。第3代以后,细胞呈典型纤维母细胞样梭形外观,开始呈漩涡状生长,MTT法检测显示,ASC的对数生长期为3-7d,第8d开始进入平台期。流式细胞术检测结果表明,ASC细胞表达CD34(-)/CD45(-)/CD90(+)/CD105(+) /CD73(+)/CD44(+),符合间充质干细胞表型。诱导分化试验表明,ASC可以向成脂、成骨和成软骨方向分化。CBMNCs分离后,单核细胞数量达到8.9×109,活性细胞计数结果显示,活性细胞数占所有有核细胞的93.16%。2. 与ASC相比,HP-ASC表达多种生长因子及其受体水平增高,与干细胞特性相关的基因Klf4和Oct4略有增高,而Nanog和Sox2表达却较后者有所降低。此外,HP-ASC中与低氧诱导表达相关的基因--血红素加氧酶-1(Heme oxygenase-1,HO-1)和基质细胞衍生因子-1(Stromal cell derived factor-1,SDF-1)均表达增高,分别为ASC组的1.38倍和1.98倍。与细胞迁移和植入相关的基因,除金属蛋白酶组织抑制剂(Tissue inhibitor of metalloproteinase,TIMP-1)下降外,趋化因子受体-4(CXCR4),细胞间粘附分子(Intercellular cell adhesion molecule, ICAM-1和ICAM-2)的表达都有所增加。体外模拟心梗环境培养可以促进HP-ASC-T表达心肌特异性标志物(CX43, FABP4, GATA-4)和促血管生长因子(VEGF),同时也可促进HP-ASC-T表达多种生长因子及其受体。3. 动物试验表明,与PBS组相比,HP-ASC组和ASC组在心梗后7d及15d均可显著减少心梗面积,增加细胞存活数量,减少凋亡细胞数量,以及显著增加新生血管数量。而HP-ASC组较ASC组改善更为明显。4.心梗大鼠进行细胞移植后30天,HP-ASC组,HP-ASC+CBMNCs组和CBMNCs组均比PBS组明显改善了心功能。其中,HP-ASC组改善效果更好。组织学检测表明,HP-ASC组的心梗比例和心梗室壁厚度,细胞存活数量和新生血管数量均比其他对照组改善更好。[结论]1.本实验所培养ASC细胞表达CD34 (-)/CD45 (-)/CD90 (+)/CD105 (+)/CD73 (+)/CD44 (+),符合间充质干细胞表型。ASC有多向分化潜能,经体外诱导可向成骨、成脂、成软骨方向分化。离心沉降法可获得临床级数量的CBMNCs,分离后细胞存活率高。2.低氧预处理后,HP-ASC表达多种生长因子及其受体升高。模拟体内心梗局部微环境可使HP-ASC表达心肌特异性标志物、促血管生成因子和生长因子水平升高。3. 与ASC相比,HP-ASC可以更有效地减少大鼠的心梗面积,增加细胞存活比例,减少心梗区细胞凋亡数量,增加新生血管数量。4. HP-ASC对心梗大鼠心功能的改善效果比HP-ASC+CBMNCs组和CBMNCs组更好,其心梗比例和心梗室壁厚度,细胞存活数量和新生血管数量均比其他对照组改善更好。
[Abstract]:[background] myocardial infarction is a serious threat to human health. About 290 million people are suffering from cardiovascular disease every year, and 3 million 500 thousand people have died. The mortality of cardiovascular disease is the first leading cause of death in urban and rural areas in China, and is increasing year by year. At present, the main treatment means of myocardial infarction are drugs and surgery. However, they can promote vascular recanalization and reduce persistent myocardial damage, but the two have no regenerative effect on the infarcted myocardium and can not effectively reverse the fibrosis after myocardial infarction. Stem cell transplantation is a new method to promote the formation of myocardial and vascularization in the ischemic region, reduce the apoptosis of myocardial cells and reduce the ventricles. Remolding. Adipose-derived mesenchymal stem cells (ASC) has become a hot spot of current research because of its unique advantages. Although the safety and effectiveness of ASC have been preliminarily recognized, the survival and effectiveness of ASC in the body are affected by many links. Hypoxia precondition (HP) is a simple and effective way to increase the survival rate of stem cells. In order to adapt to the hypoxic environment in the infarct area, ASC in vitro can be used in vitro. Cord blood mononuclear cells (CBMNCs) can promote the angiogenesis and endothelialization of blood vessels and improve the local micro level of infarct. The revascularization of endothelial cells can bring nutrients and oxygen to the transplanted stem cells and help the stem cells to survive locally. Therefore, the combination of in vitro pretreatment and the improvement of the microenvironment in the body may be more beneficial to the long-term survival of the cells. [Objective]1. to study ASC culture, identification and differentiation ability in vitro; And CBMNCs separation and cryopreservation; 2. detection of ASC gene expression changes before and after hypoxia preconditioning; and in vitro simulated myocardial infarction environment to cultivate HP-ASC and ASC, compare the changes in the level of gene expression in the two groups; 3. evaluate whether HP-ASC is superior to ASC in the treatment of myocardial infarction in rats; 4. evaluation of HP-ASC combined CBMNCs in the treatment of myocardial infarction in rats is better than that. [method]1. with the adipose tissue after liposuction of healthy adult as the experimental object, after digestion with collagenase, the lower SVF cells were centrifuged and cultured. The cell density was measured by MTT method and the growth curve of ASC was plotted, and the flow cytometry was used to detect the surface markers of ASC cells; the osteogenesis, fat formation, and cartilage induction were respectively induced by flow cytometry. ASC differentiation was induced by the guide solution, and the multidirectional differentiation potential was detected by histochemical staining and immungo-magma staining. CBMNCs was isolated from the umbilical blood centrifugation method from healthy infants. The number of cells and the proportion of active cells were detected. The frozen reserve.2.ASC was incubated with 24h under the condition of 2%O2, and the RNA was extracted and compared with the HP-ASC and the untreated ASC gene table. To simulate the local microenvironment of myocardial infarction in the body, the heart tissue of 3D after myocardial infarction was taken after the rat myocardial infarction, pulping, adding serum and essential amino acids into the mixture of myocardial infarction. HP-ASC and ASC were cultured in the mixture of 5D to detect the change of gene expression under the induction of microenvironment; 3. 2 month old Wistar female rats, according to random The experimental animals were randomly divided into 3 groups, group HP-ASC, group ASC and group PBS, each group of 10 rats. The left anterior descending branch of the coronary artery was ligated to establish the myocardial infarction damage model. After the animal myocardial infarction was injected into the myocardium immediately after the injection of stem cells (total: 2 * 106) or PBS. cells, 5 rats were killed in each group of 7D and 15d respectively, and the heart tissue was collected and frozen to make freezing. Tissue section and paraffin tissue section were used to compare the gross morphological changes of the myocardial infarction parts, the proportion of the left ventricle in the myocardial infarction area, the cell transplantation efficiency, the number of cell apoptosis and the.4. 2 month old Wistar female rats. The experimental animals were divided into 4 groups according to the random number method, group HP-ASC, HP-ASC+CBMNCs group and CBMNCs group. And group PBS, 15 rats in each group. After the model was successfully transplanted, the total cell total was 2 x 106 (1:1). Each group was killed in 7d, 15d and 30d after operation respectively. The gross morphological changes of the myocardial infarction areas were compared, the ratio of the left ventricle and the wall thickness, and the survival rate of cell transplantation were compared. After]1. primary ASC was inoculated with 6h, some cells began to grow on the wall after 6h. After 24-48h, the cells began to appear colony like growth (CFU). After the third generation, the cells showed a typical fibroblast like appearance, and began to be whirlpool. The MTT method showed that the logarithmic growth period of ASC was 3-7d, and the 8D began to enter the platform stage. Flow formula began to flow. The results of cytometry showed that ASC cells expressed CD34 (-) /CD45 (+) /CD90 (+) /CD105 (+) /CD73 (+) /CD44 (+), which conformed to the phenotype of mesenchymal stem cells. Induced differentiation test showed that ASC could be turned into fat, bone formation and chondrogenic differentiation, and the number of mononuclear cells reached 8.9 * 109, and the number of active cells showed that the number of active cells accounted for the number of active cells. Compared with ASC, HP-ASC expressed a variety of growth factors and their receptor levels in HP-ASC, and a slight increase in Klf4 and Oct4 genes related to the characteristics of stem cells, but the expression of Nanog and Sox2 was lower than that of the latter. In addition, the genes associated with hypoxia induced expression in HP-ASC, -1 (Heme oxygenase-1, HO-1), were associated with hypoxia. The expression of -1 (Stromal cell derived factor-1, SDF-1) was increased by 1.38 and 1.98 times, respectively, in the ASC group. The expression of tercellular cell adhesion molecule, ICAM-1 and ICAM-2 increased. In vitro simulated myocardial infarction environment culture can promote the expression of HP-ASC-T specific markers (CX43, FABP4, GATA-4) and vascular growth factor (VEGF), and also promote the expression of a variety of growth factors and their receptors in HP-ASC-T. Compared with group S, 7d and 15d in group HP-ASC and ASC can significantly reduce the area of myocardial infarction, increase the number of cell survival, decrease the number of apoptotic cells, and significantly increase the number of new blood vessels. The improvement of HP-ASC group is more obvious than that of the ASC group, and 30 days after the transplantation of.4. myocardial infarction rats, HP-ASC group, HP-ASC+CBMNCs group and CBMNCs group are all more than PBS group. Significantly improved cardiac function. Among them, the HP-ASC group improved the effect better. Histological examination showed that the ratio of myocardial infarction in group HP-ASC and the thickness of myocardial infarction room wall, the number of cell survival and the number of neovascularization were better than those of the other control groups. [conclusion the ASC cells cultured in this experiment are CD34 (-) /CD45 (+) /CD105 (+) /CD73 (+) /CD44 (+) (+), +) of CD34 (-) /CD45 (+) /CD105 (+). The phenotype of mesenchymal stem cells (.ASC) has multipotential differentiation potential, which can be induced to osteogenesis, fat formation and chondrogenic differentiation in vitro. Centrifuge sedimentation can obtain a clinical level of CBMNCs. After separation, the cell survival rate is high.2. hypoxia preconditioning, HP-ASC expresses a variety of growth factors and their receptors increase. The local microenvironment of myocardial infarction can be simulated in vivo. HP-ASC expressed the specific markers of cardiac muscle, and increased the level of angiogenic factor and growth factor of.3. compared with ASC. HP-ASC could reduce the area of myocardial infarction more effectively, increase the proportion of cell survival, reduce the number of apoptosis in myocardial infarction area, and increase the effect of.4. HP-ASC on cardiac function of myocardial infarction rats than HP-ASC+CBM Group NCs and group CBMNCs were better, and their myocardial infarction ratio and myocardial infarction wall thickness, cell survival and neovascularization were all better than those of other control groups.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.22

【相似文献】