脂肪间充质干细胞治疗心肺复苏后大鼠缺氧性脑病的实验研究

发布时间：2018-07-03 04:17

本文选题：脂肪间充质干细胞 + 心搏骤停　；参考：《第二军医大学》2017年硕士论文

【摘要】：近些年来,心肺复苏的成功率逐渐提高,但由于神经细胞对缺血缺氧敏感而且不能再生,大部分患者复苏后出现不可逆的神经损伤,导致失语、偏瘫、植物人等后遗症甚至导致死亡。尽管采取了亚低温治疗、药物治疗等综合措施治疗心搏骤停后的神经损伤,但是心搏骤停患者的出院生存率仍不高,在中国心搏骤停患者神经功能良好的出院率仅1%左右。如何减轻神经损伤、促进神经修复已成为现代医学的难题,在此我们尝试一种新措施治疗心搏骤停引起的神经损伤。间充质干细胞具有高度自我更新能力,可以多次传代、大量扩增;同时还具有多向分化潜能,在一定的诱导条件下能分化为脂肪细胞、成骨细胞、软骨细胞、神经细胞等。ADSCs是来源于脂肪组织的MSCs,其储备丰富,取材容易,其增殖分化能力高度稳定,不会随供者年龄产生变化。目前研究发现ADSCs在小动物脑梗塞模型中取得了良好效果,ADSCs移植能减少脑梗面积,减轻局灶性神经损伤,改善神经功能预后。而脑梗塞为局部性病灶,与心搏骤停引起的全脑范围的缺氧性脑病,在病因、病理机制、临床表现上都有所不同,ADSCs对于心搏骤停引起的全脑范围的缺氧性脑病的治疗效果及治疗机制需要进一步验证。本实验研究异种移植ADSCs对心搏骤停后缺氧性脑病的治疗效果,并探讨其作用机制,为临床治疗提供理论支持。主要内容包括:人ADSCs的培养,从细胞形态、细胞表型、多向分化能力等方面进行鉴定;采用窒息法建立大鼠心肺复苏模型并移植人ADSCs,观察ADSCs对心搏骤停后缺氧性脑病的治疗效果,海马组织内BDNF、IL-6的表达情况。第一部分ADSCs培养及鉴定研究目的从人脂肪组织中分离、培养ADSCs,并从细胞形态、细胞表型、分化能力(成骨分化、成软骨分化、成脂肪分化)等方面进行鉴定,为下一步动物实验奠定基础。材料和方法从健康供者腹部抽取脂肪组织,消化、分离出血管基质部分,在37℃、5%CO2培养箱中使用DMEM/F12+10%胎牛血清培养基中贴壁培养,逐渐纯化为ADSCs,每3天换液1次,待细胞70%~90%融合时进行传代,培养过程中观察细胞形态。取第3代ADSCs采用流式细胞术检测细胞表面抗原CD73、CD90、CD105、CD34、CD45、CD11b、CD19、HLA-DR的表达情况。使用成骨分化试剂盒诱导成骨分化,第28天茜素红染色法、磷酸苯二钠基质(NBT/BCIP)染色法检测成骨分化情况;使用成软骨分化试剂盒诱导成软骨分化,第7天阿利辛蓝染色检测成软骨分化情况;使用成脂分化试剂盒诱导成脂分化,第21天油红O染色法检测成脂分化情况。结果原代培养24小时后,镜下可见短梭形、三角形等多种形态的贴壁细胞,随着时间增长,梭形细胞逐渐增多,呈长梭形外观,细胞成集落样生长,在6~8天后达到80%左右融合。传代后始终保持着稳定的增殖速度。adscs表面抗原cd73、cd90、cd105阳性表达率高,分别为99.99%、99.99%、96.88%,同时阴性表达cd34、cd45、cd11b、cd19、hla-dr。成骨诱导后细胞生长旺盛,细胞形态由长梭形逐渐变为短梭形,7天后可见结节样结构,28天后矿化结节形成明显,矿化结节被茜素红染色法染成红色结节,胞质因含有碱性磷酸被nbt/bcip染色法染成深蓝色。成软骨诱导后,细胞形态由长梭形逐渐回缩变圆,7天后用阿利辛蓝染色检测软骨细胞基质中糖胺多糖合成情况,胞质被染成蓝色。成脂诱导后,细胞形态由长梭形逐渐变为圆形,胞浆内可见小脂滴,脂滴数量逐渐增加并相互融合,14天后油红o染色可见细胞内脂滴被染成红色。结论从人脂肪组织中分离并培养出稳定的adscs,从细胞形态、细胞表面抗原、多向分化能力3个方面对细胞进行鉴定,符合mscs的鉴定标准。第二部分观察adscs移植对心肺复苏后缺氧性脑病的治疗效果及保护机制研究研究目的观察异种adscs移植对大鼠心搏骤停后神经功能、神经元凋亡、血清s100β的影响,海马组织bdnf、il-6的表达情况材料和方法54只大鼠随机分为3组(sham组、ca组、adscs组),每组18只。sham组只进行外科操作,不进行心搏骤停及心肺复苏操作。ca组使用气管夹闭法诱导窒息性心搏骤停模型,心搏停止6分钟,复苏后1小时静脉注射1mlpbs。adscs组建立窒息性心搏骤停模型,心搏停止6分钟,复苏后1小时静脉注射人adscs1ml(细胞计数5×106)。操作完成后24小时、72小时、168小时,每组随机选取6只大鼠进行检测。使用神经功能缺损评分评价神经功能,elisa法检测血清s100β浓度,he染色观察海马组织病理变化,tunel染色检测海马神经元凋亡率,werstern-blot检测海马bdnf、il-6蛋白水平。结果1.心搏骤停前采集到的大鼠体重、平均动脉压、心率,比较差异无统计学意义(p0.05),心肺复苏期间窒息时间、按压时间及复苏后1小时map比较差异无统计学意义(p0.05)。2.神经功能缺损评分在24小时、72小时、168小时,sham组大鼠评分高于adscs组和ca组,差异具有统计学意义(p0.05),adscs组均高于ca组,差异具有统计学意义(p0.05)。3.病理变化sham组大鼠结果正常。CA组大鼠海马区锥体细胞减少,锥体细胞稀疏,大量小胶质细胞增生。ADSCs组大鼠海马区少量细胞核皱缩改变,锥体细胞排列基本正常。4.TUNEL染色在3个时间点,sham组神经元凋亡率明显低于CA组和ADSCs组,差异具有统计学意义(P0.05),而ADSCs组神经元凋亡率均低于CA组,差异具有统计学意义(P0.05)。5.血清S100β浓度在3个时间点,sham组血清S100β浓度均低于CA组和ADSCs组差异具有统计学意义(P0.01),而ADSCs组S100β浓度低于CA组,差异具有统计学意义(P0.05)。6.海马IL-6在3个时间点,sham组IL-6表达水平低于CA组和ADSCs组,差异具有统计学意义(P0.01)。在24小时、72小时,ADSCs组IL-6高于CA组,差异具有统计学意义(P0.01)。复苏后168小时,ADSCs组和CA组IL-6比较差异无统计学意义(P0.05)。7.海马BDNF在复苏后24小时和72小时,ADSCs组和CA组BDNF蛋白水平高于sham组(P0.01),而ADSCs组BDNF蛋白高于CA组,差异具有统计学意义(P0.01)。在复苏后168小时,3组BDNF蛋白表达强度接近,差异无统计学意义(P0.05)。结论心搏骤停能引起神经凋亡、神经功能障碍,ADSCs具有神经保护作用,能抑制神经细胞凋亡,改善神经功能预后。ADSCs可能是通过上调BDNF、IL-6表达发挥神经保护作用。
[Abstract]:In recent years, the success rate of cardiopulmonary resuscitation has gradually increased, but because nerve cells are sensitive to ischemia and hypoxia and can not be regenerated, most patients have irreversible nerve damage after resuscitation, resulting in aphasia, hemiplegia, vegetative sequelae and even death. Although mild hypothermia treatment, drug treatment and other comprehensive measures for the treatment of heart beat The survival rate of patients with cardiac arrest is still not high, and the discharge rate is only about 1% in patients with cardiac arrest. How to reduce nerve injury and promote nerve repair has become a difficult problem in modern medicine. Here we try a new measure to treat the nerve injury caused by cardiac arrest. Stem cells have the ability of high self renewal, which can be passaged and proliferate many times. At the same time, it also has the potential to differentiate into adipocytes under certain induction conditions. The.ADSCs of osteoblasts, chondrocytes and nerve cells is the MSCs derived from adipose tissue, which is rich in reserve and easy to obtain, and its proliferation and differentiation ability is highly stable. The present study has found that ADSCs has achieved good results in the model of cerebral infarction in small animals. ADSCs transplantation can reduce the area of cerebral infarction, reduce focal nerve damage and improve the prognosis of nerve function. Cerebral infarction is a local focus, and the whole brain area of the brain is induced by cardiac arrest, in the etiology and pathology. The mechanism and clinical manifestations are different. The therapeutic effect and treatment mechanism of ADSCs for the whole brain range of hypoxic encephalopathy caused by cardiac arrest are further verified. This experiment studies the therapeutic effect of xenotransplantation of ADSCs on hypoxic encephalopathy after cardiac arrest, and discusses its mechanism for clinical treatment. The contents include: the cultivation of human ADSCs, the identification of cell morphology, cell phenotype, and multidirectional differentiation, and the establishment of rat cardiopulmonary resuscitation model by asphyxia method and transplantation of human ADSCs, to observe the therapeutic effect of ADSCs on hypoxic encephalopathy after cardiac arrest, the expression of BDNF and IL-6 in the hippocampus. The first part of ADSCs culture and Identification Research The purpose of this study was to isolate and cultivate ADSCs from human adipose tissue, and to identify the cell morphology, cell phenotype, differentiation capacity (osteogenesis differentiation, chondrodifferentiation, adipose differentiation) and other aspects, which lay the foundation for the next animal experiment. Materials and methods were extracted from the abdominal fat tissue of the healthy donor, digested and separated from the vascular matrix, at 37, 5%. The CO2 culture box was cultured in the culture medium of DMEM/F12+10% fetal bovine serum, which was gradually purified to ADSCs and changed 1 times every 3 days. The cell morphology was observed during the cell 70%~90% fusion. The cell surface antigen CD73, CD90, CD105, CD34, CD45, CD11b, CD19, and expression were detected by flow cytometry for the third generation of ADSCs. Osteogenesis differentiation was induced by osteogenic differentiation kit, twenty-eighth days alizarin red staining, benzyl benzene two matrix (NBT/BCIP) staining method was used to detect osteogenic differentiation, chondrodifferentiation was induced by chondrogenic differentiation kit, and chondrodifferentiation was detected by alizocin blue staining at seventh days, fat differentiation was induced by fat differentiation kit and twenty-first days. Oil red O staining was used to detect the formation of lipid differentiation. Results after 24 hours of primary culture, the cells with short shuttle shape, triangle and other morphologic cells were seen under the microscope. The spindle cells gradually increased with time, and the cells grew in a long shuttle shape. The cells became colony like growth and reached about 80% after 6~8 days. The positive expression rate of.Adscs surface antigen CD73, CD90, CD105 was high, 99.99%, 99.99%, 96.88% respectively. Meanwhile, the negative expression of CD34, CD45, CD11b, CD19, and HLA-DR. became strong, and the cell morphology changed from long spindle shape to short shuttle form, and nodular structure was visible after 7 days. 28 days after the mineralization, the mineralized nodules were formed and the mineralized nodules were alizarin red. The staining method was dyed red nodules, cytoplasm was dyed deep blue by nbt/bcip staining with alkaline phosphoric acid. After induction of cartilage, the cell morphology was gradually retracted from long spindle shape, and 7 days later, the synthesis of glycosaminoglycan in chondrocyte matrix was detected by alisocen blue, and the cytoplasm was stained blue. The gradient is round, small fat droplets are visible in the cytoplasm, the number of lipid droplets gradually increases and fuses each other. 14 days after the oil red O staining, the lipid droplets in the cells are dyed red. Conclusion a stable ADSCs is separated and cultured from the human adipose tissue, and the cells are identified from the cell morphology, the cell surface antigen and the multi-directional differentiation ability, which conforms to the MSCs. The second part observed the therapeutic effect and protective mechanism of ADSCs transplantation on hypoxic encephalopathy after cardiopulmonary resuscitation. Objective To observe the effects of Xenotransplantation on nerve function, neuron apoptosis, serum S100 beta, the expression of BDNF, IL-6 in hippocampus and 54 rats randomly divided into 3 groups. (Group sham, group Ca, group ADSCs), each group of 18.Sham groups only performed surgical operation, without cardiac arrest and cardiopulmonary resuscitation, group.Ca used tracheal clamping to induce asphyxiating cardiac arrest model, cardiac arrest for 6 minutes, and 1 hours after resuscitation by intravenous injection of 1mlpbs.adscs group to establish asphyxiating cardiac arrest model, cardiac arrest for 6 minutes, and 1 small after resuscitation. Intravenous injection of human adscs1ml (cell count 5 x 106). After 24 hours, 72 hours, 168 hours after completion of the operation, 6 rats were randomly selected for each group. Nerve function defect score was used to evaluate the nerve function, ELISA method was used to detect the serum S100 beta concentration, he staining was used to observe the pathological changes of hippocampus, and TUNEL staining was used to detect the apoptosis rate of hippocampal neurons, werst Ern-blot detected the BDNF and IL-6 protein levels in the hippocampus. Results there was no statistically significant difference in weight, mean arterial pressure and heart rate before 1. cardiac arrest (P0.05). The time of asphyxia during cardiopulmonary resuscitation, compression time and 1 hour map after resuscitation were not statistically significant (P0.05).2. neural function defect score was 24 hours, 72 small At 168 hours, the score of rats in group sham was higher than that in group ADSCs and group Ca, the difference was statistically significant (P0.05), the group ADSCs was higher than that of the CA Group, the difference was statistically significant (P0.05).3. pathological changes in group sham rats, the result of the normal group of the rat hippocampus pyramidal cells decreased, the pyramidal cells were sparse, a large number of microglia proliferated the hippocampus of the.ADSCs group. A small amount of nuclear crinkle changed, the pyramidal cells were arranged basically with normal.4.TUNEL staining at 3 time points. The apoptosis rate of sham group was significantly lower than that of CA group and ADSCs group. The difference was statistically significant (P0.05), but the apoptosis rate of ADSCs group was lower than that in CA group, and the difference has statistical significance (P0.05).5. serum S100 beta concentration at 3 time points, sham The serum S100 beta concentration was lower than that of the CA group and the ADSCs group (P0.01), while the S100 beta concentration in the ADSCs group was lower than the CA group. The difference was statistically significant (P0.05).6. hippocampal IL-6 at 3 time points. The IL-6 expression level of sham group was lower than that of the CA group. In group CA, the difference was statistically significant (P0.01). 168 hours after resuscitation, there was no significant difference in IL-6 between group ADSCs and CA group (P0.05),.7. hippocampal BDNF was 24 hours and 72 hours after resuscitation, and BDNF protein level in ADSCs group and CA group was higher than that in sham group. 68 hours, the 3 groups of BDNF protein expression intensity close, the difference is not statistically significant (P0.05). Conclusion cardiac arrest can cause nerve apoptosis, neural dysfunction, ADSCs have neuroprotective effect, can inhibit the apoptosis of nerve cells, improve the prognosis of neural function.ADSCs may be through the upper modulation of BDNF, IL-6 expression to play neuroprotective effect.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

【参考文献】