胎盘间充质干细胞输注在缺氧缺血脑损伤模型中的研究

发布时间：2018-06-02 15:44

本文选题：缺氧缺血性脑损伤 + 间充质干细胞　；参考：《山东大学》2014年博士论文

【摘要】：研究背景缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)是由于围产期窒息、缺氧、缺血引致的新生儿脑损伤性疾病,围产期的窒息会导致3～5/1000的活产婴儿患中度或重度缺氧缺血性脑病,常常导致新生儿死亡和遗留一些神经功能障碍,如脑性瘫痪、癫痫以及智力低下。其中,脑瘫是儿童期最主要的运动机能伤残性疾病,可造成患儿终生的残疾,HIBD的高发病率、致残率给患儿本人、家庭和社会都造成了巨大的精神、经济负担。 HIBD的发病过程是一个十分复杂的病理过程,是多种机制综合参与的一系列连锁反应的结果,而目前确切的发病机制并不明确。目前新生儿HIBD的治疗多采用高压氧治疗、神经细胞营养药物、物理康复等措施,这对损伤程度相对较轻的患儿具有一定的效果,但对于中-重度脑病所造成的中枢神经功能障碍难以奏效。因此寻求一种有效的治疗手段对减轻HIBD患儿的病死率、致残率具有非常重要的临床意义。近年的研究表明,间充质干细胞(mesenchymal stem cells,MSCs)治疗在HIBD的动物试验及临床前期试验中都取得了很大的进展,目前研究较多的是骨髓MSCs,但是BMSCs存在干细胞含量少、增殖能力和分化潜能减弱、有创操作、病毒感染的机率增高等局限性,使其临床应用和推广受到了限制。本研究证实从孕鼠胎盘可以分离、培养胎盘来源MSCs (placenta-derived mesenchymal stem cells, PD-MSCs),经鉴定符合间充质干细胞鉴定标准,验证了从大鼠胎盘获得MSCs是完全可行的,而PD-MSCs因其自身的优点,将来可能会成为一种理想的种子细胞,具有极大的应用潜能。 HIBD的发病机制涉及炎症反应、氧化应激损伤、脑组织能量代谢紊乱等多个环节。缺血后的炎症反应已成为研究的焦点,炎性细胞可以渗入到脑实质并释放多种神经毒性物质造成炎症级联反应,多种细胞因子参与其中,辅助T细胞17(T helper cell17, Th17)和调节性T细胞(regulatory T cells,Treg)也在HIBD的发生、发展都起到至关重要的作用。另外,在脑组织缺氧缺血损伤中,氧化应激损伤也是一个核心病理过程,在机体抗氧化应激防御机制中,Keap1-Nrf2/ARE信号通路是重要的化学通路之一,可以通过综合的抗氧化、抗凋亡和抗炎特性起到保护细胞免受损害的作用,Nrf2调控下游血红素加氧酶HO-1酶的表达。脑损伤的细胞移植治疗最早被认为是一种神经细胞的替代机制,目前认为是一个多方面联合的修复机制,移植细胞不仅可以直接替代受损细胞,而且可以通过促进内源性神经干细胞、旁分泌营养因子、改善局部血供等机制。因而本研究结合HIBD的发病机理,从抗炎性反应和抗氧化应激机制两方面探讨了PD-MSCs的作用机制。研究目的第一部分：从大鼠的生长发育、神经行为学、病理改变等方面评估PD-MSCs干预治疗HIBD模型鼠的效果；第二部分：从抑制炎性反应、免疫调节、抗氧化应激等方面深入探讨PD-MSCs治疗HIBD模型鼠的作用机制。研究方法 1.从孕鼠胎盘分离、培养PD-MSCs,利用流式细胞术检测细胞表型,利用体外诱导分化技术证明其多向分化能力,验证其是否符合间充质干细胞的国际通用标准；然后利用GFP转染标记PD-MSCs,回输新生鼠并观察MSCs输注后在体内各主要组织器官的分布情况； 2.选取7日龄(postnatal day7, P7)健康Wistar大鼠,按照改良Rice法制作新生鼠HIBD模型。观察大鼠行为学改变,采用Bederson评分进行功能评分等级,标准为：0分：没有神经损伤症状；1分：抓大鼠鼠尾提起,大鼠不能完全伸展对侧前爪；2分：大鼠的前肢对对侧推力的抵抗能力下降；3分：大鼠出现向对侧转圈现象。HIBD模型建立后48h,随机分为对照组(Control)、 HIBD组(HIBD)、PD-MSCs治疗组(HIBD+PD-MSCs)和成纤维细胞治疗组(HIBD+Fibroblasts),在立体定向引导下,向大鼠脑组织内匀速注射等量的PD-MSCs、成纤维细胞。 3.细胞治疗后,观察每只实验大鼠的生长发育、皮肤外观形态、体重的增长情况及行为有无异常表现。 4.采用悬挂试验、滑棒试验评价大鼠的运动行为；水迷宫试验评价大鼠的认知功能。悬挂试验、滑棒试验在P10、P16、P22和P28完成,Morris水迷宫试验在P24～P28完成。 5.细胞治疗后5d, CD4+CD25+T淋巴细胞的检测。 6.在大鼠缺氧缺血损伤后3h、6h、24h、3d、5d以及细胞治疗后3d检测了TNF-α、 IFN-γ、IL-10、IL-17mRNA的表达。 7.缺血缺氧损伤后6h、24h、48h、72h、5d以及细胞治疗后5d时检测了HO-1、 Nrf2mRNA的表达。 8.细胞治疗后5d,采用Western blot法分别对脾脏组织中Foxp3和海马组织中HO-1、Nrf2进行检测。 9.细胞治疗后3d,应用ELISA法检测外周血清中TNF-α、IFN-γ、IL-10、IL-17的水平。 10.缺血缺氧损伤后6h,24h,48h,72h以及细胞治疗后5d,检测海马组织中MDA含量。 11.P28运动实验完成后,进行HE染色和尼氏染色并行病理分析。结果 1.从孕鼠胎盘可以分离、培养出MSCs,利用流式细胞术和体外细胞诱导分化技术,证明所获细胞符合间充质干细胞鉴定标准,验证了从大鼠胎盘获得MSCs是完全可行的。 2.新生鼠在缺氧开始时出现烦躁、紫绀,其后逐渐转为抑制,甚至出现抽搐。HIBD组与对照组相比体重明显减轻。悬挂试验、滑杆试验中,HIBD组大鼠与正常对照组比较表现出明显的运动机能落后,Morris水迷宫定位航行试验中,HIBD组与对照组相比逃避潜伏期明显延长,在第6天的空间探索试验中,HIBD组穿过原平台的频率明显降低。脑组织大体可见肿胀、苍白；后期有萎缩、梗死、液化。HE染色镜下见损伤侧皮层、海马神经细胞、小脑浦肯野细胞排列紊乱、变性、坏死。尼氏染色尼氏小体消退,出现神经元嗜酸性变、胶质细胞噬细胞现象。从生长发育、运动功能评估、病理改变等多方面判断HIBD模型是成功的。 3.PD-MSCs脑内迁移：移植后6小时,GFP阳性PD-MSCs被发现主要是在注射部位。移植后6天,绿色荧光信号明显减弱。GFP阳性细胞在接受注射的大鼠缺血侧,而对侧皮层没有发现GFP阳性细胞。PD-MSCs主要在注射针道和／或注射部位被发现。然后大量细胞迁出注射部位、扩散到整个皮层和脑室周围区域。 4.流式细胞术检测结果显示,HIBD组CD4+CD25+T细胞为11.4%,比正常对照组(8.69%)略有升高,细胞治疗后,成纤维细胞治疗组CD4+CD25+T细胞15.1%, PD-MSCs治疗组CD4+CD25+T细胞则显著升高,达18.0%。Western Blot检测大鼠脾脏Foxp3的蛋白含量,结果发现HIBD组脾脏Foxp3蛋白含量虽高于正常对照组,但无显著性差异,经过PD-MSCs治疗后,大鼠脾脏Foxp3的蛋白表达明显增多,与HIBD组比较具有统计学意义。 5.缺氧缺血损伤后,脑组织中IFN-γ、TNF-α、IL-17和IL-10mRNA的动态变化结果：HIBD组TNF-α,IFN-γ和IL-17mRNA表达大幅上调,TNF-a mRNA在损伤后3h表达开始增加,在24h时表达大幅度升高,明显高于对照组。IFN-γ mRNA、IL-17mRNA表达升高时间较TNF-α有所延迟,其二者的表达高峰在HI损伤后3d。IL-10mRNA的表达在6h开始上调,在3d后达高峰。PD-MSCs治疗前后IFN-γ、TNF-α、IL-17和IL-10细胞因子水平比较：RT-PCR法检测PD-MSCs治疗组TNF-α, IFN-γ和IL-17mRNA表达明显低于HIBD组和成纤维细胞组,在HIBD组、成纤维细胞组和PD-MSCs治疗组三个实验组,IL-10mRNA的表达与对照组比较均明显上调,在PD-MSCs治疗组升高尤为明显,明显高于HIBD组和成纤维细胞组。 6.ELISA法检测结果：HIBD组血清IFN-γ TNF-α、IL-17和IL-10水平均高于正常对照组,PD-MSCs治疗后,大鼠血清IFN-γ、TNF-α和IL-17水平较HIBD组、成纤维细胞治疗组均明显有所下降,而IL-10水平则显著有所升高,HIBD组与成纤维细胞治疗组之间IFN-γ、TNF-α、IL-17和IL-10水平没有显著差异。 7. HIBD中HO-1、Nrf2的动态变化：HIBD组HO-1和Nrf2mRNA的表达与对照组相比较在6h开始上调、48h达高峰,其后HO-1和Nrf2mRNA的表达虽然逐渐下降,但在72h和5d仍保持在较高的水平。PD-MSCs治疗前后HO-1、Nrf2表达的比较：RT-PCR法检测HIBD组、成纤维细胞组和PD-MSCs治疗组三个实验组的HO-1和Nrf2mRNA的表达均高于正常对照组,而PD-MSCs治疗组则显著高于HIBD组和成纤维细胞组。Western blot分析表明HO-1和Nrf2在HIBD组、PD-MSCs治疗组和成纤维细胞治疗组均比对照组明显增多,其中PD-MSCs治疗组较HIBD组、成纤维细胞治疗组均明显增高。 8.MDA水平动态变化及治疗前后对比：缺氧缺血性损伤后,脑组织MDA水平在HI后6h时开始显著升高,72h达峰值水平,随之MDA水平开始逐渐降低,5d时MDA水平虽显著降低,但仍然高于正常水平。PD-MSCs治疗组MDA水平与缺氧缺血组、成纤维细胞治疗组相比明显降低,具有显著性差异。结论 1.从行为学、神经运动功能、病理改变等方面证实改良Rice法建造的HIBD模型是可行的、成功的。 2.组织块贴壁法可以成功培养大鼠PD-MSCs。 3. PD-MSCs干预不但能改善HIBD大鼠近期的神经运动功能,而且能改善HIBD大鼠的远期学习记忆功能。 4. HIBD的发病机制之一是：促炎性细胞因子IFN-γ、TNF-α、IL-17和抗炎性细胞因子IL-10之间的失衡,同时存在CD4+CD25+Treg细胞功能紊乱。 5. HIBD的发病机制之一是：抗氧化(Nrf2/HO-1)／氧化(MDA)水平之间的失衡。 6. PD-MSCs治疗HIBD有效的机制之一可能是通过调节Th17/Treg细胞失衡。 7.PD-MSCs治疗HIBD的作用靶点之一可能是上调Keap1-Nrf2/ARE/HO-1通路。
[Abstract]:Research background
Hypoxic-ischemic brain damage (HIBD) is a neonatal brain injury caused by perinatal asphyxia, hypoxia, and ischemia. Perinatal asphyxia leads to moderate or severe hypoxic-ischemic encephalopathy in 3 to 5/1000 living babies, which often leads to neonatal death and some neurological dysfunction, such as brain. Paralysis, epilepsy and mental retardation. Among them, cerebral palsy is the most important motor disability disease in childhood. It can cause the disabled children for life, the high incidence of HIBD, the rate of disability to the children himself, family and society have caused great mental and economic burden.
The pathogenesis of HIBD is a very complicated pathological process, which is the result of a series of chain reactions involved in a variety of mechanisms. The exact pathogenesis is not clear at present. At present, the treatment of neonatal HIBD is mostly used in the treatment of hyperbaric oxygen, neurotrophic drugs, physical rehabilitation and so on, which are relatively light to the degree of injury. It has a certain effect, but the central nervous dysfunction caused by moderate to severe encephalopathy is difficult to work. Therefore, it is of great clinical significance to seek an effective treatment to reduce the mortality and disability rate of children with HIBD.
Recent studies have shown that mesenchymal stem cells (MSCs) has made great progress in the animal and preclinical trials of HIBD. At present, there are many studies on bone marrow MSCs, but BMSCs has less stem cell content, weak proliferation and differentiation potential, invasive operation, and increased probability of virus infection. Limitations, which make its clinical application and Promotion Limited. This study confirms that the placenta can be separated from the pregnant mouse, and the placental source MSCs (placenta-derived mesenchymal stem cells, PD-MSCs) is cultured. It is confirmed that it is suitable for the identification of mesenchymal stem cells. It is proved that it is completely feasible to obtain MSCs from the rat placenta, and PD-MSCs is superior to it for its own advantages. In the future, it may become an ideal seed cell with great potential for application.
The pathogenesis of HIBD involves inflammatory reaction, oxidative stress injury, and brain tissue energy metabolism disorder. The inflammatory response after ischemia has become the focus of research. Inflammatory cells can infiltrate into the brain parenchyma and release a variety of neurotoxic substances to cause inflammatory cascade reaction. Multiple cytokines are involved, and T cells 17 (T helper) are assisted. Cell17, Th17) and regulatory T cells (regulatory T cells, Treg) also play an important role in the development of HIBD. In addition, oxidative stress damage is also a core pathological process in the brain tissue hypoxic and ischemic injury. In the system of anti oxidative stress defense mechanism, the Keap1-Nrf2/ARE signaling pathway is an important chemical pathway. First, Nrf2 can regulate the expression of heme oxygenase HO-1 enzyme through comprehensive antioxidant, anti apoptotic and anti-inflammatory properties.
Cell transplantation for brain injury is considered as an alternative mechanism for neural cells. It is considered to be a multifaceted joint repair mechanism. Transplanted cells can not only directly replace damaged cells, but also improve local blood supply by promoting endogenous neural stem cells, paracrine nutrient factors and improving local blood supply. Combined with the pathogenesis of HIBD, the mechanism of PD-MSCs was discussed from two aspects: anti-inflammatory reaction and anti oxidative stress mechanism.
research objective
Part one: To evaluate the effect of PD-MSCs intervention on HIBD model rats from the aspects of growth and development, neurobehavioral and pathological changes.
The second part: from the aspects of inhibiting inflammatory reaction, immunoregulation and anti oxidative stress, we will further explore the mechanism of PD-MSCs in the treatment of HIBD mice.
research method
1. isolated from the placenta of pregnant rats, cultured PD-MSCs, using flow cytometry to detect cell phenotype, using in vitro differentiation technique to prove its multidirectional differentiation ability, verify whether it conforms to the international standard of mesenchymal stem cells, and then transfection of PD-MSCs with GFP transfection, retransfuse new mice and observe the main organs after MSCs infusion in the body. The distribution of officials;
2. the healthy Wistar rats of 7 days of age (postnatal Day7, P7) were used to make the HIBD model of newborn rats according to the improved Rice method. The behavioral changes were observed by the Bederson score. The standard was 0 points: no nerve injury symptoms; 1: grabbing the rat tail, the rats could not completely extend the contralateral front claw; 2 points: rats: rats: 2 points: Rats The resistance of the forelimb to the contralateral thrust decreased; 3: after the.HIBD model was established to the contralateral circle, the rats were randomly divided into the control group (Control), the HIBD group (HIBD), the PD-MSCs treatment group (HIBD+PD-MSCs) and the fibroblast treatment group (HIBD+Fibroblasts). Under the stereotactic guidance, the constant injection of the same amount to the rat brain was equal. PD-MSCs, fibroblasts.
3. after treatment, the growth, skin appearance, weight gain and behavior of each experimental rat were observed.
4. suspension test was used to evaluate the exercise behavior of rats. The water maze test was used to evaluate the cognitive function of rats. Suspension test was performed and the slide test was completed in P10, P16, P22 and P28, and the Morris water maze test was completed in P24 to P28.
5. detection of 5D and CD4+CD25+T lymphocytes after cell therapy.
6. after 3h, 6h, 24h, 3D, 5D and 3D treatment, the expressions of TNF-, IFN-, IL-10 and IL-17mRNA were detected in rats after hypoxia ischemia injury.
7. after ischemia and hypoxia, 6h, 24h, 48h, 72h, 5D and 5D expression in cells were detected for HO-1 and Nrf2mRNA expression.
8. after 5D, Western and blot were used to detect Foxp3 and HO-1 and Nrf2 in the hippocampus respectively.
9. after 3D, the levels of TNF-, IFN-, IL-10 and IL-17 in peripheral serum were detected by ELISA.
10. after ischemia and hypoxia, 6h, 24h, 48h, 72h and 5D after cell therapy were used to detect the content of MDA in hippocampus tissue.
After 11.P28 exercise test, HE staining and Nissl staining were performed and pathological analysis was performed.
Result
1. from the placenta of pregnant mice, MSCs was isolated and cultured. Using flow cytometry and in vitro cell differentiation, it was proved that the obtained cells met the criteria for identification of mesenchymal stem cells. It was proved that it was completely feasible to obtain MSCs from the rat placenta.
2. the newborn rats appeared irritable and cyanotic at the start of anoxia, and then gradually turned to inhibition, and even the group of convulsions.HIBD group had a significant reduction in weight compared with the control group. In the suspension test, the HIBD group showed obvious motor function lag compared with the normal control group. In the Morris water maze navigation test, the HIBD group and the control group In the sixth day space exploration test, the frequency of the HIBD group passed the original platform significantly decreased in the space exploration test. The brain tissue was generally swollen and pale. There was atrophy, infarction, and liquefied.HE stained lens, the injured lateral cortex, the hippocampal neurons, the cerebellum puken field cells arranged disorder, denaturation, necrosis. Nissl staining Nissl's small The body subsided, the neuron eosinophilic change, the glial cell phage phenomenon. It was successful to judge the HIBD model from many aspects, such as growth and development, motor function evaluation, pathological changes and so on.
3.PD-MSCs intracerebral migration: 6 hours after transplantation, GFP positive PD-MSCs was found mainly at the injection site. 6 days after the transplantation, the green fluorescent signal significantly weakened the.GFP positive cells in the ischemic side of the injected rat, while the GFP positive cells in the lateral cortex were not found to be found mainly in the injection and / or injection sites. The cells moved out of the injection site and spread throughout the cortex and periventricular area.
The results of 4. flow cytometry showed that CD4+CD25+T cells in group HIBD were 11.4%, slightly higher than that in normal control group (8.69%). After cell therapy, CD4+CD25+T cells in fibroblast treatment group were 15.1%, CD4+CD25+T cells in PD-MSCs treatment group increased significantly, and 18.0%.Western Blot was used to detect the protein content of spleen Foxp3 in rats, and the results were found in the HIBD group of spleen. Although the content of Foxp3 protein was higher than that of the normal control group, there was no significant difference. After PD-MSCs treatment, the expression of Foxp3 protein in the spleen increased significantly, and was statistically significant compared with that of the HIBD group.
5. the dynamic changes of IFN- gamma, TNF- a, IL-17 and IL-10mRNA in the brain tissue after the hypoxic and ischemic injury: the expression of TNF- alpha, IFN- gamma and IL-17mRNA in HIBD group increased significantly. The 3H expression of TNF-a mRNA began to increase after the injury, and the expression in 24h was significantly higher than that of the control group. The expression of 3d.IL-10mRNA was up to rise after HI injury, and the level of IFN- gamma, TNF- a, IL-17 and IL-10 cytokine before and after the peak.PD-MSCs treatment after 3D was compared: RT-PCR method was used to detect TNF- alpha in PD-MSCs treatment group, and the expression was significantly lower than that of the group and fibroblast group, and the fibroblasts were in the group of fibroblasts. In the three experimental groups of the group and the PD-MSCs treatment group, the expression of IL-10mRNA was significantly up-regulated compared with the control group, especially in the PD-MSCs treatment group, which was significantly higher than that in the HIBD group and the fibroblast group.
The results of 6.ELISA assay: serum IFN- gamma TNF- alpha, IL-17 and IL-10 levels in group HIBD were higher than that in normal control group. After PD-MSCs treatment, serum IFN- gamma, TNF- alpha and IL-17 levels were significantly lower than those in the HIBD group, while the IL-10 level was significantly higher. There was no significant difference in IL-17 and IL-10 levels.
The dynamic changes of HO-1 and Nrf2 in 7. HIBD: the expression of HO-1 and Nrf2mRNA in group HIBD compared with the control group was up up, the peak of 48h reached, and the expression of HO-1 and Nrf2mRNA decreased gradually, but the comparison between 72h and 5D still remained at a higher level. The expression of HO-1 and Nrf2mRNA in the three experimental groups of the PD-MSCs treatment group was higher than that in the normal control group, while the PD-MSCs treatment group was significantly higher than the HIBD group and the fibroblast group.Western blot analysis showed that HO-1 and Nrf2 were in the HIBD group, the PD-MSCs treatment group and the fibroblast treatment group were all significantly higher than those in the control group, and the PD-MSCs treatment group was more than that of the control group. The treatment group of fibroblast increased significantly.
8.MDA level dynamic changes and before and after treatment: after hypoxic ischemic injury, the brain tissue MDA level began to increase significantly at 6h after HI, 72h reached the peak level, and then MDA level began to decrease gradually, while MDA level decreased significantly in 5D, but still higher than normal level.PD-MSCs treatment group MDA level and hypoxia ischemia group, fibroblast treatment. The group was significantly lower than the group, and there was a significant difference.
conclusion
1. it is proved that the improved Rice method is feasible and successful in terms of ethology, neuromotor function and pathological changes. The HIBD model constructed by the improved method is feasible and successful.
2. tissue block adherence method can successfully cultivate rat PD-MSCs..
3. PD-MSCs intervention can not only improve the short-term motor function of HIBD rats, but also improve the long-term learning and memory function of HIBD rats.
One of the pathogenesis of 4. HIBD is the imbalance between proinflammatory cytokines IFN- gamma, TNF- alpha, IL-17 and anti-inflammatory cytokine IL-10, and there is a dysfunction of CD4+CD25+Treg cells.
One of the pathogenesis of 5. HIBD is the imbalance between antioxidant (Nrf2/HO-1) and oxidative (MDA) levels.
One of the effective mechanisms of 6. PD-MSCs treatment of HIBD may be to regulate Th17/Treg cell imbalance.
One of the targets of 7.PD-MSCs therapy for HIBD may be up regulation of Keap1-Nrf2/ARE/HO-1 pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742

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