下丘脑垂体肾上腺轴反应对骨髓干细胞的动员作用与分子机制

发布时间：2018-05-19 22:41

本文选题：HPA轴 + 间充质干细胞(MSCs)　；参考：《第三军医大学》2012年硕士论文

【摘要】：全球范围内由创伤引起的死亡人数持续升高，，预计到2020年会达到840万，而严重组织损伤是致死致残的关键因素，因此寻找早期有效修复损伤组织、预防和控制创伤并发症的治疗方法是提高严重创伤患者生存率、改善预后的关键。近年来，成体干细胞为严重损伤组织的修复带来新的曙光。在成年个体中，定植于骨髓中的干细胞可以动员并向损伤组织迁移，作为种子细胞直接参与损伤组织的修复。此外，成体干细胞还具有免疫调节作用，如间充质干细胞(mesenchymal stem cells, MSCs)可以抑制T、B、NK细胞的增殖和B细胞的迁移；抑制TH1细胞分泌促炎细胞因子，促进TH2细胞产生抗炎细胞因子等。虽然外源性干细胞移植在损伤组织治疗中具有重要价值，然而这种方法还有诸多伦理和技术瓶颈需要面对，如干细胞体外扩增、鉴定及其在体内的转归等等。损伤组织修复需要骨髓干细胞的动员，并作为种子细胞迁移到损伤部位。但是目前临床上所用的干细胞动员剂具有很多副作用，近30%的病人会出现骨痛、头痛、四肢乏力等症状，严重者甚至发生急性肺损伤，部分伴有基础疾患的创伤患者对干细胞动员剂不敏感。因此，探索内源性干细胞动员机制具有重要科学意义和临床价值。近年发现，神经内分泌反应对骨髓干细胞动员、增殖、分化和修复功能有潜在调控作用，如交感肾上腺髓质和其他内分泌器官源性激素(如去甲肾上腺素、雌激素)可以促进不同类型骨髓干细胞从骨髓池外流。鉴于神经内分泌反应对于骨髓干细胞数量的影响，以及效应激素在损伤微环境中的重要作用，我们推测下丘脑-垂体-肾上腺(hypothalamus-pituitary-adrenal，HPA)轴激活可能是下丘脑-骨髓-外周血三位一体的干细胞动员策略的关键环节。因此，本研究以HPA轴及其关键效应激素－糖皮质激素为切入点，探讨其对骨髓干细胞(HSCs、MSCs、EPCs)动员的影响及作用机制，进而初步探讨神经内分泌反应对骨髓干细胞的动员及后续损伤组织修复和再生的影响，旨在从骨髓干细胞动员角度为损伤组织修复另辟蹊径，从而更好地促进损伤组织实现功能修复。方法：繁育并鉴定CRH野生型(CRH+/+)小鼠和CRH基因敲除(CRH-/-)小鼠；将CRH野生型(CRH+/+)小鼠和CRH基因敲除(CRH-/-)小鼠分为强迫跑步应激组和对照组。应激后1h，通过摘眼球取血法获取足量抗凝血。ELISA法测定血浆皮质酮(corticosterone，CORT)浓度。外周血间充质干细胞(MSCs)、内皮祖细胞(EPCs)和造血干细胞(HSCs)的数量通过流式细胞分析法确定；利用微量注射泵对CRH+/+和CRH-/-小鼠分别输注不同剂量的糖皮质激素，以上述方法分别检测小鼠血浆皮质酮浓度，及其与外周血EPCs、MSCs和HSCs数量变化的关系；分离培养骨髓MSCs和EPCs，用糖皮质受体阻断剂RU486(10μM)预处理30min，通过neuro probe趋化小室法观察不同浓度(0ng/ml、75ng/ml、100ng/ml、1500ng/ml)的糖皮质激素对MSC、EPC趋化功能的影响及其趋化受体fMLP-R表达变化。结果：1.跑步应激后CRH+/+小鼠血浆皮质酮水平明显升高，同时其外周血MSCs、EPCs的数量也显著增加；但CRH-/-小鼠血浆皮质酮水平在跑步应激后没有明显变化，同时其外周血MSCs和EPCs的数量减少；2.给CRH+/+和CRH-/-小鼠注射糖皮质激素后，其血浆皮质酮水平都显著增加，同时外周血HSCs、MSCs、EPCs的数量也显著升高；3.低浓度皮质酮可以促进骨髓MSCs、EPCs向fMLP的趋化运动；RU486预处理可以逆转糖皮质激素对MSCs和EPCs趋化功能的影响；4.不同浓度的糖皮质激素均可以促进骨髓MSCs、EPCs fMLP-R mRNA表达，且低浓度的糖皮质激素可以促进fMLP-R蛋白的表达，这种作用可被RU486逆转。结论：1.急性应激条件下，间充质干细胞和内皮祖细胞发生显著的动员反应，HPA轴反应及其关键效应激素－糖皮质激素发挥了重要调节效应；2.在干细胞动员中，糖皮质激素可通过上调fMLP-R，经糖皮质激素受体基因组途径促进细胞趋化功能。
[Abstract]:The number of deaths caused by trauma continues to rise worldwide and is expected to reach 8 million 400 thousand by 2020, and severe tissue damage is the key factor for death and disability. Therefore, the search for early effective repair of damaged tissues and the prevention and control of trauma complications is the key to improving the survival rate of severe trauma patients and improving the prognosis. Adult stem cells bring new dawn to the repair of severely damaged tissues. In adult individuals, stem cells planted in bone marrow can mobilize and migrate to damaged tissues, as seed cells directly participate in the repair of damaged tissues. In addition, adult stem cells also have immunoregulation, such as mesenchymal stem cells (mesenchymal stem cells, M). SCs) can inhibit the proliferation of T, B, NK cells and the migration of B cells, inhibit the secretion of pro-inflammatory cytokines from TH1 cells and promote the production of anti-inflammatory cytokines in TH2 cells. Although exogenous stem cell transplantation is of great value in the treatment of damaged tissue, there are many ethical and technical bottlenecks, such as stem cells in vitro. The injury tissue repair requires mobilization of bone marrow stem cells and migrate as a seed cell to the site of injury. However, the current stem cell mobilization agents have many side effects, and nearly 30% of the patients have symptoms such as bone pain, headache, and weakness of the limbs and even acute lung damage. Injury, some of the trauma patients with basic diseases are not sensitive to stem cell mobilization. Therefore, it is of important scientific significance and clinical value to explore the mechanism of endogenous stem cell mobilization.
In recent years, it has been found that neuroendocrine responses have potential regulatory effects on mobilization, proliferation, differentiation and repair of bone marrow stem cells, such as sympathetic adrenal medulla and other endocrine organ derived hormones (such as norepinephrine, estrogens) that can promote the flow of different types of bone marrow stem cells from the marrow pool. The effect of the number of stem cells and the important role of the effect hormone in the damage microenvironment, we speculate that the activation of the hypothalamus pituitary adrenal (hypothalamus-pituitary-adrenal, HPA) axis may be the key link in the trinic stem cell mobilization strategy of the hypothalamus bone marrow peripheral blood. Therefore, this study is based on the HPA axis and its key effect hormones. - the effect and mechanism of glucocorticoid on the mobilization of bone marrow stem cells (HSCs, MSCs, EPCs) and the effect of neuroendocrine response on bone marrow stem cells mobilization and subsequent injury tissue repair and regeneration are discussed. To promote the function repair of the injured tissue.
Methods: CRH wild type (CRH+/+) mice and CRH gene knockout (CRH-/-) mice were bred and identified; CRH wild type (CRH+/+) mice and CRH gene knockout (CRH-/-) mice were divided into forced running stress group and control group. After stress, 1H was obtained by taking the eyeball extraction method to obtain a full amount of anticoagulant.ELISA method to determine plasma corticosterone (corticosterone, CORT) concentration. Degree. The number of peripheral blood mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) was determined by flow cytometry; CRH+/+ and CRH-/- mice were injected with different doses of glucocorticoids by microinjection pump, and the plasma corticosterone concentration in mice was detected by the above methods, and EPCs, MSCs in peripheral blood, MSCs, respectively. The relationship between the number of HSCs changes and the isolation and culture of bone marrow MSCs and EPCs and the pretreatment of 30min with the glucocorticoid receptor blocker RU486 (10 mu M) were used to observe the effects of different concentrations (0ng/ml, 75ng/ml, 100ng/ml, 1500ng/ml) on the chemotactic function and the chemotactic receptor expression changes by neuro probe chemotactic chamber method.
Results: 1. after running stress, the plasma corticosterone level of CRH+/+ mice increased significantly, while the number of peripheral blood MSCs and EPCs increased significantly, but the plasma corticosterone levels in CRH-/- mice were not significantly changed after running stress, while the number of MSCs and EPCs in peripheral blood decreased; and 2. after the injection of glucocorticoids to CRH+/+ and CRH-/- mice, The level of plasma corticosterone increased significantly, and the number of HSCs, MSCs and EPCs in peripheral blood also increased significantly; 3. low concentration of corticosterone could promote the chemotactic movement of MSCs, EPCs to fMLP; RU486 preconditioning could reverse the effect of glucocorticoid on MSCs and EPCs chemotaxis; 4. different concentrations of glucocorticoids could promote bone. Myeloid MSCs, EPCs fMLP-R mRNA expression, and low concentration of glucocorticoid can promote the expression of fMLP-R protein, which can be reversed by RU486.
Conclusion: 1. in acute stress conditions, mesenchymal stem cells and endothelial progenitor cells have a significant mobilization response. HPA axis response and its key effect hormone glucocorticoid play an important regulatory effect. 2. in stem cell mobilization, glucocorticoid can increase fMLP-R through the glucocorticoid receptor genome pathway to promote cell chemotaxis. Function.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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