本文選題：HPA軸 + 間充質(zhì)干細胞(MSCs)��； 參考：《第三軍醫(yī)大學》2012年碩士論文

【摘要】：全球范圍內(nèi)由創(chuàng)傷引起的死亡人數(shù)持續(xù)升高，，預計到2020年會達到840萬，而嚴重組織損傷是致死致殘的關(guān)鍵因素，因此尋找早期有效修復損傷組織、預防和控制創(chuàng)傷并發(fā)癥的治療方法是提高嚴重創(chuàng)傷患者生存率、改善預后的關(guān)鍵。近年來，成體干細胞為嚴重損傷組織的修復帶來新的曙光。在成年個體中，定植于骨髓中的干細胞可以動員并向損傷組織遷移，作為種子細胞直接參與損傷組織的修復。此外，成體干細胞還具有免疫調(diào)節(jié)作用，如間充質(zhì)干細胞(mesenchymal stem cells, MSCs)可以抑制T、B、NK細胞的增殖和B細胞的遷移；抑制TH1細胞分泌促炎細胞因子，促進TH2細胞產(chǎn)生抗炎細胞因子等。雖然外源性干細胞移植在損傷組織治療中具有重要價值，然而這種方法還有諸多倫理和技術(shù)瓶頸需要面對，如干細胞體外擴增、鑒定及其在體內(nèi)的轉(zhuǎn)歸等等。損傷組織修復需要骨髓干細胞的動員，并作為種子細胞遷移到損傷部位。但是目前臨床上所用的干細胞動員劑具有很多副作用，近30%的病人會出現(xiàn)骨痛、頭痛、四肢乏力等癥狀，嚴重者甚至發(fā)生急性肺損傷，部分伴有基礎(chǔ)疾患的創(chuàng)傷患者對干細胞動員劑不敏感。因此，探索內(nèi)源性干細胞動員機制具有重要科學意義和臨床價值。 近年發(fā)現(xiàn)，神經(jīng)內(nèi)分泌反應對骨髓干細胞動員、增殖、分化和修復功能有潛在調(diào)控作用，如交感腎上腺髓質(zhì)和其他內(nèi)分泌器官源性激素(如去甲腎上腺素、雌激素)可以促進不同類型骨髓干細胞從骨髓池外流。鑒于神經(jīng)內(nèi)分泌反應對于骨髓干細胞數(shù)量的影響，以及效應激素在損傷微環(huán)境中的重要作用，我們推測下丘腦-垂體-腎上腺(hypothalamus-pituitary-adrenal，HPA)軸激活可能是下丘腦-骨髓-外周血三位一體的干細胞動員策略的關(guān)鍵環(huán)節(jié)。因此，本研究以HPA軸及其關(guān)鍵效應激素－糖皮質(zhì)激素為切入點，探討其對骨髓干細胞(HSCs、MSCs、EPCs)動員的影響及作用機制，進而初步探討神經(jīng)內(nèi)分泌反應對骨髓干細胞的動員及后續(xù)損傷組織修復和再生的影響，旨在從骨髓干細胞動員角度為損傷組織修復另辟蹊徑，從而更好地促進損傷組織實現(xiàn)功能修復。 方法：繁育并鑒定CRH野生型(CRH+/+)小鼠和CRH基因敲除(CRH-/-)小鼠；將CRH野生型(CRH+/+)小鼠和CRH基因敲除(CRH-/-)小鼠分為強迫跑步應激組和對照組。應激后1h，通過摘眼球取血法獲取足量抗凝血。ELISA法測定血漿皮質(zhì)酮(corticosterone，CORT)濃度。外周血間充質(zhì)干細胞(MSCs)、內(nèi)皮祖細胞(EPCs)和造血干細胞(HSCs)的數(shù)量通過流式細胞分析法確定；利用微量注射泵對CRH+/+和CRH-/-小鼠分別輸注不同劑量的糖皮質(zhì)激素，以上述方法分別檢測小鼠血漿皮質(zhì)酮濃度，及其與外周血EPCs、MSCs和HSCs數(shù)量變化的關(guān)系；分離培養(yǎng)骨髓MSCs和EPCs，用糖皮質(zhì)受體阻斷劑RU486(10μM)預處理30min，通過neuro probe趨化小室法觀察不同濃度(0ng/ml、75ng/ml、100ng/ml、1500ng/ml)的糖皮質(zhì)激素對MSC、EPC趨化功能的影響及其趨化受體fMLP-R表達變化。 結(jié)果：1.跑步應激后CRH+/+小鼠血漿皮質(zhì)酮水平明顯升高，同時其外周血MSCs、EPCs的數(shù)量也顯著增加；但CRH-/-小鼠血漿皮質(zhì)酮水平在跑步應激后沒有明顯變化，同時其外周血MSCs和EPCs的數(shù)量減少；2.給CRH+/+和CRH-/-小鼠注射糖皮質(zhì)激素后，其血漿皮質(zhì)酮水平都顯著增加，同時外周血HSCs、MSCs、EPCs的數(shù)量也顯著升高；3.低濃度皮質(zhì)酮可以促進骨髓MSCs、EPCs向fMLP的趨化運動；RU486預處理可以逆轉(zhuǎn)糖皮質(zhì)激素對MSCs和EPCs趨化功能的影響；4.不同濃度的糖皮質(zhì)激素均可以促進骨髓MSCs、EPCs fMLP-R mRNA表達，且低濃度的糖皮質(zhì)激素可以促進fMLP-R蛋白的表達，這種作用可被RU486逆轉(zhuǎn)。 結(jié)論：1.急性應激條件下，間充質(zhì)干細胞和內(nèi)皮祖細胞發(fā)生顯著的動員反應，HPA軸反應及其關(guān)鍵效應激素－糖皮質(zhì)激素發(fā)揮了重要調(diào)節(jié)效應；2.在干細胞動員中，糖皮質(zhì)激素可通過上調(diào)fMLP-R，經(jīng)糖皮質(zhì)激素受體基因組途徑促進細胞趨化功能。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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