本文關鍵詞：體外培養(yǎng)中CSA對缺氧復氧所致MSCs凋亡的影響和機制初探　出處：《浙江大學》2007年碩士論文　論文類型：學位論文

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【摘要】： 骨髓間充質干細胞(MSCs)是組織工程研究中普遍采用的種子細胞。于1867年德國病理學家Cohnheim首次提出骨髓中存在MSCs，隨研究進一步深入，人們發(fā)現(xiàn)這種細胞能分化為多種間充質細胞并形成骨、軟骨、肌腱、脂肪等組織，甚至可被誘導形成心肌細胞，，加之其易于獲取、具有免疫耐受性、能長期存活、易于轉染并能長期表達外源基因等特點，干細胞應用研究日益引起科學家們的關注。目前MSCs在臨床上已開始實驗性用于心臟修復，但由于局部微環(huán)境促凋亡因素的存在使其在移植后不可避免出現(xiàn)凋亡。而已有許多基礎實驗和臨床研究證實環(huán)胞霉素A(CSA)可以減少多個細胞系發(fā)生的凋亡，CSA對MSCs是否也具有相近的作用目前尚不得而知。本實驗采取不同濃度CSA處理大鼠MSCs后，在體外環(huán)境中給予缺氧再復氧后應用多個方法檢測其凋亡的情況并初步測定部分有關的凋亡通路上關鍵因子初步解釋其可能的抗凋亡機制。 方法：采用密度梯度離心法分離獲取SD大鼠的MSCs，再以貼壁篩選法純化MSCs。取傳代后第三代細胞進行流式細胞學鑒定，進入實驗。先采取不同濃度(0.5-5μmol/L)CSA對MSCs進行預處理，實驗組根據CSA濃度分為(0uM CSA組，0.5uM CSA組，1uM CSA組，2.5uM CSA組，5uM CSA組)以及常規(guī)培養(yǎng)組。然后實驗組在體外環(huán)境中給予缺氧再復氧(H/R)后和常規(guī)培養(yǎng)組應用多個方法檢測其凋亡的情況，并檢測細胞色素C的釋放和caspase-3的活性，線粒體膜電位(△ψm)的變化，以及AIF的轉位等。 結果：在缺氧復氧處理后的MSCs出現(xiàn)了CSA(0.5-5μmol/L)劑量依賴下的凋亡減少。其可能的一個途徑就是通過直接或間接對抑制線粒體細胞色素C的釋放和AIF的轉位從而穩(wěn)定△ψm，控制胞漿caspase-3的活性來達到抗凋亡的作用。 結論：CSA在體外培養(yǎng)下可能通過直接或間接抑制線粒體細胞色素C的釋放和AIF的轉位從而穩(wěn)定△ψm并進一步影響胞漿caspase-3的活性來減少對缺氧再復氧培養(yǎng)所誘發(fā)MSCs的凋亡。
[Abstract]:Bone marrow mesenchymal stem cells (MSCs) is widely used as seed cells in the tissue engineering. In 1867 the German pathologist Cohnheim MSCs first proposed the existence of the bone marrow, with further research, people found that the cells can differentiate into multiple mesenchymal cells and the formation of bone, cartilage, tendon, fat, even by inducing the formation of myocardial cells, and it is easy to get, with immune tolerance, which can survive for a long time, easy transfection and long-term expression of exogenous gene characteristics of stem cells research has attracted the attention of scientists. The MSCs in clinical experiments have been started for cardiac repair, but due to the existence of local micro environment and promote apoptosis the factors in the inevitable emergence of apoptosis after transplantation. But there are many basic experiments and clinical studies confirmed that cyclosporin A (CSA) can reduce apoptosis in multiple cell lines occurred, CSA to MSCs Whether it has the same effect. This experiment is still can make nothing of it take different concentrations of CSA after MSCs in rats in vitro to hypoxia reoxygenation after application of multiple methods to detect the apoptosis and preliminary determination of key factor related apoptosis pathway on the part of preliminary interpretation of the possible mechanism of anti apoptosis.
Methods: using density gradient centrifugation to obtain SD rats MSCs, with adherence screening method for purification of MSCs. from cultured cells of the third generation of flow cytometric identification into the experiment. First take different concentration (0.5-5 mol/L) CSA pretreatment on MSCs, the experimental group was divided into 0uM (according to CSA CSA 0.5uM group, CSA group, 1uM CSA group, 2.5uM CSA group, 5uM CSA group) and the conventional culture group. Then the experimental group in vitro to hypoxia reoxygenation (H/R) and conventional culture group application of multiple methods to detect the apoptosis, and the detection of cytochrome C release and caspase-3 the activity of mitochondrial membrane potential (Delta Psi m) changes, and the translocation of AIF.
Results: in hypoxia treated MSCs CSA (0.5-5 mol/L) the dose dependent decrease of apoptosis. A possible way is through direct or indirect inhibition of mitochondrial cytochrome C release and AIF translocation to stable Delta Psi m, active control of cytoplasmic caspase-3 to resist the role of apoptosis.
Conclusion: CSA can directly or indirectly inhibit the release of cytochrome C and the translocation of AIF and m and further affect the stability of delta psi cytosolic caspase-3 activity to reduce the apoptosis of hypoxia reoxygenation induced by MSCs cultured in vitro.

【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329.2

【參考文獻】

相關期刊論文 前1條

1 陳運賢,歐瑞明,鐘雪云,徐新,趙洪云,管慧紅,陸英,韓忠朝;自體骨髓干細胞原位移植治療急性心肌梗死的臨床研究[J];中國病理生理雜志;2003年04期

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