【作者】 李晓峰；

【导师】 赵劲民；

【作者基本信息】 广西医科大学 ， 外科学， 2011， 博士

【摘要】 骨髓间充质干细胞(MSCs)是具有多向分化潜能的成体干细胞,逃避宿主免疫系统监测、免疫调节和体外培养易扩增等特点,使骨髓间充质干细胞在细胞治疗方面特别有应用前景。然而,移植细胞的死亡限制了组织再生。移植入器官的干细胞死亡主要机制之一由缺血引起。缺氧/局部缺血情况作为凋亡的一个重要的媒介和调节因素早已经得到了认识。这种情况下引发了活性氧簇(ROS)的大量产生。过多的ROS直接损伤了细胞膜,DNA和蛋白质,导致细胞功能的改变和丧失并引起细胞增殖抑制和诱导凋亡.芒果甙是一种四羟基吡啶的碳糖甙,属双苯吡酮类化合物。现代的药理和临床研究证明,芒果甙以及含芒果甙植物提取物具有多方面的生理和药理作用,如抗脂质过氧化反应、抗癌、抗糖尿病、抗炎、抗病毒、止咳、免疫调节等作用.应用骨髓间充质干细胞进行细胞移植,细胞的存活率低。通过在缺氧状态下,引入抗氧化剂对细胞进行抗氧化、抗凋亡的保护非常有意义。芒果苷成为有前景的抗凋亡剂,有望为提高细胞移植的干细胞存活提供理论依据及实践经验。尽管国内外对芒果苷抗氧化的研究已经积累了一些资料,但是对芒果苷对骨髓间充质干细胞缺氧损伤保护的机制研究却未见报道。本课题在体外实验的研究基础上,利用细胞和现代分子生物学等技术,通过氯化钴构建的缺氧模型,检测在体外环境中对大鼠骨髓间充质干细胞的生物学影响;通过芒果苷预处理,观察芒果苷对大鼠骨髓间充质干细胞的凋亡和氧化应激的保护作用;通过实时荧光相对定量RT-PCR和Western-blot等方法分别测定凋亡相关基因和氧化应激相关基因和蛋白表达,探讨芒果苷在缺氧模型下对大鼠骨髓间充质干细胞的保护机制。实验内容包括以下四部分:第一部分大鼠骨髓间充质干细胞的培养、鉴定及生物学性状分析目的:建立大鼠骨髓间充质干细胞的分离、培养、纯化方法,并进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。方法:通过全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,进行形态学观察,绘制生长曲线,细胞周期分析,流式细胞仪检测细胞表面标记物,分别向成骨、成脂方向诱导分化,进行染色体分析和荧光增殖示踪分析。结果:大鼠骨髓间充质干细胞生长以梭形细胞为主,呈放射状排列的细胞集落,细胞生长旺盛,可连续稳定传代10代以上。生长曲线及细胞周期显示骨髓间充质干细胞符合正常细胞生长特征且生长活跃。第3代骨髓间充质干细胞CD44,CD90,CD105均呈阳性表达,而CD34,CD45呈阴性表达。成脂、成骨诱导后,油红O染色、碱性磷酸酶染色、von Kossa法染色和茜素红染色均呈阳性。全骨髓贴壁培养法操作简单,可大量分离、纯化、扩增骨髓间充质干细胞,所获细胞具有间充质干细胞的一般生物学特性,经诱导培养后具有多向分化潜能。结论:实验所用的全骨髓贴壁法为组织工程提供充足的种子细胞来源具有重要的现实意义。第二部分芒果苷的提取、鉴定、代谢动力学及其对大鼠骨髓间充质干细胞生物学性状的影响目的:研究芒果苷的提取、鉴定、代谢动力学及其对大鼠骨髓间充质干细胞生物学性状的影响方法:通过乙醇回流法提取并经过萃取、预处理、再生、洗脱及重结晶等方法得到芒果苷提取物。对芒果苷提取物通过紫外及红外吸收,核磁共振,高效液相色谱(HPLC),鉴定提取物的化学结构和含量。通过口服给药,进行芒果苷在大鼠血液中药代动力学的评价。通过药物细胞毒性试验(MTT法)、细胞周期分析、细胞增殖荧光示踪等,检测芒果苷对大鼠骨髓间充质干细胞的生物学性状影响。结果:用乙醇回流法提取的芒果苷浓度可达98.0%和94.9%,其纯度已经可以满足药效试验的需要。高效液相色谱(法),可测量出芒果苷在小鼠血浆中的含量切实、可行。低浓度芒果苷(20-80μmol/l)能促进大鼠骨髓间充质干细胞生长,以40μmol/l芒果苷促进效果最好;而高浓度芒果苷可抑制细胞生长,具有浓度依赖性。结论:通过乙醇提取可获得高纯度芒果苷,并可通过高效液相色谱检测芒果苷含量。芒果苷对细胞生长有一定影响。第三部分芒果苷对大鼠骨髓间充质干细胞缺氧诱导的凋亡保护机制研究目的:研究芒果苷对大鼠骨髓间充质干细胞缺氧损伤的凋亡保护机制方法:建立细胞缺氧模型。通过MTT、流式细胞仪凋亡检测、流式细胞仪线粒体膜电位检测细胞凋亡率、实时荧光相对定量RT-PCR和Western-blot方法分别测定了细胞中Casapse3、Casapse8、Casapse9、Bcl-2及Bax等凋亡相关基因及蛋白表达情况。结果:芒果苷对大鼠骨髓间充质干细胞缺氧损伤具有保护作用,呈浓度依赖性;随着芒果苷浓度的增加,大鼠骨髓间充质干细胞在缺氧损伤模型下的凋亡率逐步减少(P<0.01)。氯化钴可引起Casapse3、Casapse8、Casapse9、Bax等凋亡相关基因及蛋白表达上调,Bcl-2基因及蛋白表达下调。芒果苷能抑制氯化钴引起的Casapse3、Casapse8、Casapse9、Bax等凋亡相关基因及蛋白表达的上调(P<0.01),及减少Bcl-2基因及蛋白表达的下调(P<0.01)。结论:芒果苷对大鼠骨髓间充质干细胞缺氧损伤诱发的凋亡具有保护作用,并呈浓度依赖性。第四部分芒果苷对大鼠骨髓间充质干细胞缺氧损伤的抗氧化保护机制研究目的:研究芒果苷对大鼠骨髓间充质干细胞缺氧损伤的抗氧化保护机制方法:建立细胞缺氧模型。通过紫外可见光分光光度计检测细胞内及细胞培养液中的氧化应激相关指标:超氧化物歧化酶、丙二醛、谷胱甘肽过氧化物酶、过氧化氢酶等。用流式细胞仪检测细胞活性氧含量。实时荧光相对定量RT-PCR和Western-blot方法分别测定了细胞中RelA、HIF-1α、Hsp70等氧化应激相关基因及蛋白表达情况。结果:芒果苷对大鼠骨髓间充质干细胞缺氧损伤具有保护作用,呈浓度依赖性;随着芒果苷浓度的增加,大鼠骨髓间充质干细胞在缺氧损伤模型下的氧化应激反应逐渐减少(P<0.01)。氯化钴可引起RelA、HIF-1α、Hsp70等氧化应激相关基因及蛋白表达上调。芒果苷能抑制氯化钴引起的RelA、Hsp70等氧化应激相关基因及蛋白表达的上调(P<0.01),但对HIF-1α基因的表达和蛋白表达没有影响(P)0.05)。结论:芒果苷对大鼠骨髓间充质干细胞缺氧损伤诱发的氧化应激具有保护作用,并呈浓度依赖性。更多还原

【Abstract】 Mesenchymal stem cells (MSCs) are multipotent differentiation potential of adult stem cells to evade the host immune system surveillance, immune modulation and in vitro culture and easy expansion characteristics, so that bone marrow mesenchymal stem cells in cell therapy particularly with applications. However, the death of transplanted cells limited tissue regeneration. one of the death mechanisms of Stem cells transplanted into the organ caused by ischemia.Hypoxic / ischemic conditions, as apoptosis is an important regulator of the media and has been recognized early. This situation led to a large number of reactive oxygen species (ROS) produced. Excessive ROS directly damage cell membranes, DNA and proteins, leading to the change and loss of cell function and cause growth inhibition and induction of apoptosisMangiferin is a four-hydroxypyridine carbon glycoside, a double benzene pyridine ketones. Modern pharmacological and clinical studies have shown that mangiferin and mangiferin containing various plant extracts with the physiological and pharmacological effects, such as lipid peroxidation, anti-cancer, anti-diabetic, anti-inflammatory anti-virus, stop-coughing, immune regulation.Using bone marrow mesenchymal stem cells for cell transplantation, cell viability is low. In hypoxia, the introduction of antioxidants on the cells exercise antioxidant, anti-apoptotic protection. Mangiferin as a promising anti-apoptotic agent, to enhance the survival of cells transplanted stem cells provide a theoretical basis and practical experience.Although the oxidation of mangiferin at home and abroad research has accumulated some data, the protected mechanism of mangiferin in bone marrow mesenchymal stem cells in in hypoxic injury has not been reported.The subject of the research base in vitro, using cell biology and modern molecular biology techniques, testing the effect hypoxia model induced by cobalt chloride in vitro environment on rat bone marrow mesenchymal stem cells biology.To investigate the protection mechanism of mangiferin on rat bone marrow mesenchymal stem cell. With mangiferin pretreatment, the protection of mangiferin on rat bone marrow mesenchymal stem cell in apoptosis and oxidative stress were observed;real-time fluorescence relative quantitative RT-PCR and Western-blot method s were used to detect the expression of gene and protein related to apoptosis and oxidative stress.Include the following four parts Part I Culture and identification of rat bone marrow mesenchymal stem cellsOBJECTIVE: To establish a method of isolation, cultivation and purification of rat BMSCs in vitro, to observe cell morphology, and to assess surface markers and multi-directional differentiation capacity.METHODS: BMSCs from rats were isolated, cultured and purified by the whole bone marrow adherence method, for morphology observations, the growth curve was drawn, cell cycle was analyzed, cell surface markers were assessed by flow cytometry, and BMSCs were induced to differentiate into osteoblasts and adipocytes.RESULTS: BMSCs from rats were spindle cell-based, showing radial colony arrangement. Cells kept strong growth and could passage in continuous and stable manner over 10 passages. The growth curve and cell cycle demonstrated that BMSCs were consistent with the growth characteristics and good activity of normal cells. At the third passage, BMSCs were negative for CD34 and CD45, but positive for CD44, CD90 and CD105. Following induction, oil red O staining, alkaline phosphatase staining, von Kossa staining and alizarin red staining produced a strong reaction in cells. Whole bone marrow adherence method is simple and can isolate, purify and amplify BMSCs in vitro. The obtained cells have general biological characteristics of mesenchymal stem cells, and also have potentiality of multi-directional differentiation.CONCLUSION:This experimental method has important practical significance to provide adequate source of seed cells for tissue engineering. Part II The extraction, identification, pharmacokinetics of Mangiferin and the biological characteristic effects of mangiferin on rat bone marrow mesenchymal stem cell.OBJECTIVE: To investigate the extraction, identification, pharmacokinetics of Mangiferin and the biological characteristic effects of mangiferin on rat bone marrow mesenchymal stem cell.METHODS: By ethanol extraction and after extraction, pretreatment, regeneration, washing and recrystallization, mangiferin was obtained. Through ultraviolet and Infrared absorption spectroscopy, nuclear magnetic resonance, high performance liquid chromatography (HPLC) , identified the chemical structure and content of the extract.By oral administration, the pharmacokinetics of mangiferin in rat blood was evaluated.By drug cytotoxicity test (MTT), cell cycle analysis, cell proliferation, the biological effects of mangiferin on rat bone marrow mesenchymal stem cells were detected.RESULTS:Extracted by refluxing with ethanol ,the concentrations of mangiferin can up to 98.0% and 94.9%, and the purity has meet the need for efficacy trials.High performance liquid chromatography is a effective and feasible method in measuring content of mangiferin in mouse plasma.The low concentrations of mangiferin (20-80μmol/l) can promote the growth of bone marrow mesenchymal stem cells ,to 40μmol / l mangiferin for the best. The high concentration of mangiferin inhibit cell growth, with a concentration-dependent.Conclusion: By using ethanol , the high purity of mangiferin was obtain, and by high performance liquid chromatography, mangiferin could be detected . Mangiferin have a certain influence on cell growth.Part III The protection mechanism of mangiferin in hypoxia induced apoptosis in rat bone marrow mesenchymal stem cellsOBJECTIVE:To investigate the protection mechanism of mangiferin in hypoxia induced apoptosis in rat bone marrow mesenchymal stem cells METHODS: Established hypoxia model of cells.The apoptosis rate by MTT,apoptosis detection of flow cytometry, detection of mitochondrial membrane potential of flow cytometry were observed.the differences of mRNA and protein expressions of Casapse3、Casapse8、Casapse9、Bcl-2 and Bax were assayed by real-time fluorescence relative quantitative RT-PCR and Western-blot method.RESULTS: Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of hypoxic injury in a dose dependent manner. With the increase of the concentration of mangiferin ,the apoptosis rate in rat bone marrow mesenchymal stem cells in hypoxic injury model were gradually reduce. (P<0.01).Cobalt chloride(CoCl2) can increase Casapse3, Casapse8, Casapse9, Bax gene and protein expression, decrease Bcl-2 gene and protein expression. Mangiferin inhibited cobalt chloride induced increasing of Casapse3, Casapse8, Casapse9, Bax gene and protein expression, and decreasing of Bcl-2 gene and protein expression(P<0.01).CONCLUSION:Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of hypoxic injury in a dose dependent manner.Part IV The antioxidant protection mechanism of mangiferin in rat bone marrow mesenchymal stem cells in hypoxiaOBJECTIVE:To investigate the antioxidant protection mechanism of mangiferin in hypoxia in rat bone marrow mesenchymal stem cellsMETHODS: Established hypoxia model of cells. By UV-Vis spectrophotometer , the relevant indicators of oxidative stress: superoxide dismutase(SOD), malondialdehyde(MDA), glutathione peroxidase(GSH-XP), catalase(CAT) were observed.in cells and cell culture medium. reactive oxygen species (ROS) were measured by flow cytometry.The differences of mRNA and protein expressions of RelA、HIF-1αand Hsp70 were assayed by real-time fluorescence relative quantitative RT-PCR and Western-blot method.RESULTS: Mangiferin in rat bone marrow mesenchymal stem cells have a protective effect of oxidative stress in a dose dependent manner.With the increase of the concentration of mangiferin ,the oxidative stress in rat bone marrow mesenchymal stem cells in hypoxic injury model were gradually reduce. ( P<0.01 ) .Cobalt chloride(CoCl2) can increase RelA、HIF-1αand Hsp70 gene and protein expression. Mangiferin inhibited CoCl2 induced increasing of RelA and Hsp70x gene and protein expression(P<0.01);but mangiferin can not inhibite HIF-1αgene and protein expression(P<0.05).CONCLUSION:Mangiferin have a protective effect on hypoxia-induced oxidative stress on rat bone marrow mesenchymal stem cells, in a dose dependent manner.更多还原