营养剥夺通过BNIP3诱导Caspase-3非依赖途径介导软骨终板干细胞凋亡研究

发布时间：2018-04-17 02:32

本文选题：软骨终板干细胞 + 凋亡　；参考：《第三军医大学》2017年硕士论文

【摘要】：一、研究背景椎间盘(Intervertebral disc,IVD)是人体重要的组织,由软骨终板,髓核及纤维环三个部分组成。IVD随着年龄的增长会发生不同程度的退变,继发引起多种临床疾病如椎管狭窄、椎间盘突出和脊柱节段不稳等,给患者和社会造成极大的经济和心理负担。IVD退变是多种因素共同作用的结果,引起其发病的原因大致可分为遗传因素、年龄因素、营养及生物力学因素等。在成年人体内,IVD是一种无血管的结缔组织,其营养来源主要是依靠软骨终板和纤维环的渗透作用。相关研究证实,营养剥夺能够诱导软骨终板细胞凋亡,进而导致软骨终板退变,最终引起整个IVD退行性改变,并且在兔髓核细胞中,营养剥夺可通过上调BNIP3(Bcl-2/adenovirus E1B 19-k Da-interacting protein 3)的表达诱导细胞凋亡进而引起IVD退变。然而营养剥夺诱导软骨终板退变过程中的分子机制尚未阐明。因此,明确营养剥夺引起软骨终板退变过程的分子机制对于防治IVD退变有重要的科学意义和社会价值。二、研究目的通过模拟椎体血供及椎体外周微循环受损的环境,在体外建立营养剥夺的细胞培养模型,研究营养剥夺对软骨终板干细胞(cartilage endplate-derived stem cells,CESCs)的影响及BNIP3信号通路在其中的可能作用,从而进一步阐明IVD的退变机制。三、研究方法在临床上获取8例退变的椎间盘软骨终板标本,从中分离提取终板的软骨细胞,用纤维连结蛋白(Fibronectin,FN)筛选获取CESCs,进行干细胞表面抗原标志物鉴定后扩大培养,选取第三代的细胞进行实验,其中对照组在正常培养条件下(DMEMF12、5m M葡萄糖、21%O2、10%血清)培养,实验组在营养剥夺条件(DMEMF12、无糖、1%O2、无血清)分别培养24 h和48 h。细胞处理后,应用流式细胞仪对细胞凋亡率进行检测,实时荧光定量PCR和Western Blot分别检测BNIP3基因和蛋白表达水平的变化,免疫荧光染色检测BNIP3蛋白与CESCs线粒体的结合情况,JC-1染色法检测线粒体膜电位(mitochondrial transmembrane potential,MMP)的变化,Caspase-3活性检测试剂盒检测Caspase-3酶的活性。进一步使用BNIP3 si RNA沉默CESCs中BNIP3基因表达,转染24 h后将细胞分别在正常条件和营养剥夺条件下培养48 h,同时设置阴性干扰对照组(使用Scramble si RNA),再次对细胞凋亡率及BNIP3的表达情况进行检测。四、研究结果1.细胞鉴定结果:使用纤维链接蛋白筛选获取CESCs,并进行表面抗原标记物检测,其中CD90、CD105、CD73和CD44为阳性,而Neg CKT(包含有HLA-DR、CD19、CD34、CD11b和CD45)为阴性,结果说明:CESCs具备干细胞的特性。2.细胞凋亡率及BNIP3表达的检测结果:实验组CESCs的凋亡率(24 h:24.47%±0.97%,48 h:28.98%±0.47%)显著高于对照组(17.26%±1.84%,p0.05);BNIP3基因及蛋白表达水平实验组显著高于对照组(p0.05),结果说明:营养剥夺可上调BNIP3的表达,诱导CESCs的凋亡率增加。3.干扰BNIP3基因的表达后,再次检测细胞凋亡率及BNIP3表达情况:干扰BNIP3表达的实验组CESCs凋亡率(23.72%±1.21%)显著低于对照si RNA实验组(34.05%±1.43%,p0.05);干扰组与对照si RNA组相比,BNIP3蛋白表达明显下调,结果说明:干扰BNIP3基因能够部分抑制由营养剥夺所导致的细胞凋亡。4.免疫荧光的结果:营养剥夺能够上调BNIP3蛋白的表达,并促进其与线粒体结合,导致线粒体膜电位下降。5.Caspase-3酶活性检测:组间的Caspase-3酶活性无统计学意义(p0.05),结果说明:营养剥夺诱导细胞凋亡是通过Caspase-3非依赖性途径发挥作用。五、结论营养剥夺通过上调CESCs中BNIP3基因和蛋白的表达水平,促进BNIP3蛋白与线粒体结合导致MMP下降,最终通过Caspase-3非依赖性途径介导CESCs的凋亡。
[Abstract]:First, the research background of intervertebral disc (Intervertebral disc IVD) is an important organization of the human body, the cartilage endplate, nucleus pulposus and annulus of three parts of.IVD with age will occur in varying degrees of degeneration, secondary causes a variety of clinical conditions such as spinal stenosis, lumbar disc herniation and lumbar segmental instability, to patients and society caused great economic and psychological burden of.IVD degeneration is the result of many factors, the cause of the disease can be divided into genetic factors, age factors, nutrition and biomechanical factors. In adults, IVD is a kind of connective tissue, its nutrition is the main source of cartilage by osmosis endplate and annulus. Related research confirmed that nutritional deprivation can induce cartilage cell apoptosis, leading to cartilage endplate degeneration, causing the entire IVD degenerative changes in rabbit nucleus pulposus and fine The cell, nutrient deprivation upregulates BNIP3 (Bcl-2/adenovirus E1B 19-k Da-interacting protein 3) expression induced apoptosis and cause IVD degeneration. However the molecular mechanism of nutrient deprivation induced cartilage endplate degeneration process has not been elucidated. Therefore, nutrient deprivation leads to clear molecular mechanism of cartilage endplate degeneration process has important scientific significance and social value for the prevention of IVD degeneration. Two, the purpose of the study by simulating the blood supply of vertebral body and the vertebral peripheral microcirculation damage to the environment, the establishment of nutrient deprivation cells in vitro model, study on nutrition deprivation of cartilage stem cells (cartilage endplate-derived stem cells, CESCs) and the effect of BNIP3 signaling pathway may play a role in them, and to further elucidate the mechanism of IVD degeneration. Three, intervertebral disc endplate cartilage specimens obtained from 8 patients with degenerative research methods in clinic, separated from Cartilage endplate extraction, with fibronectin (Fibronectin, FN) were CESCs, stem cell surface antigen markers after extended culture experiment selected third generation cells, the control group under normal culture conditions (DMEMF12,5m M glucose, serum 21%O2,10%) cultured in nutrient deprived conditions (experiment group DMEMF12, 1%O2, sugar free, no serum) were cultured for 24 h and 48 h. After treatment of cells, cell apoptosis was detected by flow cytometry, real-time fluorescence quantitative PCR and Western Blot were used to detect the BNIP3 gene and protein expression level changes, combined with immunofluorescence staining was used to detect the BNIP3 protein and mitochondrial CESCs detection of mitochondrial membrane, JC-1 staining potential (mitochondrial transmembrane potential, MMP) changes, the detection of Caspase-3 enzyme Caspase-3 activity detection kit using BNIP3 Si RNA further activity. The expression of BNIP3 silencing CESCs gene in 24 h after transfection, cells were in normal condition and nutrient deprivation cultivation under the condition of 48 h, and set the negative control group (Scramble Si interference RNA), were detected again on the apoptosis rate and BNIP3 expression. Four, the results of 1. cell identification results: screening for CESCs the use of fiber link protein, and surface antigen markers, including CD90, CD105, CD73 and CD44 were positive, while Neg CKT (including HLA-DR, CD19, CD34, CD11b and CD45) was negative. The results show that: the testing results of CESCs have the characteristics of.2. cell apoptosis rate and BNIP3 expression of stem cell. The apoptosis rate of CESCs group (24 h:24.47% h:28.98% + 0.97%, 48 + 0.47%) was significantly higher than the control group (17.26% + 1.84%, P0.05); BNIP3 gene and protein expression levels in the experimental group was significantly higher than the control group (P0.05). The results show that: nutrient deprivation upregulates BNIP3 The expression of apoptosis induced by CESCs expression increased after.3. interference of BNIP3 gene, again to detect the cell apoptosis rate and the expression of BNIP3 in experimental group: CESCs apoptosis interference BNIP3 expression rate (23.72% + 1.21%) was significantly lower than the control Si RNA experimental group (34.05% + 1.43%, P0.05); interference group compared with the control of Si RNA group, the expression of BNIP3 protein was down regulated, results show that the interference of BNIP3 gene can partially inhibit the apoptosis of.4. cells by immunofluorescence caused by nutrient deprivation results: nutrient deprivation can upregulate the expression of BNIP3 protein and promote its combination with mitochondria, decreased the mitochondrial membrane potential.5.Caspase-3 activity detection: Caspase-3 activity between groups no statistical significance (P0.05). The results showed that the nutrient deprivation induced apoptosis through Caspase-3 dependent pathway. Conclusion five, nutrient deprivation by BNIP3 upregulation of CESCs gene and egg The expression level of white, which promotes the binding of BNIP3 protein to mitochondria, leads to the decrease of MMP, and ultimately mediates the apoptosis of CESCs through the Caspase-3 non dependent pathway.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5

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