高热量摄入对大鼠骨髓间充质干细胞定向分化能力影响的研究

【作者】 毕见海；

【导师】 肖苒；

【作者基本信息】 北京协和医学院 ， 外科学， 2014， 博士

【摘要】 研究背景高热量摄入在现代社会极为常见,这种饮食习惯是造成肥胖、代谢综合征、心血管疾病等多种疾病的危险因素之一。间充质干细胞(Mesenchymal Stem Cells,MSCs)是一类中胚层来源的具有高度自我更新和多向分化潜能的干细胞,参与多种组织的再生与修复过程。大量研究证实高热量饮食对组织器官病变造成显著影响,但在这一过程中,高热量饮食对在组织器官损伤修复中起重要作用的MSCs的影响仍有待进一步研究。探讨高热量摄入状态下骨髓来源间充质干细胞(Bone Marrow MSCs, BMSCs)的生物学特性与功能的改变对了解和治疗与高热量摄入相关的疾病有重要的意义。研究目的1.建立高热量摄入的大鼠动物模型,明确高热量摄入对大鼠生理生化和组织病理的影响,并对BMSCs生物学特性的变化进行评估。2.探讨高热量摄入条件下大鼠外周血中细胞因子的变化,及其对BMSCs定向分化功能的影响。3.分析高热量摄入对大鼠BMSCs基因表达谱的影响,为高热量摄入通过改变BMSCs生物学特性,进而影响相关疾病组织器官损伤修复的机理提供理论基础。研究方法1.高热量摄入大鼠动物模型的建立与鉴定正常饮食(基础饲料)与高热量饮食(基础饲料60%,蔗糖20%,猪油10%,蛋黄粉10%)喂养6周龄雌性Sprague-Dawley(SD)大鼠,在喂养不同阶段检测体重、体长,以及空腹血糖、血脂、胰岛素水平、胰岛素耐量水平等生理生化指标,并处死后切取包括肝脏、骨和脂肪等不同组织器官进行组织学观察。2.大鼠BMSCs的分离培养与鉴定在喂养不同阶段取大鼠股骨与胫骨,低糖完全培养基冲洗并打散骨髓,置于37℃、5%C02、100%饱和湿度的条件下,采用全骨髓贴壁培养法分离培养大鼠BMSCs。细胞融合达到80%-90%时,消化并传代培养。采用流式细胞仪检测所获得细胞的干细胞表面标志物、细胞的凋亡状态以及细胞周期分布状况。采用MTT法检测所获BMSCs的增殖能力,并取P3代大鼠BMSCs用诱导培养基分别进行体外成脂和成骨诱导分化,在诱导不同时间点收集细胞并提取总RNA,Real-time PCR检测相应分化标志基因的表达,并在体外诱导成脂分化14天和成骨分化21天分别进行油红0染色和茜素红染色,分析高热量摄入后大鼠BMSCs分化能力的改变。3.大鼠外周血相关细胞因子含量的检测在喂养不同阶段,取空腹12小时后大鼠尾静脉血并分离血清,采用大鼠细胞因子抗体芯片RayBio(?) Rat Cytokine Antibody Array G-Series2检测大鼠血清中相关细胞因子含量,分析高热量摄入对大鼠血清细胞因子水平的影响。4.特定细胞因子对大鼠BMSCs体外定向分化能力的影响在正常饮食喂养4月的大鼠BMSCs诱导分化培养基中添加炎症因子CINC-1和CINC-3,对高热量饮食喂养4月的大鼠BMSCs诱导分化培养基中添加促炎症因子IL-β,在成脂和成骨诱导分化不同时间点检测定向分化标志基因的表达,分析在不同细胞因子存在条件下,BMSCs定向分化能力的改变。5.大鼠BMSCs基因表达谱检测和分析TRI Reagent提取P1代大鼠BMSCs的总RNA,采用Rat Genome2302.0Array芯片,按照表达谱芯片操作规程分别进行靶标制备、芯片杂交、清洗、染色和扫描以及数据采集,用Cluster&Tree view及Scatter对数据进行初步分析；采用博奥公司在线分析系统Bio MAS (Molecule annotation system)3.0对所得数据进行Pathways和GO (Gene Ontology)分析,对高热量摄入造成的BMSCs基因表达改变和相关主要信号通路进行分析。研究结果1.高热量摄入大鼠动物模型的建立高热量饮食实验组大鼠的体重和Lee’s肥胖指数在喂养后第3个月开始明显高于正常饮食对照组。高热量饮食组大鼠自喂养早期(1月)其空腹血清中总胆固醇和胰岛素水平,以及胰岛素耐量即有明显增高,且与对照组的差异随时间延长有增加的趋势；而空腹血糖在喂养4月后高于对照组,差异有统计学意义。2.高热量摄入导致大鼠的组织病理学改变实验组大鼠多种组织有明显的病理学改变,肝脏组织学发现高热量饮食喂养1月后,实验组大鼠肝细胞中有脂滴出现,到喂养2月时有明显的肝脂肪变现象。脂肪在皮下及腹腔有异常聚集,镜下可见腹腔大网膜处脂肪组织细胞的直径从喂养1月起就明显大于对照组,并且两者差异随时间延长逐渐增加。股骨切片中发现实验组大鼠骨髓中破骨细胞数量在喂养4月时明显增加。3.大鼠BMSCs的获取与生物学特性检测全骨髓贴壁培养12-14天后,可获得原代贴壁细胞；经过两次传代培养,实验组与对照组大鼠BMSCs在形态学上并无明显差异。经流式细胞仪检测所得到的贴壁细胞,实验组与对照组中CD90+/CD31-的细胞比例均随喂养时间延长而降低,且实验组中CD90+/CD31的细胞比例从喂养1月时即低于对照组,且二者差异有统计学意义。喂养4月后的大鼠BMSCs,实验组和对照组的细胞周期和细胞凋亡未见明显差异,但实验组BMSCs的增殖能力明显下降。4.大鼠BMSCs的体外诱导分化能力比较体外成脂诱导分化过程中,对照组BMSCs成脂相关基因CEBP-a和PPAR-y的表达随月龄增加有升高的趋势；但实验组BMSCs成脂相关基因表达在喂养1月时一过性升高后,随喂养时间的继续延长,相关标志基因表达则明显低于对照组。在成骨诱导分化过程中,对照组BMSCs成骨相关标志基因表达也有随月龄增加而升高的趋势；在喂养1月时,实验组BMSCs成骨相关基因表达与对照组相比无明显差异；喂养2月时Runx2的表达明显低于对照组；喂养4月后Runx2与OCN的表达均明显低于对照组,且两组有统计学差异。BMSCs体外诱导分化后的特异性染色结果也显示,在高热量饮食喂养4月后,实验组BMSCs的成骨和成脂分化能力比对照组降低。5.大鼠外周血中相关细胞因子含量的检测细胞因子芯片结果显示,高热量饮食喂养1个月后,MIP-3α、VEGF、TIMP1、 MMP-8、p-NGF、Fractalkine、L-Selectin和LIX的水平比对照组高出20%以上,IL-1R6和PDGF-AA的表达平均值明显降低。在喂养2个月之后,实验组CINC-1、 Leptin、TNF-α、CINC-3和Prolactin-R含量比对照组增加20%或以上,IL-1R6和PDGF-AA的血清含量回升至对照组水平；而IL-1β、MIP-3α和TIMP1的血清含量比对照组降低了20%以上,β-NGF、Fractalkine和L-Selectin血清含量降低了约15%,MMP-8和VEGF含量则降低至对照组水平。喂养4月后,实验组VEGF、CINC-1、 Leptin、TNF-α、CINC-3和Prolactin-R的血清含量均比对照组高30%以上；IL-1β和IL-1R6的含量降低至对照组的65%左右。因而Leptin、CINC-1、CINC-3和Prolactin-R含量在高热量饮食喂养过程中相对稳定的增加,IL-1β持续降低。6.添加炎症因子对大鼠BMSCs体外定向分化能力的影响在体外诱导分化液中添加CINC-3能明显降低正常饮食组大鼠BMSCs的成脂分化相关标志基因的表达,但对成骨分化相关标志基因的表达水平无明显影响；CINC-1对对照组BMSCs成脂和成骨分化标志基因表达的影响均不明显；而IL-1β能明显逆转高热量饮食组BMSCs中降低的成脂分化标志基因表达,同时进一步降低其成骨分化标志基因的表达。7.大鼠BMSCs基因表达谱分析在喂养1月后,实验组和对照组BMSCs基因表达谱即有明显差异,喂养2个月时差异更加显著,发生改变的基因数量增加,主要涉及的生物学过程集中在代谢、炎症及肿瘤发生等。其中在细胞因子-细胞因子受体相互作用、JAK-STAT信号通路、MAPK信号通路等与炎症密切相关的通路有大量基因的表达发生下调,另外参与调控干细胞功能的信号通路包括Toll样受体(TLR)信号通路、Wnt信号通路、TGF-β信号通路等均有大量的基因表达发生改变。基因芯片分析进一步证实了微环境中炎症因子水平变化导致BMSCs生物学特性改变。结论1.高热量饮食干预1月后,大鼠的血脂以及胰岛素耐量即有明显升高,相应的组织器官也有明显病理学改变,而直到喂养4月后血糖水平才发生改变。该结果提示,高热量摄入引起的相关组织器官病理改变与血脂以及胰岛素耐量变化同步,且早于血糖改变。2.高热量摄入大鼠来源的BMSCs在贴壁细胞中的比例减少,且体外的增殖能力和定向分化能力降低,提示高热量摄入会导致BMSCs的生物学特性改变,进而影响组织器官再生和损伤修复。3.高热量摄入大鼠外周血炎症相关细胞因子包括Leptin、CINC-1和CINC-3的含量明显升高,IL-1β水平则显著降低；体外诱导分化时,添加CINC-3和IL-1β明显改变BMSCs成脂分化标志基因的表达；提示微环境中细胞因子含量的变化可能是BMSCs定向分化能力改变的原因之一。BMSCs基因表达谱分析结果显示与细胞因子信号传导和干细胞功能相关的通路中有大量基因表达发生改变,进一步证实细胞因子和干细胞功能之间有重要联系。本研究中关于CINC-3降低BMSCs成脂分化能力的发现为首次报道。更多还原

【Abstract】 Background:High calorie intake has been recognized as one of the important risk factors for a variety of diseases, including obesity, cardiovascular disease and metabolic syndrome. Bone marrow mesenchymal stem cells (BMSCs) are multipotent stromal cells that hold potentials to give rise to cells of diverse lineages, participating in the regeneration and reconstructing of multiple damaged tissues. A growing body of evidence suggests that high-fat-diet (HFD) can result in significant pathological changes in tissues or organs, but the influences of HFD on the biological characteristics of BMSCs remains to be elucidated. It is imperative to uncover the changes of BMSCs under the high calorie intake circumstances for the understanding and treatment of metabolic diseases.Objectives:1. To establish a high-calorie intake animal model by feeding rats with high-fat diet (HFD) and to define the effects of HFD on physiological, biochemical, and histopathology of tissues and organs in animal model, and to assess impacts of HFD on the biological characteristics of BMSCs.2. To study the cytokine profiles of peripheral blood of rats under HFD feeding, and to detect the effects of specified cytokines on the differentiation potentials of BMSCs.3. To explore the effects of HFD on gene expression profiles of rats BMSCs so as to provide a theoretical basis for understanding the effects of HFD on BMSCs which might give further impacts on regeneration and reconstructing of multiple damaged tissues in metabolic diseases.Methods:1. Construction of rats animal models with high-calorie intakeNormal diet (ND, basal diet) and customized HFD (60%of basal diet,20%sucrose,10%lard and10%egg yolk powder) were used to feed6-week-old female Sprague-Dawley (SD) rats respectively. Studies on rat physiological and biochemical indicators (including body weight, body length, fasting blood glucose, blood lipid and insulin levels, insulin tolerance levels) and histopathological changes in the corresponding organs were performed at different feeding time points.2. Isolation and characterization of rat BMSCs in vitroBMSCs were isolated by whole bone marrow adherent method and maintained in complete medium. Cells were cultured in the incubator with the atmosphere of5%CO2and100%humidity. Cells were digested and passaged when they reached80%to90%confluency. The expression of MSC cell surface markers, percentage of apoptotic cells and distribution of cells in different phases of cell cycle of harvested adherent cells were assessed by flow cytometry. In addition, proliferation ability of BMSCs was detected by MTT assay. Total mRNA of BMSCs at specified time points was collected using TRI Reagent and real-time PCR was used to detect the expressions of relevant marker genes. BMSCs at P3were subjected to osteogenic and adipogenic differentiation in vitro. Real-time PCR analysis, Alizarin Red S staining and Oil Red O staining were performed to detect the osteogenic and adipogenic abilities of BMSCs.3. Analysis of relevant cytokines in rat serumIn order to study the impacts of HFD on the inflammatory cytokines profiles, the fasting serum was collected from tail vein of rats after1,2or4months of feeding and the levels of the relevant cytokines were measured according to the protocol of the RayBio(?) Rat Cytokine Antibody Array G-Series2.4. Impacts of the specified cytokines on the differentiation ability of BMSCs in vitroIn vitro adipogenesis and osteogenesis of BMSCs isolated from4-month HFD or ND fed rats were performed. CINC-1or CINC-3was supplemented in differentiation medium of ND BMSCs while IL-1β was added to the induction medium of HFD BMSCs during adipogenic and osteogenic differentiation in vitro. After induced differentiation, expressions of marker genes were analyzed to assess the impacts of cytokines on the differentiation potentials of BMSCs.5. Detection and analysis of gene expression profiles of BMSCsThe total mRNA of BMSCs (P1) was purified and analyzed by the Rat Genome2302.0Array (Affymetrix). The targets were prepared and hybridization followed by cleaning, dyeing and scanning was operated according to manufacturers’ instructions. Data were collected by GeneChip(?) Scanner3000and analyzed by using Bio MAS (Molecule annotation system)3.0software (CapitalBio Corporation, China). Results1. Establishment of rat model with high-calorie intakeHFD changes the physiological and biochemical properties of rats. Body mass and Lee’s obesity index of the HFD rats were significantly higher than that of ND rats after3months of feeding. The fasting total cholesterol, fasting serum insulin levels and insulin tolerance of HFD rats were significantly increased since the1st month, and the difference between both groups increased in a time-dependent manner. Interestingly, the fasting blood glucose of rats in both groups didn’t show significant differences until feeding for4months.2. HFD changed the histopathology of ratMultiple tissues of HFD rats showed obvious pathological changes. Liver biopsy demonstrated that lipid droplets were presented in the hepatocytes of1month HFD fed rat and the hepatic steatosis became more serious at2nd month. Abnormal accumulation of fat in subcutaneous and abdominal cavity was observed. The diameters of omental fat cells were significantly increased than that of control group after feeding for1month onwards, and the difference between two groups was gradually increased in a time-dependent manner. The number of osteoclasts in the bone marrow of femur in HFD rats was significantly increased when compared with ND rats after feeding for4months.3. Isolation and characterization of rat BMSCsThe adherent cells were obtained after adherent culture of12to14days and the morphologies of BMSCs isolated from HFD and ND rats were similar after two passages. Percentages of CD31-/CD90+cells in HFD and ND rats decreased along with the feeding time while the percentage of CD31-/CD90+cells in HFD rats was significantly lower than in ND rats after one month feeding. After feeding for4months, there was no significant difference in the apoptosis and cell cycle profiles of HFD and ND BMSCs, but the proliferation abilities of HFD BMSCs were dramatically decreased than ND BMSCs.4. Comparison of the differentiation abilities of rat BMSCs in vitroDuring the adipogenic differentiation, the expression of adipogenic markers in BMSCs in HFD group were significantly higher than those from ND group after1month of feeding, but rapidly decreased after2months. For osteogenic differentiation, there was no significant difference in the expressions of Runx2mRNA between HFD and ND BMSCs after feeding for2months. After feeding for4months, both the expressions of osteogenic genes expression were significantly decreased in HFD BMSCs than in ND BMSCs, suggesting that the capacities of differentiation potentials of rat BMSCs were significantly inhibited.5. Evaluation of cytokines levels in peripheral blood serum of ratsResults of cytokine antibody microarray showed that after HFD feeding for1month, levels of MIP-3a, VEGF, TIMP1, MMP-8, beta-NGF, Fractalkine, L-Selectin and LIX in HFD rats showed20%increase than in ND rats while the levels of IL-1R6and PDGF-AA were significantly reduced. After feeding for2months, levels of CINC-1, leptin, TNF-alpha, CINC-3and Prolactin-R in HFD rats were increased by20%or more when compared to ND rats while serum levels of IL-1R6and PDGF-AA went back to the same levels as ND rats. Levels of IL-1β, MIP-3a and TIMP1in HFD rats were20%lower than that in ND rats; the serum levels of beta-NGF, Fractalkine and L-Selectin in HFD rats were decreased by about15%while MMP-8and VEGF levels returned to the same level as control group. After feeding for4months, levels of VEGF, CINC-1, leptin, TNF-alpha, CINC-3and Prolactin-R in serum of HFD rat were higher than that in ND rats for more than30%while level of IL-1β continued to decrease to less than65%of ND rats.6. Effect of CINC-1, CINC-3and IL-1β on the differentiation ability of rat BMSCs in vitroSupplement of CINC-3in the differentiation medium could significantly reduce the mRNA expression of adipogenic markers of ND BMSCs, but showed no influence in the expressions of osteogenic differentiation marker genes. No significant effect of CINC-1on the expressions of adipogenic or osteogenic marker gene in ND BMSCs was observed. Supplementation of IL-1β could rescue the inhibited mRNA expressions of adipogenic markers but further inhibited the expressions of osteogenic markers in HFD BMSCs.7. Evaluation and analysis of the gene expression profiles of BMSCsAfter1month of feeding, the gene expression profiles of HFD BMSCs were significantly different from ND BMSCs. The differences of BMSCs between two groups were more pronounced after feeding for2months. Genes with significant changes were mainly involved in metabolism and inflammation signal pathways, including cytokines and cytokines receptor interaction, JAK-STAT signaling and MAPK signaling; as well as the pathways that regulated the functions of BMSCs, such as Toll-like signaling, Wnt signaling and TGF-1β signaling. The results of microarray analysis further confirmed that the changes in the serum levels of inflammatory cytokines could change the biological characteristics of BMSCs.Conclusion1. After1month of HFD intervention, blood lipids and insulin resistance were significantly increased and the histopathologies of corresponding organs were also changes. However, the blood glucose level of HFD rats did not change until feeding for4months, suggesting that the pathological changes of corresponding organs were synchronous with the changes in the blood lipids and insulin tolerance, and occurred earlier than the changes of blood glucose. These findings indicated that multiple clinical scenarios rather than blood glucose alone should be tested for early diagnosis of metabolic diseases in future.2. The BMSCs isolated from HFD rats showed reduced percentages in adherent cell population, decreased proliferation and differentiation abilities in vitro, suggesting that HFD may affect the biological characteristics of BMSCs, and thus caused impacts on the regeneration and reconstruction ability of tissues and organs in organism.3. HFD modified serum levels of inflammatory cytokines in rats, of which leptin, CINC-1and CINC-3were significantly elevated while IL-1β were reduced. Supplementation of CINC-3and IL-1β in induced differentiation culture of BMSCs in vitro significantly changed the expression of differentiation-related marker genes, indicating that changes of inflammatory cytokines in the microenvironment may be one of the factors that resulted in the change of the differentiation abilities of BMSCs. The results of gene expression microarray analysis demonstrated that a large number of genes involved in pathways that regulate cytokine signaling or stem cell functions were down or up-regulated, suggesting a correlation between cytokines and biological properties of BMSCs. In this study, we firstly reported that CINC-3could inhibit the adipogenic differentiation ability of BMSCs.更多还原