【作者】 刘玮；

【导师】 廖二元；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：构建MC3T3-E1细胞增殖期、基质分泌期、矿化期三个阶段甲基化表达差异谱并进行初步分析。验证甲基化芯片结果,探讨DNA甲基化对基因mRNA表达的影响,以及去甲基干预后基因mRNA表达情况的变化。方法：MC3T3-E1细胞使用含抗坏血酸和p-甘油磷酸钠(β-GP)的培基培养,分别在细胞培养的0天、10天、24天抽提RNA做逆转录,real-time PCR观察Ⅰ型胶原、骨钙素、骨桥蛋白、ALPmRNA表达情况。观察细胞形态变化,并行矿化结节染色。分别抽取培养0天、10天、24天的MC3T3-E1细胞gDNA,进行甲基化芯片杂交,获得三个不同分化阶段的DNA甲基化谱。挑选出细胞增殖期、基质分泌期、矿化期三个阶段高甲基化或低甲基化基因,获得DNA甲基化水平存在差异的基因分组。对差异基因进行染色体分布分析以及染色体定位,使用DAVID在线软件分别对各个分化阶段甲基化状态不同的基因进行功能分类。使用MassARRAY DNA甲基化定量分析技术对甲基化芯片中有差异的若干个基因进行验证。对部分基因进行real-time PCR,检测DNA甲基化对基因mRNA表达的影响。使用去甲基化药物RG108干预细胞后再进行MTT试验以及real-time PCR,与对照组相比较观察DNA甲基化水平改变对细胞增殖和基因mRNA表达情况的影响。结果：1.使用含抗坏血酸和β-GP的诱导培基培养MC3T3-E1细胞,]eal-timePCR显示Ⅰ型胶原mRNA在培养24天时较培养0天和10天表达量有显著增加；ALP培养第10天表达量最高；骨钙素、骨桥蛋白mRNA表达随着培养时间增加递增明显。细胞形态观察显示诱导培基培养0天时MC3T3-E1细胞形态近似梭形；培养10天时MC3T3-E1细胞近似“铺石路”样改变；培养22天时MC3T3-E1细胞重叠生长,并自发形成汇聚的细胞团,培养24天,Von Kossa染色可见典型矿化结节产生。根据ALP、骨钙素、骨桥蛋白等基因表达的变化将MC3T3-E1细胞的分化分为细胞增殖期(0天)、基质分泌期(10天)、矿化期(24天)三个阶段；2.提取诱导培基培养0天、10天、24天的MC3T3-E1细胞gDNA,进行甲基化芯片杂交。获得三个不同分化阶段的DNA甲基化谱。增殖期高甲基化基因50个,低甲基化基因93个,基质分泌期高甲基化基因96个,低甲基化基因43个,矿化期高甲基化基因52个,低甲基化基因147个。这些差异甲基化基因主要涉及程序性细胞死亡、转录调控、代谢、转运以及信号转导等广泛的生物学过程。MassARRAY DNA甲基化定量分析结果与甲基化芯片结果吻合。基因启动子区域DNA甲基化抑制Camk1d, Bmpr1b, Fbln7, Itga3, Pias2, Pcdha12, Mdm4, Tmem54 mRNA的表达,去甲基化药物RG108可促进细胞增殖,干预细胞后,DNA高甲基化基因mRNA表达明显增高。结论：培养0天、10天、24天的MC3T3-E1细胞可以代表其增殖期、基质分泌期、矿化期三个阶段。对此三个阶段细胞进行gDNA甲基化芯片杂交,获得了三个不同分化阶段的DNA甲基化谱。差异甲基化基因涉及广泛的生物学过程。基因启动子区域DNA甲基化负性调控成骨相关基因表达,去甲基化药物RG108可促进细胞增殖,促进DNA高甲基化基因的表达。目的：Taurine广泛分布于哺乳动物血浆以及组织中,是哺乳动物体内中最重要的游离beta-氨基酸。我们之前的研究发现Taurine可抑制破骨细胞的分化,但具体机制尚不十分清楚。本研究以成骨细胞与前破骨细胞共培养模型为研究对象,旨在探讨Taurine是否通过影响成骨细胞OPG与RANKL的表达来抑制破骨细胞的分化。方法：利用24小时内新生小鼠颅骨和6-8周龄小鼠四肢长骨分别分离,获得成骨细胞和骨髓细胞,将成骨细胞与骨髓细胞进行共培养,同时给予不同浓度Taurine干预。共培养6天后进行破骨细胞抗酒石酸酸性磷酸酶(TRACP)染色鉴定。同时采用real-time PCR检测Taurine对成骨细胞OPG和RANKL表达的影响。结果：Taurine可抑制成骨细胞／骨髓细胞共培养诱导的破骨细胞分化,且与浓度相关。然而Taurine不影响成骨细胞OPG和RANKL的表达。结论：Taurine可浓度依赖性抑制成骨细胞／骨髓细胞共培养诱导的破骨细胞分化,此作用与成骨细胞OPG和RANKL的表达无关。目的：我们之前的研究发现单核细胞系RAW264.7诱导的破骨细胞表达功能性TAUT (taurine transporter),并且Taurine可抑制其分化。但二者之间是否相关尚不清楚,在其他来源的破骨细胞的情况也尚不了解。本研究旨在探讨M-CSF和RANKL诱导骨髓来源巨噬细胞所获得的破骨细胞是否同样表达TAUT,其分化是否也可被Taurine抑制,进一步阻断TAUT后Taurine对破骨细胞的作用是否仍存在。方法：利用M-CSF和RANKL诱导骨髓来源巨噬细胞获得破骨细胞同时予不同浓度Taurine干预,TRACP染色计数破骨细胞鉴定分化情况。提取骨髓巨噬细胞来源破骨细胞的RNA,进行RT-PCR,检测TAUT mRNA在破骨细胞的表达情况。使用免疫细胞化学方法检测TAUT蛋白的表达。TAUT siRNA干扰RAW264.7细胞,Western blot检测干扰后TAUT蛋白合成的情况。同时[3H]taurine摄取实验评估干扰后TAUT蛋白的功能。予Taurine干预TAUTsiRNA干扰组和对照组,TRACP染色并计数破骨细胞再次检测Taurine对破骨细胞分化的作用。结果：M-CSF和RANKL诱导骨髓来源巨噬细胞获得破骨细胞中,有TAUT mRNA以及TAUT蛋白的表达。Taurine干预可显著抑制该来源破骨细胞的分化。siRNA干扰TAUT后,TAUT蛋白表达及功能均明显下降,并且Taurine失去对破骨细胞分化的抑制作用。结论：M-CSF、RANKL诱导骨髓来源巨噬细胞获得的破骨细胞有功能性TAUT表达,Taurine可抑制其分化。阻断TAUT表达后,Taurine失去对破骨细胞分化的抑制作用。更多还原

【Abstract】 Objective:Construction of the methylation profile of MC3T3-E1 cell line during it’s proliferation, extracellular matrix maturation and mineralization stage. Analysis of the effect of mRNA expression caused by DNA methylation and demethylation.Methods:MC3T3-E1 cell line was cultured with medium contain ascorbic acid and (3-Glycerophosphate disodium. The mRNA expression of type I collagen (Collal), osteocalcin(Ocn), osteopontin(Opn), alkaline phosphatase (ALP) after cultured for 0 day,10day,24day were validated by real-time PCR. The morphology of cell was observed and the characteristic mineralized node staining was performed. gDNA of MC3T3-E1 cell line after cultured for 0 day, lOday,24day were extracted, and methylation microarray was performed to obtain the methylation profile of the three stages. Hyper-and hypo-methylated genes were chosen for stage-differential expression analysis. Differentially expressed genes were counted according to the chromosome distribution. By using DAVID online gene function and classification tool, the differentially expressed genes were analyzed for gene function classification and annotation. Validation of the profile result with the DNA methylation quantitative analysis of MassARRAY technology were carried out. The relationship between DNA methylation and gene expression is further confirmed with Real-time PCR for the differentially expressed genes. The DNA methyltransferase inhibitor RG108 was applied to intervene the MC3T3-E1 of proliferation stage, MTT and Real-time PCR of the differentially expressed genes were followed, and control group(with out RG108) were also carried out to validate its potential relationship.Result:1.The Real-time PCR result showed Colla1 expression of day 24 significantly increased compared to day 0 and day 10 after cultured with medium contain ascorbic acid,β-Glycerophosphate disodium of MC3T3-E1 cell line. The highest ALP expression was examined during day 10.The expression of Opn and Ocn is significantly increased in time series. The morphology of MC3T3-E1 in day 0 showed its spindle-shape whereas in day 10 it showed the "pavement stone" characteristics. After cultured for 22 days the cell grew to form multiple layers. Typical mineralized node could be seen with Von Kossa Staining after day 24. The differentiation stage of MC3T3-E1 could be classified as proliferation stage(day 0), extracellular matrix maturation stage (day 10) and mineralization stage(day 24) based on the expression of Colla1, ALP, Ocn and Opn.2. gDNA of the MC3T3-E1 of 3 stages were extracted to perform DNA methylation microarray profile. The hyper-methylated genes of proliferation stage, extracellular matrix maturation stage and mineralization stage counted 50,96 and 52, respectively. The hypo-methylated genes of the 3 stage counted 93,43, and 147, respectively. These differentially expressed genes were mainly involved in biological processes such as cell death, transcription regulation, metabolism, transport and signal transduction. Camkld, Bmprlb, Fbln7, Itga3, Pias2, Pcdhal2, Mdm4, Tmem54 mRNA expression were negatively correlated with methylation statue. Under the intervention of DNA methyltransferase inhibitor RG108, the hypermethylated genes expression were significantly upregulated.Conclusion:Cultured MC3T3-E1 cell line in day 0, day 10 and day 24 could represent the stage of proliferation, extracellular matrix maturation and mineralization stage, respectively. DNA methylation microarray captured the DNA methylation profile of the 3 stages. Analysis of the profiles showed the differentially expressed hyper-and hypo-methylated genes are involved in board range of biological process and function. During the differentiation process of MC3T3-E1 cell line, The methylation of promoter region caused down-regulation of the osteogenesis-related gene. RG108, the DNA methyltransferase inhibitor, could promote the proliferation of MC3T3-E1 cell line and upregulate the expression of DNA hypermethylated genes. Objective:Taurine is widely distributed in mammalian plasma and tissues, it is the most important free beta-amino acids in mammals. Our previous studies have found that Taurine could inhibit osteoclastogenesis, but the exact mechanism is still unknown. In this study, osteoblasts and bone marrow cells were co-cultured, in order to investigate whether Taurine inhibits osteoclastogenesis by affecting the expression of OPG and RANKL in osteoblasts.Methods:Skull of 24 hour newborn mouse and long bone of 6-8week aged mouse were isolated for osteoblast and bone marrow cell, respectively.The osteoblast and bone marrow cells were then co-cultured with different concentrations of Taurine for 6 days TRACP staining were performed for validation and counting of the osteoclasts. The expression of OPG and RANKL in osteoblast were then validated by real-time PCRResults:Taurine dose-relatively inhibited osteoclastogenesis in co-culture of osteoblasts and bone marrow cells, but Taurine showed no influential effect to the expression of OPG and RANKL in osteoblasts.Conclusion:Taurine inhibits osteoclastogenesis in a dose-dependent manner in co-culture of osteoblast and bone marrow cell. This inhibitory effect had no relationship with respect of OPG and RANKL in osteoblast.Objective:Our previous study found that RAW264.7 monocyte cell line expressed functional TAUT(taurine transporter), and Taurine could inhibit its differentiation. However, the relevance between the two is not clear. In this study, mature osteoclasts were obtained from M-CSF and RANKL induced bone marrow-derived macrophages, TAUT mRNA and Protein expression was measured by RT-PCR and immunocytochemistry. TAUT was blocked and then measured the Taurine effect on osteoclasts.Methods:Mature osteoclasts were obtained from M-CSF and RANKL induced bone marrow-derived macrophages. TAUT mRNA and Protein expression was measured by RT-PCR and Immunocytochemistry. RAW264.7 is transfected with siRNA of TAUT. TAUT Protein expression was measured by western blot and TAUT function with or without siRNA interference were then measured by [3H] taurine uptake examination. TRACP staining were applied for counting the number of osteoclasts.Results:M-CSF and RANKL induced bone marrow-derived macrophages expressed TAUT mRNA and TAUT protein. Taurine intervention could significantly inhibit osteoclastogenes. while TAUT siRNA relieved this effect.Conclusion:Taurine played important role in bone homeostasis by inhibiting osteoclastogenesis directly through TAUT. Taurine lost the ability to inhibit osteoclastogenesis when TAUT expression was blocked.更多还原