p75NTR敲除小鼠外胚间充质干细胞矿化能力的研究

发布时间：2018-03-19 14:03

  本文选题：p75神经营养因子受体　切入点：敲除　出处：《第三军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景牙齿缺失是人一生中常见的口腔问题,牙齿缺失不仅会对人体健康产生影响,同时还会对患者面部美观、心理健康产生影响。传统的牙齿缺失修复方式即为义齿修复,但其在功能及感觉方面,都无法与天然牙相媲美。因而组织工程牙齿无疑被认为是将来解决牙齿缺失最为理想的方式。颅神经嵴源性的外胚间充质干细胞(ectomesenchymal stem cells,EMSCs)被认为是除牙釉质外所有牙齿组织始祖生成细胞。本课题组前期研究发现,处于牙胚钟状期的EMSCs较其他时期的EMSCs具有更好的体外矿化能力。p75NTR(p75 neurotrophin receptor,神经营养因子受体)为神经营养因子低亲和力受体,不仅是颅神经嵴源性外胚间充质干细胞的经典表面标记物,同时还参与了细胞增殖、迁移、分化、生存、凋亡等生物学过程的调控。p75NTR参与神经系统的发生发育中的调控作用已被充分探究,但其参与神经系统以外组织(如肝脏、肌肉、脂肪、牙齿等)的发生发育过程的研究却仍处于起步阶段。尤其是在牙齿发育中的可能的调控作用鲜有报道。本研究借助于来源于美国Jackson实验室的p75NTR敲除小鼠,利用E18.5d处于牙胚钟状期的胚胎进行p75NTR(-/-)EMSCs及p75NTR(+/+)EMSCs的矿化分化能力差异的比较,探究p75NTR敲除对EMSCs矿化能力的影响,为进一步明确p75NTR影响EMSCs矿化发育可能的分子机制,揭示牙齿发生发育的分子生物学机制提供理论依据及实验支持。研究内容1、E18.5d颌突EMSCs的获取、培养、基因型鉴定和生物学特性检测将p75NTR(-/+)雌性小鼠及p75NTR(-/+)雄性小鼠配笼,常规饲养,小鼠从见阴栓午间为胚胎0.5d,并于孕18.5d时行2%戊巴比妥钠(50mg/kg)小鼠腹腔麻醉注射,5-10分钟后行腹腔手术,无菌条件下取出胚胎,于生物安全柜中PBS冲洗并切取颌突组织,尽量切碎组织使其为0.1mm*0.1mm*0.1mm的组织块,1%Ⅰ型胶原酶消化,中和离心,去除上清后加入约2ml含10%胎牛血清、1%青霉素、链霉素的DMEMF12培养基,重悬于六孔板中,放置于含5%CO2的37℃孵育箱中培养。切取胎鼠的尾巴及四肢组织行基因型鉴定。并分别从细胞形态学、流式细胞检测细胞表面抗原及细胞周期等方面对两种基因型EMSCs进行检测、鉴定。2、E18.5d p75NTR(-/-)和p75NTR(+/+)EMSCs矿化能力的检测分别对p75NTR(-/-)和p75NTR(+/+)EMSCs进行体外矿化诱导,以检测其矿化分化能力的差异。矿化诱导液为10-8M地塞米松、50 g/L抗坏血酸、100ml/L胎牛血清及10 mmol/Lβ甘油磷酸钠配制的α-MEM培养液。每3天换一次液。分别矿化诱导第7天行ALP染色,第21天行茜素红染色,并分别收集诱导第7天、14天、21天的细胞RNA,以检测矿化相关基因ALP、Col I随矿化诱导的变化,明确p75NTR(-/-)和p75NTR(+/+)EMSCs体外矿化诱导能力的差异。3、p75NTR(-/-)和p75NTR(+/+)小鼠骨矿化发育情况的研究选取同窝p75NTR(-/-)小鼠及p75NTR(+/+)小鼠,常规饲养至4个月。挑选雄性小鼠,以50mg/kg的2%戊巴比妥钠行腹腔注射麻醉后,脱颈处死。剥脱分离左侧股骨,放于4%多聚甲醛中固定组织,借助Micro-CT(viva ct40 Scanco Medical AG;Brüttisellen,Switzerland)进行扫描,并利用其配套软件后期进行三维图像重建及数据分析;选取同窝p75NTR(-/-)小鼠及p75NTR(+/+)雄性小鼠,常规饲养40天时行钙黄绿素荧光标记实验,即每间隔5天注射钙黄绿素(25mg/kg,sigma),共4次,并于最后一次腹腔注射2天后脱颈处死小鼠,剥脱分离出小鼠左侧股骨,行硬组织切片,并观察计算荧光带之间的距离,计算出每种小鼠股骨各自的平均每天矿化沉积率(mineral apposition rate,MAR),单位为um/d;提取4周龄大p75NTR(-/-)和p75NTR(+/+)小鼠股骨总RNA及蛋白质,通过RT-PCR和Western blot法检测矿化相关基因ALP、Runx2的表达水平。结果1、(1)切除颌突组织后剩余组织进行基因型鉴定,结果在280bp处单条带者为p75NTR(-/-),345bp处单条带者为p75NTR(+/+),在280bp、345bp处双条带者为p75NTR(-/+)。(2)E18.5d的p75NTR(-/-)和p75NTR(+/+)EMSCs分别从相应的p75NTR(-/-)和p75NTR(+/+)胚胎颌突组织中获取,两种基因型EMSCs在细胞形态上均为明显的长梭形的成纤维细胞形态,无明显差异。(3)流式细胞检测细胞表面抗原检测结果:p75NTR(-/-)EMSCs的CD14(99.07%)、CD146(95.79%)、CD166(95.79%);p75NTR(+/+)EMSCs的CD14(97.57%)、CD146(96.32%)、CD166(97.90%)。两种基因型细胞的CD45均为阴性表达结果,分别为0.28%和0.50%。(4)细胞周期检测结果显示p75NTR(-/-)EMSCs的G1期(77.65%)、G2期(17.01%)、S期(7.56%);p75NTR(+/+)EMSCs的G1期(79.25%)、G2期(15.27%)、S期(8.90%),二者之间细胞周期无明显差异。2、(1)碱性磷酸酶染色结果:矿化诱导7d后,p75NTR(+/+)EMSCs组染色较p75NTR(-/-)EMSCs组深。(2)茜素红染色结果:矿化诱导21天后,各组均有矿化结节形成,p75NTR(+/+)EMSCs组染色较p75NTR(-/-)EMSCs组深,且矿化结节量多。(3)RT-PCR结果:矿化诱导7天、14天、21天后,各组提取总RNA,逆转录后进行PCR反应,结果发现矿化诱导7天、14天和21天时均为p75NTR(+/+)EMSCs组ALP表达水平明显高于p75NTR(-/-)EMSCs组(P�d0.05);Col I也表现为相同结果(P�d0.05)。3、(1)Micro-CT扫描4月龄大同窝p75NTR(-/-)和p75NTR(+/+)小鼠左侧股骨,扫描后经三维重建及数据分析结果:松质骨的BV(骨量),BV/TV(骨体积分数),Tb.N(骨小梁数),Tb.Th(骨小梁厚度),均为p75NTR(-/-)小鼠较p75NTR(+/+)小鼠为低,而BS/BV(骨表面积/骨量),Tb.Sp(骨小梁分离度),则为p75NTR(-/-)组小鼠高于p75NTR(+/+)组小鼠;同段股骨皮质骨的分析结果:Ct.TBTh(骨皮质厚度)及Ct.BV(骨皮质骨量)均为p75NTR(-/-)组小鼠较p75NTR(+/+)小鼠组为低,但Ct.BS/BV(骨表面积/骨量)则为p75NTR(-/-)组小鼠高于p75NTR(+/+)小鼠组。(2)钙黄绿素荧光标记检测结果显示p75NTR(-/-)组小鼠股骨的平均矿化速率明显低于同窝p75NTR(+/+)组小鼠(P0.01);(3)PCR及Western blot结果显示p75NTR(-/-)组小鼠的ALP、Runx2基因表达水平明显较p75NTR(+/+)组小鼠组低(p0.01)。结论1、通过两种基因型EMSCs的培养及矿化能力检测,发现p75NTR参与了小鼠外胚间充干细胞EMSCs的矿化分化能力的调控,并且p75NTR敲除对EMSCs的矿化分化能力存在负性调控作用;两种基因型细胞之间细胞周期无明显差异,说明p75NTR敲除后对EMSCs矿化分化能力负性调控作用的实现并不涉及细胞增殖能力、凋亡情况的改变。2、p75NTR敲除小鼠不表达具有结合NGF能力的p75NTR,Micro-CT检测、钙黄绿素荧光标记实验、RT-PCR及Western blot检测发现p75NTR(-/-)组小鼠股骨矿化形成能力明显低于p75NTR(+/+)小鼠组,因此我们初步猜测p75NTR对小鼠骨的矿化形成能力的调控是通过结合NGF实现的。
[Abstract]:The research background of missing teeth is oral common problems in life, tooth loss will not only affect human health, but also in patients with facial appearance, affect mental health. The traditional way of repairing missing teeth for denture, but its function and feeling, and are not comparable. Thus the organization of natural teeth the project is considered to be the solution to the teeth is the most ideal way of missing teeth. Embryo cranial neural crest derived mesenchymal stem cells (ectomesenchymal stem cells, EMSCs) is considered in addition to all teeth group ancestor enamel producing cells. Previous studies showed that EMSCs in the bell stage of tooth germ compared with other periods of EMSCs has better in vitro mineralization ability of.P75NTR (p75 neurotrophin receptor, neurotrophic factor receptor) is a low affinity receptor of neurotrophic factor, not only is the cranial nerve Crest derived classical ectomesenchymal stem cell surface markers, and is also involved in cell proliferation, migration, differentiation, survival, apoptosis and regulation of.P75NTR in the development of nervous system in the development of regulation has been fully explored, but its role in the nervous system outside the organization (such as the liver, muscle fat, teeth etc.) occur during the development of the research is still in its infancy stage. Especially in tooth development possible regulation is rarely reported. This study with the help from the United States Jackson laboratory p75NTR knockout mice, the use of E18.5d in tooth germ bell stage embryos were p75NTR and EMSCs (- / -) p75NTR (+ / +) differences in mineralization differentiation ability of EMSCs, explore the effect of p75NTR knockout on EMSCs mineralization, to further clarify the effect of p75NTR EMSCs mineralization of the molecular mechanism of the occurrence and development of the teeth, revealing To provide a theoretical basis and experimental support for molecular biology mechanism. 1 research contents, acquisition, training E18.5d gnathic EMSCs, genotype identification and biological characteristics of p75NTR (- / +) mice and p75NTR (- / +) male mice with cage, conventional breeding, mice from the vaginal plug at noon for embryonic 0.5d, and 18.5d in pregnancy by 2% pentobarbital sodium (50mg/kg) intraperitoneal anesthesia injection, 5-10 minutes after abdominal surgery, embryos removed under sterile conditions, in a biological safety cabinet PBS rinse and cut Gnathism tissue, try to make it for the organization organization chop block 0.1mm*0.1mm*0.1mm, 1% of type I collagenase digestion and centrifugation, the supernatant was added about removal 2ml containing 10% fetal bovine serum, 1% DMEMF12 penicillin, streptomycin medium and resuspended in 6-well plates, placed in 5%CO2 containing 37 C were incubated in the incubator. Cut tail and limbs organization for genotype identification of fetal rat. And from the cell Morphology, flow cytometry, cell surface antigen and cell cycle was detected in two genotypes of EMSCs, identification of.2, E18.5d and p75NTR (- / -) p75NTR (+ / +) EMSCs mineralization ability of detection respectively for p75NTR (- / -) and p75NTR (+ / +) EMSCs in vitro induced mineralization, to detect differences in the mineralization differentiation ability. Mineralized induced liquid 10-8M dexamethasone, 50 g/L ascorbic acid, alpha -MEM 100ml/L fetal bovine serum and 10 mmol/L beta glycerophosphate mixed culture liquid. Once every 3 days. Seventh days were mineralized induced liquid ALP staining, alizarin red staining and twenty-first days, were collected and cultured for seventh days, 14 days 21 days, RNA cells, ALP gene related to detect the changes of Col I mineralization, with mineralization induction, clear p75NTR (- / -) and p75NTR (+ / +) between.3 EMSCs in vitro induced mineralization ability, p75NTR (- / -) and p75NTR (+ / +) on bone mineralization and development of mice from the same litter P75NTR (- / -) mice and p75NTR (+ / +) mice, conventional breeding to 4 months. Choose male mice, with 50mg/kg anesthesia with 2% sodium pentobarbital intraperitoneal injection after sacrificed. Stripping separation of the left femur, placed in 4% poly formaldehyde fixed tissues, using Micro-CT (Viva ct40 Scanco Medical AG Br; ttisellen, Switzerland) were scanned, and the use of the software later analysis of 3D image reconstruction and data from the same litter; p75NTR (- / -) mice and p75NTR (+ / +) mice were fed for 40 days, the conventional calcein fluorescence labeling experiments, i.e. every 5 days after injection of calcein (25mg/kg, sigma), a total of 4 times, and in the last 2 days after intraperitoneal injection of sacrificed mice, stripping the isolated mouse left femur, for hard tissue slices, and calculate the distance between the observed fluorescence band, calculated each mouse femoral average daily mineralization of respective rate (mineral apposition rate,MAR),鍗曚綅涓簎m/d;鎻愬彇4鍛ㄩ緞澶�p75NTR(-/-)鍜宲75NTR(+/+)灏忛紶鑲￠�ㄦ�籖NA鍙婅泲鐧借川,閫氳繃RT-PCR鍜學estern blot娉曟��娴嬬熆鍖栫浉鍏冲熀鍥燗LP,Runx2鐨勮〃杈炬按骞，

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