p75NTR、RUNX2基因在鼠胚牙发育过程中的时空表达规律研究

发布时间：2018-06-04 16:03

本文选题：p75NTR + RUNX2　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:组织工程化牙齿的研究无疑是近年来口腔医学重要的研究热点之一,但目前牙齿发生发育过程中的众多生物学机制尚不清楚,仍无法获得真正意义上的牙齿再生。而模拟牙发育的外胚间充质-上皮细胞重聚技术为牙齿组织工程带来了新的希望。来源于颅神经嵴的外胚间充质干细胞(ectomesenchymal stem cell,EMSCs)作为一种牙源性的干细胞,是细胞重聚技术获得牙再生的理想细胞之一,被认为是除牙釉质以外牙齿各组织发育的重要来源细胞。p75NTR(p75 neurotrophin receptor,p75NTR)是一种低亲和性神经营养素受体,被认为是鉴定来源于神经嵴EMSCs的主要膜受体之一,很可能参与牙齿形成,有研究发现非牙源性细胞中p75NTR可能参与促进矿化,且与矿化因子RUNX2(Runt-related transcription factor 2,RUNX2)有一定联系。牙源性细胞的矿化可能不同于其他细胞,牙源性细胞中p75NTR是否参与矿化以及与RUNX2的关系报道较少。因此本实验通过观察p75NTR及RUNX2在SD大鼠下颌第一磨牙成牙发育初期不同发育阶段的表达分布情况及矿化诱导后二者的变化,探讨两者在牙齿发育初期的作用及二者与矿化的联系。有助于揭示牙齿发生发育机制,推动牙齿组织工程学发展,选出适合的种子细胞。方法:1.HE染色观察下颌磨牙发育初期大鼠牙胚的形态2%戊巴比妥钠(40mg/kg)注射受孕E13.5d、E14.5d、E15.5d、E16.5d、E18.5d、P0.5d SD大鼠腹腔,等待5min左右,取出胚胎,4%多聚甲醛固定1d,取下颌组织包埋,按冠状面方向切片,厚℃为6μm,苏木素染色5 min,反蓝后伊红染色1 min,梯度酒精脱水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性树胶封片显微镜观察、拍照。2.P75NTR在大鼠胚胎下颌磨牙发育初期牙胚的时空表达取上述E13.5d、E14.5d、E15.5d、E16.5d、E18.5d、P0.5d含有SD大鼠下颌第一磨牙牙胚的冰冻切片复温30 min,PBS冲洗;滴加试剂A(5%山羊血清)封闭1h;倾去血清,滴加一抗工作液比例(p75NTR1:1500),放入湿盒,4℃过夜;倾去一抗,PBS冲洗;滴加试剂B(二抗工作液)室温30min;PBS冲洗;滴加试剂C(辣根酶标记链霉卵白素工作液)室温30min;PBS冲洗;滴入配好的DAB显色剂(A:B=50:1000)出现明显阳性流水终止反应;苏木素复染1min;1%盐酸酒精10s;蒸馏水冲洗5min;梯度酒精脱水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性树胶封片显微镜观察、拍照。3.RUNX2在大鼠胚胎下颌磨牙发育初期牙胚的时空表达取上述E13.5d、E14.5d、E15.5d、E16.5d、E18.5d、P0.5d含有SD大鼠下颌第一磨牙牙胚的冰冻切片复温30 min,后放入丙酮10min;PBS冲洗;滴加试剂A(5%山羊血清)封闭1h;倾去血清,滴加一抗工作液比例(p75NTR 1:1500),放入湿盒,4℃过夜;倾去一抗,PBS冲洗;滴加试剂B(二抗工作液)室温30min;PBS冲洗;滴加试剂C(辣根酶标记链霉卵白素工作液)室温30min;PBS冲洗;滴入配好的DAB显色剂(A:B=50:1000)出现明显阳性流水终止反应;苏木素复染1min;1%盐酸酒精10s;蒸馏水冲洗5min;梯度酒精脱水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性树胶封片显微镜观察、拍照。4.P0.5d EMSCs的获取及矿化诱导4.1 P0.5d颌突外胚间充质干细胞获得、体外培养及鉴定2%戊巴比妥钠(40mg/kg)注射受孕P0.5d SD大鼠腹腔,等待5左右,无菌条件下取出胎鼠,切取下颌突,寻找下颌第一磨牙牙胚,胰酶消化、离心,经不锈钢筛网过滤后分别加入含100ml/L胎牛血清的培养基,置于5%CO2 37℃孵育箱中培养。流式细胞技术检测细胞表面抗原对P0.5d SD大鼠EMSCs进行生物学鉴定。4.2 P0.5d EMSCs体外矿化诱导及分析利用矿化诱导液(以50 g/L抗坏血酸、10 mmol/Lβ甘油磷酸钠、10-8M地塞米松及100ml/L胎牛血清配制α-MEM矿化诱导液)每3天换液1次。矿化诱导第0d、7d、14d、21d收集蛋白进行western blot检测相关蛋白趋势。统计学方法:对本实验样本的灰℃值数据进行统计分析,采用均数(标准差)进行统计描述,各结果不同时间点的比较采用单组重复测量方差分析,并采用LSD方法进行两两比较,各指标的相关性分析采用Pearson相关分析方法。所有统计分析在SPSS 22.0软件中完成,检验水准α=0.05,双侧检验。结果:1.E13.5d大鼠的磨牙牙胚处于蕾状期,镜下可见牙板末端膨大,上皮增生,上皮的下方,周围外胚间叶细胞增生、聚集。E14.5d大鼠磨牙胚的上皮芽继续向外胚间充质生长,体积变大,基底向内凹陷,为帽状初期。E15.5d牙胚继续发育进入为帽状末期,成釉器分成3层—外釉上皮层、内釉上皮层和星网状层。周围的外胚间充质细胞密度增大,形成为细胞凝聚区,为牙乳头。包围牙乳头边缘和成釉器的外胚间充质细胞聚集形成结缔组织层,为牙囊。E16.5d进入钟状早期。E18.5d为钟状期,成釉器逐渐成熟,逐渐分化为4层—外釉上皮层、内釉上皮层、星网状层和中间层。P0.5d(钟状晚期)时,牙乳头附近的内釉上皮细胞分化为前成釉细胞,一些基底膜相邻的牙乳头细胞也开始分化成为前成牙本质细胞,而另一些分化为具有分泌功能的成牙本质细胞。在牙乳头的尖端,一部分成牙本质细胞分泌为前期牙本质基质。2.E13.5d大鼠磨牙胚处于蕾状期,p75NTR在上皮近舌侧靠近成釉器的间质高表达,此时成釉器及其下面的间质无表达。E14.5d帽状初期p75NTR染色沿着上皮爬升到牙乳头处,此时内釉上皮(釉结)出现高表达。E15.5d帽状末期p75NTR在内釉上皮层表达为分明的两部分,为次级釉结;星网状层,次级釉结,牙乳头、牙囊高表达。E16.5d钟状早期p75NTR在内釉上皮层依旧表达在次级釉结处;下方牙乳头、牙囊高表达。E18.5d钟状期p75NTR在内釉上皮全层表达。下方牙乳头、牙囊高表达。P0.5d钟状晚期,p75NTR在前成釉细胞,前成牙本质细胞,成牙本质细胞。在牙尖乳头尖处,高表达。3.E13.5d大鼠磨牙胚处于蕾状期,RUNX2成釉器下方的间质表达,此时上皮组织内无表达。E14.5d帽状初期RUNX2成釉器下方的牙乳头间质表达,上皮组织内无表达。E15.5d帽状末期RUNX2与p75NTR表达类似在内釉上皮层中的次级釉结高表达。下方牙乳头、牙囊高表达。E16.5d钟状早期RUNX2与p75NTR表达类似的表达在内釉上皮的次级釉结处。下方牙乳头、牙囊高表达。E18.5d钟状期RUNX2与p75NTR类似表达在在内釉上皮全层表达。下方牙乳头、牙囊高表达。P0.5d钟状晚期,RUNX2在各个区域表达下降。4.(1)体外分离、培养的P0.5d EMSCs均为长梭型的成纤维样细胞,胞体丰满,细胞增殖能力强。流式细胞检测:P0.5d EMSCs的CD29、CD90、CD146、CD166、p75NTR、CD45表达率分别为:96.22%、95.36%、96.47%、96.12%、97.27%、0.52%,阳性表达CD14、CD29、CD90、CD146、CD166、p75NTR,阴性表达CD45。(2)经矿化诱导液处理7d、14d、21d后的EMSCs,p75NTR和RUNX2的表达量随着诱导时间的增加而增加。以未进行矿化诱导的EMSCs作为对照,矿化诱导第7d与对照组相比,RUNX2的表达呈显著上升(P0.05)。矿化诱导14d和21d后,细胞中的p75NTR、RUNX2表达量都显著升高(P0.05)。在矿化过程中p75NTR与RUNX2的表达随着诱导时间的增加而上升,p75NTR与RUNX2的表达量在不同矿化时间点的相对表达量存在成正相关关系(r=0.992,P0.001)。结论:p75NTR和RUNX2在成牙初期不同天数表达在在上皮-间充质相互作用区域,且高度相似,具有时空特异性,可能与牙齿矿化发育相关,矿化诱导后变化趋势趋于一致等说明二者在SD大鼠成牙发育初期下颌第一磨牙的发育及矿化中都起到一定的作用,可能存在正相关关系。
[Abstract]:Objective: the study of tissue engineered teeth is undoubtedly one of the most important research hot spots in oral medicine in recent years. However, many biological mechanisms in the process of tooth development are still not clear, and the real tooth regeneration can not be obtained. Ectomesenchymal stem cell (EMSCs) from cranial neural crest, as a odontogenic stem cell, is one of the ideal cells for cell repolymerization, which is considered to be an important source of.P75NTR (p75 neurotrophin receptor, P), except for the tooth enamel. 75NTR) is a low affinity neurotrophic receptor, which is considered to be one of the major membrane receptors identified from the neural crest EMSCs. It is likely to be involved in dental formation. Some studies have found that p75NTR in non odontogenic cells may be involved in promoting mineralization and is associated with the mineralized factor RUNX2 (Runt-related transcription factor 2, RUNX2). The mineralization of sexual cells may be different from other cells. Whether p75NTR is involved in mineralization in odontogenic cells and reports on the relationship with RUNX2 are less. Therefore, the distribution of p75NTR and RUNX2 in the different developmental stages of the first molar development of the mandibular first molar of SD rats and the changes of the two after mineralization induction were investigated. The relationship between the early stage of tooth development and the relationship between the two and mineralization can help to reveal the mechanism of tooth development, promote the development of dental tissue engineering and select the suitable seed cells. Methods: 1.HE staining was used to observe the morphology of the tooth embryo in the early stage of mandibular molar development by 2% pentobarbital sodium (40mg/kg) injection of E13.5d, E14.5d, E15.5d, E16.5d, E18.5. D, P0.5d SD rat abdominal cavity, wait for 5min, take out the embryo, 4% polyoxymethylene fixed 1D, take the mandibular tissue embedded, slice the mandible, sliced in the direction of the coronal plane, the thickness is 6 mu, 5 min with hematoxin, 1 min in the anti blue eosin, and the gradient alcohol dehydration (70%, 80%, 90%, 100% I, 100% II); dimethylbenzene (I, II); neutral gum seal microscope observation, taking photos.2.P75N TR, E13.5d, E14.5d, E15.5d, E16.5d, E18.5d, E14.5d, E15.5d, E16.5d, E18.5d, and P0.5d containing SD rats' mandibular first molar teeth were reheated by 30 min, PBS flushing, and the reagent A (5% goat serum) closed 1H. Overnight; tilting one resistance, PBS flushing; B (two anti working fluid) room temperature 30min; PBS flushing; C (horseradish enzyme labelled chylum oval working liquid) 30min; PBS flushing; PBS rinsing; A:B=50:1000 (A:B=50:1000) showed an obvious positive flow termination reaction; hematoxylin redyeing 1min; 1% hydrochloric alcohol 10s; distilled water flushing 5min; gradient Alcohol dehydration (70%, 80%, 90%, 100% I, 100% II); dimethylbenzene (I, II); neutral gum seal microscopes, taking photo.3.RUNX2 for the expression of E13.5d, E14.5d, E15.5d, E16.5d, E18.5d, and P0.5d containing the frozen section of the first molar of the rat in SD rats, and 30 min, and then put into acetone in SD rats. 10min; PBS flushing; A (5% goat sera) closed 1H; dip the serum, add an anti working liquid ratio (p75NTR 1:1500), put into a wet box, stay overnight; dip one anti, PBS rinse; drop reagent B (two anti working liquid) room temperature 30min; PBS rinse; adding reagent C (horseradish enzyme labelled oval whiten working liquid) room room room 30min; drip irrigation; drop in match well The DAB chromogenic agent (A:B=50:1000) showed an obvious positive water terminating reaction; hematoxylin restained 1min; 1% hydrochloric acid 10s; distilled water to rinse 5min; gradient alcohol dehydration (70%, 80%, 90%, 100%, 100% II); dimethylbenzene (I, II); neutral gum seal microscopes; acquisition of.4.P0.5d EMSCs and mineralization induced 4.1 P0.5d exotomical mesenchymal transition. Stem cells were obtained, in vitro culture and identification of 2% pentobarbital sodium (40mg/kg) injection in the abdominal cavity of pregnant P0.5d SD rats, waiting for about 5. Under aseptic conditions, fetal rats were removed, mandibular process was removed, mandibular first molar tooth embryo was found, trypsin digestion, centrifugation, and after filtration of stainless steel screen net, the culture medium containing 100ml/L fetal bovine serum was added to 5%CO2 37 degrees centigrade. Cell culture in incubators. Flow cytometry detection of cell surface antigen on P0.5d SD rat EMSCs biological identification of.4.2 P0.5d EMSCs in vitro mineralization induction and analysis using mineralization inducer (50 g/L ascorbic acid, 10 mmol/L beta glycerol phosphate, 10-8M dexamethasone and 100ml/L fetal bovine serum preparation of alpha -MEM mineralization inducer) every 3 days for 1 times Mineralization induced 0d, 7d, 14d, 21d collecting protein for Western blot detection related protein trend. Statistical method: statistical analysis of the gray value data of the experimental samples, using the mean number (standard deviation) for statistical description, the comparison of the results at different time points using a single group of repeated measurements of variance analysis, and LSD method for 22 The correlation analysis of each index was analyzed by Pearson correlation analysis. All statistical analysis was completed in SPSS 22 software. The test level was alpha =0.05 and bilateral test. Results: the molar teeth of the 1.E13.5d rats were in the bud stage. The end expansion of the dental plate, the epithelial hyperplasia, the lower epithelium, the proliferation of peripheral lobar cells and the aggregation of.E14. were found in the 1.E13.5d rats. The epithelial buds of the molar embryo of 5D rats continue to grow in the outer embryo, the volume becomes larger, the basement is inward, and the early.E15.5d tooth germ of the cap continues to develop into the end of the cap, and the glaze apparatus is divided into 3 layers - the outer glaze epithelium, the inner glaze epithelium and the star reticular layer. The ectoembryonic mesenchymal cells that encircled the edge of the dental papilla and the enamel formed a connective tissue layer and formed a connective tissue layer for the early bell like.E18.5d of the tooth sac.E16.5d, and the glaze matured gradually and gradually differentiated into 4 layers of outer glaze epithelium, the inner glaze epithelium, the star reticular layer and the middle layer of.P0.5d (late bell shaped), and the inner glaze near the dental papilla. The skin cells differentiated into preameloblastoma, and some of the dental papilla cells adjacent to the basement membrane began to differentiate into preodontoblast cells, while others differentiated into odontoblasts with secretory function. At the tip of the dental papilla, a part of odontoblast cells were secreted into the bud stage of the molars embryo of the early dentine matrix.2.E13.5d rats, p75 NTR is highly expressed in the epithelia near the lingual side of the enamel. At this time, the glaze device and its underneath the underneath the interstitial.E14.5d hat like initial p75NTR stain climb up the epithelium to the tooth nipple, and the enamel epithelium (glaze knot) appears high expression of the.E15.5d cap end p75NTR of the inner glaze epithelium as a distinct two part, the secondary enamel junction; the star net. Layer, secondary enamel junction, dental papilla, high expression of.E16.5d bell shape early p75NTR inner glaze epithelial layer still expressed in the secondary enamel junction, the lower tooth nipple, the tooth sac high expression.E18.5d bell shape p75NTR inner glaze epithelium all layer expression. Lower tooth nipple, the tooth sac high expression.P0.5d bell form late, p75NTR in the former ameloblastoma, the former odontoblast cells, At the tip of the cusp nipple, the high expression of the.3.E13.5d rat molar embryo was in the bud stage, and the interstitial expression under the RUNX2 glaze device was expressed. At this time, there was no expression of the interstitial expression of the dental papilla below the initial RUNX2 glaze device of the.E14.5d cap in the epithelial tissue, and the expression of the.E15.5d like end RUNX2 in the epithelial tissue was similar to the expression of the inner glaze with the expression of p75NTR in the epithelial tissue. The secondary enamel junction is highly expressed. The lower dentate papilla, the high expression of the.E16.5d bell shaped early RUNX2 and p75NTR expression, is similar to the expression of the secondary enamel junction of the inner enamel epithelium. The lower tooth nipple, the high expression of the tooth sac and the.E18.5d bell like phase RUNX2 and p75NTR expression are expressed in the entire layer of the inner enamel epithelium. The lower tooth nipple and the tooth sac express.P0.5d high. In the late bell form, the expression of RUNX2 in each region was reduced by.4. (1) in vitro, and the cultured P0.5d EMSCs were all spindle shaped fibroblast like cells, the cell body was plump and the cell proliferation was strong. The flow cytometry: CD29, CD90, CD146, CD166, p75NTR, P0.5d EMSCs: 96.22%, 95.36%, 96.47%, 96.12%, 97.27%, 0.52%, respectively. CD29, CD90, CD146, CD166, p75NTR, negative expression CD45. (2) the expression of 7D after mineralization, 14d, EMSCs, p75NTR and RUNX2 increased with the increase of induction time. The expression of p75NTR and RUNX2 in the cells increased significantly (P0.05). During the mineralization, the expression of p75NTR and RUNX2 increased with the increase of induction time. The relative expression of p75NTR and RUNX2 expression at different mineralization time points was positive correlation (r=0.992, P0.001). Conclusion: p75NTR and RUNX2 are different days table in the early stage of tooth formation. In the area of epithelial mesenchymal interaction, and highly similar, with time and space specificity, it may be related to the mineralization development of the teeth, and the trend of the mineralization induced change tends to be consistent. The two people play a certain role in the development and mineralization of the first molar in the early stage of SD rat tooth development, which may have a positive correlation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R78

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