本文選題：p75NTR + RUNX2�。� 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:組織工程化牙齒的研究無疑是近年來口腔醫(yī)學(xué)重要的研究熱點(diǎn)之一,但目前牙齒發(fā)生發(fā)育過程中的眾多生物學(xué)機(jī)制尚不清楚,仍無法獲得真正意義上的牙齒再生。而模擬牙發(fā)育的外胚間充質(zhì)-上皮細(xì)胞重聚技術(shù)為牙齒組織工程帶來了新的希望。來源于顱神經(jīng)嵴的外胚間充質(zhì)干細(xì)胞(ectomesenchymal stem cell,EMSCs)作為一種牙源性的干細(xì)胞,是細(xì)胞重聚技術(shù)獲得牙再生的理想細(xì)胞之一,被認(rèn)為是除牙釉質(zhì)以外牙齒各組織發(fā)育的重要來源細(xì)胞。p75NTR(p75 neurotrophin receptor,p75NTR)是一種低親和性神經(jīng)營養(yǎng)素受體,被認(rèn)為是鑒定來源于神經(jīng)嵴EMSCs的主要膜受體之一,很可能參與牙齒形成,有研究發(fā)現(xiàn)非牙源性細(xì)胞中p75NTR可能參與促進(jìn)礦化,且與礦化因子RUNX2(Runt-related transcription factor 2,RUNX2)有一定聯(lián)系。牙源性細(xì)胞的礦化可能不同于其他細(xì)胞,牙源性細(xì)胞中p75NTR是否參與礦化以及與RUNX2的關(guān)系報(bào)道較少。因此本實(shí)驗(yàn)通過觀察p75NTR及RUNX2在SD大鼠下頜第一磨牙成牙發(fā)育初期不同發(fā)育階段的表達(dá)分布情況及礦化誘導(dǎo)后二者的變化,探討兩者在牙齒發(fā)育初期的作用及二者與礦化的聯(lián)系。有助于揭示牙齒發(fā)生發(fā)育機(jī)制,推動(dòng)牙齒組織工程學(xué)發(fā)展,選出適合的種子細(xì)胞。方法:1.HE染色觀察下頜磨牙發(fā)育初期大鼠牙胚的形態(tài)2%戊巴比妥鈉(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,取下頜組織包埋,按冠狀面方向切片,厚℃為6μm,蘇木素染色5 min,反藍(lán)后伊紅染色1 min,梯度酒精脫水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性樹膠封片顯微鏡觀察、拍照。2.P75NTR在大鼠胚胎下頜磨牙發(fā)育初期牙胚的時(shí)空表達(dá)取上述E13.5d、E14.5d、E15.5d、E16.5d、E18.5d、P0.5d含有SD大鼠下頜第一磨牙牙胚的冰凍切片復(fù)溫30 min,PBS沖洗;滴加試劑A(5%山羊血清)封閉1h;傾去血清,滴加一抗工作液比例(p75NTR1:1500),放入濕盒,4℃過夜;傾去一抗,PBS沖洗;滴加試劑B(二抗工作液)室溫30min;PBS沖洗;滴加試劑C(辣根酶標(biāo)記鏈霉卵白素工作液)室溫30min;PBS沖洗;滴入配好的DAB顯色劑(A:B=50:1000)出現(xiàn)明顯陽性流水終止反應(yīng);蘇木素復(fù)染1min;1%鹽酸酒精10s;蒸餾水沖洗5min;梯度酒精脫水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性樹膠封片顯微鏡觀察、拍照。3.RUNX2在大鼠胚胎下頜磨牙發(fā)育初期牙胚的時(shí)空表達(dá)取上述E13.5d、E14.5d、E15.5d、E16.5d、E18.5d、P0.5d含有SD大鼠下頜第一磨牙牙胚的冰凍切片復(fù)溫30 min,后放入丙酮10min;PBS沖洗;滴加試劑A(5%山羊血清)封閉1h;傾去血清,滴加一抗工作液比例(p75NTR 1:1500),放入濕盒,4℃過夜;傾去一抗,PBS沖洗;滴加試劑B(二抗工作液)室溫30min;PBS沖洗;滴加試劑C(辣根酶標(biāo)記鏈霉卵白素工作液)室溫30min;PBS沖洗;滴入配好的DAB顯色劑(A:B=50:1000)出現(xiàn)明顯陽性流水終止反應(yīng);蘇木素復(fù)染1min;1%鹽酸酒精10s;蒸餾水沖洗5min;梯度酒精脫水(70%、80%、90%、100%Ⅰ、100%Ⅱ);二甲苯(Ⅰ、Ⅱ);中性樹膠封片顯微鏡觀察、拍照。4.P0.5d EMSCs的獲取及礦化誘導(dǎo)4.1 P0.5d頜突外胚間充質(zhì)干細(xì)胞獲得、體外培養(yǎng)及鑒定2%戊巴比妥鈉(40mg/kg)注射受孕P0.5d SD大鼠腹腔,等待5左右,無菌條件下取出胎鼠,切取下頜突,尋找下頜第一磨牙牙胚,胰酶消化、離心,經(jīng)不銹鋼篩網(wǎng)過濾后分別加入含100ml/L胎牛血清的培養(yǎng)基,置于5%CO2 37℃孵育箱中培養(yǎng)。流式細(xì)胞技術(shù)檢測(cè)細(xì)胞表面抗原對(duì)P0.5d SD大鼠EMSCs進(jìn)行生物學(xué)鑒定。4.2 P0.5d EMSCs體外礦化誘導(dǎo)及分析利用礦化誘導(dǎo)液(以50 g/L抗壞血酸、10 mmol/Lβ甘油磷酸鈉、10-8M地塞米松及100ml/L胎牛血清配制α-MEM礦化誘導(dǎo)液)每3天換液1次。礦化誘導(dǎo)第0d、7d、14d、21d收集蛋白進(jìn)行western blot檢測(cè)相關(guān)蛋白趨勢(shì)。統(tǒng)計(jì)學(xué)方法:對(duì)本實(shí)驗(yàn)樣本的灰℃值數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,采用均數(shù)(標(biāo)準(zhǔn)差)進(jìn)行統(tǒng)計(jì)描述,各結(jié)果不同時(shí)間點(diǎn)的比較采用單組重復(fù)測(cè)量方差分析,并采用LSD方法進(jìn)行兩兩比較,各指標(biāo)的相關(guān)性分析采用Pearson相關(guān)分析方法。所有統(tǒng)計(jì)分析在SPSS 22.0軟件中完成,檢驗(yàn)水準(zhǔn)α=0.05,雙側(cè)檢驗(yàn)。結(jié)果:1.E13.5d大鼠的磨牙牙胚處于蕾狀期,鏡下可見牙板末端膨大,上皮增生,上皮的下方,周圍外胚間葉細(xì)胞增生、聚集。E14.5d大鼠磨牙胚的上皮芽繼續(xù)向外胚間充質(zhì)生長,體積變大,基底向內(nèi)凹陷,為帽狀初期。E15.5d牙胚繼續(xù)發(fā)育進(jìn)入為帽狀末期,成釉器分成3層—外釉上皮層、內(nèi)釉上皮層和星網(wǎng)狀層。周圍的外胚間充質(zhì)細(xì)胞密度增大,形成為細(xì)胞凝聚區(qū),為牙乳頭。包圍牙乳頭邊緣和成釉器的外胚間充質(zhì)細(xì)胞聚集形成結(jié)締組織層,為牙囊。E16.5d進(jìn)入鐘狀早期。E18.5d為鐘狀期,成釉器逐漸成熟,逐漸分化為4層—外釉上皮層、內(nèi)釉上皮層、星網(wǎng)狀層和中間層。P0.5d(鐘狀晚期)時(shí),牙乳頭附近的內(nèi)釉上皮細(xì)胞分化為前成釉細(xì)胞,一些基底膜相鄰的牙乳頭細(xì)胞也開始分化成為前成牙本質(zhì)細(xì)胞,而另一些分化為具有分泌功能的成牙本質(zhì)細(xì)胞。在牙乳頭的尖端,一部分成牙本質(zhì)細(xì)胞分泌為前期牙本質(zhì)基質(zhì)。2.E13.5d大鼠磨牙胚處于蕾狀期,p75NTR在上皮近舌側(cè)靠近成釉器的間質(zhì)高表達(dá),此時(shí)成釉器及其下面的間質(zhì)無表達(dá)。E14.5d帽狀初期p75NTR染色沿著上皮爬升到牙乳頭處,此時(shí)內(nèi)釉上皮(釉結(jié))出現(xiàn)高表達(dá)。E15.5d帽狀末期p75NTR在內(nèi)釉上皮層表達(dá)為分明的兩部分,為次級(jí)釉結(jié);星網(wǎng)狀層,次級(jí)釉結(jié),牙乳頭、牙囊高表達(dá)。E16.5d鐘狀早期p75NTR在內(nèi)釉上皮層依舊表達(dá)在次級(jí)釉結(jié)處;下方牙乳頭、牙囊高表達(dá)。E18.5d鐘狀期p75NTR在內(nèi)釉上皮全層表達(dá)。下方牙乳頭、牙囊高表達(dá)。P0.5d鐘狀晚期,p75NTR在前成釉細(xì)胞,前成牙本質(zhì)細(xì)胞,成牙本質(zhì)細(xì)胞。在牙尖乳頭尖處,高表達(dá)。3.E13.5d大鼠磨牙胚處于蕾狀期,RUNX2成釉器下方的間質(zhì)表達(dá),此時(shí)上皮組織內(nèi)無表達(dá)。E14.5d帽狀初期RUNX2成釉器下方的牙乳頭間質(zhì)表達(dá),上皮組織內(nèi)無表達(dá)。E15.5d帽狀末期RUNX2與p75NTR表達(dá)類似在內(nèi)釉上皮層中的次級(jí)釉結(jié)高表達(dá)。下方牙乳頭、牙囊高表達(dá)。E16.5d鐘狀早期RUNX2與p75NTR表達(dá)類似的表達(dá)在內(nèi)釉上皮的次級(jí)釉結(jié)處。下方牙乳頭、牙囊高表達(dá)。E18.5d鐘狀期RUNX2與p75NTR類似表達(dá)在在內(nèi)釉上皮全層表達(dá)。下方牙乳頭、牙囊高表達(dá)。P0.5d鐘狀晚期,RUNX2在各個(gè)區(qū)域表達(dá)下降。4.(1)體外分離、培養(yǎng)的P0.5d EMSCs均為長梭型的成纖維樣細(xì)胞,胞體豐滿,細(xì)胞增殖能力強(qiáng)。流式細(xì)胞檢測(cè):P0.5d EMSCs的CD29、CD90、CD146、CD166、p75NTR、CD45表達(dá)率分別為:96.22%、95.36%、96.47%、96.12%、97.27%、0.52%,陽性表達(dá)CD14、CD29、CD90、CD146、CD166、p75NTR,陰性表達(dá)CD45。(2)經(jīng)礦化誘導(dǎo)液處理7d、14d、21d后的EMSCs,p75NTR和RUNX2的表達(dá)量隨著誘導(dǎo)時(shí)間的增加而增加。以未進(jìn)行礦化誘導(dǎo)的EMSCs作為對(duì)照,礦化誘導(dǎo)第7d與對(duì)照組相比,RUNX2的表達(dá)呈顯著上升(P0.05)。礦化誘導(dǎo)14d和21d后,細(xì)胞中的p75NTR、RUNX2表達(dá)量都顯著升高(P0.05)。在礦化過程中p75NTR與RUNX2的表達(dá)隨著誘導(dǎo)時(shí)間的增加而上升,p75NTR與RUNX2的表達(dá)量在不同礦化時(shí)間點(diǎn)的相對(duì)表達(dá)量存在成正相關(guān)關(guān)系(r=0.992,P0.001)。結(jié)論:p75NTR和RUNX2在成牙初期不同天數(shù)表達(dá)在在上皮-間充質(zhì)相互作用區(qū)域,且高度相似,具有時(shí)空特異性,可能與牙齒礦化發(fā)育相關(guān),礦化誘導(dǎo)后變化趨勢(shì)趨于一致等說明二者在SD大鼠成牙發(fā)育初期下頜第一磨牙的發(fā)育及礦化中都起到一定的作用,可能存在正相關(guān)關(guān)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R78

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