本文選題：p75神經(jīng)營養(yǎng)因子受體　切入點：敲除　出處：《第三軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究背景牙齒缺失是人一生中常見的口腔問題,牙齒缺失不僅會對人體健康產生影響,同時還會對患者面部美觀、心理健康產生影響。傳統(tǒng)的牙齒缺失修復方式即為義齒修復,但其在功能及感覺方面,都無法與天然牙相媲美。因而組織工程牙齒無疑被認為是將來解決牙齒缺失最為理想的方式。顱神經(jīng)嵴源性的外胚間充質干細胞(ectomesenchymal stem cells,EMSCs)被認為是除牙釉質外所有牙齒組織始祖生成細胞。本課題組前期研究發(fā)現(xiàn),處于牙胚鐘狀期的EMSCs較其他時期的EMSCs具有更好的體外礦化能力。p75NTR(p75 neurotrophin receptor,神經(jīng)營養(yǎng)因子受體)為神經(jīng)營養(yǎng)因子低親和力受體,不僅是顱神經(jīng)嵴源性外胚間充質干細胞的經(jīng)典表面標記物,同時還參與了細胞增殖、遷移、分化、生存、凋亡等生物學過程的調控。p75NTR參與神經(jīng)系統(tǒng)的發(fā)生發(fā)育中的調控作用已被充分探究,但其參與神經(jīng)系統(tǒng)以外組織(如肝臟、肌肉、脂肪、牙齒等)的發(fā)生發(fā)育過程的研究卻仍處于起步階段。尤其是在牙齒發(fā)育中的可能的調控作用鮮有報道。本研究借助于來源于美國Jackson實驗室的p75NTR敲除小鼠,利用E18.5d處于牙胚鐘狀期的胚胎進行p75NTR(-/-)EMSCs及p75NTR(+/+)EMSCs的礦化分化能力差異的比較,探究p75NTR敲除對EMSCs礦化能力的影響,為進一步明確p75NTR影響EMSCs礦化發(fā)育可能的分子機制,揭示牙齒發(fā)生發(fā)育的分子生物學機制提供理論依據(jù)及實驗支持。研究內容1、E18.5d頜突EMSCs的獲取、培養(yǎng)、基因型鑒定和生物學特性檢測將p75NTR(-/+)雌性小鼠及p75NTR(-/+)雄性小鼠配籠,常規(guī)飼養(yǎng),小鼠從見陰栓午間為胚胎0.5d,并于孕18.5d時行2%戊巴比妥鈉(50mg/kg)小鼠腹腔麻醉注射,5-10分鐘后行腹腔手術,無菌條件下取出胚胎,于生物安全柜中PBS沖洗并切取頜突組織,盡量切碎組織使其為0.1mm*0.1mm*0.1mm的組織塊,1%Ⅰ型膠原酶消化,中和離心,去除上清后加入約2ml含10%胎牛血清、1%青霉素、鏈霉素的DMEMF12培養(yǎng)基,重懸于六孔板中,放置于含5%CO2的37℃孵育箱中培養(yǎng)。切取胎鼠的尾巴及四肢組織行基因型鑒定。并分別從細胞形態(tài)學、流式細胞檢測細胞表面抗原及細胞周期等方面對兩種基因型EMSCs進行檢測、鑒定。2、E18.5d p75NTR(-/-)和p75NTR(+/+)EMSCs礦化能力的檢測分別對p75NTR(-/-)和p75NTR(+/+)EMSCs進行體外礦化誘導,以檢測其礦化分化能力的差異。礦化誘導液為10-8M地塞米松、50 g/L抗壞血酸、100ml/L胎牛血清及10 mmol/Lβ甘油磷酸鈉配制的α-MEM培養(yǎng)液。每3天換一次液。分別礦化誘導第7天行ALP染色,第21天行茜素紅染色,并分別收集誘導第7天、14天、21天的細胞RNA,以檢測礦化相關基因ALP、Col I隨礦化誘導的變化,明確p75NTR(-/-)和p75NTR(+/+)EMSCs體外礦化誘導能力的差異。3、p75NTR(-/-)和p75NTR(+/+)小鼠骨礦化發(fā)育情況的研究選取同窩p75NTR(-/-)小鼠及p75NTR(+/+)小鼠,常規(guī)飼養(yǎng)至4個月。挑選雄性小鼠,以50mg/kg的2%戊巴比妥鈉行腹腔注射麻醉后,脫頸處死。剝脫分離左側股骨,放于4%多聚甲醛中固定組織,借助Micro-CT(viva ct40 Scanco Medical AG;Brüttisellen,Switzerland)進行掃描,并利用其配套軟件后期進行三維圖像重建及數(shù)據(jù)分析;選取同窩p75NTR(-/-)小鼠及p75NTR(+/+)雄性小鼠,常規(guī)飼養(yǎng)40天時行鈣黃綠素熒光標記實驗,即每間隔5天注射鈣黃綠素(25mg/kg,sigma),共4次,并于最后一次腹腔注射2天后脫頸處死小鼠,剝脫分離出小鼠左側股骨,行硬組織切片,并觀察計算熒光帶之間的距離,計算出每種小鼠股骨各自的平均每天礦化沉積率(mineral apposition rate,MAR),單位為um/d;提取4周齡大p75NTR(-/-)和p75NTR(+/+)小鼠股骨總RNA及蛋白質,通過RT-PCR和Western blot法檢測礦化相關基因ALP、Runx2的表達水平。結果1、(1)切除頜突組織后剩余組織進行基因型鑒定,結果在280bp處單條帶者為p75NTR(-/-),345bp處單條帶者為p75NTR(+/+),在280bp、345bp處雙條帶者為p75NTR(-/+)。(2)E18.5d的p75NTR(-/-)和p75NTR(+/+)EMSCs分別從相應的p75NTR(-/-)和p75NTR(+/+)胚胎頜突組織中獲取,兩種基因型EMSCs在細胞形態(tài)上均為明顯的長梭形的成纖維細胞形態(tài),無明顯差異。(3)流式細胞檢測細胞表面抗原檢測結果:p75NTR(-/-)EMSCs的CD14(99.07%)、CD146(95.79%)、CD166(95.79%);p75NTR(+/+)EMSCs的CD14(97.57%)、CD146(96.32%)、CD166(97.90%)。兩種基因型細胞的CD45均為陰性表達結果,分別為0.28%和0.50%。(4)細胞周期檢測結果顯示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%),二者之間細胞周期無明顯差異。2、(1)堿性磷酸酶染色結果:礦化誘導7d后,p75NTR(+/+)EMSCs組染色較p75NTR(-/-)EMSCs組深。(2)茜素紅染色結果:礦化誘導21天后,各組均有礦化結節(jié)形成,p75NTR(+/+)EMSCs組染色較p75NTR(-/-)EMSCs組深,且礦化結節(jié)量多。(3)RT-PCR結果:礦化誘導7天、14天、21天后,各組提取總RNA,逆轉錄后進行PCR反應,結果發(fā)現(xiàn)礦化誘導7天、14天和21天時均為p75NTR(+/+)EMSCs組ALP表達水平明顯高于p75NTR(-/-)EMSCs組(P㩳0.05);Col I也表現(xiàn)為相同結果(P㩳0.05)。3、(1)Micro-CT掃描4月齡大同窩p75NTR(-/-)和p75NTR(+/+)小鼠左側股骨,掃描后經(jīng)三維重建及數(shù)據(jù)分析結果:松質骨的BV(骨量),BV/TV(骨體積分數(shù)),Tb.N(骨小梁數(shù)),Tb.Th(骨小梁厚度),均為p75NTR(-/-)小鼠較p75NTR(+/+)小鼠為低,而BS/BV(骨表面積/骨量),Tb.Sp(骨小梁分離度),則為p75NTR(-/-)組小鼠高于p75NTR(+/+)組小鼠;同段股骨皮質骨的分析結果:Ct.TBTh(骨皮質厚度)及Ct.BV(骨皮質骨量)均為p75NTR(-/-)組小鼠較p75NTR(+/+)小鼠組為低,但Ct.BS/BV(骨表面積/骨量)則為p75NTR(-/-)組小鼠高于p75NTR(+/+)小鼠組。(2)鈣黃綠素熒光標記檢測結果顯示p75NTR(-/-)組小鼠股骨的平均礦化速率明顯低于同窩p75NTR(+/+)組小鼠(P0.01);(3)PCR及Western blot結果顯示p75NTR(-/-)組小鼠的ALP、Runx2基因表達水平明顯較p75NTR(+/+)組小鼠組低(p0.01)。結論1、通過兩種基因型EMSCs的培養(yǎng)及礦化能力檢測,發(fā)現(xiàn)p75NTR參與了小鼠外胚間充干細胞EMSCs的礦化分化能力的調控,并且p75NTR敲除對EMSCs的礦化分化能力存在負性調控作用;兩種基因型細胞之間細胞周期無明顯差異,說明p75NTR敲除后對EMSCs礦化分化能力負性調控作用的實現(xiàn)并不涉及細胞增殖能力、凋亡情況的改變。2、p75NTR敲除小鼠不表達具有結合NGF能力的p75NTR,Micro-CT檢測、鈣黃綠素熒光標記實驗、RT-PCR及Western blot檢測發(fā)現(xiàn)p75NTR(-/-)組小鼠股骨礦化形成能力明顯低于p75NTR(+/+)小鼠組,因此我們初步猜測p75NTR對小鼠骨的礦化形成能力的調控是通過結合NGF實現(xiàn)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75NTR(-/-)鍜宲75NTR(+/+)灝忛紶鑲￠鎬籖NA鍙婅泲鐧借川,閫氳繃RT-PCR鍜學estern blot娉曟嫻嬬熆鍖栫浉鍏沖熀鍥燗LP,Runx2鐨勮〃杈炬按騫，

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