本文關(guān)鍵詞： 成牙本質(zhì)細(xì)胞 細(xì)胞極性 細(xì)胞連接 細(xì)胞分裂周期蛋白42　出處：《第四軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：成牙本質(zhì)細(xì)胞是位于牙髓組織最外層的高柱狀細(xì)胞，頂端能夠分泌牙本質(zhì)基質(zhì)，底端位于牙髓組織內(nèi)，是一種典型的極性化細(xì)胞。在成牙本質(zhì)細(xì)胞的發(fā)生過程中，經(jīng)歷了從無極性化細(xì)胞分化發(fā)育成為極性化細(xì)胞的過程。該過程的進(jìn)行同時(shí)也伴隨著成牙本質(zhì)細(xì)胞的成熟與牙本質(zhì)基質(zhì)的分泌。在細(xì)胞頂?shù)讟O性建立過程中，細(xì)胞連接的形成和極性蛋白復(fù)合物的定位是兩個(gè)必要的細(xì)胞生物學(xué)行為。細(xì)胞連接作為相鄰細(xì)胞間的錨定結(jié)構(gòu)，起到阻隔外來物質(zhì)通透細(xì)胞層的作用，同時(shí)作為細(xì)胞膜分隔條帶，將細(xì)胞膜區(qū)域分為頂端和底端。細(xì)胞連接的建立也為細(xì)胞極性復(fù)合物在細(xì)胞膜上的定位提供了錨定結(jié)構(gòu)，是細(xì)胞極性形成的前提。細(xì)胞極性復(fù)合物的發(fā)生及其在細(xì)胞膜特定區(qū)域的定位則是細(xì)胞極性形成的分子基礎(chǔ)。極性分子在細(xì)胞膜的定位決定了細(xì)胞膜相應(yīng)區(qū)域的功能。同時(shí)，，細(xì)胞連接的建立和極性分子的定位也可以影響細(xì)胞器在胞漿內(nèi)的分布。發(fā)育成熟的成牙本質(zhì)細(xì)胞，其細(xì)胞核位于細(xì)胞的底端，內(nèi)質(zhì)網(wǎng)、高爾基體等細(xì)胞器則朝向細(xì)胞的分泌端，即細(xì)胞頂端。這種細(xì)胞形態(tài)的形成正是其細(xì)胞極性化分泌功能的形態(tài)學(xué)基礎(chǔ)。本課題采用多種分子生物學(xué)方法證實(shí)了與成牙本質(zhì)細(xì)胞極性建立密切相關(guān)的部分蛋白的表達(dá)，并對其功能進(jìn)行了初步探討，以期為相關(guān)研究提供一定的基礎(chǔ)。 1．成牙本質(zhì)細(xì)胞部分極性蛋白的表達(dá) 提取小鼠牙髓組織和OLCs細(xì)胞的總RNA和總蛋白后，用PCR檢測cdc42、ZO-1、occludin、claudin-1、E-cadherin mRNA的表達(dá)，用western blot檢測cdc42蛋白的表達(dá)。OLCs細(xì)胞經(jīng)固定、脫水、通透后，用細(xì)胞免疫熒光檢測cdc42在OLCs中的表達(dá)定位。出生后2周小鼠及出生前14天、16天、18天胚胎小鼠經(jīng)固定、脫水、浸蠟、包埋、切片后，用組織免疫熒光檢測cdc42在牙胚發(fā)育過程中的表達(dá)。結(jié)果顯示，cdc42、ZO-1、occludin、claudin-1、E-cadherin mRNA及cdc42蛋白在牙髓組織和OLCs中皆有表達(dá)。在OLCs細(xì)胞膜和細(xì)胞漿中，cdc42皆有表達(dá)并且在細(xì)胞核周圍表達(dá)量明顯高于其他部位。在牙胚發(fā)育過程中，cdc42參與了成牙本質(zhì)細(xì)胞極性化的發(fā)生過程。 2．成牙本質(zhì)細(xì)胞細(xì)胞連接的體外建立及其與LPS的關(guān)系 OLCs接種于含有α-MEM培養(yǎng)液的transwell小室中培養(yǎng)兩周后，通過掃描電鏡觀察細(xì)胞排列狀態(tài)，通過透射電鏡觀察細(xì)胞間連接的建立。OLCs接種于6孔板中，各組培養(yǎng)液中加入不同濃度LPS（10ng/ml、100ng/ml、和1000ng/ml），以條件培養(yǎng)液（10ng/ml LPS）中同時(shí)加入NF-κB抑制劑PDTC和LPS組作為對照；分別培養(yǎng)0.5h、1h、2h、6h，各時(shí)間點(diǎn)采用Western blot觀察細(xì)胞連接蛋白E-cadherin在蛋白水平的變化；以實(shí)時(shí)定量PCR觀察細(xì)胞連接蛋白ZO-1、occludin、claudin-1和E-cadherin在mRNA水平的改變。結(jié)果顯示，經(jīng)過立體培養(yǎng)的OLCs細(xì)胞間形成了明顯的細(xì)胞連接。經(jīng)過LPS處理后，ZO-1、occludin、claudin-1和E-cadherin的mRNA表達(dá)水平明顯降低，且在一定的作用時(shí)間范圍內(nèi)呈明顯的時(shí)間依賴性，但與濃度（10-1000ng/ml）無明顯依賴性。同時(shí)，E-cadherin在蛋白水平隨著LPS作用時(shí)間的延長其表達(dá)水平逐漸降低，而NF-κB抑制劑PDTC時(shí)能夠抑制這種效應(yīng)。 3．cdc42與牙源性細(xì)胞礦化的關(guān)系 OLCs細(xì)胞接種培養(yǎng)至融合80%后，實(shí)驗(yàn)組分別更換為礦化誘導(dǎo)液、含10ng/mlLPS的α-MEM培養(yǎng)液和含10ng/ml LPS礦化誘導(dǎo)液經(jīng)一定時(shí)間培養(yǎng)后，通過westernblot測定其中cdc42含量的變化；礦化誘導(dǎo)液組和含ML141的礦化誘導(dǎo)液組培養(yǎng)一定時(shí)間后，通過茜素紅染色觀察礦化結(jié)節(jié)形成情況并進(jìn)行定量分析。HDPSCs在含不同濃度LPS的礦化誘導(dǎo)液和含不同濃度ML141的礦化誘導(dǎo)液中培養(yǎng)一定時(shí)間后，通過茜素紅染色觀察礦化結(jié)節(jié)形成并定量分析。Western blot結(jié)果顯示，OLCs中cdc42的表達(dá)在礦化誘導(dǎo)液的作用下未發(fā)生明顯改變，在LPS作用下明顯上調(diào)。茜素紅染色結(jié)果顯示，OLCs礦化結(jié)節(jié)的形成量與時(shí)間正相關(guān)，與ML141的濃度呈負(fù)相關(guān)；HDPSCs礦化結(jié)節(jié)的形成與LPS濃度正相關(guān)，與ML141濃度負(fù)相關(guān)。
[Abstract]:Odontoblasts are tall columnar cells in the pulp tissue of the outermost layer top can secrete dentin matrix, the bottom end is located in the pulp, is a typical polar cell. In the event of the odontoblast process, experienced from the polarity of cell differentiation and development process become polarized cells. That process was accompanied by a secreted mature odontoblasts and dentin matrix. At the top of the bottom cell in the process of the establishment, positioning and formation of cell junctions and polarity protein complexes are two essential cell biology. Cell junctions between adjacent cells as the anchor structure to isolate a foreign substance, transparent cell layer, and at the same time as the cell membrane separating strip, the membrane area is divided into the top and bottom. The connection for the establishment of cell cell polarity complexes located on the cell membrane provided The anchor structure is a prerequisite for cell polarity formation. Cell polarity complexes and its localization in the cell membrane in specific regions is the molecular basis of cell polarity formation. The positioning of polar molecules in the cell membrane determines the corresponding membrane area function. At the same time, the establishment of cell connection and positioning the molecule can also influence the distribution of the cytoplasmic organelles. Development of odontoblast cells into mature cells, the nucleus is located at the bottom of the endoplasmic reticulum and Golgi bodies toward the end of the secretion of cells, i.e. cells. This cell apical shape is the morphology of cell polarity the basic secretion function. This issue using a variety of molecular biology methods confirmed the expression of some proteins closely related to odontoblast cell polarity establishment, and its function are discussed, in order to provide some related research Basics.
Expression of partial polar protein in 1. odontoblast cells
Total RNA was extracted from mouse dental pulp tissue and OLCs cells and the total protein detected by PCR, Cdc42, ZO-1, occludin, claudin-1, E-cadherin mRNA expression, Cdc42 protein expression in.OLCs cells by detection of Western blot by fixed, dehydrated, transparent, with immunofluorescence detection of Cdc42 expression in OLCs after birth location. 2 and 14 days before the birth of Zhou Xiaoshu, 16 days, 18 days of embryonic mice after fixation, dehydration, paraffin embedding, slicing, immunohistochemistry, expression of Cdc42 during tooth development. The results showed that Cdc42, ZO-1, occludin, claudin-1, E-cadherin mRNA and Cdc42 protein in dental pulp and OLCs is expressed in OLCs cell membrane and cytoplasm, Cdc42 is expressed and the expression level was significantly higher than that of other parts in the vicinity of the nucleus. During tooth development, Cdc42 is involved in the generation process of dentin cell polarity.
Establishment of 2. odontoblast cell connection in vitro and its relationship with LPS
OLCs inoculated in -MEM culture liquid containing alpha Transwell cells in culture after two weeks, by using scanning electron microscope arrangement of cells observed through transmission electron microscope, the connection between the establishment of.OLCs were inoculated in 6 well plates, with different concentrations of LPS in liquid culture groups (10ng/ml, 100ng/ml, and 1000ng/ml), with conditioned medium (10ng/ml LPS) in both NF- and B inhibitors PDTC and LPS as the control group were cultured in 0.5h, 1H; 2h, 6h, changes in each time point using Western blot to observe cell junction protein E-cadherin in protein level; real-time quantitative PCR to observe cell junction proteins ZO-1, occludin, claudin-1 and E-cadherin at mRNA level. The results showed that after three-dimensional cultured OLCs cells formed obvious cell connection. After treatment by LPS, ZO-1, occludin, claudin-1 and E-cadherin mRNA expression level significantly decreased, and in the There was a significant time dependence in time range, but no significant dependence on concentration (10-1000ng/ml). At the same time, E-cadherin level decreased with the prolongation of LPS action time, while NF- kappa B inhibitor could inhibit this effect.
Relationship between 3.cdc42 and odontogenic cell mineralization
OLCs cells were cultured to confluence after 80%, the experimental group were replaced with mineralized induced liquid, alpha -MEM containing 10ng/mlLPS culture medium and culture medium induced by a certain period of time with 10ng/ml LPS after mineralization, determined the change of Cdc42 content by Westernblot; mineralization induced group and ML141 induced group mineralization after cultured for a certain time, the formation and quantitative analysis by.HDPSCs and ML141 in solution containing different concentration with different LPS concentrations of mineralized induced liquid in a certain period of training by alizarin red staining of mineralized nodules after alizarin red staining was used to observe the formation of mineralized nodules and the quantitative analysis of.Western blot showed that the expression of OLCs Cdc42 in mineralized induced liquid under the action of not changed significantly in the presence of LPS was obviously up-regulated. Alizarin red staining showed that OLCs mineralized nodule formation was related to quantity and time, and ML141 The concentration was negatively correlated, and the formation of HDPSCs mineralized nodules was positively related to the concentration of LPS, and was negatively correlated with the concentration of ML141.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.3

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