成牙本质细胞极性相关蛋白cdc42和E-cadherin的表达及功能

发布时间：2018-02-02 02:35

本文关键词： 成牙本质细胞细胞极性细胞连接细胞分裂周期蛋白42　出处：《第四军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：成牙本质细胞是位于牙髓组织最外层的高柱状细胞，顶端能够分泌牙本质基质，底端位于牙髓组织内，是一种典型的极性化细胞。在成牙本质细胞的发生过程中，经历了从无极性化细胞分化发育成为极性化细胞的过程。该过程的进行同时也伴随着成牙本质细胞的成熟与牙本质基质的分泌。在细胞顶底极性建立过程中，细胞连接的形成和极性蛋白复合物的定位是两个必要的细胞生物学行为。细胞连接作为相邻细胞间的锚定结构，起到阻隔外来物质通透细胞层的作用，同时作为细胞膜分隔条带，将细胞膜区域分为顶端和底端。细胞连接的建立也为细胞极性复合物在细胞膜上的定位提供了锚定结构，是细胞极性形成的前提。细胞极性复合物的发生及其在细胞膜特定区域的定位则是细胞极性形成的分子基础。极性分子在细胞膜的定位决定了细胞膜相应区域的功能。同时，，细胞连接的建立和极性分子的定位也可以影响细胞器在胞浆内的分布。发育成熟的成牙本质细胞，其细胞核位于细胞的底端，内质网、高尔基体等细胞器则朝向细胞的分泌端，即细胞顶端。这种细胞形态的形成正是其细胞极性化分泌功能的形态学基础。本课题采用多种分子生物学方法证实了与成牙本质细胞极性建立密切相关的部分蛋白的表达，并对其功能进行了初步探讨，以期为相关研究提供一定的基础。 1．成牙本质细胞部分极性蛋白的表达提取小鼠牙髓组织和OLCs细胞的总RNA和总蛋白后，用PCR检测cdc42、ZO-1、occludin、claudin-1、E-cadherin mRNA的表达，用western blot检测cdc42蛋白的表达。OLCs细胞经固定、脱水、通透后，用细胞免疫荧光检测cdc42在OLCs中的表达定位。出生后2周小鼠及出生前14天、16天、18天胚胎小鼠经固定、脱水、浸蜡、包埋、切片后，用组织免疫荧光检测cdc42在牙胚发育过程中的表达。结果显示，cdc42、ZO-1、occludin、claudin-1、E-cadherin mRNA及cdc42蛋白在牙髓组织和OLCs中皆有表达。在OLCs细胞膜和细胞浆中，cdc42皆有表达并且在细胞核周围表达量明显高于其他部位。在牙胚发育过程中，cdc42参与了成牙本质细胞极性化的发生过程。 2．成牙本质细胞细胞连接的体外建立及其与LPS的关系 OLCs接种于含有α-MEM培养液的transwell小室中培养两周后，通过扫描电镜观察细胞排列状态，通过透射电镜观察细胞间连接的建立。OLCs接种于6孔板中，各组培养液中加入不同浓度LPS（10ng/ml、100ng/ml、和1000ng/ml），以条件培养液（10ng/ml LPS）中同时加入NF-κB抑制剂PDTC和LPS组作为对照；分别培养0.5h、1h、2h、6h，各时间点采用Western blot观察细胞连接蛋白E-cadherin在蛋白水平的变化；以实时定量PCR观察细胞连接蛋白ZO-1、occludin、claudin-1和E-cadherin在mRNA水平的改变。结果显示，经过立体培养的OLCs细胞间形成了明显的细胞连接。经过LPS处理后，ZO-1、occludin、claudin-1和E-cadherin的mRNA表达水平明显降低，且在一定的作用时间范围内呈明显的时间依赖性，但与浓度（10-1000ng/ml）无明显依赖性。同时，E-cadherin在蛋白水平随着LPS作用时间的延长其表达水平逐渐降低，而NF-κB抑制剂PDTC时能够抑制这种效应。 3．cdc42与牙源性细胞矿化的关系 OLCs细胞接种培养至融合80%后，实验组分别更换为矿化诱导液、含10ng/mlLPS的α-MEM培养液和含10ng/ml LPS矿化诱导液经一定时间培养后，通过westernblot测定其中cdc42含量的变化；矿化诱导液组和含ML141的矿化诱导液组培养一定时间后，通过茜素红染色观察矿化结节形成情况并进行定量分析。HDPSCs在含不同浓度LPS的矿化诱导液和含不同浓度ML141的矿化诱导液中培养一定时间后，通过茜素红染色观察矿化结节形成并定量分析。Western blot结果显示，OLCs中cdc42的表达在矿化诱导液的作用下未发生明显改变，在LPS作用下明显上调。茜素红染色结果显示，OLCs矿化结节的形成量与时间正相关，与ML141的浓度呈负相关；HDPSCs矿化结节的形成与LPS浓度正相关，与ML141浓度负相关。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.3

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