Cadherin-11对大鼠牙髓细胞迁移、黏附和分化的影响
发布时间：2019-06-07 07:57
【摘要】：目的牙髓细胞是一类存在于机体的牙髓组织中,有多向分化潜能及自我更新能力的未分化细胞,也称为前体细胞,具备同其他组织干细胞相似的生物学特性。当牙本质-牙髓复合体受到伤害后,这些牙髓细胞就会向受损的地方迁移,发生黏附,并分化成成牙本质细胞,分泌矿化组织形成修复性牙本质修复损伤。钙粘蛋白家族是一类钙离子依赖性介导嗜同性细胞-细胞间黏附的单链跨膜糖蛋白,分子量约120～140ku,与免疫球蛋白超家族、整合素家族、选择素家族等统称为细胞黏附分子。基于结构上的差异,钙粘蛋白分为Ⅰ型和Ⅱ型,Cadherin-11属于后者,介导的细胞间粘附连接在细胞识别、迁移、增生、分化和程序性死亡等生理过程以及调控组织器官形态发生中起重要作用。该种钙粘附蛋白在骨组织特别丰富,在骨组织分化早期高表达。新近研究显示,cadherin-11参与牙根的吸收过程,在根吸收过程中起调节作用。wu等的基因芯片研究显示,在大鼠牙髓组织中有cadherin-11mRNA的表达,提示cadherin-11可能在大鼠牙髓组织的生长发育中有重要作用。成骨细胞和成牙本质细胞同为可分泌矿化组织的细胞,我们有理由相信cadherin-11在这两种细胞可能有着相似的功能作用。关于cadherin-11对牙髓细胞的迁移、黏附和成牙/成骨向分化能力的影响,目前尚未见文献报道。因此,探讨cadherin-11在大鼠牙髓细胞的迁移、黏附以及成牙本质分化过程中的作用及其相关的分子机制具有重要意义。本研究目的探讨过表达cadherin-11对大鼠牙髓细胞迁移、黏附和成牙本质分化能力的影响,为牙齿的损伤修复提供新思路。方法1.组织块酶消化法分离培养大鼠牙髓细胞,收集第3-5代牙髓细胞行波形丝蛋白、角蛋白免疫组化染色,鉴定细胞来源。2.用RT-PCR检测大鼠切牙牙髓组织cadherin-11mRNA的表达,用免疫组化法检测其在牙髓组织中的分布,初步探讨其与牙齿发育的关系。3.腺病毒感染：取实验室培养的3～5代牙髓细胞,并用pDC316-mCMV-EGFP-Cadherin11重组腺病毒进行转染。通过RT-PCR、免疫组化法检测大鼠牙髓细胞中cadherin-11基因及蛋白的表达。实验将细胞分为三组,包括经pDC316-mCMV-EGFP-Cadherin11重组腺病毒感染的实验组,经空载体腺病毒感染的阴性对照组和未经腺病毒感染的正常细胞为空白对照组。4.细胞迁移、黏附和分化的检测：通过Transwe11实验探索cadherin-11对大鼠牙髓细胞迁移的作用,用细胞黏附实验确定cadherin-11对大鼠牙髓细胞黏附能力的影响。矿化诱导14d后,通过碱性磷酸酶试剂盒进行ALP染色检测ALP活性。矿化诱导21d,进行茜素红钙结节染色及定量检测其成牙本质分化情况。半定量PCR检测牙髓细胞分化相关基因DMP1、DSPP、ALP的表达。结果1.利用组织块酶消化法分离培养出大鼠牙髓细胞,成功建立大鼠牙髓细胞体外研究模型。免疫组化结果显示波形丝蛋白染色阳性,而角蛋白染色阴性,提示牙髓细胞来源于中胚层。2. RT-PCR显示在大鼠切牙牙髓组织有cadherin-11mRNA的表达。大鼠牙髓组织免疫组化染色可见cadherin-11广泛表达于牙髓细胞、成牙本质细胞的细胞浆和细胞膜；在根尖部牙髓细胞表现为弱阳性表达,往牙冠方向,表达增强,在牙髓冠方为强阳性表达。cadherin-11在成牙本质细胞为强阳性表达,比牙髓细胞表达强。3. RT-PCR、免疫组化染色分别示cadherin-11基因及蛋白高表达,提示cadherin-11基因转染成功。4. Cadherin-11可增加细胞的黏附性和迁移能力(P＜0.05)。与阴性对照组和空白对照组相比,实验组在矿化诱导14d可增强大鼠牙髓细胞ALP表达,诱导21d,形成钙化结节量增加(P0.05),同时半定量PCR示牙髓细胞分化相关基因DMP1、 DSPP、ALP表达增高(P0.05)。结论1.从大鼠牙髓组织中分离、培养获得的大鼠牙髓细胞长期传代仍能够保持稳定的生长能力,为cadherin-11转染大鼠牙髓细胞的实验奠定了基础。2. Cadherin-11在大鼠牙髓组织中选择性的表达,提示cadherin-11可能在牙齿发育中发挥重要的调控作用。这为进一步深入研究cadherin-11在牙齿硬组织发生和发育中的作用奠定实验基础。3. Cadherin-11能提高其迁移和黏附能力,同时促进大鼠牙髓细胞成牙本质分化能力。
[Abstract]:Objective The pulp cell is a kind of non-differentiated cell which is present in the pulp tissue of the body, which has multi-directional differentiation potential and self-renewing ability, also called the precursor cell, and has the same biological characteristics as the other tissue stem cells. When the dentin-pulp complex is damaged, the dental pulp cells will migrate to the damaged areas, adhere, and differentiate into odontoblast cells, and secrete the mineralized tissue to form a repair dentin repair injury. The calcium-binding protein family is a class of calcium-ion-dependent, single-chain transmembrane glycoproteins that mediate the adhesion of the same-sex cell-cells, with a molecular weight of about 120-140 ku, and are collectively referred to as cell adhesion molecules with the immunoglobulin superfamily, the integrin family, the selectin family, and the like. Based on the structural differences, the cadherin is divided into type I and type II, and Cadherin-11 belongs to the latter, and the mediated intercellular adhesion connection plays an important role in the physiological processes of cell identification, migration, proliferation, differentiation and programmed death. The calcium adhesion protein is particularly rich in bone tissue and is highly expressed in the early stage of bone tissue differentiation. Recent studies have shown that cadherin-11 is involved in the absorption process of the root, and plays an important role in the root absorption process. The gene chip of wu et al. showed that the expression of the cadherin-11 mRNA in the pulp tissue of the rat suggested that the cadherin-11 might play an important role in the growth and development of the rat dental pulp tissue. Osteoblasts and odontoblasts are also cells that secrete mineralized tissue, and we have reason to believe that cadherin-11 may have similar functional effects in both of these cells. The effect of cadherin-11 on the migration, adhesion and dental/ osteogenesis of dental pulp cells has not been reported in the literature. Therefore, it is of great significance to study the role of the cadherin-11 in the migration and adhesion of pulp cells in the rat and the role of the related molecular mechanism in the process of dentine differentiation. Objective To study the effects of the expression of cadherin-11 on the migration, adhesion and dentinal differentiation of dental pulp cells in rats, and to provide a new way to repair the damage of the teeth. Method 1. The rat dental pulp cells were isolated and cultured by the tissue-block enzyme digestion method, and the 3-5-generation dental pulp cell line-shaped silk protein was collected, and the cytokeratin was stained with immunohistochemical staining to identify the cell source. The expression of cadherin-11 mRNA was detected by RT-PCR, and its distribution in dental pulp was detected by immunohistochemistry. Adenovirus infection:3 ~ 5 generations of dental pulp cells were cultured in the laboratory, and the adenovirus was transfected with the recombinant adenovirus of pDC316-mCMV-EGFP-Cadherin11. The expression of cadherin-11 gene and protein in dental pulp cells was detected by RT-PCR and immunohistochemistry. The cells were divided into three groups, including the experimental group infected with the recombinant adenovirus of pDC316-mCMV-EGFP-Cadherin11, the negative control group infected with the empty vector adenovirus and the normal cells infected with the adenovirus as the blank control group. Cell migration, adhesion and differentiation: The effect of caadherin-11 on the rat dental pulp cell migration was explored by the Transwe11 experiment, and the effect of the cadherin-11 on the rat dental pulp cell adhesion was determined by cell adhesion assay. After the mineralization induction for 14 days, the ALP activity was detected by ALP staining by the alkaline phosphatase kit. The mineralization was induced for 21 days, and the red calcium nodules were stained and the dentinal differentiation was detected quantitatively. The expression of DP1, DSPP and ALP was detected by semi-quantitative PCR. Results 1. The rat dental pulp cells were isolated and cultured by the tissue-block enzyme digestion method, and the rat dental pulp cell in vitro study model was successfully established. The results showed that the staining of the silk protein was positive, and the staining of the keratin was negative, indicating that the pulp cells were derived from the mesoderm. RT-PCR showed the expression of cadherin-11 mRNA in the dental pulp of the rat. The immunohistochemical staining of dental pulp in the rat showed that the cadherin-11 was widely expressed in the pulp cells, the cytoplasm and the cell membrane of the odontoblast cells, and the pulp cells in the apical part showed a weak positive expression, and in the direction of the crown, the expression was enhanced, and the dental pulp crown was a strong positive expression. Cadherin-11 is a strong positive expression in odontoblast cells, and is stronger than that of dental pulp cells. The expression of the cadherin-11 gene and the protein was shown by RT-PCR and immunohistochemical staining, suggesting that the transfection of the cadherin-11 gene was successful. Cadherin-11 increased the adhesion and migration ability of the cells (P <0.05). Compared with the negative control group and the blank control group, the experimental group increased the ALP expression of the pulp cells in the rat dental pulp cells by the mineralization induction for 14 days, and the amount of the calcified nodules was increased (P0.05), and the expression of the DP1, DSPP and ALP in the dental pulp cells was increased by semi-quantitative PCR (P0.05). Conclusion 1. It was found that the long-term passage of dental pulp cells from rat dental pulp was able to maintain a stable growth ability and lay a foundation for the experiment of the transfection of the rat dental pulp cells with the cadherin-11. The selective expression of Cadherin-11 in the rat dental pulp tissue suggests that the cadherin-11 may play an important regulatory role in the development of teeth. This provides an experimental basis for further study of the role of the cadherin-11 in the development and development of dental hard tissues. Cadherin-11 can improve its migration and adhesion, while promoting the ability of dental pulp cells to differentiate into dentin.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R781
本文编号：2494638
[Abstract]:Objective The pulp cell is a kind of non-differentiated cell which is present in the pulp tissue of the body, which has multi-directional differentiation potential and self-renewing ability, also called the precursor cell, and has the same biological characteristics as the other tissue stem cells. When the dentin-pulp complex is damaged, the dental pulp cells will migrate to the damaged areas, adhere, and differentiate into odontoblast cells, and secrete the mineralized tissue to form a repair dentin repair injury. The calcium-binding protein family is a class of calcium-ion-dependent, single-chain transmembrane glycoproteins that mediate the adhesion of the same-sex cell-cells, with a molecular weight of about 120-140 ku, and are collectively referred to as cell adhesion molecules with the immunoglobulin superfamily, the integrin family, the selectin family, and the like. Based on the structural differences, the cadherin is divided into type I and type II, and Cadherin-11 belongs to the latter, and the mediated intercellular adhesion connection plays an important role in the physiological processes of cell identification, migration, proliferation, differentiation and programmed death. The calcium adhesion protein is particularly rich in bone tissue and is highly expressed in the early stage of bone tissue differentiation. Recent studies have shown that cadherin-11 is involved in the absorption process of the root, and plays an important role in the root absorption process. The gene chip of wu et al. showed that the expression of the cadherin-11 mRNA in the pulp tissue of the rat suggested that the cadherin-11 might play an important role in the growth and development of the rat dental pulp tissue. Osteoblasts and odontoblasts are also cells that secrete mineralized tissue, and we have reason to believe that cadherin-11 may have similar functional effects in both of these cells. The effect of cadherin-11 on the migration, adhesion and dental/ osteogenesis of dental pulp cells has not been reported in the literature. Therefore, it is of great significance to study the role of the cadherin-11 in the migration and adhesion of pulp cells in the rat and the role of the related molecular mechanism in the process of dentine differentiation. Objective To study the effects of the expression of cadherin-11 on the migration, adhesion and dentinal differentiation of dental pulp cells in rats, and to provide a new way to repair the damage of the teeth. Method 1. The rat dental pulp cells were isolated and cultured by the tissue-block enzyme digestion method, and the 3-5-generation dental pulp cell line-shaped silk protein was collected, and the cytokeratin was stained with immunohistochemical staining to identify the cell source. The expression of cadherin-11 mRNA was detected by RT-PCR, and its distribution in dental pulp was detected by immunohistochemistry. Adenovirus infection:3 ~ 5 generations of dental pulp cells were cultured in the laboratory, and the adenovirus was transfected with the recombinant adenovirus of pDC316-mCMV-EGFP-Cadherin11. The expression of cadherin-11 gene and protein in dental pulp cells was detected by RT-PCR and immunohistochemistry. The cells were divided into three groups, including the experimental group infected with the recombinant adenovirus of pDC316-mCMV-EGFP-Cadherin11, the negative control group infected with the empty vector adenovirus and the normal cells infected with the adenovirus as the blank control group. Cell migration, adhesion and differentiation: The effect of caadherin-11 on the rat dental pulp cell migration was explored by the Transwe11 experiment, and the effect of the cadherin-11 on the rat dental pulp cell adhesion was determined by cell adhesion assay. After the mineralization induction for 14 days, the ALP activity was detected by ALP staining by the alkaline phosphatase kit. The mineralization was induced for 21 days, and the red calcium nodules were stained and the dentinal differentiation was detected quantitatively. The expression of DP1, DSPP and ALP was detected by semi-quantitative PCR. Results 1. The rat dental pulp cells were isolated and cultured by the tissue-block enzyme digestion method, and the rat dental pulp cell in vitro study model was successfully established. The results showed that the staining of the silk protein was positive, and the staining of the keratin was negative, indicating that the pulp cells were derived from the mesoderm. RT-PCR showed the expression of cadherin-11 mRNA in the dental pulp of the rat. The immunohistochemical staining of dental pulp in the rat showed that the cadherin-11 was widely expressed in the pulp cells, the cytoplasm and the cell membrane of the odontoblast cells, and the pulp cells in the apical part showed a weak positive expression, and in the direction of the crown, the expression was enhanced, and the dental pulp crown was a strong positive expression. Cadherin-11 is a strong positive expression in odontoblast cells, and is stronger than that of dental pulp cells. The expression of the cadherin-11 gene and the protein was shown by RT-PCR and immunohistochemical staining, suggesting that the transfection of the cadherin-11 gene was successful. Cadherin-11 increased the adhesion and migration ability of the cells (P <0.05). Compared with the negative control group and the blank control group, the experimental group increased the ALP expression of the pulp cells in the rat dental pulp cells by the mineralization induction for 14 days, and the amount of the calcified nodules was increased (P0.05), and the expression of the DP1, DSPP and ALP in the dental pulp cells was increased by semi-quantitative PCR (P0.05). Conclusion 1. It was found that the long-term passage of dental pulp cells from rat dental pulp was able to maintain a stable growth ability and lay a foundation for the experiment of the transfection of the rat dental pulp cells with the cadherin-11. The selective expression of Cadherin-11 in the rat dental pulp tissue suggests that the cadherin-11 may play an important regulatory role in the development of teeth. This provides an experimental basis for further study of the role of the cadherin-11 in the development and development of dental hard tissues. Cadherin-11 can improve its migration and adhesion, while promoting the ability of dental pulp cells to differentiate into dentin.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R781
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