Cadherin-11對大鼠牙髓細胞遷移、黏附和分化的影響
發(fā)布時間：2019-06-07 07:57
【摘要】：目的牙髓細胞是一類存在于機體的牙髓組織中,有多向分化潛能及自我更新能力的未分化細胞,也稱為前體細胞,具備同其他組織干細胞相似的生物學特性。當牙本質(zhì)-牙髓復(fù)合體受到傷害后,這些牙髓細胞就會向受損的地方遷移,發(fā)生黏附,并分化成成牙本質(zhì)細胞,分泌礦化組織形成修復(fù)性牙本質(zhì)修復(fù)損傷。鈣粘蛋白家族是一類鈣離子依賴性介導(dǎo)嗜同性細胞-細胞間黏附的單鏈跨膜糖蛋白,分子量約120～140ku,與免疫球蛋白超家族、整合素家族、選擇素家族等統(tǒng)稱為細胞黏附分子�；诮Y(jié)構(gòu)上的差異,鈣粘蛋白分為Ⅰ型和Ⅱ型,Cadherin-11屬于后者,介導(dǎo)的細胞間粘附連接在細胞識別、遷移、增生、分化和程序性死亡等生理過程以及調(diào)控組織器官形態(tài)發(fā)生中起重要作用。該種鈣粘附蛋白在骨組織特別豐富,在骨組織分化早期高表達。新近研究顯示,cadherin-11參與牙根的吸收過程,在根吸收過程中起調(diào)節(jié)作用。wu等的基因芯片研究顯示,在大鼠牙髓組織中有cadherin-11mRNA的表達,提示cadherin-11可能在大鼠牙髓組織的生長發(fā)育中有重要作用。成骨細胞和成牙本質(zhì)細胞同為可分泌礦化組織的細胞,我們有理由相信cadherin-11在這兩種細胞可能有著相似的功能作用。關(guān)于cadherin-11對牙髓細胞的遷移、黏附和成牙/成骨向分化能力的影響,目前尚未見文獻報道。因此,探討cadherin-11在大鼠牙髓細胞的遷移、黏附以及成牙本質(zhì)分化過程中的作用及其相關(guān)的分子機制具有重要意義。本研究目的探討過表達cadherin-11對大鼠牙髓細胞遷移、黏附和成牙本質(zhì)分化能力的影響,為牙齒的損傷修復(fù)提供新思路。方法1.組織塊酶消化法分離培養(yǎng)大鼠牙髓細胞,收集第3-5代牙髓細胞行波形絲蛋白、角蛋白免疫組化染色,鑒定細胞來源。2.用RT-PCR檢測大鼠切牙牙髓組織cadherin-11mRNA的表達,用免疫組化法檢測其在牙髓組織中的分布,初步探討其與牙齒發(fā)育的關(guān)系。3.腺病毒感染：取實驗室培養(yǎng)的3～5代牙髓細胞,并用pDC316-mCMV-EGFP-Cadherin11重組腺病毒進行轉(zhuǎn)染。通過RT-PCR、免疫組化法檢測大鼠牙髓細胞中cadherin-11基因及蛋白的表達。實驗將細胞分為三組,包括經(jīng)pDC316-mCMV-EGFP-Cadherin11重組腺病毒感染的實驗組,經(jīng)空載體腺病毒感染的陰性對照組和未經(jīng)腺病毒感染的正常細胞為空白對照組。4.細胞遷移、黏附和分化的檢測：通過Transwe11實驗探索cadherin-11對大鼠牙髓細胞遷移的作用,用細胞黏附實驗確定cadherin-11對大鼠牙髓細胞黏附能力的影響。礦化誘導(dǎo)14d后,通過堿性磷酸酶試劑盒進行ALP染色檢測ALP活性。礦化誘導(dǎo)21d,進行茜素紅鈣結(jié)節(jié)染色及定量檢測其成牙本質(zhì)分化情況。半定量PCR檢測牙髓細胞分化相關(guān)基因DMP1、DSPP、ALP的表達。結(jié)果1.利用組織塊酶消化法分離培養(yǎng)出大鼠牙髓細胞,成功建立大鼠牙髓細胞體外研究模型。免疫組化結(jié)果顯示波形絲蛋白染色陽性,而角蛋白染色陰性,提示牙髓細胞來源于中胚層。2. RT-PCR顯示在大鼠切牙牙髓組織有cadherin-11mRNA的表達。大鼠牙髓組織免疫組化染色可見cadherin-11廣泛表達于牙髓細胞、成牙本質(zhì)細胞的細胞漿和細胞膜；在根尖部牙髓細胞表現(xiàn)為弱陽性表達,往牙冠方向,表達增強,在牙髓冠方為強陽性表達。cadherin-11在成牙本質(zhì)細胞為強陽性表達,比牙髓細胞表達強。3. RT-PCR、免疫組化染色分別示cadherin-11基因及蛋白高表達,提示cadherin-11基因轉(zhuǎn)染成功。4. Cadherin-11可增加細胞的黏附性和遷移能力(P＜0.05)。與陰性對照組和空白對照組相比,實驗組在礦化誘導(dǎo)14d可增強大鼠牙髓細胞ALP表達,誘導(dǎo)21d,形成鈣化結(jié)節(jié)量增加(P0.05),同時半定量PCR示牙髓細胞分化相關(guān)基因DMP1、 DSPP、ALP表達增高(P0.05)。結(jié)論1.從大鼠牙髓組織中分離、培養(yǎng)獲得的大鼠牙髓細胞長期傳代仍能夠保持穩(wěn)定的生長能力,為cadherin-11轉(zhuǎn)染大鼠牙髓細胞的實驗奠定了基礎(chǔ)。2. Cadherin-11在大鼠牙髓組織中選擇性的表達,提示cadherin-11可能在牙齒發(fā)育中發(fā)揮重要的調(diào)控作用。這為進一步深入研究cadherin-11在牙齒硬組織發(fā)生和發(fā)育中的作用奠定實驗基礎(chǔ)。3. Cadherin-11能提高其遷移和黏附能力,同時促進大鼠牙髓細胞成牙本質(zhì)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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R781
本文編號：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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R781
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