贻贝足丝蛋白在牙龈上皮细胞粘结中的应用研究

发布时间：2018-04-23 18:17

本文选题：牙龈上皮细胞 + 细胞粘附　；参考：《福建医科大学》2012年硕士论文

【摘要】：目的随着科学技术的发展，越来越多的粘结相关材料被广泛应用于医学科学研究，并为临床科学研究作出许多重要贡献。然而，现有材料不仅在量或质方面无法满足日新月异的科学需求，且生物相容性差。因此，寻找一种行之有效、可大量生产、生物相容性好的粘结材料是目前亟需解决的问题。本研究根据贻贝能够超强粘附在各种物体表面的特性，选用贻贝足丝蛋白（Mussel Foot Protein, Mefp）及其粘附关键因子L-3,4-二羟基苯丙氨酸（L-Dioxyphenylalanine, L-DOPA）进行牙龈上皮细胞的粘附实验，同时对比几种牙龈上皮细胞原代培养的方法，通过观察细胞形态、绘制细胞生长曲线、测量细胞粘附量以及后续钙离子浓度的测定评价Mefp和L-DOPA对牙龈上皮细胞的粘结作用，旨在寻找一种利于牙龈上皮细胞原代及传代培养的方法，为临床进行牙龈上皮细胞的研究提供理论与技术支持。方法 1.分别采用单纯Dispaes酶消化、单纯玻片覆盖组织块法以及Dispase酶消化联合玻片覆盖组织块法培养Beagle犬牙龈上皮细胞，通过观察细胞形态以及细胞生长时间，对比寻找最适细胞培养方法。 2.培养皿表面分别经Mefp、L-DOPA、多聚赖氨酸(Poly-L-Lysine,PLL)、蒸馏水处理后接种一定量细胞，通过二眯基苯基吲哚(4,6-Diamidino-2-Phenylindole,DAPI)染色计数计算细胞粘附量；酶标仪测量光密度值(Optical Density, OD)，，绘制细胞生长曲线；扫描电子显微镜(Scanning Electron Microscopy, SEM)观察细胞形态；细胞内钙离子浓度测定分析细胞增殖能力。并与空白组的作用进行对比。结果 1.单纯Dispase酶消化培养的牙龈上皮细胞贴壁少，培养成功率为0.0%。单纯玻片覆盖组织块法培养细胞贴壁多，但成纤维细胞污染严重，培养成功率仅为30.5%。Dispase酶消化联合玻片覆盖组织块法培养，短时间内可获得大量牙龈上皮细胞，且不存在成纤维细胞污染，成功率可达91.6%。卡方检验表明，各组细胞培养成功率有统计学意义（P0.05）。 2. Mefp处理组与空白组相比，镜下DAPI染色细胞多，细胞呈扁圆形，棘明显可见，48h细胞OD值高（P0.05），呈典型S型生长，细胞内钙离子浓度也较空白组高（P0.05）；但与PLL处理组相比并无显著差异（P0.05）。L-DOPA处理组与空白组相比，镜下DAPI染色细胞多，细胞呈扁圆形，伪足明显，48h细胞OD值高（P0.05），呈典型S型生长，细胞内钙离子浓度也较高（P0.05）；而与Mefp处理组及PLL处理组相比，DAPI染色细胞及细胞内钙离子浓度亦显著增多（P0.05）。结论 1. Dispase酶消化联合玻片覆盖组织块法进行牙龈上皮细胞培养，可短时间获得大量牙龈上皮细胞，此法简单、有效。 2. Mefp及L-DOPA可显著提高牙龈上皮细胞的粘附，其作用同PLL相似。
[Abstract]:objective
With the development of science and technology, more and more adhesive related materials have been widely used in medical science research, and make many important contributions to clinical scientific research. However, the existing materials are not only unable to meet the new scientific needs in quantity or quality, but also have poor biocompatibility. Therefore, it is effective to find a kind of effective and large amount of production. The adhesive material with good biocompatibility is an urgent problem to be solved at present. In this study, the adhesion of Mussel Foot Protein (Mefp) and L-3,4- two hydroxy phenylalanine (L-Dioxyphenylalanine, L-DOPA), which is the key factor of mussels, can be used to adhere to the surface of various objects. At the same time, the methods of primary culture of several gingival epithelial cells were compared. By observing cell morphology, plotting cell growth curve, measuring cell adhesion and measuring the subsequent calcium concentration, the binding effect of Mefp and L-DOPA on gingival epithelial cells was evaluated in order to find a kind of proproic and subculture culture of gingival epithelial cells. Methods to provide theoretical and technical support for the clinical study of gingival epithelial cells.
Method
1. the Beagle canine gingival epithelial cells were cultured by simple Dispaes enzyme digestion, simple glass covered tissue block method and Dispase enzyme digestion combined with slide covered tissue block method. The optimum cell culture method was compared by observing cell morphology and cell growth time.
2. the surface of the Petri dish was treated with Mefp, L-DOPA, Poly-L-Lysine (PLL) and distilled water to inoculate a certain amount of cells and calculate the cell adhesion by two squinyl phenyl indole (4,6-Diamidino-2-Phenylindole, DAPI), and the density value (Optical Density, OD) was measured by the enzyme labeling instrument, and the cell growth curve was plotted; scanning electron The morphology of the cells was observed by Scanning Electron Microscopy (SEM), and the intracellular calcium concentration was used to determine the cell proliferation ability and to compare the effect of the blank group.
Result
1. the gingival epithelial cells cultured with simple Dispase enzyme digestion were less adherent, and the success rate of culture was 0.0%. simple glass covered tissue block method, but the fibroblasts were more polluted. The success rate of the culture was only 30.5%.Dispase enzyme digestion combined with glass covered tissue block method, and a large number of gingival epithelial cells could be obtained in a short time. There was fibroblast contamination, and the success rate was 91.6%.. Chi square test showed that the success rate of cell culture was statistically significant (P0.05).
2. Mefp treatment group compared with the blank group, the DAPI staining cells were many, the cells showed flat circle, the spines were obviously visible, the 48h cell OD value was high (P0.05), the growth of S type was typical, the intracellular calcium concentration was higher than that of the blank group (P0.05), but there was no significant difference compared with the PLL treatment group (P0.05) the.L-DOPA treatment group was compared with the blank group, the microscope DAPI stained cells were compared. The cells showed a flat circle, the pseudo foot was obvious, the 48h cell OD value was high (P0.05), which showed a typical S type growth, and the intracellular calcium concentration was also higher (P0.05). Compared with the Mefp treatment group and the PLL treatment group, the DAPI staining cells and the intracellular calcium concentration were also significantly increased (P0.05).
conclusion
1. gingival epithelial cells were cultured by Dispase enzyme digestion combined with slide covering tissue block method, and a large number of gingival epithelial cells could be obtained in a short time. This method is simple and effective.
2. Mefp and L-DOPA can significantly enhance the adhesion of gingival epithelial cells, which is similar to PLL.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R783.1

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