多元掺杂纳米羟基磷灰石粉体体外细胞毒性的相关研究

发布时间：2018-01-19 12:32

本文关键词： 纳米羟基磷灰石多元掺杂细胞凋亡 L929细胞人牙髓细胞　出处：《第二军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：【研究背景】羟基磷灰石具有与人体骨组织和牙齿矿物质相似的化学组成和晶体结构,而被用作硬组织植入材料。虽然羟基磷灰石有着诸多的优点和良好的应用前景,但是其同样存在着生物学活性不足和不易降解等缺点,从而限制了其临床应用。研究发现人体骨组织磷灰石中除了含有钙和磷之外,还含有大量的微量元素。目前,通过水热法可以合成多元素掺杂的纳米羟基磷灰石粉体。但多元素掺杂是否会对纳米羟基磷灰石粉体的毒性产生影响尚不明确。本研究将对这一问题展开研究。【研究目的】研究多元掺杂对纳米羟基磷灰石粉体体外细胞毒性的影响及相关机制;研究多元掺杂纳米羟基磷灰石粉体浸提液体外细胞毒性及相关机制;初步探索多元掺杂纳米羟基磷灰石粉体对牙髓细胞的影响为继续探索临床应用以及深入研究硬组织植入材料多元掺杂纳米羟基磷灰石涂层的制备和生物学效应提供一定的理论依据。【研究方法】第一部分:多元掺杂纳米羟基磷灰石粉体体外细胞毒性及相关机制。1.以CCK-8比色法研究多元掺杂纳米羟基磷灰石粉体对L929细胞增殖的影响;2.以Annexin V-PI双染法经流式细胞仪检测、以Hochest33342-PI双染法经荧光显微镜检测细胞凋亡;3.以ROS荧光探针标记经荧光显微镜和流式细胞仪检测活性氧;4.以JC-1荧光探针标记经流式细胞仪检测线粒体膜电位变化;5.以Western-Bolt法检测相关蛋白的表达。第二部分:多元掺杂纳米羟基磷灰石粉体浸提液体外细胞毒性及相关机制。1.以完全培养液制备多元掺杂纳米羟基磷灰石粉体浸提液;2.以CCK-8比色法研究多元掺杂纳米羟基磷灰石粉体浸提液对L929细胞增殖的影响;3.以Annexin V-PI双染法经流式细胞仪检测细胞凋亡;4.以Western-Bolt法检测相关蛋白的表达。第三部分:多元掺杂纳米羟基磷灰石粉体对牙髓细胞的影响。1.人牙髓原代细胞的培养;2.以CCK-8比色法研究多元掺杂纳米羟基磷灰石粉体对人牙髓细胞增殖的影响。【结果】第一部分:多元掺杂纳米羟基磷灰石粉体体外细胞毒性及相关机制。多元掺杂纳米羟基磷灰石粉体与非掺杂粉体均可以抑制L929细胞增殖,促进细胞凋亡,并诱导L929细胞产生ROS和线粒体膜电位降低,同时促进p53蛋白表达,刺激Caspase 3蛋白活化。另外,通过细胞凋亡检测多元掺杂纳米羟基磷灰石粉体具有较高的凋亡率,且ROS水平和线粒体膜电位降低的比例也较高。通过细胞增殖检测结果可知虽然多元掺杂纳米羟基磷灰石粉体对于L929细胞增殖的抑制作用更强,但两者之间差异没有统计学意义(p=0.9716)。第二部分:多元掺杂纳米羟基磷灰石粉体浸提液体外细胞毒性及相关机制。多元掺杂纳米羟基磷灰石粉体与非掺杂粉体浸提液均可以抑制L929细胞增殖,促进凋亡,同时促进p53蛋白表达,刺激Caspase 3蛋白活化;通过细胞增殖检测结果可知,两者之间差异没有统计学意义(p=0.9728)。第三部分:多元掺杂纳米羟基磷灰石粉体对牙髓细胞的影响。多元掺杂纳米羟基磷灰石粉体与非掺杂粉体与目前临床常用的两种氢氧化钙盖髓材料(Calcimol LC、氢氧化钙根管消毒材料)对人牙髓细胞的增殖均无明显影响,各处理组与对照组OD值相比差异均没有统计学意义;且两种纳米羟基磷灰石材料之间的差异同样没有统计学意义(p=0.9411)。【结论】1.多元掺杂纳米羟基磷灰石粉体与非掺杂粉体均可以通过氧化应激促进p53蛋白表达,并通过线粒体途径引起细胞凋亡;多元掺杂对于纳米羟基磷灰石粉体的细胞毒性没有明显的促进作用。2.多元掺杂纳米羟基磷灰石粉体与非掺杂粉体浸提液均存在细胞毒性;多元素掺杂并未明显增加其粉体浸提液的细胞毒性。3.多元掺杂纳米羟基磷灰石粉体与非掺杂粉体对人牙髓细胞的增殖没有明显的抑制作用;多元素掺杂并未明显增加纳米羟基磷灰石材料对牙髓细胞增殖的抑制作用。
[Abstract]:[background] hydroxyapatite has to human bone and teeth similar mineral composition and crystal structure, which is used as hard tissue implants. Although hydroxyapatite has many advantages and good application prospect, but it also has a biological activity and not easy to be degraded and other shortcomings, thus limiting its clinical application the study found that human bone apatite. In addition to containing calcium and phosphorus, also contains trace elements. At present, through the hydrothermal method to nano hydroxyapatite powder synthesized doped. But whether the multiple doping of nano hydroxyapatite powder toxicity effect is not clear. This study will conduct a study on this problem. [Objective] to study effect of doped nano hydroxyapatite powder in vitro cytotoxicity and mechanism of multi doped; Hybrid nano hydroxyapatite powder extraction liquid cell toxicity and related mechanism; to explore the influence of multi doped nano hydroxyapatite powder on dental pulp cells in order to provide a theoretical basis to explore the clinical application and further study of hard tissue implant materials doped with nano hydroxyapatite coating preparation and biological effect. [Methods] the first part: multi doped nano hydroxyapatite powder in vitro cytotoxicity and mechanism of.1. to CCK-8 than the influence of color on multi doped nano hydroxyapatite on the proliferation of L929 cells; 2. with Annexin V-PI double staining by flow cytometry with Hochest33342-PI double staining method to detect apoptosis by fluorescence microscope; 3. with fluorescent probe ROS fluorescence microscopy and flow cytometry to active oxygen; 4. JC-1 fluorescent probe labeled by flow cytometry The changes of mitochondrial membrane potential; 5. to detect the expression of related protein by Western-Bolt. The second part: multi doped nano hydroxyapatite powder extraction liquid cell toxicity and mechanism of.1. complete culture medium for preparation of doped nano hydroxyapatite powder extract; 2. by CCK-8 colorimetry of doped nano hydroxyapatite powder leaching effect of extract on the proliferation of L929 cells; 3. with Annexin V-PI double staining by flow cytometry to detect apoptosis related protein; 4. was detected by using Western-Bolt method. The third part: the primary cultured human dental pulp cells in.1. doped nano hydroxyapatite powder effect on dental pulp cells; 2. to CCK-8 study on the influence of color method of multi doped nano hydroxyapatite on the proliferation of human dental pulp cells. [results] the first part: multi doped nano hydroxyapatite powder and cytotoxicity in vitro The related mechanism. Multi doped nano hydroxyapatite powder and non doped powder can inhibit the proliferation of L929 cells, promote cell apoptosis, and induce L929 cells to produce ROS and decreased mitochondrial membrane potential, and promote the expression of p53 protein, Caspase protein activation stimulated 3. In addition, apoptosis has high through detection of apoptosis of multiple doped nano hydroxyapatite powder the rate and level of ROS and decreased mitochondrial membrane potential was higher. The cell proliferation results show that although the multi doped nano hydroxyapatite on the proliferation of L929 cells inhibited by stronger, but there is no significant difference between them (p=0.9716). The second part: multi doped nano hydroxyapatite powder extraction liquid the cytotoxicity and mechanism of nano hydroxyapatite powders. Multi doped and non doped powder extracts could inhibit L929 cells Cell proliferation, promote apoptosis, and promote the expression of p53 protein, Caspase protein activated by stimulation of 3; cell proliferation test results, the difference between the two groups was not statistically significant (p=0.9728). The third part: multi doped nano hydroxyapatite powder on pulp cells. The influence of doped nano hydroxyapatite powder and powder doped with current two kinds of calcium hydroxide pulp capping materials used clinically (Calcimol LC, calcium hydroxide root canal disinfection materials) had no significant effect on the proliferation of human dental pulp cells, each treatment group compared with the control group OD value differences were not statistically significant; and the difference between the two kinds of nano hydroxyapatite materials also have no statistical significance (p=0.9411). [Conclusion] 1. multi doped nano hydroxyapatite powder and non doped powder can promote p53 protein expression by oxidative stress, and mitochondrial pathway caused by Cell apoptosis; multiple doping has no obvious effect on.2. doped nano hydroxyapatite powder and non doped powder extracts are cytotoxic to the cytotoxicity of nano hydroxyapatite powders; cytotoxicity of.3. doped nano hydroxyapatite powder and non doped powders with multiple doping did not significantly increase the powder extract on the proliferation of human dental pulp cells had no obvious inhibitory effect; multiple doping did not significantly increase the inhibitory effect of nano hydroxyapatite on the proliferation of dental pulp cells.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R783.1

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