本文關(guān)鍵詞： 納米羥基磷灰石 多元摻雜 細(xì)胞凋亡 L929細(xì)胞 人牙髓細(xì)胞　出處：《第二軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：【研究背景】羥基磷灰石具有與人體骨組織和牙齒礦物質(zhì)相似的化學(xué)組成和晶體結(jié)構(gòu),而被用作硬組織植入材料。雖然羥基磷灰石有著諸多的優(yōu)點(diǎn)和良好的應(yīng)用前景,但是其同樣存在著生物學(xué)活性不足和不易降解等缺點(diǎn),從而限制了其臨床應(yīng)用。研究發(fā)現(xiàn)人體骨組織磷灰石中除了含有鈣和磷之外,還含有大量的微量元素。目前,通過水熱法可以合成多元素?fù)诫s的納米羥基磷灰石粉體。但多元素?fù)诫s是否會(huì)對(duì)納米羥基磷灰石粉體的毒性產(chǎn)生影響尚不明確。本研究將對(duì)這一問題展開研究。【研究目的】研究多元摻雜對(duì)納米羥基磷灰石粉體體外細(xì)胞毒性的影響及相關(guān)機(jī)制;研究多元摻雜納米羥基磷灰石粉體浸提液體外細(xì)胞毒性及相關(guān)機(jī)制;初步探索多元摻雜納米羥基磷灰石粉體對(duì)牙髓細(xì)胞的影響為繼續(xù)探索臨床應(yīng)用以及深入研究硬組織植入材料多元摻雜納米羥基磷灰石涂層的制備和生物學(xué)效應(yīng)提供一定的理論依據(jù)。【研究方法】第一部分:多元摻雜納米羥基磷灰石粉體體外細(xì)胞毒性及相關(guān)機(jī)制。1.以CCK-8比色法研究多元摻雜納米羥基磷灰石粉體對(duì)L929細(xì)胞增殖的影響;2.以Annexin V-PI雙染法經(jīng)流式細(xì)胞儀檢測(cè)、以Hochest33342-PI雙染法經(jīng)熒光顯微鏡檢測(cè)細(xì)胞凋亡;3.以ROS熒光探針標(biāo)記經(jīng)熒光顯微鏡和流式細(xì)胞儀檢測(cè)活性氧;4.以JC-1熒光探針標(biāo)記經(jīng)流式細(xì)胞儀檢測(cè)線粒體膜電位變化;5.以Western-Bolt法檢測(cè)相關(guān)蛋白的表達(dá)。第二部分:多元摻雜納米羥基磷灰石粉體浸提液體外細(xì)胞毒性及相關(guān)機(jī)制。1.以完全培養(yǎng)液制備多元摻雜納米羥基磷灰石粉體浸提液;2.以CCK-8比色法研究多元摻雜納米羥基磷灰石粉體浸提液對(duì)L929細(xì)胞增殖的影響;3.以Annexin V-PI雙染法經(jīng)流式細(xì)胞儀檢測(cè)細(xì)胞凋亡;4.以Western-Bolt法檢測(cè)相關(guān)蛋白的表達(dá)。第三部分:多元摻雜納米羥基磷灰石粉體對(duì)牙髓細(xì)胞的影響。1.人牙髓原代細(xì)胞的培養(yǎng);2.以CCK-8比色法研究多元摻雜納米羥基磷灰石粉體對(duì)人牙髓細(xì)胞增殖的影響。【結(jié)果】第一部分:多元摻雜納米羥基磷灰石粉體體外細(xì)胞毒性及相關(guān)機(jī)制。多元摻雜納米羥基磷灰石粉體與非摻雜粉體均可以抑制L929細(xì)胞增殖,促進(jìn)細(xì)胞凋亡,并誘導(dǎo)L929細(xì)胞產(chǎn)生ROS和線粒體膜電位降低,同時(shí)促進(jìn)p53蛋白表達(dá),刺激Caspase 3蛋白活化。另外,通過細(xì)胞凋亡檢測(cè)多元摻雜納米羥基磷灰石粉體具有較高的凋亡率,且ROS水平和線粒體膜電位降低的比例也較高。通過細(xì)胞增殖檢測(cè)結(jié)果可知雖然多元摻雜納米羥基磷灰石粉體對(duì)于L929細(xì)胞增殖的抑制作用更強(qiáng),但兩者之間差異沒有統(tǒng)計(jì)學(xué)意義(p=0.9716)。第二部分:多元摻雜納米羥基磷灰石粉體浸提液體外細(xì)胞毒性及相關(guān)機(jī)制。多元摻雜納米羥基磷灰石粉體與非摻雜粉體浸提液均可以抑制L929細(xì)胞增殖,促進(jìn)凋亡,同時(shí)促進(jìn)p53蛋白表達(dá),刺激Caspase 3蛋白活化;通過細(xì)胞增殖檢測(cè)結(jié)果可知,兩者之間差異沒有統(tǒng)計(jì)學(xué)意義(p=0.9728)。第三部分:多元摻雜納米羥基磷灰石粉體對(duì)牙髓細(xì)胞的影響。多元摻雜納米羥基磷灰石粉體與非摻雜粉體與目前臨床常用的兩種氫氧化鈣蓋髓材料(Calcimol LC、氫氧化鈣根管消毒材料)對(duì)人牙髓細(xì)胞的增殖均無明顯影響,各處理組與對(duì)照組OD值相比差異均沒有統(tǒng)計(jì)學(xué)意義;且兩種納米羥基磷灰石材料之間的差異同樣沒有統(tǒng)計(jì)學(xué)意義(p=0.9411)�！窘Y(jié)論】1.多元摻雜納米羥基磷灰石粉體與非摻雜粉體均可以通過氧化應(yīng)激促進(jìn)p53蛋白表達(dá),并通過線粒體途徑引起細(xì)胞凋亡;多元摻雜對(duì)于納米羥基磷灰石粉體的細(xì)胞毒性沒有明顯的促進(jìn)作用。2.多元摻雜納米羥基磷灰石粉體與非摻雜粉體浸提液均存在細(xì)胞毒性;多元素?fù)诫s并未明顯增加其粉體浸提液的細(xì)胞毒性。3.多元摻雜納米羥基磷灰石粉體與非摻雜粉體對(duì)人牙髓細(xì)胞的增殖沒有明顯的抑制作用;多元素?fù)诫s并未明顯增加納米羥基磷灰石材料對(duì)牙髓細(xì)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R783.1

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