碘在体外培养成纤维细胞模型中的作用研究

发布时间：2018-01-12 20:33

本文关键词：碘在体外培养成纤维细胞模型中的作用研究　出处：《山东大学》2008年硕士论文　论文类型：学位论文

【摘要】： 【目的】本实验利用体外培养成纤维细胞模型,运用形态学观察、免疫细胞化学技术、流式细胞术及相关生化指标检测等多种方法研究不同浓度的碘离子及碘分子对体外培养成纤维细胞增殖活性与功能的影响,探讨碘对甲状腺外组织的作用,丰富微量元素碘的实验研究。【方法】 1.细胞株:人皮肤成纤维(HSF)细胞株,购于协和医科大学基础医学细胞中心。常规细胞培养,待传代稳定后进行以下实验。 2.试剂配制及实验分组:碘化钾,分析纯,以纯水配制成为1000mgI/L母液,避光保存,以DMEM培养液稀释待用。碘,分析纯,以纯水配制成饱和溶液,以化学滴定法测定其浓度,现配现用。本实验中,碘离子设1个空白对照组及50-1500μg/L中若干实验组;碘分子设1个空白对照组及50-400μg/L中若干实验组。 3.细胞计数及形态学观察:种24孔板,待细胞贴壁后加入不同浓度的碘离子及碘分子,每板均设对照组,于作用4、12、24、48小时后进行细胞计数及观察细胞形态学改变。 4.MTT:种96孔培养板,不同浓度碘离子及碘分子处理4、12、24、48小时后,每孔添加20μl(5mg/ml)MTT试剂,37℃孵育4小时,小心弃去培养液,每孔加入150μl二甲基亚砜(DMSO),振荡10分钟。以酶标仪在490nm波长下测其光吸收值(吸光度A值),可间接反映细胞数量。 5.流式细胞术:不同浓度碘离子及碘分子处理细胞48小时,经离心—PBS冲洗—吹匀—固定—再离心—冲洗—吹打—染色(4℃,20-30分钟)后,以400目尼龙网过滤后上机检测。ModFit分析软件分析,用细胞分裂增殖指数表示相对增殖活力。 6.免疫细胞化学:将洁净无菌的盖玻片放入6孔培养板中,制备单细胞悬液,种6孔板,以不同浓度的碘离子及碘分子处理48小时后取出盖玻片,固定细胞,检测细胞中Ki-67的表达。 7.SOD、MDA检测:利用试剂盒对培养细胞的上清液中丙二醛(MDA)和超氧化物岐化酶(SOD)进行检测。【结果】 1.细胞计数及MTT检测:碘离子浓度及碘分子浓度较低(如48小时:碘离子≤3000μg/L,碘分子≤400μg/L)时,细胞计数随浓度增加而增加,细胞活性也随浓度增加而升高;碘离子浓度及碘分子浓度较高(如48小时:碘离子≥3000μg/L,碘分子≥400μg/L)时,细胞计数随浓度增加而减少,细胞增殖活性也随浓度增加而降低,且可见细胞形态学改变。 2.流式细胞术:碘离子浓度100-4000μg/L(碘分子浓度50-700μg/L)实验组细胞增殖指数明显高于对照组,同时细胞凋亡率明显低于对照组;碘离子浓度5000μg/L(碘分子浓度800μg/L)实验组细胞增殖指数与对照组比较没有明显差别,而细胞凋亡率明显高于对照组;碘离子浓度≥6000μg/L(碘分子浓度≥900μg/L)实验组细胞增殖指数明显低于对照组,同时细胞凋亡率明显高于对照组。 3.免疫细胞化学:碘离子浓度100-3000μg/L(碘分子浓度50-500μg/L)实验组Ki-67的阳性率明显高于对照组;碘离子浓度≥500μg/L(碘分子浓度≥700μg/L)各实验组Ki-67的表达率明显低于对照组。 4.SOD、MDA检测:碘离子浓度50-600μg/L(碘分子浓度50-300μg/L)实验组随碘离子浓度的增加SOD含量增加,碘离子浓度800-10000μg/L(碘分子浓度600-4000μg/L)实验组随碘离子浓度的增加SOD含量减少;碘离子浓度50-800μg/L(碘分子浓度50-500μg/L除200μg/L外)各实验组MDA含量与对照组比较无明显差别,碘离子浓度1000-1000μg/L(碘分子浓度700-4000μg/L)各实验组MDA含量与对照组比较明显高于对照组。【结论】 1.碘对成纤维细胞增殖活性具有促进和抑制两方面的作用,其作用效果与作用时间作用浓度密切相关,即高碘对成纤维细胞增殖活性存在时间-剂量-效应关系。 2.初步分析碘主要是通过对成纤维细胞氧化-抗氧化体系、细胞周期和细胞凋亡的调控来影响成纤维细胞增殖活性。 3.碘分子对成纤维细胞增殖活性的作用明显强于碘离子,可能与碘分子本身的强氧化性有关。
[Abstract]:[Objective]
This experiment using fibroblasts cultured in vitro model, using morphological observation, immunocytochemistry, flow cytometry and various methods of detection and other related biochemical indexes of different concentrations of iodine and iodine ions on cultured fibroblasts proliferation and function, to explore the effects of iodine on thyroid tissue, rich in trace the experimental study of iodine.
[method]
1. cell line: human skin fibroblast (HSF) cell line, purchased at the basic medical cell center of Union Medical College. After routine cell culture, the following experiments were carried out after stable passage.
2. reagent preparation and experimental grouping: potassium iodide, analytically pure, formulated in pure water to become 1000mgI/L mother liquor, keep away from light and dilute in DMEM culture solution. Iodine, analytically pure, make saturated solution with pure water, determine its concentration by chemical titration, and make use of it now.
In this experiment, iodide ions were set up in 1 blank control groups and 50-1500 g/L experimental groups, and iodine molecules were set up in 1 blank control groups and several experimental groups in 50-400 g/L.
3. cell counts and morphological observation: 24 holes and plates. After adding cell walls, iodide and iodine molecules were added. Each plate was set up in the control group. After 4,12,24,48 hours, cell counts and morphological changes were observed.
4.MTT: 96 hole culture plate, different concentrations of iodide and iodine after 4,12,24,48 hours of treatment per hole to add 20 l (5mg/ml) MTT kit, 37 C 4 h incubation, careful to discard the culture medium, each hole by adding 150 l two dimethyl sulfoxide (DMSO), 10 minute oscillation by enzyme. Standard instrument for measuring the optical absorbance in the wavelength of 490nm (value A), which indirectly reflects the number of cells.
5. flow cytometry: different concentrations of iodide and iodine treated cells 48 hours after centrifugation, PBS washing - - Fixed - centrifugal blowing uniform - Rinse - percussion - staining (4 C, 20-30 minutes), with 400 mesh nylon filter detection.ModFit analysis software, with cell proliferation the relative proliferation index.
6. immunocytochemistry: clean and sterile coverslips in 6 well plates and single cell suspension was prepared, 6 hole plate, with different concentrations of iodine and iodine treatment after 48 hours the coverslips were taken out and fixed cells, detect the expression of Ki-67 in the cells.
7.SOD, MDA detection: detection of malondialdehyde (MDA) and superoxide dismutase (SOD) in the supernatant of cultured cells by using a kit.
[results]
1. cell counting and MTT detection: the concentration of iodide and iodine concentration is low (e.g. 48 hours: the iodine ion is less than or equal to 3000 mu g/L, molecular iodine is less than or equal to 400 mu g/L), cell count and cell activity increased with the increase of concentration, with the increase of concentration also increased; the concentration of iodide and iodine concentration was high (if 48 hours: the iodine ion of more than 3000 g/L, the iodine of more than 400 g/L), cell count decreased with the increase of concentration, the cell proliferation activity also decreased with the increase of concentration, and visible change cell morphology.
2. flow cytometry: iodine ion concentration of 100-4000 g/L (molecular iodine concentration 50-700 g/L) experimental group cell proliferation index was significantly higher than the control group, while the apoptosis rate was significantly lower than the control group; the iodine ion concentration of 5000 g/L (molecular iodine concentration 800 g/L) refers to the number of cell proliferation in the experimental group compared with the control group no obvious difference, but the apoptosis rate was significantly higher than the control group; the iodine ion concentration of more than 6000 g/L (molecular iodine concentration of more than 900 g/L) experimental group cell proliferation index was significantly lower than the control group, while the apoptosis rate was significantly higher than the control group.
3. immunocytochemistry: iodine ion concentration 100-3000 g/L (iodine concentration 50-500 g/L) the positive rate of Ki-67 in the experimental group was significantly higher than the control group; the iodine ion concentration of more than 500 g/L (molecular iodine concentration of more than 700 g/L) the expression of Ki-67 in experimental group were significantly lower than the control group.
4.SOD, MDA: detection of iodine ion concentration of 50-600 g/L (molecular iodine concentration 50-300 g/L) experimental group with the increase of iodine ion concentration increased SOD content, iodine concentration of 800-10000 g/L (molecular iodine concentration 600-4000 g/L) experimental group with iodine ion concentration increased SOD decreased; iodine ion concentration of 50-800 g/L (iodine concentration of 50-500 g/L in 200 g/L) MDA content in each experimental group compared with the control group had no significant difference, iodine ion concentration of 1000-1000 g/L (molecular iodine concentration of 700-4000 g/L) in the experimental group compared with control group, the content of MDA was significantly higher than the control group.
[Conclusion]
1. iodine can promote and inhibit the proliferation of fibroblasts in two aspects, and its effect is closely related to the concentration of action time. That is, there is a time dose effect relationship between high iodine and fibroblast proliferation activity.
2. preliminary analysis of iodine mainly affects the proliferation activity of fibroblasts by regulating the oxidation antioxidant system of fibroblasts, cell cycle and apoptosis.
The effect of 3. iodine molecules on the proliferation activity of fibroblasts is stronger than that of iodide ions, which may be related to the strong oxidation of the iodine molecule itself.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【引证文献】