【作者】 张海涛；

【导师】 郭成浩；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2008， 硕士

【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。更多还原

【Abstract】 [Objective]To research the effect of different concentrations of iodine ion and iodine molecule on cultured fibroblast cells by various kinds of methods,including:cell culture,immunocytochemistry technique,flow cytometry and other interrelated biochemical techniques.This work can observe the effect of iodine on the proliferation cultured fibroblast cells and enrich the study on trace element.[Material and Methods]1.Cell strain:The fibroblast cells(Human Skin Fibroblast,HSF)were bought from Cell Culture Facility,Peking Union Medical College.The following expressions proceed when the cells passage steadily.2.Reagent and grouping:Potassium Iodide which is analytical reagent was dissolved to 1000mg/L by purified water and was diluted by DMEM for using. Iodine which is analytical reagent was dissolved by purified water.The concentration was determined by titrimetric method.It was prepared when it was demanded.There were one control group and many groups among 50～15000μg/L of iodine ion;There were one control group and many groups among 50～4000μg/L of iodine molecule.3.Cell counting and observation of morphology:The cells were seeded in 24-well culture plates and cultured in the DMEM which contains different concentration of iodine.Cell counting and observation of morphology were done after 4-hour,12-hour,24-hour and 48-hour. 4.MTT experimentation:The cells were seeded in 96-well culture plates and cultured in the DMEM which contains different concentration of iodine for 4-hour,12-hour,24-hour and 48-hour.20μl MT-F solution(5mg/ml)was added in each well,and continued to culture at 37℃for 4 hours.The supernatant was discarded subsequently,and 150μl DMSO was added to each well.The plates were vibrated to dissolve the crystals in the cells.The absorbance was determined by the microplate reader at 490nm.5.Flow Cytometry:The cells were cultured in the DMEM which contains different concentration of iodine for 48-hour.The single cell suspension was prepared and collected into centrifuge tube.The suspension was centrifuged to discard the supernatant.Cells were fixed by 70%alcohol.Supernatant was discarded after centrifuging.Cells were washed twice with PBS and stained with synthetic staining solution.Flow cytometer was used to analyze the cells.Data analysis was performed by ModFit software.6.Immunocytochemistry:Coverslips were sterilized and put in 6-well plates.Cells were seeded on the coverslips and cultured in the DMEM which contains different concentration iodine ion/molecule for 48-hour.The cells were washed with PBS buffer and fixed with acetone.The expression of Ki-67 was detected by PV method.7.Detection of SOD and MDA:Maleic Dialdehyde(MDA)and Superoxide dismutase(SOD)in supernate was detected by kits.[Results]1.Cell counting and MTT experimentation:When the iodine concentration was low (such as,iodine ion concentration was lower than 3000μg/L in 48-hour,and iodine molecule concentration was lower than 400μg/L in 48-hour),cell counting and proliferation activity increased with the increase of concentration. When the concentration of iodine was high(such as,iodine ion concentration was higher than 3000μg/L in 48-hour and iodine molecule concentration was higher than 400μg/L in 48-hour),cell counting and proliferation activity decreased with the increase of concentration,and change of cell morphology can be observed.2.Flow Cytometry:Proliferation index of 100 to 4000μg/L iodine ion or 50 to 700μg/L iodine molecule in experimental group were significantly higher than in the control group,while apoptosis rate were significantly lower than in the control group.There was no significant difference between proliferation index of 5000μg/L iodine ion or 800μg/L iodine molecule experimental group and proliferation index of the control group,while apoptosis rate were significantly higher in the experimental group.When iodine ion concentration is higher than 6000μg/L or iodine molecular concentration is much higher than 900μg/L,the proliferation index of experimental group cells was lower than the control group cells’,while apoptosis rate was significantly high.3.Immunocytochemistry:Expression of Ki-67 in the experimental groups of 100 to 3000μg/L iodine ion or 50 to 500μg/L iodine molecular was much higher than in the control group;and expression of Ki-67 in the experimental group that iodine ion concentrations were higher than 5000μg/L or iodine molecular concentrations were higher than 700μg/L were lower than in the control group.4.Detection of SOD and MDA:Concentration of SOD in the experimental groups of 50 to 600μg/L iodine ion or 50 to 300μg/L iodine molecular increased with the concentration iodine increase,but it decreased with the concentration iodine increase when the concentration of iodine ion were higher than 800μg/L or iodine molecular were higher than 600μg/L.There was no significant difference of concentration of MDA between in the experimental group and in the control group when the iodine ion concentrations were lower than 800μg/L or the iodine molecular concentrations were lower than 500μg/L,while it was much higher when the iodine ion concentrations were higher than 1000μg/L or iodine molecular concentrations were higher than 700μg/L.[Conclusions]1.Iodine has the effects of improving and decreasing proliferation activities of fibroblast.The effects are related to time and concentration.In other words, there exists the relation of time-dose-effect.2.The effect of different concentrations of iodine ion or iodine molecule on proliferation activity of cultured fibroblast cells may be related to oxidation-antioxygen system,cell cycle and apoptosis.3.The effect of iodine molecular on fibroblast proliferation activity was much more obvious than the role of iodine ion,and it may be related to the oxidative of iodine molecular.更多还原