發(fā)布時(shí)間：2018-01-12 20:33

  本文關(guān)鍵詞：碘在體外培養(yǎng)成纖維細(xì)胞模型中的作用研究　出處：《山東大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 碘 成纖維細(xì)胞 增殖 流式細(xì)胞術(shù)

【摘要】： 【目的】 本實(shí)驗(yàn)利用體外培養(yǎng)成纖維細(xì)胞模型,運(yùn)用形態(tài)學(xué)觀察、免疫細(xì)胞化學(xué)技術(shù)、流式細(xì)胞術(shù)及相關(guān)生化指標(biāo)檢測(cè)等多種方法研究不同濃度的碘離子及碘分子對(duì)體外培養(yǎng)成纖維細(xì)胞增殖活性與功能的影響,探討碘對(duì)甲狀腺外組織的作用,豐富微量元素碘的實(shí)驗(yàn)研究。 【方法】 1.細(xì)胞株:人皮膚成纖維(HSF)細(xì)胞株,購于協(xié)和醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)細(xì)胞中心。常規(guī)細(xì)胞培養(yǎng),待傳代穩(wěn)定后進(jìn)行以下實(shí)驗(yàn)。 2.試劑配制及實(shí)驗(yàn)分組:碘化鉀,分析純,以純水配制成為1000mgI/L母液,避光保存,以DMEM培養(yǎng)液稀釋待用。碘,分析純,以純水配制成飽和溶液,以化學(xué)滴定法測(cè)定其濃度,現(xiàn)配現(xiàn)用。 本實(shí)驗(yàn)中,碘離子設(shè)1個(gè)空白對(duì)照組及50-1500μg/L中若干實(shí)驗(yàn)組;碘分子設(shè)1個(gè)空白對(duì)照組及50-400μg/L中若干實(shí)驗(yàn)組。 3.細(xì)胞計(jì)數(shù)及形態(tài)學(xué)觀察:種24孔板,待細(xì)胞貼壁后加入不同濃度的碘離子及碘分子,每板均設(shè)對(duì)照組,于作用4、12、24、48小時(shí)后進(jìn)行細(xì)胞計(jì)數(shù)及觀察細(xì)胞形態(tài)學(xué)改變。 4.MTT:種96孔培養(yǎng)板,不同濃度碘離子及碘分子處理4、12、24、48小時(shí)后,每孔添加20μl(5mg/ml)MTT試劑,37℃孵育4小時(shí),小心棄去培養(yǎng)液,每孔加入150μl二甲基亞砜(DMSO),振蕩10分鐘。以酶標(biāo)儀在490nm波長(zhǎng)下測(cè)其光吸收值(吸光度A值),可間接反映細(xì)胞數(shù)量。 5.流式細(xì)胞術(shù):不同濃度碘離子及碘分子處理細(xì)胞48小時(shí),經(jīng)離心—PBS沖洗—吹勻—固定—再離心—沖洗—吹打—染色(4℃,20-30分鐘)后,以400目尼龍網(wǎng)過濾后上機(jī)檢測(cè)。ModFit分析軟件分析,用細(xì)胞分裂增殖指數(shù)表示相對(duì)增殖活力。 6.免疫細(xì)胞化學(xué):將潔凈無菌的蓋玻片放入6孔培養(yǎng)板中,制備單細(xì)胞懸液,種6孔板,以不同濃度的碘離子及碘分子處理48小時(shí)后取出蓋玻片,固定細(xì)胞,檢測(cè)細(xì)胞中Ki-67的表達(dá)。 7.SOD、MDA檢測(cè):利用試劑盒對(duì)培養(yǎng)細(xì)胞的上清液中丙二醛(MDA)和超氧化物岐化酶(SOD)進(jìn)行檢測(cè)。 【結(jié)果】 1.細(xì)胞計(jì)數(shù)及MTT檢測(cè):碘離子濃度及碘分子濃度較低(如48小時(shí):碘離子≤3000μg/L,碘分子≤400μg/L)時(shí),細(xì)胞計(jì)數(shù)隨濃度增加而增加,細(xì)胞活性也隨濃度增加而升高;碘離子濃度及碘分子濃度較高(如48小時(shí):碘離子≥3000μg/L,碘分子≥400μg/L)時(shí),細(xì)胞計(jì)數(shù)隨濃度增加而減少,細(xì)胞增殖活性也隨濃度增加而降低,且可見細(xì)胞形態(tài)學(xué)改變。 2.流式細(xì)胞術(shù):碘離子濃度100-4000μg/L(碘分子濃度50-700μg/L)實(shí)驗(yàn)組細(xì)胞增殖指數(shù)明顯高于對(duì)照組,同時(shí)細(xì)胞凋亡率明顯低于對(duì)照組;碘離子濃度5000μg/L(碘分子濃度800μg/L)實(shí)驗(yàn)組細(xì)胞增殖指數(shù)與對(duì)照組比較沒有明顯差別,而細(xì)胞凋亡率明顯高于對(duì)照組;碘離子濃度≥6000μg/L(碘分子濃度≥900μg/L)實(shí)驗(yàn)組細(xì)胞增殖指數(shù)明顯低于對(duì)照組,同時(shí)細(xì)胞凋亡率明顯高于對(duì)照組。 3.免疫細(xì)胞化學(xué):碘離子濃度100-3000μg/L(碘分子濃度50-500μg/L)實(shí)驗(yàn)組Ki-67的陽性率明顯高于對(duì)照組;碘離子濃度≥500μg/L(碘分子濃度≥700μg/L)各實(shí)驗(yàn)組Ki-67的表達(dá)率明顯低于對(duì)照組。 4.SOD、MDA檢測(cè):碘離子濃度50-600μg/L(碘分子濃度50-300μg/L)實(shí)驗(yàn)組隨碘離子濃度的增加SOD含量增加,碘離子濃度800-10000μg/L(碘分子濃度600-4000μg/L)實(shí)驗(yàn)組隨碘離子濃度的增加SOD含量減少;碘離子濃度50-800μg/L(碘分子濃度50-500μg/L除200μg/L外)各實(shí)驗(yàn)組MDA含量與對(duì)照組比較無明顯差別,碘離子濃度1000-1000μg/L(碘分子濃度700-4000μg/L)各實(shí)驗(yàn)組MDA含量與對(duì)照組比較明顯高于對(duì)照組。 【結(jié)論】 1.碘對(duì)成纖維細(xì)胞增殖活性具有促進(jìn)和抑制兩方面的作用,其作用效果與作用時(shí)間作用濃度密切相關(guān),即高碘對(duì)成纖維細(xì)胞增殖活性存在時(shí)間-劑量-效應(yīng)關(guān)系。 2.初步分析碘主要是通過對(duì)成纖維細(xì)胞氧化-抗氧化體系、細(xì)胞周期和細(xì)胞凋亡的調(diào)控來影響成纖維細(xì)胞增殖活性。 3.碘分子對(duì)成纖維細(xì)胞增殖活性的作用明顯強(qiáng)于碘離子,可能與碘分子本身的強(qiáng)氧化性有關(guān)。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 肖培瑞;生活環(huán)境高碘、飲食習(xí)慣與布—加綜合征發(fā)病關(guān)系探討[D];山東大學(xué);2011年

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本文編號(hào)：1415887