【作者】 杨高松；

【导师】 王一兵； 王明青；

【作者基本信息】 山东大学 ， 外科学， 2010， 硕士

【摘要】 碘是人体必需的微量元素之一,为卤族元素之一,又有“智力元素”之称,是人体合成甲状腺激素、调节中枢神经系统及内分泌的主要原料；碘的主要功能是作为甲状腺激素的组成成分而对机体生长发育产生重要影响。对碘的研究,大部分是和甲状腺激素有关的研究。近年来,仍以碘或者其化合物对甲状腺激素水平的影响方面的研究居多。在碘对创伤修复是否有促进作用的研究方面比较少,且国内外关于碘伏、卡地姆碘和碘化钾等的研究,争议较多。碘的作用机制尚待进一步研究。为深入研究碘对创面愈合的影响及其分子机制,本课题进行了以下三个方面的工作,通过观察碘对成纤维细胞增殖活性,胶原分泌、凋亡等方面研究碘对成纤维细胞的影响。第一部分不同碘制剂对体外培养人皮肤成纤维细胞粘附增殖活性影响的研究目的本实验通过综合运用体外细胞培养技术、免疫荧光细胞化学技术及相关指标检测等多种方法,初次区别针对性的研究碘化钾(KI)和碘伏(PI)浓度高低对体外培养人皮肤成纤维细胞(human skin fibroblast, HSF)的生长与功能的影响,观察不同碘浓度在相同时间段对人皮肤成纤维细胞的影响,为探讨微量元素碘对人体的影响与作用提供有效的实验支持,丰富对碘的实验研究,初步探索碘对成纤维细胞的作用机制。方法1.细胞株：人皮肤成纤维(HSF)细胞株,购于中国医学科学院昆明细胞库。常规细胞培养,待传代稳定后进行以下实验,用于实验的是6-9代细胞。2.实验分组：实验分为9组：空白对照组、3 mg／L KI组、30 mg／L KI组、300 mg／L KI组、3000 mg／L KI组、1 mg／L PI组、2 mg／L PI组、3 mg／L PI组、4 mg／L PI组3.形态学观察：以每孔1×105个细胞将细胞接种于6孔板中,按实验设计分组,药物作用96 h后,在倒置显微镜下观察、照相。4.细胞粘附特性测定：将第6代细胞接种于6孔板中,24 h细胞完全贴壁后,按实验设计分组,置于37℃、饱和湿度、5%CO2培养箱培养96 h,消化计数,以4×103个细胞／孔接种于96孔板,每组设5个复孔,1h后吸去培养基,D-Hanks轻洗3次,洗除未贴壁细胞,四唑氮蓝(MTT)比色法测定孔底粘附的细胞量,每组结果取5个复孔计数的平均值。5.细胞增殖测定：将传至第6代细胞以4×104／mL密度接种于96孔培养板,每孔加入完全培养基100 uL,在37℃、饱和湿度、5%CO2培养箱培养。24h细胞完全贴壁后吸去培养基,按实验设计分组,每组5孔,分别于第2、4、6天用四唑氮蓝(MTT)比色法分析HSF的存活和增殖能力。6.增殖细胞核抗原(PCNA)表达情况：将盖玻片置于6孔培养板内,每孔接种1×105个细胞,按实验设计分组,置于37℃、饱和湿度、5%CO2培养箱培养,药物作用96 h后,用免疫荧光细胞化学方法染色,在荧光显微镜下观察,并随机选择5个视野照相,用Image-pro plus 6.0软件分析平均光密度值。结果1.普通光学显微镜下观察结果：显微镜下观察,培养的人皮肤成纤维细胞为细长梭形,部分细胞可见1-2个突起,或呈三角形,轮廓清晰。不同浓度KI作用后,与对照组比较3 mg／L KI组和30 mg／L KI组可见细胞折光性增强,细胞密度增大,300 mg／L KI组可见个别细胞变圆脱落,折光性稍减弱,3000 mg／L KI组细胞皱裂,大部分细胞破碎。1 mg／L PI组可见细胞密度较对照组小,形态多呈多角形,细胞排列紊乱,2 mg／L PI组出现细胞脱水并伴有部分细胞脱落,当浓度增加至3 mg／L时,细胞出现明显脱水伴破裂,并有大片脱落。4 mg／L PI组细胞全部死亡,视野中仅见细胞碎片。2.细胞粘附特性测定结果：3 mg／L、KI组、30 mg／L KI组、300 mg／L KI组细胞粘附活性明显高于对照组,但三组组间比较无统计学差异。3000 mg／LKI组、1 mg／L PI组、2 mg／L PI组、3 mg／L PI组、4 mg／L PI组,在药物作用96h后,细胞大部分死亡,无法测定其粘附活性。3.细胞增殖测定结果：2d时,3 mg／L KI组增殖活性与对照组差异无统计学意义,30mg／L KI组和300 mg／L KI组增殖活性增加,但两组组间细胞增殖活性无明显差别,3000 mg／L KI组增殖活性明显低于对照组。1 mg／L PI组、2 mg／L PI组、3 mg／L PI组、4 mg／L PI组组间细胞增殖活性随碘浓度的增加而降低,与对照组比较,差异有统计学意义(P<0.05或P<0.01)；4 d时,与对照组比较,3 mg／L KI组、30 mg／L KI组、300 mg／L KI组细胞增殖活性随碘浓度的增加而增加,差异有统计学意义(P<0.05),30 mg／L KI组和300 mg／L KI组组间无明显差异,3000 mg／L KI组增殖活性明显低于对照组。1 mg／L PI组、2 mg／L PI组、3 mg／L PI组、4 mg／L PI组组间细胞增殖活性随碘浓度的增加而降低,与对照组比较,差异有统计学意义(P<0.01)；6 d时,3 mg／L KI组增殖活性与对照组差异无统计学意义,30 mg／L KI组和300 mg/L KI组增殖活性增加,但两组组间细胞增殖活性无明显差别,3000 mg／L KI组细胞完全死亡,1 mg／L PI组和2 mg／L PI组细胞增殖活性随碘浓度的增加而降低,与对照组比较,差异有统计学意义(P<0.01)；3 mg／L PI组、4 mg／L PI组细胞完全死亡。4.增殖细胞核抗原(PCNA)表达情况：与对照组比较,3 mg／L KI组、30 mg／L KI组和300 mg／L KI组平均光密度值均增加,其中以3 mg／L KI组增加最明显,3000 mg／L KI组细胞完全死亡,未做免疫荧光染色,实验中的阴性对照未见胞核呈阳性反应。结论：1.适当浓度碘化钾使体外培养人皮肤成纤维细胞粘附和增殖活性增强,高浓度的碘化钾导致细胞凋亡；2.稀释浓度的碘伏对体外培养成纤维细胞有明显毒性作用。第二部分碘对人皮肤成纤维细胞胶原合成的影响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、real-time PCR技术和3H-脯氨酸比色法研究不同浓度的碘离子对体外培养人皮肤成纤维细胞(human skin fibroblast, HSF)胶原蛋白转录、表达情况的影响,探讨碘对成纤维细胞的作用,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,购于中国医学科学院中国科学院昆明细胞库。常规细胞培养,待传代稳定后进行以下实验,用于实验的是6～9代细胞。2.药物配制和分组：碘化钾溶液的配制：用K1分析纯和双蒸水按照分组浓度配制1g／L、10g／L、100g／L高浓度KI母液,过滤除菌,闭光、4℃保存。以临床常用的碘伏碘浓度为依据,将实验分为5组：空白对照组、bFGF(3mg／L)组、3mg／L KI组、30mg／L KI组、300mg／L KI组,其中空白对照组加入无菌生理盐水。3.人皮肤成纤维细胞形态学观察：取对数生长期状态良好的人皮肤成纤维细胞,以细胞密度1×105／mL接种于6孔培养板中,每孔2mL,培养24h后,设空白对照组和实验组,每个浓度做3个复孔。继续培养96h后,在倒置显微镜下观察、照相。4. real-time PCR:①以细胞密度1×105／mL接种于6孔培养板中,每孔2mL,24小时后按实验分组设立空白对照组和实验组,37℃、饱和湿度、5%CO2培养箱培养96h后,用Trizol提取总RNA,按照说明书进行操作.②反转录按Fermentas反转录试剂盒说明书方法进行cDNA合成,根据酶标仪测的RNA浓度,取总RNA lug, RNase FreeH2O补足11μL,并加入随机六聚体引物1μL,5×reaction Buffer 4 u L, Ribolock Rnase Inhibitor 1μL, lOmM dNTP Mix 2uL, Revert AidTM M-Mulv RT 1μL,反应体系为20μL。③引物合成引物由上海生工生物技术有限公司合成。④实时荧光定量PCR用SYBR Green荧光定量试剂盒进行聚合酶链式反应,总体系20μL。SYBR Green混合液10μL,Primer 1 (0.8μu mol/L)5μL,Primer2(0.8μmol／L)5μL,cDNA 5μL(已稀释),反应扩增条件为：50℃2min,95℃10min,(95℃15s,60℃1min,)：40个扩增循环,60℃1min,95℃15s。以GAPDH为内参,每个样品重复3管。⑤结果分析：实时荧光定量PCR产物利用ABI公司自带的7500PCR系统软件进行分析,可观察扩增曲线,并对cDNA的含量进行相对定量分析,计算样本的△△Ct值。5.3H-脯氨酸比色法测定细胞上清液中的胶原含量：取对数生长期状态良好的人皮肤成纤维细胞,以细胞密度1×105／mL接种于6孔培养板中,每孔2mL,置37℃、饱和湿度、5%CO2培养箱中培养24h后,弃15%胎牛血清的DMEM培养基,以无血清DMEM同步化24h,弃去各孔培养液,加入含5%胎牛血清的DMEM培养基,按实验计划设立对照组和实验组,37℃、饱和湿度、5%CO2培养箱培养96h,取培养液上清50μL于玻璃试管中,按羟脯氨酸测定试剂盒说明书方法,用spectra Max M2型酶标仪于550 nm波长测定吸光度(A),实验重复3次。测定胞浆中的胶原含量：将上步骤取完培养液上清的细胞分别用0.25%胰酶消化制成细胞悬液,1000 r／min(离心半径15cm)离心5 min,将细胞收集于离心管中,弃上清,反复冻融法提取细胞内蛋白,同法于550 nm波长测定吸光度(A),实验重复3次。结果1人皮肤成纤维细胞形态学观察：培养的人皮肤成纤维细胞为细长梭形,部分细胞可见1-2个突起,或呈三角形,轮廓清晰。不同浓度KI作用后,显微镜下观察无明显差异,细胞生长快,活性好,细胞形态正常,与对照组比较3mg／L KI组和30mg／LKI组可见细胞折光性增强,细胞密度增大,300mg／L KI组可见个别细胞变圆脱落,折光性稍减弱,2 Real-Time PCR测Ⅰ型胶原和Ⅲ型胶原mRNA表达情况：Ⅲ型胶原mRNA表达情况：与对照组比较,3mg／L KI组Ⅲ型胶原mRNA表达明显增强(P<0.05)30mg／L KI组和300mg／L KI组差异无统计学意义。工型胶原mRNA表达情况：各组间差异无统计学意义,阳性对照组：bFGF组Ⅰ型和Ⅲ型胶原mRNA表达均明显高于空白对照组3. 3H-脯氨酸掺入法测定成纤维细胞胶原的合成：细胞上清液中的胶原含量：3mg／L KI组和30mg／L KI组可使分泌到细胞培养上清中的胶原含量明显增加,以3mg／L KI组作用最为显著(t=4.285,P<0.05),300mg／L KI组与对照组比较差异无统计学意义。胞浆中的胶原含量：三种不同浓度KI组细胞胞浆中胶原的含量均明显增多,以3mg／L浓度组作用最为显著(t=4.901,P<0.05)。结论适量浓度的碘化钾通过对细胞基因水平的调控作用,使细胞合成和分泌胶原增加,在不影响Ⅰ型胶原合成的情况下,有效地提高Ⅲ型胶原含量。第三部分碘离子对人皮肤成纤维细胞凋亡相关基因的影响目的研究碘离子人皮肤成纤维细胞凋亡相关基因的影响,探讨碘离子影响细胞增殖活性和凋亡的可能机制。方法体外培养人皮肤成纤维细胞.采用western blotting方法检测不同浓度碘离子刺激后的人皮肤成纤维细胞凋亡相关蛋白bcl-2、bax的变化。结果不同浓度的碘离子(0,3 mg／L,30 mg／L,300 mg／L)处理人皮肤成纤维细胞48h后,收集各组细胞总蛋白,用Western Blotting检测bax和bcl-2的蛋白表达。结果表明3 mg／L碘化钾组明显上调bcl-2的表达,随药物浓度升高表达量逐渐降低；3 mg／L碘化钾组和30 mg／L碘化钾组对细胞bax表达无明显影响,300 mg／L碘化钾组明显上调细胞bax的表达。结论碘离子的变化对人皮肤成纤维细胞的影响与凋亡相关基因bcl-2和bax蛋白密切相关。更多还原

【Abstract】 PART ONEBiological effects of iodine preparations on the adhesion and proliferation of human skin fibroblasts in vitroObjectiveIn this study, we use several kind of techniques including cell culture in vitro, immunofluorescence cytochemistry and MTT for detection, to investigate the biological effects of potassium iodide and povidone-iodine (PI).on the adhesion and proliferation of human skin fibroblasts (HSF) in vitro and to explore the role of iodine in wound healing. The research "may enrich experimental research of iodine, providing an effective experimental support for the human body.Methods1. Cell strain:The cells(Human Skin Fibroblast, HSF)were bought from kunming cellbank of Chinese Academy of Sciences. The following expressions proceed when the cells passage steadily.2. groups:Human skin fibroblasts were cultured in vitro, and were divided into 9 groups:control group,3 mg/L KI group,30 mg/L KI group,300 mg/L KI group,3000 mg/L KI group,1 mg/L PI group,2 mg/L PI group,3 mg/L PI group,4 mg/L PI group.3. observation of morphology:The cells were seeded in 6-well culture plates and cultured in the DMEM according to experimental design. Cell were observed after 96 hours.4. MTT for cell adhesion:The cells were cultured in the DMEM which contains different concentration of iodine according to experimental design. After 96 hours inoculated in 96-well plates, each set up five re-holes, 1h later sucking the medium, using D-Hanks lightly washed three times.Human skin fibroblasts adhesion were measured with MTT5. MTT cell proliferation:The cells were cultured in the DMEM which contains different concentration of iodine according to experimental design, cell proliferation were determined by MTT in2d,4d and 6d.6. immunofluorescence: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 for 96 hour. Stained By immunofluorescence cytochemistrying method,then the cells were observed under fluorescence microscope.the photos were analyzed by Image-pro plus 6.0.ResultsIn 3 mg/L KI group,30 mg/L KI group and 300 mg/L KI group, the adhesion and proliferation of skin fibroblasts were remarkably increased, especially in 3 mg/L group the differences were significant compared with the iodine free control (P< 0.05), but the proliferation activity was progressively retarded in 3000 mg/L concentration. In povidone iodine groups, Fibroblast growth was totally inhibited by povidone iodine solutions.ConclusionProper concentration of KI can promote fibroblast adhesion and proliferation, but high concentration of KI lead to apoptosis; Even dilute solutions of povidone-iodine were toxic to human fibroblasts.PART TWOEffects of KI on collagen synthesis of skin fibroblasts in vitroObjectiveTo study the effects of KI on the collagen synthesis of skin fibroblasts in vitro and to explore the role of iodine in wound healing.MethodsHuman skin fibroblasts were cultured in vitro, then our experiment is divided into 5 groups:control group,3mg/L KI group,30mg/L KI group,300mg/L KI group and bFGF, after being stimulated by KI with different concentration for 96 hours, collagen synthesis of human skin fibroblasts were examined by hydroxyproline colorimetric analysis and Real-Time PCR respectively.Results①3mg/L KI and 30mg/L KI group remarkably increase collagen content in supernatant of cell and in endochylema, especially in 3mg/L group (P< 0.05).②Compared with the control group, typeⅢcollagen mRNA expression in 3mg/L KI group was significantly enhanced (P<0.05),and 30mg/L KI group and 300mg/L KI group had no statistical significance, the difference of typeⅠcollagen mRNA expression were not statistically significant between each groups.ConclusionIn the gene level of regulation,proper concentration of KI promote cell synthesis and secretion of collagen. Furthermore without affecting the synthesis of typeⅠcollagen, Proper concentration of KI effectively increase typeⅢcollagen content, thereby it may promote wound healing, and reduce scar formation in the wound healing process.PART THREEEffect of iodine on apoptosis in cultured human skin fibroblasts in vitro ObjectiveTo investigate the influence of iodine on apoptosis of human skin fibroblasts in vitro and to explore the mechanism that iodide ions affect cell proliferation and apoptosis. MethodsHuman skin fibroblasts were cultured in vitro, then our experiment is divided into 4 groups:control group,3mg/L KI group,30mg/L KI group,300mg/L KI group, after being stimulated by KI with different concentration for 96 hours. Apoptosis gene Bcl-2 and Bax expression were detected by Western blotting.Results3 mg/L KI concentrations significantly increased bcl-2 expression, but bcl-2 expression was gradually inhibited when using the higher iodine concentrations. 300mg/L KI increased the expression of Bax expression. ConclusionApoptosis is influenced by iodine in cultured human skin fibroblasts through the modulation of Bcl-2 and Bax expression. Low concentration of iodine inhibits apoptosis, whereas high concentrations of iodine increase the rate of apoptosis.更多还原