柚皮苷对体外培养人牙髓细胞增殖及分化影响的研究

发布时间：2018-03-26 01:02

  本文选题：柚皮苷　切入点：人牙髓细胞　出处：《河北医科大学》2014年硕士论文

【摘要】：目的：本实验通过采用人牙髓细胞体外培养的方法，探讨不同浓度柚皮苷对体外培养人牙髓细胞增殖、分化、矿化、细胞周期及碱性磷酸酶(ALP)活性的影响。 方法：选择因正畸或阻生新鲜拔除的健康、完整、无龋、无隐裂的恒牙或第三磨牙，在无菌的条件下取出牙髓组织，采用组织块酶解法获取人牙髓细胞并进行原代培养。待细胞长出后在倒置显微镜下观察细胞形态及生长情况。当细胞生长至铺满孔底80%时进行传代，细胞传代成功后，取第4代对数生长期人牙髓细胞进行细胞爬片，用SABC法进行波形丝蛋白和角蛋白免疫组织化学染色鉴定细胞来源。将处于对数生长期的第4代人牙髓细胞用0.25%的胰蛋白酶消化后制成细胞悬液，以2×104cells/ml密度分别接种于96孔培养板中，每板余出一列培养孔不加液，其余各孔每孔液量200μl，培养箱中培养。观察细胞贴壁情况良好后弃上清液，PBS缓冲液清洗3次后吸干。将细胞随机分为5个实验组、1个对照组和1个空白对照组，每组选5个孔。实验组：在相应的有细胞孔内加入200μl用含15%标准胎牛血清的高糖DMEM培养液配置的终末浓度分别为10-5、10-6、10-7、10-8、10-9mol/L的柚皮苷。对照组：在相应的有细胞孔内加入200μl含15%标准胎牛血清的高糖DMEM培养液。空白对照组：在相应的无细胞孔内加入200μl含15%标准胎牛血清的高糖DMEM培养液。 MTT比色法检测柚皮苷对人牙髓细胞增殖能力的影响：分别于培养24h、48h和72h时随机取出一个96孔培养板，向所测孔内加入5mg/mlMTT20μl后继续孵育4h，弃孔内液体，每孔加入150μl二甲基亚砜（DMSO），振荡5min，以空白对照孔调零，在酶标仪上测定492nm波长下各孔的吸光度值。 CCK-8法检测柚皮苷对人牙髓细胞增殖能力的影响：分别于培养24h、48h和72h时随机取出一个96孔培养板，弃孔内液体，向所测孔内加入110μl经DMEM稀释后的CCK-8溶液（DMEM和CCK-8以10：1配比），将培养板在培养箱内孵育2小时，用酶标仪测定在450nm处的吸光度值。 流式细胞术检测柚皮苷对人牙髓细胞的细胞周期的影响：选用含15%标准胎牛血清的高糖DMEM培养液配置的终末浓度为10-7mol/L的柚皮苷作为试验组，与对照组（15%标准胎牛血清的高糖DMEM培养液）进行细胞周期试验。按照细胞周期试剂盒说明书操作，进行流式细胞仪分析。 检测柚皮苷对人牙髓细胞的碱性磷酸酶活性的影响：分别在药物作用24h、48h和72h随机取出一个96孔培养板，弃上清液，PBS缓冲液清洗3次后吸干，每孔加入50μl0.1%TritonX-100，置于4℃冰箱过夜。观察细胞已无完整结构，经震荡后，按碱性磷酸酶试剂盒说明书加入碱性磷酸酶底物，每孔100μl，在培养箱内置30min，最后每孔加入0.2mol/LNaOH50μl终止反应，以空白对照孔调零，在酶标仪上测定410nm下各孔的吸光度值。 茜素红染色法检测柚皮苷对人牙髓细胞矿化结节形成的影响：选用第4代生长良好的牙髓细胞，制备2×104cells/ml细胞悬液，接种于六孔板内。分为对照组和10-7mol/L浓度的柚皮苷处理后的实验组，每组设3个复孔。分别培养14d、28d后，进行茜素红染色，观察矿化结节的形成。 柚皮苷对人牙髓细胞牙本质涎磷蛋白mRNA表达的影响：选用第4代生长良好的牙髓细胞，制备2×104cells/ml细胞悬液，，接种于六孔板内。以含15%标准胎牛血清的高糖DMEM培养液配置的终末浓度为10-7mol/L的柚皮苷作为试验组，以15%标准胎牛血清的高糖DMEM培养液作为对照组，每组设3个复孔。分别培养24h、48h、72h后PBS清洗3遍，加入Trizol试剂1ml，反复吹打至细胞完全溶解，收至无酶EP管中，-80℃冰箱保存。按照试剂盒说明书提取细胞总RNA，RT-PCR检测牙本质涎磷蛋白基因的表达。 采用SPSS13.0统计软件作单因素方差分析，多重比较采用S-N-K法。实验数据以均数±标准差表示，检验水准α=0.05，P0.05为有统计学意义。 结果： 1牙髓细胞的免疫组化染色结果显示为抗波形蛋白染色阳性，角蛋白染色阴性。从而证实牙髓细胞的组织来源为中胚层间充质细胞并且无上皮细胞混杂，符合实验要求。 2与对照组相比，在一定浓度范围内的柚皮苷能促进人牙髓细胞的增殖和细胞周期进程，提高其碱性磷酸酶活性，促进人牙髓细胞牙本质涎磷蛋白mRNA表达，并且增强人牙髓细胞的矿化能力，其中以浓度为10-7mol/L的柚皮苷作用最为明显。 结论：柚皮苷可促进体外培养的人牙髓细胞增殖，推进细胞周期进程；柚皮苷可以提高人牙髓细胞碱性磷酸酶的活性，矿化能力和分化能力。
[Abstract]:Objective: To explore the effects of different concentrations of naringin on the proliferation, differentiation, mineralization, cell cycle and alkaline phosphatase (ALP) activity of human dental pulp cells in vitro.
Methods: fresh extracted for orthodontic or impacted health, integrity, no caries, no cracked teeth or third molar, remove the pulp tissue under sterile conditions, using tissue enzymatic acquisition of human dental pulp cells were cultured in vitro. After the cells grow to observe the cell morphology and growth in inversion under the microscope. Passaged when cell growth to fill the hole at the end of 80%, after the success of cell passage, the fourth generation of the logarithmic growth phase of human dental pulp cells seeded, using SABC method of vimentin and cytokeratin immunohistochemical staining. Cells in the logarithmic growth phase of the fourth generation human dental pulp cells by trypsin digestion after 0.25% cell suspension with 2 * 104cells/ml density were inoculated into 96 well culture plates, each plate more than a series of training with the rest of the pore fluid, each hole hole liquid volume 200 L, incubator training observation. The adherent cells in good condition after discarding the supernatant and washed with PBS buffer after 3 times of dry. The cells were randomly divided into 5 experimental groups and 1 control group and 1 control group, each group selected 5 holes. The experimental group: in the final concentration of corresponding cells in hole add 200 mu l medium the configuration standard containing 15% fetal bovine serum glucose DMEM were naringin 10-5,10-6,10-7,10-8,10-9mol/L. Control group: Cultured in the corresponding cell hole join 200 L containing 15% FBS. DMEM high glucose control group: adding 200 L containing 15% fetal bovine serum glucose standard in DMEM cells no holes within the corresponding medium.
The detection effect of naringin MTT colorimetric method on the ability of human dental pulp cell proliferation: 24h were cultured, 48h and 72h were selected from a 96 hole culture plate, measured by adding 5mg/mlMTT20 l to the hole after incubating for 4h, liquid abandoned holes, each hole by adding 150 l two dimethyl sulfoxide (DMSO), oscillator 5min, the blank control wells zero, the enzyme labelling instrument determination wavelength of 492nm absorbance value of each hole.
Effect of naringin detection method of CCK-8 ability of human dental pulp cells were cultured 24h proliferation: 72h, 48h and random out of a 96 hole culture plate, liquid waste hole, the hole to join 110 L by CCK-8 DMEM solution diluted (DMEM and CCK-8 to 10:1 ratio), the culture in the incubator incubation for 2 hours, measured absorbance at 450nm values by ELISA.
Cell cycle effects of naringin by flow cytometry on human dental pulp cells: using standard containing 15% fetal bovine serum high glucose DMEM medium configuration final concentration of 10-7mol/L naringin as experimental group, and control group (15% standard fetal bovine serum high glucose DMEM medium) in cell cycle test. According to cell cycle kit, were analyzed by flow cytometry.
Effect of alkaline phosphatase activity detection of naringin on human dental pulp cells: role in drug 24h, 48h and 72h were selected from a 96 hole culture plate, the supernatant was removed, washed with PBS buffer after 3 times of dry, each hole by adding 50 l0.1%TritonX-100, 4 C in the refrigerator overnight. Observe the cell has no complete structure. After shocks, according to the instructions of the alkaline phosphatase kit with alkaline phosphatase substrate, each hole of 100 L in the incubator built in 30min, 0.2mol/LNaOH50 l finally added to each well to stop the reaction, the blank control wells zero, ELISA was determined in each hole under the 410nm value of the instrument.
The detection effect of naringin alizarin red stain on the formation of mineralized nodules in human dental pulp cells: the fourth generation of good growth of dental pulp cells, preparation of 2 * 104cells/ml cell suspension and inoculated in 6-well plates. Divided into experimental group and control group with naringin and 10-7mol/L concentration of 3 wells per group. Culture respectively 14d, 28d, alizarin red staining, observe the formation of mineralized nodules.
Effect of naringin on the expression of human dental pulp cells of dentin sialophosphoprotein mRNA: the fourth generation of good growth of dental pulp cells, preparation of 2 * 104cells/ml cell suspension and inoculated in 6-well plates. With 15% fetal bovine serum high glucose DMEM medium configuration final concentration of 10-7mol/L naringin as experimental group in 15%, the standard of fetal bovine serum high glucose DMEM medium as the control group, with 3 wells in each group. 48h, 72h were cultured in 24h, PBS after washing 3 times, adding Trizol reagent 1ml, repeated pipetting to cells completely dissolved, close to the free enzyme in the EP tube, -80 C refrigerator preservation. Cell extraction according to the total RNA kit, detect the expression of dentin sialophosphoprotein gene RT-PCR.
SPSS13.0 statistical software was used for one-way ANOVA. Multiple comparisons were made by S-N-K method. The experimental data were expressed by mean + standard deviation. The test level =0.05 and P0.05 were statistically significant.
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