本文選題：柚皮苷　切入點(diǎn)：人牙髓細(xì)胞　出處：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：本實(shí)驗(yàn)通過采用人牙髓細(xì)胞體外培養(yǎng)的方法，探討不同濃度柚皮苷對體外培養(yǎng)人牙髓細(xì)胞增殖、分化、礦化、細(xì)胞周期及堿性磷酸酶(ALP)活性的影響。 方法：選擇因正畸或阻生新鮮拔除的健康、完整、無齲、無隱裂的恒牙或第三磨牙，在無菌的條件下取出牙髓組織，采用組織塊酶解法獲取人牙髓細(xì)胞并進(jìn)行原代培養(yǎng)。待細(xì)胞長出后在倒置顯微鏡下觀察細(xì)胞形態(tài)及生長情況。當(dāng)細(xì)胞生長至鋪滿孔底80%時(shí)進(jìn)行傳代，細(xì)胞傳代成功后，取第4代對數(shù)生長期人牙髓細(xì)胞進(jìn)行細(xì)胞爬片，用SABC法進(jìn)行波形絲蛋白和角蛋白免疫組織化學(xué)染色鑒定細(xì)胞來源。將處于對數(shù)生長期的第4代人牙髓細(xì)胞用0.25%的胰蛋白酶消化后制成細(xì)胞懸液，以2×104cells/ml密度分別接種于96孔培養(yǎng)板中，每板余出一列培養(yǎng)孔不加液，其余各孔每孔液量200μl，培養(yǎng)箱中培養(yǎng)。觀察細(xì)胞貼壁情況良好后棄上清液，PBS緩沖液清洗3次后吸干。將細(xì)胞隨機(jī)分為5個(gè)實(shí)驗(yàn)組、1個(gè)對照組和1個(gè)空白對照組，每組選5個(gè)孔。實(shí)驗(yàn)組：在相應(yīng)的有細(xì)胞孔內(nèi)加入200μl用含15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液配置的終末濃度分別為10-5、10-6、10-7、10-8、10-9mol/L的柚皮苷。對照組：在相應(yīng)的有細(xì)胞孔內(nèi)加入200μl含15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液�？瞻讓φ战M：在相應(yīng)的無細(xì)胞孔內(nèi)加入200μl含15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液。 MTT比色法檢測柚皮苷對人牙髓細(xì)胞增殖能力的影響：分別于培養(yǎng)24h、48h和72h時(shí)隨機(jī)取出一個(gè)96孔培養(yǎng)板，向所測孔內(nèi)加入5mg/mlMTT20μl后繼續(xù)孵育4h，棄孔內(nèi)液體，每孔加入150μl二甲基亞砜（DMSO），振蕩5min，以空白對照孔調(diào)零，在酶標(biāo)儀上測定492nm波長下各孔的吸光度值。 CCK-8法檢測柚皮苷對人牙髓細(xì)胞增殖能力的影響：分別于培養(yǎng)24h、48h和72h時(shí)隨機(jī)取出一個(gè)96孔培養(yǎng)板，棄孔內(nèi)液體，向所測孔內(nèi)加入110μl經(jīng)DMEM稀釋后的CCK-8溶液（DMEM和CCK-8以10：1配比），將培養(yǎng)板在培養(yǎng)箱內(nèi)孵育2小時(shí)，用酶標(biāo)儀測定在450nm處的吸光度值。 流式細(xì)胞術(shù)檢測柚皮苷對人牙髓細(xì)胞的細(xì)胞周期的影響：選用含15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液配置的終末濃度為10-7mol/L的柚皮苷作為試驗(yàn)組，與對照組（15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液）進(jìn)行細(xì)胞周期試驗(yàn)。按照細(xì)胞周期試劑盒說明書操作，進(jìn)行流式細(xì)胞儀分析。 檢測柚皮苷對人牙髓細(xì)胞的堿性磷酸酶活性的影響：分別在藥物作用24h、48h和72h隨機(jī)取出一個(gè)96孔培養(yǎng)板，棄上清液，PBS緩沖液清洗3次后吸干，每孔加入50μl0.1%TritonX-100，置于4℃冰箱過夜。觀察細(xì)胞已無完整結(jié)構(gòu)，經(jīng)震蕩后，按堿性磷酸酶試劑盒說明書加入堿性磷酸酶底物，每孔100μl，在培養(yǎng)箱內(nèi)置30min，最后每孔加入0.2mol/LNaOH50μl終止反應(yīng)，以空白對照孔調(diào)零，在酶標(biāo)儀上測定410nm下各孔的吸光度值。 茜素紅染色法檢測柚皮苷對人牙髓細(xì)胞礦化結(jié)節(jié)形成的影響：選用第4代生長良好的牙髓細(xì)胞，制備2×104cells/ml細(xì)胞懸液，接種于六孔板內(nèi)。分為對照組和10-7mol/L濃度的柚皮苷處理后的實(shí)驗(yàn)組，每組設(shè)3個(gè)復(fù)孔。分別培養(yǎng)14d、28d后，進(jìn)行茜素紅染色，觀察礦化結(jié)節(jié)的形成。 柚皮苷對人牙髓細(xì)胞牙本質(zhì)涎磷蛋白mRNA表達(dá)的影響：選用第4代生長良好的牙髓細(xì)胞，制備2×104cells/ml細(xì)胞懸液，，接種于六孔板內(nèi)。以含15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液配置的終末濃度為10-7mol/L的柚皮苷作為試驗(yàn)組，以15%標(biāo)準(zhǔn)胎牛血清的高糖DMEM培養(yǎng)液作為對照組，每組設(shè)3個(gè)復(fù)孔。分別培養(yǎng)24h、48h、72h后PBS清洗3遍，加入Trizol試劑1ml，反復(fù)吹打至細(xì)胞完全溶解，收至無酶EP管中，-80℃冰箱保存。按照試劑盒說明書提取細(xì)胞總RNA，RT-PCR檢測牙本質(zhì)涎磷蛋白基因的表達(dá)。 采用SPSS13.0統(tǒng)計(jì)軟件作單因素方差分析，多重比較采用S-N-K法。實(shí)驗(yàn)數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差表示，檢驗(yàn)水準(zhǔn)α=0.05，P0.05為有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1牙髓細(xì)胞的免疫組化染色結(jié)果顯示為抗波形蛋白染色陽性，角蛋白染色陰性。從而證實(shí)牙髓細(xì)胞的組織來源為中胚層間充質(zhì)細(xì)胞并且無上皮細(xì)胞混雜，符合實(shí)驗(yàn)要求。 2與對照組相比，在一定濃度范圍內(nèi)的柚皮苷能促進(jìn)人牙髓細(xì)胞的增殖和細(xì)胞周期進(jìn)程，提高其堿性磷酸酶活性，促進(jìn)人牙髓細(xì)胞牙本質(zhì)涎磷蛋白mRNA表達(dá)，并且增強(qiáng)人牙髓細(xì)胞的礦化能力，其中以濃度為10-7mol/L的柚皮苷作用最為明顯。 結(jié)論：柚皮苷可促進(jìn)體外培養(yǎng)的人牙髓細(xì)胞增殖，推進(jìn)細(xì)胞周期進(jìn)程；柚皮苷可以提高人牙髓細(xì)胞堿性磷酸酶的活性，礦化能力和分化能力。
[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.
Result錛

本文編號：1665640