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

【摘要】：目的：本實驗通過采用人牙髓細胞體外培養(yǎng)的方法，探討不同濃度柚皮苷對體外培養(yǎng)人牙髓細胞增殖、分化、礦化、細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胞并進行原代培養(yǎng)。待細胞長出后在倒置顯微鏡下觀察細胞形態(tài)及生長情況。當細胞生長至鋪滿孔底80%時進行傳代，細胞傳代成功后，取第4代對數(shù)生長期人牙髓細胞進行細胞爬片，用SABC法進行波形絲蛋白和角蛋白免疫組織化學染色鑒定細胞來源。將處于對數(shù)生長期的第4代人牙髓細胞用0.25%的胰蛋白酶消化后制成細胞懸液，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。將細胞隨機分為5個實驗組、1個對照組和1個空白對照組，每組選5個孔。實驗組：在相應的有細胞孔內加入200μl用含15%標準胎牛血清的高糖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：在相應的有細胞孔內加入200μl含15%標準胎牛血清的高糖DMEM培養(yǎng)液。空白對照組：在相應的無細胞孔內加入200μl含15%標準胎牛血清的高糖DMEM培養(yǎng)液。 MTT比色法檢測柚皮苷對人牙髓細胞增殖能力的影響：分別于培養(yǎng)24h、48h和72h時隨機取出一個96孔培養(yǎng)板，向所測孔內加入5mg/mlMTT20μl后繼續(xù)孵育4h，棄孔內液體，每孔加入150μl二甲基亞砜（DMSO），振蕩5min，以空白對照孔調零，在酶標儀上測定492nm波長下各孔的吸光度值。 CCK-8法檢測柚皮苷對人牙髓細胞增殖能力的影響：分別于培養(yǎng)24h、48h和72h時隨機取出一個96孔培養(yǎng)板，棄孔內液體，向所測孔內加入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)箱內孵育2小時，用酶標儀測定在450nm處的吸光度值。 流式細胞術檢測柚皮苷對人牙髓細胞的細胞周期的影響：選用含15%標準胎牛血清的高糖DMEM培養(yǎng)液配置的終末濃度為10-7mol/L的柚皮苷作為試驗組，與對照組（15%標準胎牛血清的高糖DMEM培養(yǎng)液）進行細胞周期試驗。按照細胞周期試劑盒說明書操作，進行流式細胞儀分析。 檢測柚皮苷對人牙髓細胞的堿性磷酸酶活性的影響：分別在藥物作用24h、48h和72h隨機取出一個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。觀察細胞已無完整結構，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)箱內置30min，最后每孔加入0.2mol/LNaOH50μl終止反應，以空白對照孔調零，在酶標儀上測定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胞礦化結節(jié)形成的影響：選用第4代生長良好的牙髓細胞，制備2×104cells/ml細胞懸液，接種于六孔板內。分為對照組和10-7mol/L濃度的柚皮苷處理后的實驗組，每組設3個復孔。分別培養(yǎng)14d、28d后，進行茜素紅染色，觀察礦化結節(jié)的形成。 柚皮苷對人牙髓細胞牙本質涎磷蛋白mRNA表達的影響：選用第4代生長良好的牙髓細胞，制備2×104cells/ml細胞懸液，，接種于六孔板內。以含15%標準胎牛血清的高糖DMEM培養(yǎng)液配置的終末濃度為10-7mol/L的柚皮苷作為試驗組，以15%標準胎牛血清的高糖DMEM培養(yǎng)液作為對照組，每組設3個復孔。分別培養(yǎng)24h、48h、72h后PBS清洗3遍，加入Trizol試劑1ml，反復吹打至細胞完全溶解，收至無酶EP管中，-80℃冰箱保存。按照試劑盒說明書提取細胞總RNA，RT-PCR檢測牙本質涎磷蛋白基因的表達。 采用SPSS13.0統(tǒng)計軟件作單因素方差分析，多重比較采用S-N-K法。實驗數(shù)據(jù)以均數(shù)±標準差表示，檢驗水準α=0.05，P0.05為有統(tǒng)計學意義。 結果： 1牙髓細胞的免疫組化染色結果顯示為抗波形蛋白染色陽性，角蛋白染色陰性。從而證實牙髓細胞的組織來源為中胚層間充質細胞并且無上皮細胞混雜，符合實驗要求。 2與對照組相比，在一定濃度范圍內的柚皮苷能促進人牙髓細胞的增殖和細胞周期進程，提高其堿性磷酸酶活性，促進人牙髓細胞牙本質涎磷蛋白mRNA表達，并且增強人牙髓細胞的礦化能力，其中以濃度為10-7mol/L的柚皮苷作用最為明顯。 結論：柚皮苷可促進體外培養(yǎng)的人牙髓細胞增殖，推進細胞周期進程；柚皮苷可以提高人牙髓細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