【作者】 赵旭；

【导师】 于灵芝；

【作者基本信息】 山东大学 ， 麻醉学， 2015， 硕士

【摘要】 目的在不同时间点采用不同浓度的IL-17F干预大鼠原代成骨细胞,观察对大鼠原代成骨细胞的增殖能力和ALP活性的影响,以及对大鼠成骨细胞的转录因子Runx2和Osterix mRNA的表达的影响。探讨IL-17F对大鼠成骨细胞增殖和分化功能的作用,观察成骨细胞特异性转录因子Osterix以及Runx2 mRNA在IL-17F干预下表达的变化,探寻炎症因子IL-17F在骨形成中的作用,以期为临床治疗骨质疏松和相关骨代谢疾病提供理论依据。方法1.细胞提取、培养和鉴定：取出生24小时以内Wistar大鼠8只,雌雄不限,采用拉颈法处死,在无菌条件下取其颅骨,分别采用胰酶-胶原酶消化法和组织块反复贴壁法提取原代成骨细胞,37℃、5%CO2、饱和湿度培养箱内进行细胞培养,待细胞生长至培养瓶底面积80%左右时进行细胞传代,并采用差异贴壁法纯化成骨细胞。采用第4代成骨细胞进行实验。成骨细胞的鉴定：(1)采用倒置相差显微镜观察成骨细胞形态和生长情况,定期拍照记录。(2)采用碱性磷酸酶(Alkaline phosphatase, ALP)染色法(钙钴法)鉴定成骨细胞。2.实验分组及处理：将第3代成骨细胞,胰酶消化、离心后制成细胞悬液,细胞计数并调整细胞浓度至5×105/ml,加入到6孔板中培养24小时。待细胞贴壁后换用无血清培养基培养24小时,使细胞同步化。将6孔板内细胞随机分为对照组和实验组,对照组采用含10%胎牛血清(Fatal bovine serum, FBS)低糖DMEM培养基培养,前期预实验分别用Ing/ml、10ng/ml、20ng/ml、50ng/ml和100ng/ml的IL-17F干预成骨细胞,结果显示浓度为20ng/ml、50ng/ml、100ng/ml的IL-17F对成骨细胞的作用明显,因此实验组分别应用含有20ng/ml、50ng/ml、100ng/ml、IL-17F的10% FBS低糖DMEM培养基进行培养。分别在IL-17F干预后的第1、3天留取细胞上清液待测,-80℃保存,成骨细胞进行以下检测。3.指标检测(1)采用CCK-8法检测成骨细胞增殖。(2)采用微量酶标法检测成骨细胞培养上清液中ALP活性。(3)采用RT-PCR法检测成骨细胞转录因子Runx2和Osterix mRNA的表达变化。结果1.成骨细胞的分离、培养和鉴定：胰酶-胶原酶消化法和组织块反复贴壁法均可获取大鼠成骨细胞,其细胞形态特征和生长情况符合成骨细胞特点。通过倒置相差显微镜观察ALP染色结果,发现成骨细胞内的ALP反应生成不溶性染料,呈现黑色颗粒或团块,证明所培养细胞为成骨细胞。2.CCK-8检测细胞增殖：与对照组相比,第1天细胞增殖率实验组50ng/ml组和100ng/ml组均明显升高,差异有统计学意义(P<0.05),20ng/ml组也有升高,但差异无统计学意义；第3天实验组20ng/ml、50ng/ml组和100ng/ml组均高于对照组(P<0.05)；第5天的实验组20ng/ml组、50ng/ml组和100ng/ml组细胞增殖率增加,差异有统计学意义(P<0.05)。与实验组20ng/ml组相比,实验组100ng/ml组在第1、3、5天细胞增值率升高,差异有统计学意义(P<0.05)；第3天50ng/ml组和100ng/ml组均高于21ng/ml组,差异有统计学意义(P<0.05)。与实验组50ng/ml组相比,100ng/ml组在1、3和5天均明显升高,差异有统计学意义(P<0.05)。3.微量酶标法检测细胞上清液ALP活性与对照组相比,第1天实验组20ng/ml和50ng/ml组ALP活性降低,100ng/ml组ALP活性增加,差异具有统计学意义(P<0.05)；第3天实验组20ng/ml和50ng/ml组ALP活性较对照组降低,而100ng/ml组ALP活性与对照组、20ng/ml组和50ng/ml组相比均增加,差异具有统计学意义(P<0.05)。4. Runx2和Osterix mRNA表达变化与对照组相比,1、3、5天50 ng/ml组和100ng/ml组Runx2mRNA表达增高(P<0.05)：与实验组20ng/ml组相比,第1、3天100ng/ml组Runx2 mRNA表达均增加(P<0.05),第5天50ng/ml组和100ng/ml组Runx2mRNA表达均升高(P<0.05)；与50 ng/ml组相比,实验组1、3、5天100ng/ml组Runx2mRNA表达均增加,差异具有统计学意义(P<0.05)。与对照组相比,实验组第1、3和5天的50ng/ml组和100ng/ml组Osterix mRNA表达均明显增加,差异具有统计学意义(P<0.05)；第1、3、5天50ng/ml组和100ng/ml组Osterix mRNA表达高于实验组20ng/ml组,差异具有统计学意义(P<0.05)；各时间点实验组100ng/ml组Osterix mRNA表达均高于50ng/ml组,差异有统计学意义(P<0.05)。结论IL-17F在20ng/ml～100ng/ml浓度范围内可促进成骨细胞的增殖活性,提高ALP活性,这种促进作用很可能与IL-17F上调成骨细胞特异性转录因子Osterix及Runx2mRNA表达有关,浓度为100ng/ml的IL-17F促进作用尤为显著。更多还原

【Abstract】 ObjectiveTo detect the cells proliferation, ALP activity, and the changes of mRNA expression of transcription factors Runx2 and Osterix at different concentrations of IL-17F intervened rat osteoblasts and different times. By discussing the influence of IL-17F on osteoblasts proliferation, differentiation, and the changes of mRNA expression of osteoblast specific transcription factor Osterix and Runx2 under the intervention of IL-17F, explore the role of inflammatory cytokines IL-17F in bone formation to provide a theoretical basis for the clinical treatment of osteoporosis and related bone metabolic diseases.Methods1. Cell extraction, cell culture and identificationEight new-born Wistar rats younger than 24 hours were taken skulls under the aseptic conditions after sacrificed. Osteoblasts were harvested from the calvarias of neonatal rats and isolated via trypsin and type II collagenase and improved tissue-culture method. Osteoblasts were incubated in 5% CO2 at 37℃ and 100% relative humidified. After the monolayer culture grew to confluence of 80% of culture flask, cells were harvested with a 0.25% trypsin solution, split, resuspended in the culture medium, and filled into new culture flasks. The osteoblasts were purified by difference-speed adherence method. After confluent monolayer cells were achieved by the fourth passage, cells were used for experiment.The identification of osteoblasts(1) Morphological characteristics and growth situation of cells were observed and photographed by an inverted phase contrast microscope.(2) Alkaline phosphatase staining verified the osteoblastic phenotype.2. Grouping and treatmentThe fourth passage osteoblasts were counted and cell concentration was adjusted to 5 × 105/ml. One day prior to cell seeding in 6-well plates, adherent cells were cultured with serum-free medium for another 24 hours in order to cell synchronization. Cells in the 6-well plates were randomly divided into the control group and the experimental group. The control group was applied with low glucose DMEM culture medium supplemented with 10% fatal bovine serum (FBS). In pre-experiment, the concentration of lng/ml,10 ng/ml,20ng/ml,50ng/ml and 100ng/ml IL-17F was used to intervene in osteoblasts, which showed that 20ng/ml,50ng/ml and 100ng/ml IL-17F affected osteoblasts significantly. Therefore the experimental groups were applied with low glucose DMEM culture medium supplemented with 10%FBS and 20ng/ml, 50ng/ml and 100ng/ml IL-17F, respectively. Cell supernatant specimens were collected on the first and the third day after the intervention of IL-17F, respectively. Cell supernatant specimens were preserved in -80℃.3. Index detection(1) CCK-8 analysis was used to detect the proliferation of osteoblasts.(2) Trace amounts of cell culture supernatant of ALP activity was detected by ELIS A method.(3) The mRNA expression changes of the osteoblast specific transcription factor Osterix and Runx2 were measured by RT-PCR.Results1. Cell extraction, cell culture and identification of osteoblastCells can be harvested with the method of trypsin-collagenase digestion and improved tissue-culture method. The morphology and growth characters of cells were consistent with the features of osteoblasts. ALP staining had proved that the cultured cells were osteoblasts which showed black particles or clumps in the cells observed by inverted phase contrast microscope.2. The proliferation of osteoblasts measured by CCK-8Compared with the control group, cell proliferation supplemented with 50ng/ml and 100ng/ml IL-17F was significantly increased on the first day, there was significant statistically difference (P<0.05); cell proliferation supplemented with 20ng/ml IL-17F was also developed, however, there was no statistical significance; cell proliferation supplemented with 20ng/ml,50ng/ml and 100ng/ml IL-17F was higher than that of control group on the third day (P<0.05); cell proliferation supplemented with 20ng/ml, 50ng/ml and 100ng/ml IL-17F was significantly developed than that of control group on the fifth day, which was significant statistically difference (P<0.05).Compared with the experimental group supplemented with 20ng/ml IL-17F, cell proliferation in the experimental group supplemented with 100ng/ml IL-17F was obviously raised on the first, third and fifth day, which was significant statistically difference (P<0.05); cell proliferation supplemented with 20ng/ml IL-17F was significantly lower than that supplemented with 50ng/ml and 100ng/ml IL-17F, there was significant statistically difference (P<0.05).Compared with the experimental group supplemented with 50ng/ml IL-17F, cell proliferation supplemented with 100ng/ml IL-17F was remarkable elevated at the first, third and fifth day, which was significant statistically difference (P<0.05).3. The ALP activity measured by ELISA assayCompared with the control group, ALP activity of the experimental group supplemented with 20ng/ml and 50ng/ml IL-17F was declined, and 100ng/ml IL-17F was conspicuously increased at the first day, there was significant statistically difference (P<0.05); ALP activity supplemented with 20ng/ml and 50ng/ml IL-17F was lower than that of the control group, but ALP activity supplemented with 100ng/ml IL-17F was outstanding raised than that of the control group and the experimental group supplemented with 20ng/ml and 50ng/ml IL-17F at the third day, which was significant statistically difference (P<0.05).4. The changes of expressions of Osterix and Runx2 mRNACompared with control group, the mRNA expression of Runx2 supplemented with 50ng/ml and 100ng/ml IL-17F was increased at the first, third and fifth day (P<0.05). The mRNA expression of Runx2 supplemented with 100ng/ml IL-17F was striking increased than that with 20ng/ml IL-17F at the first and third day (P<0.05). At the fifth day the mRNA expression of Runx2 supplemented with 50ng/ml and 100ng/ml IL-17F was increased than that of the control group (P<0.05). In comparison to experimental group supplemented with 50ng/ml IL-17F, the mRNA expression of Runx2 supplemented with 100ng/ml IL-17F was higher at the first, third and fifth day, which was prominent statistically significant difference (P<0.05).The mRNA expression of Osterix intervened in 50ng/ml and 100ng/ml IL-17F was significantly increased than that of control group at the first, third and fifth day, which was prominent statistically significant difference (P<0.05). The expression of Osterix mRNA intervened in 50ng/ml and 100ng/ml IL-17F was higher than that of the experimental group with 20ng/ml IL-17F at the first, third and fifth day, respectively, which was prominent statistically significant difference (P<0.05). The mRNA expression of Osterix intervened in 100ng/ml IL-17F was significantly higher than that with 20ng/ml and 50ng/ml IL-17F, there was prominent statistically significant difference (P<0.05).ConclusionAt the concentration of 20-100ng/ml cytokine IL-17F can promote the proliferation, differentiation, ALP activity of osteoblasts. This promotion effect may be related to that IL-17F can up regulate the mRNA expression of osteoblast-specific transcription factor Osterix and Runx2, and supplemented with 100ng/ml, the effect may be the most significant.更多还原