木犀草素和橙皮素对人牙周膜细胞群增殖和分化能力影响的初步研究

发布时间：2018-06-09 16:12

本文选题：牙周膜细胞群 + 木犀草素　；参考：《郑州大学》2014年硕士论文

【摘要】：目的本研究拟通过体外分离、培养人牙周膜细胞群，探索及检测木犀草素和橙皮素对人牙周膜细胞群增殖及骨向分化能力的影响，探索此两种中药有效组分应用于牙周疾病治疗的可能性，为科学合理的推进中药有效组分在牙周病的应用，促进中药的国际化提供理论基础和实践参考。方法本研究采用盖玻片覆盖组织块法对牙周膜细胞进行原代培养，在倒置显微镜下观察牙周膜细胞的生长状况；MTT法绘制细胞生长曲线；免疫组织化学法鉴定细胞来源；并对细胞进行成骨诱导及茜素红染色；采用MTT四唑盐比色法、考马斯亮蓝染色法、ALP碱性磷酸酶检测试剂盒、Q-PCR检测成骨相关基因等方法观察不同浓度木犀草素、橙皮素对牙周膜细胞群增殖、蛋白含量及骨向分化的影响。结果 1、镜下观察牙周膜细胞第3-9d从组织块中游出，细胞排列无序，大部分呈长梭形，胞体饱满，具有细而长的胞突，胞质均匀，细胞核位于细胞中央、呈圆形或卵圆形。少量细胞较小且形态不规则；细胞生长曲线测定显示第3代牙周膜细胞群（periodontal ligament cell population, PDLP）生长曲线呈“S”型；免疫组化结果显示：第3代PDLP波形丝蛋白强阳性表达，角蛋白染色阴性表达；PDLP成骨诱导：矿化诱导组大量矿化结节形成。 2、不同木犀草素和橙皮素对PDLP增殖及蛋白含量的影响 ①MTT结果显示：木犀草素、橙皮素作用于PDLP24、48、72h，各浓度组（100μM、10μM、1μM、0.1μM、0.01μM）均可促进牙周膜细胞的增殖（P0.05）。 ②考马斯亮蓝染色法结果显示： 1）木犀草素各实验组细胞内总蛋白含量均高于对照组（P0.05），1μM木犀草素总蛋白含量较其他实验组高（P0.05） 2）橙皮素各实验组细胞内总蛋白含量均高于对照组（P0.05），0.1μM木犀草素总蛋白含量较其他实验组高（P0.05）。 3、木犀草素和橙皮素对PDLP骨向分化的影响 ①ALP碱性磷酸酶检测试剂盒结果显示： 1）木犀草素各实验组ALP活性均高于对照组（P0.05），1μM橙皮素对应OD值较其他各实验组高（P0.05），10μM浓度木犀草素对应OD值交其他各实验组低（P0.05） 2）橙皮素各实验组ALP活性均高于对照组（P0.05），0.1μM橙皮素ALP活性较其他各实验组高（P0.05）。 ②Q-PCR检测结果显示： 1）木犀草素作用72h，实验组ALP表达均呈上调趋势，其中1μM组上调最为显著，0.01μM组ALP表达上调无统计学差异（P0.05），其余实验组ALP表达上调均有统计学差异（P0.05）。1μM和0.1μM组RUNX2表达上调，，具有统计学差异（P0.05）。 2）橙皮素作用72h，实验组ALP表达均呈上调趋势，其中0.1μM组上调最为显著，0.01μM组ALP表达上调无统计学差异（P0.05），其余实验组ALP表达上调均有统计学差异（P0.05）。0.1μM和00.1μM组RUNX2表达上调，具有统计学差异（P0.05）。结论 1、盖玻片覆盖组织块法可成功培养出牙周膜细胞群，成功率高，来源可靠，细胞生长状态良好，且在一定条件下可以骨向分化。 2、一定浓度的木犀草素可以促进牙周膜细胞群的增殖和骨向分化。 3、一定浓度的橙皮素可以促进牙周膜细胞群的增殖和骨向分化。 4、本实验为木犀草素和橙皮素进一步应用于牙周组织再生修复提供了一定的参考价值，但是二者的作用机制还需要进一步的实验研究。
[Abstract]:objective
This study aims to explore and detect the effects of luteolin and hesperidin on the proliferation of human periodontal ligament cells and the ability of bone differentiation by isolation of the human periodontal membrane cells in vitro, and to explore the possibility of applying the two effective components in the treatment of periodontal diseases to promote the scientific and rational application of the effective components of traditional Chinese medicine in periodontitis. It provides theoretical basis and practical reference for internationalization of Chinese medicine.
Method
In this study, the periodontal membrane cells were cultured by the method of cover glass covering tissue. The growth of the periodontal ligament cells was observed under the inverted microscope; the cell growth curve was plotted by the MTT method; the cell origin was identified by immunohistochemistry; and the cells were induced by the osteogenesis and alizarin red, and the MTT four azoles colorimetric method was used. The effects of different concentrations of luteolin on the proliferation, protein content and bone differentiation of periodontal ligament cells were observed by ALP alkaline phosphatase detection kit and Q-PCR detection of bone related genes.
Result
1, under the microscope, the periodontal ligament cells, 3-9d, were observed from the tissue block. Most of the cells were arranged in disorder. Most of the cells were long spindle shaped, and the cells were full, with thin and long cytosks and homogeneous cytoplasm. The nuclei were in the center of the cells, and the cells were round or oval. A small number of cells were small and irregular. Cell growth curves showed the third generation periodontal cell group (peri The growth curve of odontal ligament cell population, PDLP was "S", and the results of immunohistochemical staining showed that the third generation PDLP was strongly positive and negative for keratin; PDLP induced bone induction: a large number of mineralized nodules were formed in the mineralization induced group.
2, the effects of luteolin and hesperidin on PDLP proliferation and protein content.
(1) MTT results showed that luteolin and hesperidin acted on PDLP24,48,72h, and each concentration group (100 mu M, 10 mu M, 1 mu M, 0.1 mu M, 0.01 mu M) could promote the proliferation of periodontal ligament cells (P0.05).
The results of Coomassie brilliant blue staining showed that:
1) the total protein content of luteolin in each experimental group was higher than that in the control group (P0.05), and the total protein content of luteolin in 1 micron M was higher than that in other experimental groups (P0.05).
2) the total protein content of hesperidin in each experimental group was higher than that in the control group (P0.05), and the total protein content of luteolin in 0.1 M was higher than that in other experimental groups (P0.05).
3, luteolin and hesperidin affect the differentiation of PDLP into bone.
The results of ALP alkaline phosphatase test kit showed that:
1) the activity of ALP in the experimental group of luteolin was higher than that of the control group (P0.05), and the corresponding OD value of 1 u M was higher than that of the other experimental groups (P0.05), and the concentration of luteolin with 10 mu M was lower than that of the other experimental groups (P0.05).
2) the activity of ALP in hesperidin experimental group was higher than that in control group (P0.05), and the activity of hesperidin 0.1 ALP was higher than that in other experimental groups (P0.05). The activity of ALP in hesperidin group was higher than that in control group (P0.05).
2. The results of Q-PCR detection showed that:
1) the effect of luteolin on 72h, the expression of ALP in the experimental group was up trend, and the up regulation of the 1 mu M group was the most significant, and the expression of ALP in the 0.01 mu M group was up to no statistical difference (P0.05). The up regulation of ALP expression in the rest of the experimental group was statistically different (P0.05),.1 mu M and 0.1 mu M group up up, with statistical differences.
2) the expression of ALP in the experimental group was up-regulated by the action of hesperin on 72h, and the up regulation of the 0.1 mu M group was the most significant, and there was no statistical difference between the 0.01 mu M group (P0.05). The up regulation of the ALP expression in the rest of the experimental groups was statistically different (P0.05) in.0.1 mu M and the 0.1 mu M group.
conclusion
1, the cover glass covering tissue block method can successfully develop the periodontal cell group, with high success rate, reliable source, good cell growth state and bone differentiation under certain conditions.
2, luteolin can promote the proliferation and osteogenic differentiation of periodontal ligament cells.
3, a certain concentration of hesperidin can promote the proliferation and osteogenic differentiation of periodontal ligament cell population.
4, this experiment provides a certain reference value for the further application of luteolin and hesperidin to periodontal tissue regeneration, but the mechanism of the two is still needed further experimental research.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R780.2

【参考文献】