本文選題：牙周膜細胞群 + 木犀草素　； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：目的 本研究擬通過體外分離、培養(yǎng)人牙周膜細胞群，探索及檢測木犀草素和橙皮素對人牙周膜細胞群增殖及骨向分化能力的影響，探索此兩種中藥有效組分應(yīng)用于牙周疾病治療的可能性，為科學(xué)合理的推進中藥有效組分在牙周病的應(yīng)用，促進中藥的國際化提供理論基礎(chǔ)和實踐參考。 方法 本研究采用蓋玻片覆蓋組織塊法對牙周膜細胞進行原代培養(yǎng)，在倒置顯微鏡下觀察牙周膜細胞的生長狀況；MTT法繪制細胞生長曲線；免疫組織化學(xué)法鑒定細胞來源；并對細胞進行成骨誘導(dǎo)及茜素紅染色；采用MTT四唑鹽比色法、考馬斯亮藍染色法、ALP堿性磷酸酶檢測試劑盒、Q-PCR檢測成骨相關(guān)基因等方法觀察不同濃度木犀草素、橙皮素對牙周膜細胞群增殖、蛋白含量及骨向分化的影響。 結(jié)果 1、鏡下觀察牙周膜細胞第3-9d從組織塊中游出，細胞排列無序，大部分呈長梭形，胞體飽滿，具有細而長的胞突，胞質(zhì)均勻，細胞核位于細胞中央、呈圓形或卵圓形。少量細胞較小且形態(tài)不規(guī)則；細胞生長曲線測定顯示第3代牙周膜細胞群（periodontal ligament cell population, PDLP）生長曲線呈“S”型；免疫組化結(jié)果顯示：第3代PDLP波形絲蛋白強陽性表達，角蛋白染色陰性表達；PDLP成骨誘導(dǎo)：礦化誘導(dǎo)組大量礦化結(jié)節(jié)形成。 2、不同木犀草素和橙皮素對PDLP增殖及蛋白含量的影響 ①MTT結(jié)果顯示：木犀草素、橙皮素作用于PDLP24、48、72h，各濃度組（100μM、10μM、1μM、0.1μM、0.01μM）均可促進牙周膜細胞的增殖（P0.05）。 ②考馬斯亮藍染色法結(jié)果顯示： 1）木犀草素各實驗組細胞內(nèi)總蛋白含量均高于對照組（P0.05），1μM木犀草素總蛋白含量較其他實驗組高（P0.05） 2）橙皮素各實驗組細胞內(nèi)總蛋白含量均高于對照組（P0.05），0.1μM木犀草素總蛋白含量較其他實驗組高（P0.05）。 3、木犀草素和橙皮素對PDLP骨向分化的影響 ①ALP堿性磷酸酶檢測試劑盒結(jié)果顯示： 1）木犀草素各實驗組ALP活性均高于對照組（P0.05），1μM橙皮素對應(yīng)OD值較其他各實驗組高（P0.05），10μM濃度木犀草素對應(yīng)OD值交其他各實驗組低（P0.05） 2）橙皮素各實驗組ALP活性均高于對照組（P0.05），0.1μM橙皮素ALP活性較其他各實驗組高（P0.05）。 ②Q-PCR檢測結(jié)果顯示： 1）木犀草素作用72h，實驗組ALP表達均呈上調(diào)趨勢，其中1μM組上調(diào)最為顯著，0.01μM組ALP表達上調(diào)無統(tǒng)計學(xué)差異（P0.05），其余實驗組ALP表達上調(diào)均有統(tǒng)計學(xué)差異（P0.05）。1μM和0.1μM組RUNX2表達上調(diào)，，具有統(tǒng)計學(xué)差異（P0.05）。 2）橙皮素作用72h，實驗組ALP表達均呈上調(diào)趨勢，其中0.1μM組上調(diào)最為顯著，0.01μM組ALP表達上調(diào)無統(tǒng)計學(xué)差異（P0.05），其余實驗組ALP表達上調(diào)均有統(tǒng)計學(xué)差異（P0.05）。0.1μM和00.1μM組RUNX2表達上調(diào)，具有統(tǒng)計學(xué)差異（P0.05）。 結(jié)論 1、蓋玻片覆蓋組織塊法可成功培養(yǎng)出牙周膜細胞群，成功率高，來源可靠，細胞生長狀態(tài)良好，且在一定條件下可以骨向分化。 2、一定濃度的木犀草素可以促進牙周膜細胞群的增殖和骨向分化。 3、一定濃度的橙皮素可以促進牙周膜細胞群的增殖和骨向分化。 4、本實驗為木犀草素和橙皮素進一步應(yīng)用于牙周組織再生修復(fù)提供了一定的參考價值，但是二者的作用機制還需要進一步的實驗研究。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R780.2

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