本文選題：脫氧雪腐鐮刀菌烯醇 + 成骨細胞��； 參考：《泰山醫(yī)學院》2012年碩士論文

【摘要】：目的 利用小鼠成骨細胞(mouseosteoblast)為體外試驗模型，探討脫氧雪腐鐮刀菌烯醇(deoxynivalenol, DON)對小鼠成骨細胞增殖和凋亡的影響；及其對成骨細胞Pax1基因表達的影響，進而揭示DON干擾成骨細胞生長和分化的分子機制，為防治DON所致的骨骼畸形奠定理論基礎。 方法 體外培養(yǎng)小鼠成骨細胞，將不同濃度的DON分別作用于對數(shù)生長期的成骨細胞，噻唑藍(MTT)比色法檢測DON對成骨細胞增殖的影響；流式細胞儀和熒光染色檢測DON誘導的細胞凋亡，Western blot檢測凋亡蛋白Bax、Bcl-2表達量變化；Trizol法裂解細胞，提取成骨細胞總RNA，普通半定量PCR和熒光定量PCR法檢測DON對Pax1基因轉錄水平的影響；用RIPA細胞裂解液提取成骨細胞總蛋白，通過Westernblot檢測DON對Pax1蛋白表達水平的影響。 結果 1.DON對成骨細胞增殖的影響： 將含有不同濃度DON的培養(yǎng)液作用于對數(shù)生長期的成骨細胞，，觀察DON對成骨細胞增殖的影響。隨DON濃度的增加，作用時間的延長，吸光度值呈逐漸降低的趨勢。當DON濃度為100ng/ml、200ng/ml時，吸光度值變化不明顯。當DON濃度為500ng/ml、1000ng/ml、1500ng/ml時，表現(xiàn)為明顯的細胞毒性作用，吸光度值明顯降低，與空白對照組兩兩比較有統(tǒng)計學意義（P＜0.05）。且同一濃度的DON作用組，培養(yǎng)時間越長，吸光度值越低。從細胞增殖抑制率折線狀圖可見：隨DON濃度的增加和作用時間的延長，細胞的數(shù)量逐漸減少，表明DON可明顯抑制成骨細胞的增殖。顯微鏡下觀察500ng/ml、1000ng/ml、1500ng/ml組細胞數(shù)量明顯減少，細胞周圍的突起減少甚至消失，可見細胞碎片、核碎裂等細胞凋亡表現(xiàn)。 2.DON對成骨細胞凋亡的影響： 流式細胞儀檢測結果顯示：將含有不同濃度DON的培養(yǎng)液作用于對數(shù)生長期的成骨細胞，隨DON濃度的增加，細胞增殖指數(shù)降低。與對照組相比，有顯著性差異。凋亡率隨DON濃度的增加而升高，呈現(xiàn)劑量依賴關系。 AO-EB熒光染色觀察結果顯示：對照組細胞核呈均染綠色熒光，形態(tài)結構正常。DON500、1000ng/ml作用48h，大部分細胞胞核染色增強，熒光更為明亮，但細胞核開始固縮或呈串珠狀，表現(xiàn)為早期凋亡細胞核，也有部分細胞核染色質呈橘紅色熒光，且胞核出現(xiàn)碎片，表現(xiàn)為晚期凋亡細胞核。1500ng/mlDON作用成骨細胞48h后，細胞數(shù)目明顯減少，細胞大部分表現(xiàn)為晚期凋亡。 Western blot檢測顯示：500、1000、1500ng/mlDON作用組，Bcl-2蛋白表達減少，Bax蛋白表達增加，Bax/Bcl-2比值增大。以上試驗表明DON具有明顯的誘導成骨細胞凋亡的作用。 3. DON對PAX1基因表達的影響： RT-PCR凝膠電泳圖顯示：DON可明顯增加成骨細胞中Pax1基因的mRNA表達水平。且隨DON濃度增加，Pax1基因表達量逐漸升高，Pax1條帶的亮度逐漸增強。1500ng/ml組與1000ng/ml組相比，Pax1基因表達量稍下降。DON500ng/ml、1000ng/ml、1500ng/ml組與空白對照組相比，差異有統(tǒng)計學意義（P0.05）。 實時熒光定量PCR結果顯示：DON可明顯增加成骨細胞中Pax1基因的相對表達量。且隨DON濃度增加，Pax1基因表達量逐漸升高。500ng/ml、1000ng/ml、1500ng/ml組與空白對照組相比，差異有統(tǒng)計學意義（P0.01）。 Western blot檢測顯示：DON可明顯增加成骨細胞中Pax1基因的蛋白表達水平。且隨DON濃度增加，Pax1蛋白條帶的灰度值逐漸增強。DON500ng/ml、1000ng/ml、1500ng/ml組與空白對照組相比，差異有統(tǒng)計學意義（P0.05）。 結論 1.DON對成骨細胞有直接毒性作用，能抑制成骨細胞的增殖，誘導成骨細胞發(fā)生凋亡。 2. DON作用成骨細胞48h后，Pax1基因表達量升高，。
[Abstract]:objective
The effect of deoxynivalenol (DON) on the proliferation and apoptosis of osteoblasts in mice was investigated by using mouse osteoblast (mouseosteoblast) in vitro, and the effect on the expression of Pax1 gene in osteoblasts was investigated, and the molecular mechanism of DON interfering with the growth and differentiation of osteoblasts was revealed, which was caused by the prevention and control of DON. Bone malformation lays a theoretical foundation.
Method
Mouse osteoblasts were cultured in vitro, and different concentrations of DON were acted on logarithmic long-term osteoblasts, and thiazolium (MTT) colorimetric assay was used to detect the effect of DON on the proliferation of osteoblasts; flow cytometry and fluorescence staining were used to detect apoptosis induced by DON; Western blot was used to detect the expression of Bax, Bcl-2 expression and Trizol method. The total RNA of osteoblasts was extracted, the effect of DON on the transcriptional level of Pax1 gene was detected by ordinary semi quantitative PCR and fluorescence quantitative PCR, and the total protein of osteoblast was extracted with RIPA cell lysate, and the effect of DON on the expression of Pax1 protein was detected by Westernblot.
Result
The effect of 1.DON on the proliferation of osteoblast:
The effects of DON on the proliferation of osteoblasts were observed with the culture solution containing different concentrations of DON. With the increase of DON concentration and the prolongation of the action time, the absorbance value decreased gradually. When the concentration of DON was 100ng/ml and 200ng/ml, the change of absorbency was not obvious. When the concentration of DON was 500ng/ml, 1000ng/ml, 1500 At ng/ml, the apparent cytotoxic effect, the absorbance value decreased obviously, compared with the blank control group 22 (P < 0.05). And the longer the culture time, the lower the absorbance value of the same concentration group, the longer the cell proliferation inhibition rate showed: with the increase of the concentration of DON and the prolongation of the action time, the cell The number gradually decreased, indicating that DON could obviously inhibit the proliferation of osteoblasts. Under the microscope, the number of cells in 500ng/ml, 1000ng/ml, 1500ng/ml group decreased obviously, the protrusions around the cells decreased or even disappeared, and cell fragmentation and nuclear fragmentation could be seen.
The effect of 2.DON on osteoblast apoptosis:
The results of flow cytometry showed that the culture medium containing different concentrations of DON acted on the logarithmic long-term osteoblasts, and the proliferation index decreased with the increase of DON concentration. Compared with the control group, there was a significant difference. The rate of apoptosis increased with the increase of DON concentration, which showed the dose dependence.
The results of AO-EB fluorescence staining showed that the nuclei of the control group were stained with green fluorescence, and the morphology and structure of normal.DON5001000ng/ml were 48h. Most of the cell nuclei were stained enhanced and the fluorescence was brighter, but the nuclei began to shrink or beaded, showing the early apoptotic nuclei, and some of the nuclei of the nuclei were orange red fluorescence. The nucleus appeared fragmented, showing late apoptotic nucleus.1500ng/mlDON. After osteoblast 48h, the number of cells decreased significantly. Most of the cells showed late apoptosis.
Western blot detection showed that the expression of Bcl-2 protein decreased, the expression of Bax protein increased and the ratio of Bax/Bcl-2 increased in the 50010001500ng/mlDON group. These experiments showed that DON has a significant effect on inducing apoptosis of osteoblasts.
The effect of 3. DON on the expression of PAX1 gene:
RT-PCR gel electrophoresis showed that DON could significantly increase the mRNA expression level of Pax1 gene in osteoblasts. With the increase of DON concentration, the expression of Pax1 gene increased gradually, and the brightness of Pax1 bands increased gradually in.1500ng/ml group compared with 1000ng/ml group, Pax1 gene expression decreased slightly.DON500ng/ml, 1000ng/ml, group and blank control group The difference was statistically significant (P0.05).
The results of real time fluorescence quantitative PCR showed that DON could significantly increase the relative expression of Pax1 gene in osteoblasts, and with the increase of DON concentration, the expression of Pax1 gene increased gradually,.500ng/ml, 1000ng/ml, and 1500ng/ml group, compared with the blank control group, the difference was statistically significant (P0.01).
Western blot detection showed that DON could significantly increase the protein expression level of Pax1 gene in osteoblasts. And with the increase of DON concentration, the gray value of Pax1 protein bands gradually increased.DON500ng/ml, 1000ng/ml, and 1500ng/ml group compared with the blank control group, the difference was statistically significant (P0.05).
conclusion
1.DON has direct toxicity on osteoblasts, inhibits proliferation of osteoblasts and induces osteoblasts apoptosis.
After 2. DON osteoblast 48h, the expression of Pax1 gene increased.
【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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