脱氧雪腐镰刀菌烯醇对小鼠成骨细胞PAX1基因表达的影响

发布时间：2018-05-17 15:42

本文选题：脱氧雪腐镰刀菌烯醇 + 成骨细胞　；参考：《泰山医学院》2012年硕士论文

【摘要】：目的利用小鼠成骨细胞(mouseosteoblast)为体外试验模型，探讨脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)对小鼠成骨细胞增殖和凋亡的影响；及其对成骨细胞Pax1基因表达的影响，进而揭示DON干扰成骨细胞生长和分化的分子机制，为防治DON所致的骨骼畸形奠定理论基础。方法体外培养小鼠成骨细胞，将不同浓度的DON分别作用于对数生长期的成骨细胞，噻唑蓝(MTT)比色法检测DON对成骨细胞增殖的影响；流式细胞仪和荧光染色检测DON诱导的细胞凋亡，Western blot检测凋亡蛋白Bax、Bcl-2表达量变化；Trizol法裂解细胞，提取成骨细胞总RNA，普通半定量PCR和荧光定量PCR法检测DON对Pax1基因转录水平的影响；用RIPA细胞裂解液提取成骨细胞总蛋白，通过Westernblot检测DON对Pax1蛋白表达水平的影响。结果 1.DON对成骨细胞增殖的影响：将含有不同浓度DON的培养液作用于对数生长期的成骨细胞，，观察DON对成骨细胞增殖的影响。随DON浓度的增加，作用时间的延长，吸光度值呈逐渐降低的趋势。当DON浓度为100ng/ml、200ng/ml时，吸光度值变化不明显。当DON浓度为500ng/ml、1000ng/ml、1500ng/ml时，表现为明显的细胞毒性作用，吸光度值明显降低，与空白对照组两两比较有统计学意义（P＜0.05）。且同一浓度的DON作用组，培养时间越长，吸光度值越低。从细胞增殖抑制率折线状图可见：随DON浓度的增加和作用时间的延长，细胞的数量逐渐减少，表明DON可明显抑制成骨细胞的增殖。显微镜下观察500ng/ml、1000ng/ml、1500ng/ml组细胞数量明显减少，细胞周围的突起减少甚至消失，可见细胞碎片、核碎裂等细胞凋亡表现。 2.DON对成骨细胞凋亡的影响：流式细胞仪检测结果显示：将含有不同浓度DON的培养液作用于对数生长期的成骨细胞，随DON浓度的增加，细胞增殖指数降低。与对照组相比，有显著性差异。凋亡率随DON浓度的增加而升高，呈现剂量依赖关系。 AO-EB荧光染色观察结果显示：对照组细胞核呈均染绿色荧光，形态结构正常。DON500、1000ng/ml作用48h，大部分细胞胞核染色增强，荧光更为明亮，但细胞核开始固缩或呈串珠状，表现为早期凋亡细胞核，也有部分细胞核染色质呈橘红色荧光，且胞核出现碎片，表现为晚期凋亡细胞核。1500ng/mlDON作用成骨细胞48h后，细胞数目明显减少，细胞大部分表现为晚期凋亡。 Western blot检测显示：500、1000、1500ng/mlDON作用组，Bcl-2蛋白表达减少，Bax蛋白表达增加，Bax/Bcl-2比值增大。以上试验表明DON具有明显的诱导成骨细胞凋亡的作用。 3. DON对PAX1基因表达的影响： RT-PCR凝胶电泳图显示：DON可明显增加成骨细胞中Pax1基因的mRNA表达水平。且随DON浓度增加，Pax1基因表达量逐渐升高，Pax1条带的亮度逐渐增强。1500ng/ml组与1000ng/ml组相比，Pax1基因表达量稍下降。DON500ng/ml、1000ng/ml、1500ng/ml组与空白对照组相比，差异有统计学意义（P0.05）。实时荧光定量PCR结果显示：DON可明显增加成骨细胞中Pax1基因的相对表达量。且随DON浓度增加，Pax1基因表达量逐渐升高。500ng/ml、1000ng/ml、1500ng/ml组与空白对照组相比，差异有统计学意义（P0.01）。 Western blot检测显示：DON可明显增加成骨细胞中Pax1基因的蛋白表达水平。且随DON浓度增加，Pax1蛋白条带的灰度值逐渐增强。DON500ng/ml、1000ng/ml、1500ng/ml组与空白对照组相比，差异有统计学意义（P0.05）。结论 1.DON对成骨细胞有直接毒性作用，能抑制成骨细胞的增殖，诱导成骨细胞发生凋亡。 2. DON作用成骨细胞48h后，Pax1基因表达量升高，。
[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.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【参考文献】