启动子区域甲基化状态与炎症细胞TNF-α分泌关系及调控因素的研究
发布时间：2018-07-18 14:07
【摘要】：研究目的： 测定人单核细胞系THP-1细胞在炎症刺激时肿瘤坏死因子α(TNF-a)表达分泌的规律,确定其分泌峰值时间；研究TNF-α基因启动子区域散在CpG双核苷酸结构甲基化状态与蛋白分泌和基因表达的关系；探讨TNF-α基因启动子区域散在CpG双核苷酸结构甲基化状态影响其基因表达的调控因素。 研究方法： 采用酶联免疫分析(ELISA)法检测人单核细胞系THP-1细胞在未受到炎症因子刺激时,其TNF-α表达分泌水平；使用终浓度为1μg/ml的脂多糖(LPS)刺激人单核细胞系THP-1细胞,采用ELISA法检测刺激不同时间时细胞培养液上清TNF-α分泌水平。对不同刺激时间分泌量与未刺激时分泌量进行比较,确定THP-1细胞在受到终浓度为1μg/ml的脂多糖刺激后,TNF-α分泌高峰时间和分泌下降时间。独立进行4次上述测定,结果以刺激不同时间的分泌量与未刺激时分泌量比值的平均数±标准误表示。对刺激后分泌高峰时间和分泌下降时间其分泌水平进行统计学分析,P0.05为差异具有统计学意义。同时采用实时定量PCR方法测定THP-1细胞TNF-α基因表达水平。采用重亚硫酸盐转化基因测序法测定THP-1细胞在未受到炎症刺激时TNF-α基因启动子区域的甲基化状态。使用终浓度为1μg/ml LPS刺激THP-1细胞,采用重亚硫酸盐转化基因测序法测定THP-1细胞在LPS刺激分泌高峰时TNF-α基因启动子区域的甲基化状态,以及受刺激后分泌下降时TNF-α基因启动子区域的甲基化状态。每个时间点样本选取5个克隆进行测序,结果以所测得CpG双核苷酸结构甲基化数占扩增区域总CpG双核苷酸结构数的百分率表示。采用卡方检验对测序结果进行统计学分析,P＜0.05为差异具有统计学意义。将未刺激时、刺激分泌高峰时期、刺激分泌下降时期TNF-α表达分泌量与对应时间点其基因启动子区域的甲基化状态进行对比。使用实时定量PCR方法测定甲基化CpG结合蛋白表达量。并与相应时间点TNF-α基因启动子区域甲基化状态及TNF-α分泌的关系进行研究。 实验结果： 受到终浓度为1μg/ml的LPS刺激后,THP-1细胞迅速开始产生TNF-α,1 h过后升幅明显增加,在4h达到分泌高峰水平,浓度为未刺激时的48.9±1.06倍。之后快速下降,至8h,已有明显下降,浓度为未刺激时的36.3±1.65倍。至12h,浓度已降至峰值一半以下。刺激4h与未刺激时相比,TNF-α分泌量明显升高(P＜0.01),其差别有统计学意义。刺激8h与刺激4h相比,TNF-α分泌量明显下降(P0.01),其差别有统计学意义。TNF-α基因启动子区域无典型CpG岛结构,其启动子-344bp到+57 bp区域内,存在12个散在CpG双核昔酸结构,其中-344bp到转录起始位点(TSS)区域内,存在11个散在CpG双核苷酸结构。未受到LPS刺激时,该11个散在CpG双核苷酸均处于甲基化状态；刺激4h时,有4个处于甲基化状态；刺激8h时,有9个处于甲基化状态。未刺激时,5个克隆总体甲基化率为100%,刺激4h时,5个克隆总体甲基化率为21.8%,刺激8h时,5个克隆总体甲基化率为41.8%。刺激4h时与未刺激时相比,TNF-α基因启动子区域甲基化率明显下降,差别具有统计学意义(P0.01)；刺激8h时与刺激4h时相比,TNF-α基因启动子区域甲基化率明显上升,差别具有统计学意义(P＜0.05)。各时间点甲基化率与相应时间点TNF-α分泌量比值之间呈明显负相关(r=-0.99,P0.01)。未受到LPS刺激时,甲基化结合蛋白(MBD1、MBD2、MeCP2)表达量均较高；刺激4h后,MeCP2表达量明显降低。MBD1、MBD2表达量较低；刺激8h后,MBD1、MBD2、MeCP2表达量又升高。 实验结论： 1. THP-1细胞TNF-α表达分泌量与LPS刺激明显相关。未受到LPS刺激时,TNF-α表达分泌量较低。受到LPS刺激后迅速开始产生TNF-α,1h过后升幅明显增加,在4h时达到分泌高峰水平,之后快速下降,至8h时,已有明显下降。TNF-α表达分泌呈现快速开始,快速升高并快速下降的规律。 2. TNF-α基因启动子区域散在CpG双核苷酸结构甲基化状态与THP-1细胞是否受到炎症刺激,以及受刺激时间明显相关。即未刺激时,总体甲基化水平很高；刺激4h,总体甲基化水平较低；刺激8h,总体甲基化水平较高。 3. THP-1细胞TNF-α表达分泌量与TNF-α基因启动子区域散在CpG双核苷酸结构甲基化状态呈明显负相关性。 4. TNF-α基因启动子区域散在CpG双核苷酸结构甲基化状态可能通过甲基化CpG结合蛋白影响其基因表达。
[Abstract]:The purpose of the study is:
The regulation of the expression and secretion of tumor necrosis factor alpha (TNF-a) in human mononuclear cell line THP-1 cells was determined and its peak time was determined. The relationship between the methylation of the TNF- alpha gene promoter region and the protein secretion and gene expression of the CpG binucleate acid structure was studied. The TNF- alpha gene promoter region was scattered in the CpG binuclear region. The methylation status of glucosides affects the regulation of gene expression.
Research methods:
Enzyme linked immunosorbent assay (ELISA) was used to detect the expression and secretion of TNF- alpha in human mononuclear cell line THP-1 cells when no inflammatory factors were stimulated; the THP-1 cells of human mononuclear cell line were stimulated by the lipopolysaccharide (LPS) with a final concentration of 1 g/ml. The secretion level of TNF- alpha in cell culture liquid supernatant was detected by ELISA method. The secretory quantity of the same stimulation time and the unstimulated secretion were compared, and the peak time and the decrease time of TNF- alpha secretion were determined after the THP-1 cells were stimulated by the lipopolysaccharide at the final concentration of 1 g/ml. The above results were performed independently for 4 times, and the result was an average error table of the average number of secretory quantities at different time and the ratio of unstimulated secretion. The secretion level of the secretory peak time and secretory time after stimulation was statistically analyzed. The difference of P0.05 was statistically significant. The TNF- alpha gene expression level in THP-1 cells was measured by real-time quantitative PCR method. The THP-1 cells were determined by heavy sulfite transformation gene sequencing method to determine the TNF- alpha base of THP-1 cells in the absence of inflammatory stimulation. The methylation status of the promoter region was stimulated with the final concentration of 1 g/ml LPS to stimulate THP-1 cells, and the methylation status of TNF- alpha promoter region at the peak of LPS stimulation was measured by heavy sulfite transformation gene sequencing, and the methylation status of the TNF- a gene promoter region when the secretion decreased after the stimulation was stimulated. 5 clones were sequenced at the time point sample. The results showed that the number of CpG binucleate acid structure methylation number accounted for the percentage of the total CpG binucleate acid structure in the amplified region. The results were statistically analyzed by chi square test, and P < 0.05 was statistically significant. The expression of TNF- alpha expression was compared with the methylation status of the gene promoter region at the corresponding time point. The expression of methylated CpG binding protein was measured by real-time quantitative PCR method. The relationship between methylation status and TNF- alpha secretion in the promoter region of TNF- a gene at corresponding time points was studied.
Experimental results:
After the final concentration of 1 u g/ml LPS stimulation, THP-1 cells quickly began to produce TNF- alpha, after 1 h, the increase was obviously increased, and reached the peak level in 4h, the concentration was 48.9 + 1.06 times that of unstimulated, and then decreased rapidly to 8h, and the concentration was 36.3 + 1.65 times when the concentration was not stimulated. To 12h, the concentration had dropped to below half the peak. Compared with unstimulated 4h, the secretion of TNF- alpha increased significantly (P < 0.01), and the difference was statistically significant. The secretion of TNF- alpha was significantly decreased (P0.01) compared with stimulation of 4H (P0.01). The difference was statistically significant in the.TNF- a gene promoter region with no typical CpG island structure, and there were 12 scattered in -344bp to +57 BP region. The acid structure, in which the -344bp to the transcriptional starting site (TSS) region, has 11 scattered in the CpG binucleate acid structure. When unstimulated by LPS, the 11 scattered CpG dinucleotides in the methylation state; when stimulating 4h, 4 are in the methylation state; at the time of stimulating 8h, there are 9 methylation states. 5 clones are generally methylation when unstimulated. The rate of 100%, when stimulating 4h, the total methylation rate of 5 clones was 21.8%. When the overall methylation rate of the 5 clones was 41.8%. stimulated 4h, the methylation rate of the TNF- alpha gene promoter region decreased significantly when compared with the unstimulated 4h, and the difference was statistically significant (P0.01). The promoter region methylation of the TNF- alpha promoter region was compared to the stimulation of 4H. The difference was statistically significant (P < 0.05). There was a significant negative correlation between the rate of methylation and the ratio of TNF- alpha secretion at the corresponding time points (r=-0.99, P0.01). The expression of methylated binding protein (MBD1, MBD2, MeCP2) was higher without LPS stimulation; the expression of MeCP2 was significantly lower than that of.MBD1, and the expression of MBD2 was higher than that of 4H. The expression level of MBD1, MBD2 and MeCP2 increased after stimulation of 8h.
Experimental conclusions:
The secretion of TNF- alpha expression in 1. THP-1 cells was significantly related to LPS stimulation. Without LPS stimulation, the expression of TNF- alpha was low. After LPS stimulation, TNF- alpha began to produce TNF- a, and the ascending amplitude of 1H increased obviously after 1h, and then reached the peak level at 4h, and then decreased rapidly. To 8h, the expression of.TNF- alpha expression was obviously reduced to a rapid start. The law of rapid rise and rapid decline.
The methylation of the 2. TNF- alpha gene promoter region dispersed in the CpG binucleate acid structure was significantly related to whether the THP-1 cells were stimulated by inflammation and the time to be stimulated. That is, the overall methylation level was high when the stimulus was not stimulated; the overall methylation level was low in stimulating 4h, and 8h was stimulated, and the overall methylation level was higher.
3. the expression of TNF- alpha in THP-1 cells was negatively correlated with the methylation status of CpG dinucleotide in TNF- promoter region.
4. methylation status of CpG binucleotide structure in the promoter region of TNF- alpha gene may affect its gene expression through methylation of CpG binding protein.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2132140
[Abstract]:The purpose of the study is:
The regulation of the expression and secretion of tumor necrosis factor alpha (TNF-a) in human mononuclear cell line THP-1 cells was determined and its peak time was determined. The relationship between the methylation of the TNF- alpha gene promoter region and the protein secretion and gene expression of the CpG binucleate acid structure was studied. The TNF- alpha gene promoter region was scattered in the CpG binuclear region. The methylation status of glucosides affects the regulation of gene expression.
Research methods:
Enzyme linked immunosorbent assay (ELISA) was used to detect the expression and secretion of TNF- alpha in human mononuclear cell line THP-1 cells when no inflammatory factors were stimulated; the THP-1 cells of human mononuclear cell line were stimulated by the lipopolysaccharide (LPS) with a final concentration of 1 g/ml. The secretion level of TNF- alpha in cell culture liquid supernatant was detected by ELISA method. The secretory quantity of the same stimulation time and the unstimulated secretion were compared, and the peak time and the decrease time of TNF- alpha secretion were determined after the THP-1 cells were stimulated by the lipopolysaccharide at the final concentration of 1 g/ml. The above results were performed independently for 4 times, and the result was an average error table of the average number of secretory quantities at different time and the ratio of unstimulated secretion. The secretion level of the secretory peak time and secretory time after stimulation was statistically analyzed. The difference of P0.05 was statistically significant. The TNF- alpha gene expression level in THP-1 cells was measured by real-time quantitative PCR method. The THP-1 cells were determined by heavy sulfite transformation gene sequencing method to determine the TNF- alpha base of THP-1 cells in the absence of inflammatory stimulation. The methylation status of the promoter region was stimulated with the final concentration of 1 g/ml LPS to stimulate THP-1 cells, and the methylation status of TNF- alpha promoter region at the peak of LPS stimulation was measured by heavy sulfite transformation gene sequencing, and the methylation status of the TNF- a gene promoter region when the secretion decreased after the stimulation was stimulated. 5 clones were sequenced at the time point sample. The results showed that the number of CpG binucleate acid structure methylation number accounted for the percentage of the total CpG binucleate acid structure in the amplified region. The results were statistically analyzed by chi square test, and P < 0.05 was statistically significant. The expression of TNF- alpha expression was compared with the methylation status of the gene promoter region at the corresponding time point. The expression of methylated CpG binding protein was measured by real-time quantitative PCR method. The relationship between methylation status and TNF- alpha secretion in the promoter region of TNF- a gene at corresponding time points was studied.
Experimental results:
After the final concentration of 1 u g/ml LPS stimulation, THP-1 cells quickly began to produce TNF- alpha, after 1 h, the increase was obviously increased, and reached the peak level in 4h, the concentration was 48.9 + 1.06 times that of unstimulated, and then decreased rapidly to 8h, and the concentration was 36.3 + 1.65 times when the concentration was not stimulated. To 12h, the concentration had dropped to below half the peak. Compared with unstimulated 4h, the secretion of TNF- alpha increased significantly (P < 0.01), and the difference was statistically significant. The secretion of TNF- alpha was significantly decreased (P0.01) compared with stimulation of 4H (P0.01). The difference was statistically significant in the.TNF- a gene promoter region with no typical CpG island structure, and there were 12 scattered in -344bp to +57 BP region. The acid structure, in which the -344bp to the transcriptional starting site (TSS) region, has 11 scattered in the CpG binucleate acid structure. When unstimulated by LPS, the 11 scattered CpG dinucleotides in the methylation state; when stimulating 4h, 4 are in the methylation state; at the time of stimulating 8h, there are 9 methylation states. 5 clones are generally methylation when unstimulated. The rate of 100%, when stimulating 4h, the total methylation rate of 5 clones was 21.8%. When the overall methylation rate of the 5 clones was 41.8%. stimulated 4h, the methylation rate of the TNF- alpha gene promoter region decreased significantly when compared with the unstimulated 4h, and the difference was statistically significant (P0.01). The promoter region methylation of the TNF- alpha promoter region was compared to the stimulation of 4H. The difference was statistically significant (P < 0.05). There was a significant negative correlation between the rate of methylation and the ratio of TNF- alpha secretion at the corresponding time points (r=-0.99, P0.01). The expression of methylated binding protein (MBD1, MBD2, MeCP2) was higher without LPS stimulation; the expression of MeCP2 was significantly lower than that of.MBD1, and the expression of MBD2 was higher than that of 4H. The expression level of MBD1, MBD2 and MeCP2 increased after stimulation of 8h.
Experimental conclusions:
The secretion of TNF- alpha expression in 1. THP-1 cells was significantly related to LPS stimulation. Without LPS stimulation, the expression of TNF- alpha was low. After LPS stimulation, TNF- alpha began to produce TNF- a, and the ascending amplitude of 1H increased obviously after 1h, and then reached the peak level at 4h, and then decreased rapidly. To 8h, the expression of.TNF- alpha expression was obviously reduced to a rapid start. The law of rapid rise and rapid decline.
The methylation of the 2. TNF- alpha gene promoter region dispersed in the CpG binucleate acid structure was significantly related to whether the THP-1 cells were stimulated by inflammation and the time to be stimulated. That is, the overall methylation level was high when the stimulus was not stimulated; the overall methylation level was low in stimulating 4h, and 8h was stimulated, and the overall methylation level was higher.
3. the expression of TNF- alpha in THP-1 cells was negatively correlated with the methylation status of CpG dinucleotide in TNF- promoter region.
4. methylation status of CpG binucleotide structure in the promoter region of TNF- alpha gene may affect its gene expression through methylation of CpG binding protein.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
相关期刊论文 前2条
1 丘创华;侯敢;黄迪南;;TNF-α信号传导通路的分子机理[J];中国生物化学与分子生物学报;2007年06期
2 张方;李子玲;施毅;赵明;辛晓峰;钱桂生;;LPS致大鼠肺泡巨噬细胞NF-κB促进TNF-α分泌[J];中国病理生理杂志;2007年07期
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