啟動(dòng)子區(qū)域甲基化狀態(tài)與炎癥細(xì)胞TNF-α分泌關(guān)系及調(diào)控因素的研究
發(fā)布時(shí)間：2018-07-18 14:07
【摘要】：研究目的： 測(cè)定人單核細(xì)胞系THP-1細(xì)胞在炎癥刺激時(shí)腫瘤壞死因子α(TNF-a)表達(dá)分泌的規(guī)律,確定其分泌峰值時(shí)間；研究TNF-α基因啟動(dòng)子區(qū)域散在CpG雙核苷酸結(jié)構(gòu)甲基化狀態(tài)與蛋白分泌和基因表達(dá)的關(guān)系；探討TNF-α基因啟動(dòng)子區(qū)域散在CpG雙核苷酸結(jié)構(gòu)甲基化狀態(tài)影響其基因表達(dá)的調(diào)控因素。 研究方法： 采用酶聯(lián)免疫分析(ELISA)法檢測(cè)人單核細(xì)胞系THP-1細(xì)胞在未受到炎癥因子刺激時(shí),其TNF-α表達(dá)分泌水平；使用終濃度為1μg/ml的脂多糖(LPS)刺激人單核細(xì)胞系THP-1細(xì)胞,采用ELISA法檢測(cè)刺激不同時(shí)間時(shí)細(xì)胞培養(yǎng)液上清TNF-α分泌水平。對(duì)不同刺激時(shí)間分泌量與未刺激時(shí)分泌量進(jìn)行比較,確定THP-1細(xì)胞在受到終濃度為1μg/ml的脂多糖刺激后,TNF-α分泌高峰時(shí)間和分泌下降時(shí)間。獨(dú)立進(jìn)行4次上述測(cè)定,結(jié)果以刺激不同時(shí)間的分泌量與未刺激時(shí)分泌量比值的平均數(shù)±標(biāo)準(zhǔn)誤表示。對(duì)刺激后分泌高峰時(shí)間和分泌下降時(shí)間其分泌水平進(jìn)行統(tǒng)計(jì)學(xué)分析,P0.05為差異具有統(tǒng)計(jì)學(xué)意義。同時(shí)采用實(shí)時(shí)定量PCR方法測(cè)定THP-1細(xì)胞TNF-α基因表達(dá)水平。采用重亞硫酸鹽轉(zhuǎn)化基因測(cè)序法測(cè)定THP-1細(xì)胞在未受到炎癥刺激時(shí)TNF-α基因啟動(dòng)子區(qū)域的甲基化狀態(tài)。使用終濃度為1μg/ml LPS刺激THP-1細(xì)胞,采用重亞硫酸鹽轉(zhuǎn)化基因測(cè)序法測(cè)定THP-1細(xì)胞在LPS刺激分泌高峰時(shí)TNF-α基因啟動(dòng)子區(qū)域的甲基化狀態(tài),以及受刺激后分泌下降時(shí)TNF-α基因啟動(dòng)子區(qū)域的甲基化狀態(tài)。每個(gè)時(shí)間點(diǎn)樣本選取5個(gè)克隆進(jìn)行測(cè)序,結(jié)果以所測(cè)得CpG雙核苷酸結(jié)構(gòu)甲基化數(shù)占擴(kuò)增區(qū)域總CpG雙核苷酸結(jié)構(gòu)數(shù)的百分率表示。采用卡方檢驗(yàn)對(duì)測(cè)序結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析,P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。將未刺激時(shí)、刺激分泌高峰時(shí)期、刺激分泌下降時(shí)期TNF-α表達(dá)分泌量與對(duì)應(yīng)時(shí)間點(diǎn)其基因啟動(dòng)子區(qū)域的甲基化狀態(tài)進(jìn)行對(duì)比。使用實(shí)時(shí)定量PCR方法測(cè)定甲基化CpG結(jié)合蛋白表達(dá)量。并與相應(yīng)時(shí)間點(diǎn)TNF-α基因啟動(dòng)子區(qū)域甲基化狀態(tài)及TNF-α分泌的關(guān)系進(jìn)行研究。 實(shí)驗(yàn)結(jié)果： 受到終濃度為1μg/ml的LPS刺激后,THP-1細(xì)胞迅速開(kāi)始產(chǎn)生TNF-α,1 h過(guò)后升幅明顯增加,在4h達(dá)到分泌高峰水平,濃度為未刺激時(shí)的48.9±1.06倍。之后快速下降,至8h,已有明顯下降,濃度為未刺激時(shí)的36.3±1.65倍。至12h,濃度已降至峰值一半以下。刺激4h與未刺激時(shí)相比,TNF-α分泌量明顯升高(P＜0.01),其差別有統(tǒng)計(jì)學(xué)意義。刺激8h與刺激4h相比,TNF-α分泌量明顯下降(P0.01),其差別有統(tǒng)計(jì)學(xué)意義。TNF-α基因啟動(dòng)子區(qū)域無(wú)典型CpG島結(jié)構(gòu),其啟動(dòng)子-344bp到+57 bp區(qū)域內(nèi),存在12個(gè)散在CpG雙核昔酸結(jié)構(gòu),其中-344bp到轉(zhuǎn)錄起始位點(diǎn)(TSS)區(qū)域內(nèi),存在11個(gè)散在CpG雙核苷酸結(jié)構(gòu)。未受到LPS刺激時(shí),該11個(gè)散在CpG雙核苷酸均處于甲基化狀態(tài)；刺激4h時(shí),有4個(gè)處于甲基化狀態(tài)；刺激8h時(shí),有9個(gè)處于甲基化狀態(tài)。未刺激時(shí),5個(gè)克隆總體甲基化率為100%,刺激4h時(shí),5個(gè)克隆總體甲基化率為21.8%,刺激8h時(shí),5個(gè)克隆總體甲基化率為41.8%。刺激4h時(shí)與未刺激時(shí)相比,TNF-α基因啟動(dòng)子區(qū)域甲基化率明顯下降,差別具有統(tǒng)計(jì)學(xué)意義(P0.01)；刺激8h時(shí)與刺激4h時(shí)相比,TNF-α基因啟動(dòng)子區(qū)域甲基化率明顯上升,差別具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。各時(shí)間點(diǎn)甲基化率與相應(yīng)時(shí)間點(diǎn)TNF-α分泌量比值之間呈明顯負(fù)相關(guān)(r=-0.99,P0.01)。未受到LPS刺激時(shí),甲基化結(jié)合蛋白(MBD1、MBD2、MeCP2)表達(dá)量均較高；刺激4h后,MeCP2表達(dá)量明顯降低。MBD1、MBD2表達(dá)量較低；刺激8h后,MBD1、MBD2、MeCP2表達(dá)量又升高。 實(shí)驗(yàn)結(jié)論： 1. THP-1細(xì)胞TNF-α表達(dá)分泌量與LPS刺激明顯相關(guān)。未受到LPS刺激時(shí),TNF-α表達(dá)分泌量較低。受到LPS刺激后迅速開(kāi)始產(chǎn)生TNF-α,1h過(guò)后升幅明顯增加,在4h時(shí)達(dá)到分泌高峰水平,之后快速下降,至8h時(shí),已有明顯下降。TNF-α表達(dá)分泌呈現(xiàn)快速開(kāi)始,快速升高并快速下降的規(guī)律。 2. TNF-α基因啟動(dòng)子區(qū)域散在CpG雙核苷酸結(jié)構(gòu)甲基化狀態(tài)與THP-1細(xì)胞是否受到炎癥刺激,以及受刺激時(shí)間明顯相關(guān)。即未刺激時(shí),總體甲基化水平很高；刺激4h,總體甲基化水平較低；刺激8h,總體甲基化水平較高。 3. THP-1細(xì)胞TNF-α表達(dá)分泌量與TNF-α基因啟動(dòng)子區(qū)域散在CpG雙核苷酸結(jié)構(gòu)甲基化狀態(tài)呈明顯負(fù)相關(guān)性。 4. TNF-α基因啟動(dòng)子區(qū)域散在CpG雙核苷酸結(jié)構(gòu)甲基化狀態(tài)可能通過(guò)甲基化CpG結(jié)合蛋白影響其基因表達(dá)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 丘創(chuàng)華;侯敢;黃迪南;;TNF-α信號(hào)傳導(dǎo)通路的分子機(jī)理[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2007年06期
2 張方;李子玲;施毅;趙明;辛?xí)苑?錢桂生;;LPS致大鼠肺泡巨噬細(xì)胞NF-κB促進(jìn)TNF-α分泌[J];中國(guó)病理生理雜志;2007年07期
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