與ZNF446同源的LT-7在布魯菌侵染THP-1中對(duì)MAPK通路影響的研究
發(fā)布時(shí)間：2018-12-15 19:47
【摘要】：目的：布病（Brucellosis）是一種嚴(yán)重危害人和動(dòng)物健康的人獸共患傳染病。巨噬細(xì)胞是布魯菌生存和繁殖的靶細(xì)胞之一，布魯菌的胞內(nèi)生存與抑制宿主細(xì)胞凋亡有關(guān)，外膜蛋白OMP25(OuterMembrane Protein25)在布魯菌的毒力及胞內(nèi)寄生中起重要作用。前期，本課題組應(yīng)用噬菌體展示技術(shù)篩選出與布魯菌OMP25特異性結(jié)合的42個(gè)環(huán)形七肽，通過生物信息學(xué)分析，LT-7C與鋅指蛋白ZNF446（Zinc Finger Protein Peptide446）對(duì)應(yīng)區(qū)域有100%的同源性。本研究以布魯菌RB51(Brucellaabortus strain RB51)侵染THP-1(human leukemia cell line)細(xì)胞，研究LT-7C在不同階段對(duì)MAPK通路(MAPK signal pathway)的影響，進(jìn)而探討LT-7C對(duì)細(xì)胞凋亡的影響。 方法：本研究利用合成的環(huán)形七肽與THP-1共孵育，采用MTT法進(jìn)行多肽的毒性檢測(cè)，選擇合適的七肽濃度與THP-1共孵育，實(shí)驗(yàn)設(shè)未感染組和感染組，包括未感染對(duì)照組、未感染線性七肽組、未感染環(huán)形七肽組、感染對(duì)照組、感染線性七肽組和感染環(huán)形七肽組。以MOI(multiplicity of infection)為50:1建立RB51感染THP-1細(xì)胞的感染模型。分別收集4、8、12、24h和48h的感染細(xì)胞，利用流式細(xì)胞儀檢測(cè)不同時(shí)間段的早期凋亡率；感染4h和48h時(shí)，曲拉通裂解細(xì)胞，進(jìn)行細(xì)胞內(nèi)布魯菌計(jì)數(shù)；采用qRT-PCR技術(shù)和Western blot檢測(cè)4h和48h MAPK通路中ERK1/2、JNK1/2、P38的mRNA水平和蛋白的表達(dá)變化。 結(jié)果：(1)合成的環(huán)形七肽對(duì)THP-1無毒副作用；(2)流式細(xì)胞術(shù)檢測(cè)顯示：在未感染組中環(huán)形七肽組和線性七肽組與對(duì)照組比較早期凋亡率隨著時(shí)間的變化呈上升趨勢(shì)，統(tǒng)計(jì)學(xué)分析各組差異無統(tǒng)計(jì)學(xué)意義（P0.05）；在感染組中環(huán)形七肽組和線性七肽組與感染對(duì)照組比較12h前呈上升趨勢(shì)，12h后呈下降趨勢(shì)，其中4h時(shí)感染環(huán)形組高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；48h時(shí)感染環(huán)形組低于對(duì)照組，，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。(3)CFU計(jì)數(shù)結(jié)果顯示感染環(huán)形七肽組早期4h時(shí)胞內(nèi)細(xì)菌量低于感染對(duì)照組，并且差異有統(tǒng)計(jì)學(xué)意義(P0.05)；線性七肽組4h胞內(nèi)含菌量高于感染對(duì)照組，差異無統(tǒng)計(jì)學(xué)意義；48h時(shí)各組差異無統(tǒng)計(jì)學(xué)意義。(4) qRT-PCR檢測(cè)結(jié)果顯示4h時(shí)感染線性組LT-7組和感染環(huán)形LT-7C組erk1的拷貝數(shù)低于感染對(duì)照組，ekr2、p38、jnk1和jnk2拷貝數(shù)在各感染組無變化，未感染組4h時(shí)各基因的拷貝數(shù)無變化；48h時(shí)感染環(huán)形組中jnk1/2拷貝數(shù)低于感染對(duì)照組；erk1、ekr2、p38拷貝數(shù)在各感染組無變化，未感染組48h時(shí)各基因的拷貝數(shù)無變化；（5）Western blot結(jié)果與qRT-PCR一致。 結(jié)論：(1)以MOI為50:1的RB51感染的環(huán)形七肽組中，環(huán)形七肽4h時(shí)有抑制胞內(nèi)入侵作用，48h時(shí)其對(duì)胞內(nèi)入侵無作用。(2)環(huán)形七肽組4h時(shí)ERK1/2的低表達(dá)與凋亡率增高有關(guān)，48h時(shí)JNK1/2的低表達(dá)與凋亡率降低有關(guān)。
[Abstract]:Objective: (Brucellosis) is a zoonotic infectious disease which seriously endangers human and animal health. Macrophages are one of the target cells for brucellosis survival and reproduction. The intracellular survival of Brucella is related to the inhibition of host cell apoptosis. The outer membrane protein OMP25 (OuterMembrane Protein25) plays an important role in the virulence and intracellular parasitism of Brucella. In the early stage, the phage display technique was used to screen 42 cyclic heptapeptides specifically bound to Brucella OMP25. By bioinformatics analysis, 100% homology was found between LT-7C and zinc finger protein (ZNF446 (Zinc Finger Protein Peptide446). In this study, THP-1 (human leukemia cell line) cells were infected with brucellosis (RB51 (Brucellaabortus strain RB51) to study the effect of LT-7C on (MAPK signal pathway) of MAPK pathway at different stages, and then to explore the effect of LT-7C on apoptosis. Methods: in this study, the synthetic ring heptapeptide was co-incubated with THP-1, the toxicity of the peptide was detected by MTT method, and the appropriate concentration of heptapeptide was co-incubated with THP-1. The experiment was divided into two groups: uninfected group and infected group, including uninfected control group. No infection linear heptapeptide group, no infection ring heptapeptide group, infection control group, infection linear heptapeptide group and infection ring heptapeptide group. The infection model of THP-1 cells infected with RB51 was established by using MOI (multiplicity of infection) as 50:1. The rate of early apoptosis was detected by flow cytometry at 24 h and 48 h, respectively, and at 4 h and 48 h after infection, the cells were lysed by traitone, and the intracellular brucellosis was counted. QRT-PCR and Western blot were used to detect the expression of ERK1/2,JNK1/2,P38 mRNA and protein in MAPK pathway at 4h and 48h. Results: (1) the synthetic ring heptapeptide had no side effect on THP-1; (2) flow cytometry showed that in the uninfected group, the early apoptosis rate of the ring heptapeptide group and the linear heptapeptide group showed an increasing trend with the change of time compared with the control group, and there was no significant difference between the two groups (P0.05). In the infection group, the ring heptapeptide group and the linear heptapeptide group showed an upward trend before 12 h and a downward trend after 12 h, and the infection ring group was higher than the control group at 4 h, the difference was statistically significant (P0.05). 48 h infection ring group was lower than the control group, the difference was statistically significant (P0.05). (3) CFU count results showed that the number of intracellular bacteria in the infection ring heptapeptide group was lower than that in the infected control group at 4 h, and the difference was statistically significant (P0.05). The intracellular bacterial content in the linear heptapeptide group was higher than that in the infected control group at 4 h, and the difference was not statistically significant. (4) the results of qRT-PCR detection showed that the copy number of erk1 in LT-7 group and ring LT-7C group was lower than that in infected control group at 4 h, but the copy numbers of ekr2,p38,jnk1 and jnk2 were not changed in each infection group. The number of copies of each gene did not change at 4 hours in the uninfected group. The copy number of jnk1/2 in the infection ring group was lower than that in the infected control group at 48 h, but the copy number of erk1,ekr2,p38 did not change in each infection group, but the copy number of each gene in the non-infected group did not change at 48 h. (5) the result of) Western blot was consistent with that of qRT-PCR. Conclusion: (1) in the ring heptapeptide group infected with MOI 50:1 RB51, the circular heptapeptide can inhibit the invasion of the cells at 4 h. (2) the low expression of ERK1/2 was related to the increase of apoptosis rate at 4 h, and the low expression of JNK1/2 was related to the decrease of apoptosis rate at 48 h.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R516.7
[Abstract]:Objective: (Brucellosis) is a zoonotic infectious disease which seriously endangers human and animal health. Macrophages are one of the target cells for brucellosis survival and reproduction. The intracellular survival of Brucella is related to the inhibition of host cell apoptosis. The outer membrane protein OMP25 (OuterMembrane Protein25) plays an important role in the virulence and intracellular parasitism of Brucella. In the early stage, the phage display technique was used to screen 42 cyclic heptapeptides specifically bound to Brucella OMP25. By bioinformatics analysis, 100% homology was found between LT-7C and zinc finger protein (ZNF446 (Zinc Finger Protein Peptide446). In this study, THP-1 (human leukemia cell line) cells were infected with brucellosis (RB51 (Brucellaabortus strain RB51) to study the effect of LT-7C on (MAPK signal pathway) of MAPK pathway at different stages, and then to explore the effect of LT-7C on apoptosis. Methods: in this study, the synthetic ring heptapeptide was co-incubated with THP-1, the toxicity of the peptide was detected by MTT method, and the appropriate concentration of heptapeptide was co-incubated with THP-1. The experiment was divided into two groups: uninfected group and infected group, including uninfected control group. No infection linear heptapeptide group, no infection ring heptapeptide group, infection control group, infection linear heptapeptide group and infection ring heptapeptide group. The infection model of THP-1 cells infected with RB51 was established by using MOI (multiplicity of infection) as 50:1. The rate of early apoptosis was detected by flow cytometry at 24 h and 48 h, respectively, and at 4 h and 48 h after infection, the cells were lysed by traitone, and the intracellular brucellosis was counted. QRT-PCR and Western blot were used to detect the expression of ERK1/2,JNK1/2,P38 mRNA and protein in MAPK pathway at 4h and 48h. Results: (1) the synthetic ring heptapeptide had no side effect on THP-1; (2) flow cytometry showed that in the uninfected group, the early apoptosis rate of the ring heptapeptide group and the linear heptapeptide group showed an increasing trend with the change of time compared with the control group, and there was no significant difference between the two groups (P0.05). In the infection group, the ring heptapeptide group and the linear heptapeptide group showed an upward trend before 12 h and a downward trend after 12 h, and the infection ring group was higher than the control group at 4 h, the difference was statistically significant (P0.05). 48 h infection ring group was lower than the control group, the difference was statistically significant (P0.05). (3) CFU count results showed that the number of intracellular bacteria in the infection ring heptapeptide group was lower than that in the infected control group at 4 h, and the difference was statistically significant (P0.05). The intracellular bacterial content in the linear heptapeptide group was higher than that in the infected control group at 4 h, and the difference was not statistically significant. (4) the results of qRT-PCR detection showed that the copy number of erk1 in LT-7 group and ring LT-7C group was lower than that in infected control group at 4 h, but the copy numbers of ekr2,p38,jnk1 and jnk2 were not changed in each infection group. The number of copies of each gene did not change at 4 hours in the uninfected group. The copy number of jnk1/2 in the infection ring group was lower than that in the infected control group at 48 h, but the copy number of erk1,ekr2,p38 did not change in each infection group, but the copy number of each gene in the non-infected group did not change at 48 h. (5) the result of) Western blot was consistent with that of qRT-PCR. Conclusion: (1) in the ring heptapeptide group infected with MOI 50:1 RB51, the circular heptapeptide can inhibit the invasion of the cells at 4 h. (2) the low expression of ERK1/2 was related to the increase of apoptosis rate at 4 h, and the low expression of JNK1/2 was related to the decrease of apoptosis rate at 48 h.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R516.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 楊建偉;曹維偉;;奶牛布魯氏菌病的檢測(cè)技術(shù)研究進(jìn)展[J];中國(guó)牛業(yè)科學(xué);2010年05期
2 郝宗宇;鄧文斌;何俊滌;沈元珊;;布氏菌外膜蛋白的研究進(jìn)展[J];地方病譯叢;1992年01期
3 尚德秋;布魯氏菌病再度肆虐及其原因[J];中國(guó)地方病防治雜志;2001年01期
4 黃辰;劉利英;宋土生;倪磊;宋麗萍;司履生;;siRNA介導(dǎo)的MA PK p42沉默誘導(dǎo)HeLa細(xì)胞凋亡[J];南方醫(yī)科大學(xué)學(xué)報(bào);2006年01期
5 殷文武;孫輝;;中國(guó)布魯氏菌病疫情形勢(shì)及對(duì)策建議[J];疾病監(jiān)測(cè);2009年07期
6 張亞麗;陳創(chuàng)夫;黃小強(qiáng);張科;張t
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