弓形蟲(chóng)TgCtwh3和RH株調(diào)控宿主巨噬細(xì)胞凋亡的研究
發(fā)布時(shí)間：2019-07-05 05:46
【摘要】：背景弓形蟲(chóng)是一種專(zhuān)性寄生于有核細(xì)胞內(nèi)的頂復(fù)門(mén)原蟲(chóng)，具有機(jī)會(huì)致病性。貓科動(dòng)物為其終宿主，中間宿主為包括人在內(nèi)的幾乎所有溫血?jiǎng)游�。已知北美和歐洲的弓形蟲(chóng)基因型主要為I、II和III型；我國(guó)的優(yōu)勢(shì)基因型為Chinese1型。大量研究證實(shí)，弓形蟲(chóng)通過(guò)抑制宿主細(xì)胞的凋亡在宿主體內(nèi)建立廣泛而有效的感染，維持自身的生存與繁殖。目前，，針對(duì)弓形蟲(chóng)調(diào)控宿主細(xì)胞凋亡機(jī)制的研究主要集中于死亡受體和線粒體兩個(gè)經(jīng)典凋亡途徑，而關(guān)于內(nèi)質(zhì)網(wǎng)應(yīng)激途徑的相關(guān)研究較少。此外，研究的蟲(chóng)株多為I型的RH強(qiáng)毒株，而針對(duì)我國(guó)流行的優(yōu)勢(shì)基因型Chinese1型（TgCtwh3，強(qiáng)毒株）調(diào)控宿主細(xì)胞凋亡的研究較為罕見(jiàn)。 目的觀察感染弓形蟲(chóng)兩種分離株（TgCtwh3和RH）的巨噬細(xì)胞（Raw264.7）凋亡，觀察各種凋亡相關(guān)因子的轉(zhuǎn)錄表達(dá)，旨在探討我國(guó)流行的弓形蟲(chóng)TgCtwh3株與I型的RH株調(diào)控宿主細(xì)胞凋亡機(jī)制的差異，以期為我國(guó)弓形蟲(chóng)病的防治提供理論與實(shí)驗(yàn)依據(jù)。 方法1）將研究對(duì)象分為3組：正常對(duì)照組、TgCtwh3感染組和RH感染組，分別給予凋亡誘導(dǎo)劑星狀孢子素（staurosporine）處理，通過(guò)流式細(xì)胞術(shù)檢測(cè)3組巨噬細(xì)胞的凋亡；2）應(yīng)用qRT-PCR檢測(cè)3組細(xì)胞的促凋亡家族成員（Bax，Bad）、抗凋亡家族成員（Bcl-2，Bcl-xl），內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志物CHOP的轉(zhuǎn)錄水平；3）應(yīng)用Western blotting檢測(cè)3組巨噬細(xì)胞的促凋亡蛋白（Bax，Bad）、抗凋亡蛋白（Bcl-2，Bcl-xl）、內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志物CHOP以及凋亡關(guān)鍵酶Caspase9的蛋白表達(dá)水平。結(jié)果1）感染組巨噬細(xì)胞早期凋亡率較正常對(duì)照均明顯下降。2）qRT-PCR結(jié)果顯示，感染組Bcl-2，Bcl-xl及CHOP的轉(zhuǎn)錄水平均明顯升高，Bax和Bad的轉(zhuǎn)錄水平無(wú)明顯變化。此外，TgCtwh3組的Bcl-xl轉(zhuǎn)錄水平明顯高于RH組，而兩組的Bcl-2轉(zhuǎn)錄水平無(wú)明顯差異。3）Western Blot結(jié)果顯示，感染組巨噬細(xì)胞的Bcl-2，Bcl-xl及CHOP蛋白表達(dá)水平均顯著升高，Bax和Bad的蛋白表達(dá)水平無(wú)明顯變化。其中，TgCtwh3組的Bcl-xl蛋白表達(dá)水平明顯高于RH感染組，而兩組的Bcl-2的蛋白表達(dá)水平無(wú)明顯差異。 結(jié)論在弓形蟲(chóng)感染早期階段，TgCtwh3和RH分離株通過(guò)上調(diào)Bcl-2，Bcl-xl的表達(dá)均顯著抑制了宿主細(xì)胞凋亡；TgCtwh3株抑制凋亡的作用較RH強(qiáng)，雖同為強(qiáng)毒株，TgCtwh3株毒力稍弱。 促凋亡相關(guān)基因除CHOP外，Bax、Bad、Caspase9均無(wú)明顯變化。感染早期抑制凋亡作用強(qiáng)于促凋亡作用。
[Abstract]:Background Toxoplasma gondii (Toxoplasma gondii) is a kind of parasite parasitic in nucleated cells, which has the opportunity to be pathogenic. Cats are their final hosts, and the intermediate hosts are almost all warm-blooded animals, including humans. It is known that the main genotypic of Toxoplasma gondii in North America and Europe is I, type II and type III, and the dominant genotype in China is Chinese1. A large number of studies have confirmed that Toxoplasma gondii can maintain its own survival and reproduction by inhibiting the apoptosis of host cells and establishing extensive and effective infection in host body. At present, the mechanism of Toxoplasma gondii regulating host cell apoptosis mainly focuses on two classical apoptotic pathways: death receptor and mitochondria, but there are few studies on endoplasmic reticulum stress pathway. In addition, most of the insect strains studied were type I RH virulent strains, but the dominant genotype Chinese1 (TgCtwh3, virulent strain) in China was rare to regulate host cell apoptosis. Objective to observe the apoptosis of macrophages (Raw264.7) infected with two isolates of Toxoplasma gondii (TgCtwh3 and RH) and to observe the transcriptional expression of various apoptosis related factors in order to explore the difference of apoptosis mechanism between TgCtwh3 strain and type I strain of Toxoplasma gondii in China, so as to provide theoretical and experimental basis for the prevention and treatment of Toxoplasma gondii in China. Methods 1) the subjects were divided into three groups: normal control group, TgCtwh3 infection group and RH infection group, treated with (staurosporine), an apoptosis inducer, respectively, and the apoptosis of macrophages in three groups was detected by flow cytometry. 2) the transcription levels of apoptosis family members (Bax,Bad), anti-apoptosis family members (Bcl-2,Bcl-xl) and endoplasmic reticulum stress marker CHOP in the three groups were detected by qRT-PCR. 3) the expression of apoptosis promoting protein (Bax,Bad), anti-apoptosis protein (Bcl-2,Bcl-xl), endoplasmic reticulum stress marker CHOP and apoptosis key enzyme Caspase9 in macrophages of three groups were detected by Western blotting. Results 1) the early apoptosis rate of macrophages in the infected group was significantly lower than that in the normal control group. 2) qRT-PCR results showed that the transcriptional levels of Bcl-2,Bcl-xl and CHOP in the infected group were significantly increased, but the transcriptional levels of Bax and Bad were not significantly changed. In addition, the transcription level of Bcl-xl in TgCtwh3 group was significantly higher than that in RH group, but there was no significant difference in Bcl-2 transcription level between the two groups. 3 the results of) Western Blot showed that the expression levels of Bcl-2,Bcl-xl and CHOP protein in macrophages of infected group were significantly increased, but the protein expression levels of Bax and Bad had no significant change. The expression level of Bcl-xl protein in TgCtwh3 group was significantly higher than that in RH infection group, but there was no significant difference in Bcl-2 protein expression level between the two groups. Conclusion in the early stage of Toxoplasma gondii infection, TgCtwh3 and RH isolates significantly inhibited the apoptosis of host cells by up-regulating the expression of Bcl-2,Bcl-xl, and the inhibitory effect of TgCtwh3 strain on apoptosis was stronger than that of RH strain, although it was both virulent, but the toxicity of TgCtwh3 strain was slightly weaker. There was no significant change in Bax,Bad,Caspase9 except CHOP. The effect of inhibiting apoptosis in the early stage of infection was stronger than that of promoting apoptosis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R531.8
[Abstract]:Background Toxoplasma gondii (Toxoplasma gondii) is a kind of parasite parasitic in nucleated cells, which has the opportunity to be pathogenic. Cats are their final hosts, and the intermediate hosts are almost all warm-blooded animals, including humans. It is known that the main genotypic of Toxoplasma gondii in North America and Europe is I, type II and type III, and the dominant genotype in China is Chinese1. A large number of studies have confirmed that Toxoplasma gondii can maintain its own survival and reproduction by inhibiting the apoptosis of host cells and establishing extensive and effective infection in host body. At present, the mechanism of Toxoplasma gondii regulating host cell apoptosis mainly focuses on two classical apoptotic pathways: death receptor and mitochondria, but there are few studies on endoplasmic reticulum stress pathway. In addition, most of the insect strains studied were type I RH virulent strains, but the dominant genotype Chinese1 (TgCtwh3, virulent strain) in China was rare to regulate host cell apoptosis. Objective to observe the apoptosis of macrophages (Raw264.7) infected with two isolates of Toxoplasma gondii (TgCtwh3 and RH) and to observe the transcriptional expression of various apoptosis related factors in order to explore the difference of apoptosis mechanism between TgCtwh3 strain and type I strain of Toxoplasma gondii in China, so as to provide theoretical and experimental basis for the prevention and treatment of Toxoplasma gondii in China. Methods 1) the subjects were divided into three groups: normal control group, TgCtwh3 infection group and RH infection group, treated with (staurosporine), an apoptosis inducer, respectively, and the apoptosis of macrophages in three groups was detected by flow cytometry. 2) the transcription levels of apoptosis family members (Bax,Bad), anti-apoptosis family members (Bcl-2,Bcl-xl) and endoplasmic reticulum stress marker CHOP in the three groups were detected by qRT-PCR. 3) the expression of apoptosis promoting protein (Bax,Bad), anti-apoptosis protein (Bcl-2,Bcl-xl), endoplasmic reticulum stress marker CHOP and apoptosis key enzyme Caspase9 in macrophages of three groups were detected by Western blotting. Results 1) the early apoptosis rate of macrophages in the infected group was significantly lower than that in the normal control group. 2) qRT-PCR results showed that the transcriptional levels of Bcl-2,Bcl-xl and CHOP in the infected group were significantly increased, but the transcriptional levels of Bax and Bad were not significantly changed. In addition, the transcription level of Bcl-xl in TgCtwh3 group was significantly higher than that in RH group, but there was no significant difference in Bcl-2 transcription level between the two groups. 3 the results of) Western Blot showed that the expression levels of Bcl-2,Bcl-xl and CHOP protein in macrophages of infected group were significantly increased, but the protein expression levels of Bax and Bad had no significant change. The expression level of Bcl-xl protein in TgCtwh3 group was significantly higher than that in RH infection group, but there was no significant difference in Bcl-2 protein expression level between the two groups. Conclusion in the early stage of Toxoplasma gondii infection, TgCtwh3 and RH isolates significantly inhibited the apoptosis of host cells by up-regulating the expression of Bcl-2,Bcl-xl, and the inhibitory effect of TgCtwh3 strain on apoptosis was stronger than that of RH strain, although it was both virulent, but the toxicity of TgCtwh3 strain was slightly weaker. There was no significant change in Bax,Bad,Caspase9 except CHOP. The effect of inhibiting apoptosis in the early stage of infection was stronger than that of promoting apoptosis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R531.8
【共引文獻(xiàn)】
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