HBV宫内传播机制的蛋白质组学和相关miRNA研究
摘要
我国是乙型肝炎病毒(hepatitis B virus,HBV)感染高流行区,HBV感染及其导致的肝硬化和肝癌等疾病严重影响人们的健康。HBV主要通过血液、母婴及性接触传播,其中母婴垂直传播主要发生在围产期,阻断母婴传播是目前控制HBV感染流行的关键,而HBV宫内跨胎盘传播机制是母婴传播阻断研究的重点和难点。
HBV宫内传播机制主要有胎盘渗漏学说、胎盘感染学说及外周血单核细胞感染学说等几种假说,但病毒感染宿主细胞是一个复杂的内化过程,多层次多个分子可能参与该过程,目前有关HBV跨胎盘宫内感染的机制研究尚不够深入也远未阐明,亦无有效阻断措施,深入探讨HBV宫内传播机制对于HBV宫内跨胎盘传播的有效阻断具有理论和实际意义。
蛋白质组学技术可用来比较疾病与正常蛋白质样品间的表达水平,是揭示疾病分子机制、发现诊断标志物以及寻找药物新靶标的有效平台,尚未见HBV宫内跨胎盘传播机制的蛋白质组学研究报道。此外,病毒感染宿主细胞后,可导致宿主细胞中的miRNA表达谱发生变化,HBV和宿主细胞之间通过miRNA进行的相互调节对于HBV宫内感染的发生发展可能具有一定作用,亦未见相关研究报道。因此,本论文利用蛋白质组学技术筛选HBV阳性产妇胎盘差异表达蛋白,并建立胎盘滋养层细胞体外HBV感染模型,探讨HBV感染后细胞中miRNA的表达变化,从蛋白质组和miRNA两个层次深入探讨HBV如何跨胎盘宫内传播,可望从新的角度对HBV宫内跨胎盘传播的机制予以揭示。
方法:
1.以HBV阳性产妇胎盘为研究对象,以正常产妇胎盘为对照,利用MALDI-TOF和ESI-MS/MS进行分析,建立正常胎盘蛋白双向电泳(2-DE)参考图谱;采用比较蛋白质组学方法,筛选HBV阳性与正常产妇胎盘组织的差异表达蛋白,分析评价部分差异蛋白在HBV跨胎盘传播中的功能及意义。
2.体外培养胎盘滋养层细胞(JEG-3),应用HBV阳性血清处理,采用ELISA法检测感染细胞培养上清中的HBsAg、免疫组织化学SP法检测细胞中HBsAg蛋白的表达、PCR法检测细胞中HBV DNA的表达、AnnexinⅤ/PI双标法检测细胞凋亡和坏死,透射电镜观察细胞超微结构的变化并寻找病毒颗粒。
3.应用HBV阳性血清处理JEG-3细胞,采用miRNA芯片进行处理前后细胞miRNA表达谱的比较,并对差异miRNA的靶基因进行预测和生物学功能分析。
结果:
1.建立了正常足月胎盘总蛋白2-DE参考图谱;在pH4-10的合成图谱上检测到405个蛋白质点,鉴定出157个蛋白点对应的104个蛋白,其中24个蛋白对应多个蛋白质点,包括细胞结构蛋白、细胞支架蛋白等;所鉴定蛋白质中有部分功能不明蛋白质存在。共筛选出HBV感染产妇胎盘蛋白中的56个差异表达蛋白点,鉴定出24个差异蛋白;差异蛋白中有14个表达上调,10个下调;差异蛋白质中包括了酶类、调节蛋白、转运蛋白、结构蛋白、支架蛋白、保护蛋白及功能未知蛋白等。
2.HBV阳性血清处理后,胎盘滋养层细胞JEG-3培养上清中HBsAg呈阳性;JEG-3细胞中HBsAg和HBV DNA呈阳性;HBV阳性血清处理后细胞坏死率显著增加,凋亡率无明显变化;HBV阳性血清处理后JEG-3细胞超微结构的改变表现为胞浆内出现较多空泡状结构,溶酶体明显增多,并可见病毒样颗粒。
3.与正常血清处理细胞相比,HBV阳性血清处理后JEG-3细胞中高表达的miRNA包括:has-miR-197、has-miR-32、has-miR-125a-3p、has-miR-20a、has-miR-210和has-miR-574-3p;未发现差异显著的低表达has-miRNA。
结论:
1.成功建立了正常足月胎盘总蛋白2-DE参考图谱;鉴定得到104个蛋白质,其中部分蛋白质可能存在较多翻译后修饰,其修饰的多样性反映出这些蛋白在胎盘发育中的重要性;发现功能不明蛋白在胎盘发育过程中的存在。筛选鉴定得到HBV感染产妇胎盘蛋白表达谱中的24个差异蛋白;其中包括多种类别,热休克蛋白、肌动蛋白、膜联蛋白等差异蛋白可能与HBV跨胎盘传播有关。
2.HBV阳性血清可导致体外培养胎盘滋养层细胞的HBV感染,建立与体内HBV感染胎盘滋养层细胞条件接近的细胞感染模型;HBV阳性血清可导致胎盘滋养层细胞出现溶酶体大量增多等形态结构改变,并可导致细胞坏死。
3.HBV阳性血清处理胎盘滋养层细胞后,细胞miRNA表达谱发生变化,差异表达的miRNA包括:has-miR-197、has-miR-32、has-miR-125a-3p、has-miR-20a、has-miR-210和has-miR-574-3p,表明miRNA可能在HBV感染中发挥调控作用。
Hepatitis B virus (HBV), widely spread in China, which causes acute or chronic HBV infection and leads to hepatoma cirrhosis and eventually hepatocellular carcinoma has been a main threaten to public health. HBV could spread by blood, mother-infant transmission or sexual contact. Mother-infant transmission of HBV occurred in the perinatal period, so the blockade of the mother-infant transmission of HBV is the key to control HBV spread. The exploration of the mechanisms of HBV intra-uterine transplacental transmission is the emphasis and difficulty in the prevention of mother-infant transmission of HBV.
HBV intra-uterine transmission could be caused by placenta leakage, placenta infection or peripheral blood mononuclear cell infection. However, the process of virus infects host cells is complicated, many molecules may play a role in this process. Till now, the underlying mechanisms of HBV intra-uterine transplacental transmission remain unclear and there are no effective measures to block the transmission. Therefore, it is requisite to explore the mechanisms of HBV intra-uterine transplacental transmission, which will conduce to the blockade of the HBV intra-uterine transmission.
Proteome methods, which may be used to detecet the protein expression patterns in the diseased samples compared to the normal samples, are effectively means to clarify the mechanisms of disease and find novel therapeutic targets or biomarkers to serve as diagnostic indicators of disease. Besides that, the infection of virus may lead to the changes of miRNA(microRNA) expression in the host cells, so the interaction of HBV with host cells regulated by miRNA may play a role in HBV infection. This study is to detect protein expression patterns in the placentas derived from HBV-positive pregnant women by proteome methods compared to that of normal pregnant women, and then to explore the changes of miRNA expressions in placental trophoblasts infected with HBV on the basis of the establishment of cell model infected by HBV. Therefore, the mechanisms of HBV intra-uterine transmission may be clarified from new points of view on both proteome and miRNA.
Methods
1.The placentas derived from HBV-positive pregnant women were used as experimental objects and the placentas derived from HBV-negative pregnant women were used as control. By MALDI-TOF and ESI–MS/MS, the 2-DE map of normal placentas was established. The comparative proteome methods were used to detect protein expression patterns in the HBV-positive and negative placentas, and then the functions and roles of the proteins different in expression were analyzed.
2.Placental trophoblast cells JEG-3 were treated with HBV positive or negative serum, HBsAg in the cell supernatant or in cells was determined by ELISA or immunohistochemistry SP method respectively, and HBV DNA in JEG-3 was detected by PCR. Besides that, apoptosis and necrosis of JEG-3 was determined by AnnexinⅤ/PI staining and then FACS detection. Transmission Electro Microscope was used to observe cell ultra structure and find virus granule.
3.JEG-3 were treated with HBV positive or normal serum, miRNA chip was used to detect the differences in miRNA expression between the HBV positive serum treated cells and normal serum treated cells. Then the target genes of the miRNA different in expression were predicted and then their biological functions were analyzed.
Results
1.The 2-DE map of normal mature placenta was established. 405 protein spots were detected in the pH4-10 compositive map, 104 proteins correspond to 157 protein spots were identified, 24 proteins of which were found to correspond to multi-protein spots in the map, including structural proteins and scaffolding protein, et al. The functions of some identified proteins were unknown. 56 proteins displayed significant differences in expression in the placenta derived from HBV-positive placenta compared to the control placenta, and then 24 proteins were identified, 14 of these proteins were up-regulated while 10 proteins displayed reduced expression. These proteins different in expression include enzymes, regulated proteins, transport proteins, structural proteins, scaffolding protein, protective proteins and some proteins with unknown functions.
2.HBsAg in cell supernatant of placental trophoblast cells JEG-3 treated with HBV-positive serum was positive. HBsAg and HBV DNA in JEG-3 cells were also positive. Besides that, the necrosis rate of JEG-3 cells treated with HBV-positive serum significantly increased compared to control cells, but the apoptosis rate was not changed. The changes of ultra structure of JEG-3 cells treated with HBV-positive serum was as follows: many vacuoles appeared in cytoplasma and lysosome increased significantly, besides that, virus-like granules were found in cytoplasma.
3.Compared to cells treated with normal serum, some miRNA in JEG-3 cells treated with HBV-positive serum were up-regulated, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, there was no significantly down-regulated miRNA was detected.
Conclusions
1.The 2-DE map of normal mature placenta was established and 104 proteins were identified. Some proteins may have post-translational modifications, suggesting the important role of these proteins in the development of placenta. 24 proteins different in expression in placenta derived from HBV–positive pregnant women compare to control placenta were identified. HSPs, actin and Annecxin may play a role in the HBV intra-uterine transmission
2.Placental trophoblast cells treated with HBV-positive serum could be infected by HBV, so the HBV infection cell model was established with the method which was most consistent to the conditions of HBV infection in vivo. HBV-positive serum may cause significant increase of lysosome and cell necrosis.
3.miRNA expressed in JEG-3 cells treated with HBV-positive serum changed compared to control cells. Some up-regulated miRNA were detected, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, suggesting miRNA may play a role in the regulation of HBV infection.
HBV宫内传播机制主要有胎盘渗漏学说、胎盘感染学说及外周血单核细胞感染学说等几种假说,但病毒感染宿主细胞是一个复杂的内化过程,多层次多个分子可能参与该过程,目前有关HBV跨胎盘宫内感染的机制研究尚不够深入也远未阐明,亦无有效阻断措施,深入探讨HBV宫内传播机制对于HBV宫内跨胎盘传播的有效阻断具有理论和实际意义。
蛋白质组学技术可用来比较疾病与正常蛋白质样品间的表达水平,是揭示疾病分子机制、发现诊断标志物以及寻找药物新靶标的有效平台,尚未见HBV宫内跨胎盘传播机制的蛋白质组学研究报道。此外,病毒感染宿主细胞后,可导致宿主细胞中的miRNA表达谱发生变化,HBV和宿主细胞之间通过miRNA进行的相互调节对于HBV宫内感染的发生发展可能具有一定作用,亦未见相关研究报道。因此,本论文利用蛋白质组学技术筛选HBV阳性产妇胎盘差异表达蛋白,并建立胎盘滋养层细胞体外HBV感染模型,探讨HBV感染后细胞中miRNA的表达变化,从蛋白质组和miRNA两个层次深入探讨HBV如何跨胎盘宫内传播,可望从新的角度对HBV宫内跨胎盘传播的机制予以揭示。
方法:
1.以HBV阳性产妇胎盘为研究对象,以正常产妇胎盘为对照,利用MALDI-TOF和ESI-MS/MS进行分析,建立正常胎盘蛋白双向电泳(2-DE)参考图谱;采用比较蛋白质组学方法,筛选HBV阳性与正常产妇胎盘组织的差异表达蛋白,分析评价部分差异蛋白在HBV跨胎盘传播中的功能及意义。
2.体外培养胎盘滋养层细胞(JEG-3),应用HBV阳性血清处理,采用ELISA法检测感染细胞培养上清中的HBsAg、免疫组织化学SP法检测细胞中HBsAg蛋白的表达、PCR法检测细胞中HBV DNA的表达、AnnexinⅤ/PI双标法检测细胞凋亡和坏死,透射电镜观察细胞超微结构的变化并寻找病毒颗粒。
3.应用HBV阳性血清处理JEG-3细胞,采用miRNA芯片进行处理前后细胞miRNA表达谱的比较,并对差异miRNA的靶基因进行预测和生物学功能分析。
结果:
1.建立了正常足月胎盘总蛋白2-DE参考图谱;在pH4-10的合成图谱上检测到405个蛋白质点,鉴定出157个蛋白点对应的104个蛋白,其中24个蛋白对应多个蛋白质点,包括细胞结构蛋白、细胞支架蛋白等;所鉴定蛋白质中有部分功能不明蛋白质存在。共筛选出HBV感染产妇胎盘蛋白中的56个差异表达蛋白点,鉴定出24个差异蛋白;差异蛋白中有14个表达上调,10个下调;差异蛋白质中包括了酶类、调节蛋白、转运蛋白、结构蛋白、支架蛋白、保护蛋白及功能未知蛋白等。
2.HBV阳性血清处理后,胎盘滋养层细胞JEG-3培养上清中HBsAg呈阳性;JEG-3细胞中HBsAg和HBV DNA呈阳性;HBV阳性血清处理后细胞坏死率显著增加,凋亡率无明显变化;HBV阳性血清处理后JEG-3细胞超微结构的改变表现为胞浆内出现较多空泡状结构,溶酶体明显增多,并可见病毒样颗粒。
3.与正常血清处理细胞相比,HBV阳性血清处理后JEG-3细胞中高表达的miRNA包括:has-miR-197、has-miR-32、has-miR-125a-3p、has-miR-20a、has-miR-210和has-miR-574-3p;未发现差异显著的低表达has-miRNA。
结论:
1.成功建立了正常足月胎盘总蛋白2-DE参考图谱;鉴定得到104个蛋白质,其中部分蛋白质可能存在较多翻译后修饰,其修饰的多样性反映出这些蛋白在胎盘发育中的重要性;发现功能不明蛋白在胎盘发育过程中的存在。筛选鉴定得到HBV感染产妇胎盘蛋白表达谱中的24个差异蛋白;其中包括多种类别,热休克蛋白、肌动蛋白、膜联蛋白等差异蛋白可能与HBV跨胎盘传播有关。
2.HBV阳性血清可导致体外培养胎盘滋养层细胞的HBV感染,建立与体内HBV感染胎盘滋养层细胞条件接近的细胞感染模型;HBV阳性血清可导致胎盘滋养层细胞出现溶酶体大量增多等形态结构改变,并可导致细胞坏死。
3.HBV阳性血清处理胎盘滋养层细胞后,细胞miRNA表达谱发生变化,差异表达的miRNA包括:has-miR-197、has-miR-32、has-miR-125a-3p、has-miR-20a、has-miR-210和has-miR-574-3p,表明miRNA可能在HBV感染中发挥调控作用。
Hepatitis B virus (HBV), widely spread in China, which causes acute or chronic HBV infection and leads to hepatoma cirrhosis and eventually hepatocellular carcinoma has been a main threaten to public health. HBV could spread by blood, mother-infant transmission or sexual contact. Mother-infant transmission of HBV occurred in the perinatal period, so the blockade of the mother-infant transmission of HBV is the key to control HBV spread. The exploration of the mechanisms of HBV intra-uterine transplacental transmission is the emphasis and difficulty in the prevention of mother-infant transmission of HBV.
HBV intra-uterine transmission could be caused by placenta leakage, placenta infection or peripheral blood mononuclear cell infection. However, the process of virus infects host cells is complicated, many molecules may play a role in this process. Till now, the underlying mechanisms of HBV intra-uterine transplacental transmission remain unclear and there are no effective measures to block the transmission. Therefore, it is requisite to explore the mechanisms of HBV intra-uterine transplacental transmission, which will conduce to the blockade of the HBV intra-uterine transmission.
Proteome methods, which may be used to detecet the protein expression patterns in the diseased samples compared to the normal samples, are effectively means to clarify the mechanisms of disease and find novel therapeutic targets or biomarkers to serve as diagnostic indicators of disease. Besides that, the infection of virus may lead to the changes of miRNA(microRNA) expression in the host cells, so the interaction of HBV with host cells regulated by miRNA may play a role in HBV infection. This study is to detect protein expression patterns in the placentas derived from HBV-positive pregnant women by proteome methods compared to that of normal pregnant women, and then to explore the changes of miRNA expressions in placental trophoblasts infected with HBV on the basis of the establishment of cell model infected by HBV. Therefore, the mechanisms of HBV intra-uterine transmission may be clarified from new points of view on both proteome and miRNA.
Methods
1.The placentas derived from HBV-positive pregnant women were used as experimental objects and the placentas derived from HBV-negative pregnant women were used as control. By MALDI-TOF and ESI–MS/MS, the 2-DE map of normal placentas was established. The comparative proteome methods were used to detect protein expression patterns in the HBV-positive and negative placentas, and then the functions and roles of the proteins different in expression were analyzed.
2.Placental trophoblast cells JEG-3 were treated with HBV positive or negative serum, HBsAg in the cell supernatant or in cells was determined by ELISA or immunohistochemistry SP method respectively, and HBV DNA in JEG-3 was detected by PCR. Besides that, apoptosis and necrosis of JEG-3 was determined by AnnexinⅤ/PI staining and then FACS detection. Transmission Electro Microscope was used to observe cell ultra structure and find virus granule.
3.JEG-3 were treated with HBV positive or normal serum, miRNA chip was used to detect the differences in miRNA expression between the HBV positive serum treated cells and normal serum treated cells. Then the target genes of the miRNA different in expression were predicted and then their biological functions were analyzed.
Results
1.The 2-DE map of normal mature placenta was established. 405 protein spots were detected in the pH4-10 compositive map, 104 proteins correspond to 157 protein spots were identified, 24 proteins of which were found to correspond to multi-protein spots in the map, including structural proteins and scaffolding protein, et al. The functions of some identified proteins were unknown. 56 proteins displayed significant differences in expression in the placenta derived from HBV-positive placenta compared to the control placenta, and then 24 proteins were identified, 14 of these proteins were up-regulated while 10 proteins displayed reduced expression. These proteins different in expression include enzymes, regulated proteins, transport proteins, structural proteins, scaffolding protein, protective proteins and some proteins with unknown functions.
2.HBsAg in cell supernatant of placental trophoblast cells JEG-3 treated with HBV-positive serum was positive. HBsAg and HBV DNA in JEG-3 cells were also positive. Besides that, the necrosis rate of JEG-3 cells treated with HBV-positive serum significantly increased compared to control cells, but the apoptosis rate was not changed. The changes of ultra structure of JEG-3 cells treated with HBV-positive serum was as follows: many vacuoles appeared in cytoplasma and lysosome increased significantly, besides that, virus-like granules were found in cytoplasma.
3.Compared to cells treated with normal serum, some miRNA in JEG-3 cells treated with HBV-positive serum were up-regulated, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, there was no significantly down-regulated miRNA was detected.
Conclusions
1.The 2-DE map of normal mature placenta was established and 104 proteins were identified. Some proteins may have post-translational modifications, suggesting the important role of these proteins in the development of placenta. 24 proteins different in expression in placenta derived from HBV–positive pregnant women compare to control placenta were identified. HSPs, actin and Annecxin may play a role in the HBV intra-uterine transmission
2.Placental trophoblast cells treated with HBV-positive serum could be infected by HBV, so the HBV infection cell model was established with the method which was most consistent to the conditions of HBV infection in vivo. HBV-positive serum may cause significant increase of lysosome and cell necrosis.
3.miRNA expressed in JEG-3 cells treated with HBV-positive serum changed compared to control cells. Some up-regulated miRNA were detected, including has-miR-197, has-miR-32, has-miR-125a-3p, has-miR-20a, has-miR-210 and has-miR-574-3p, suggesting miRNA may play a role in the regulation of HBV infection.
引文
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