乙肝胎盘P-gp/MDR1和ABCG2/BCRP的表达
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摘要
P-gp/MDR1和ABCG2/BCRP是ABC膜转运蛋白家族成员,表达于足月胎盘合体滋养层的母体面胞膜,,可以通过主动转运的方式,特异性地清除一些内源性的毒物或异生物,从而影响抗病毒药物等底物在胎盘滋养细胞的转运。因此,通过调控P-gp/MDR1和ABCG2/BCRP的表达,可提高抗病毒药物进入滋养细胞和胎儿血循环的浓度,更有效地阻断乙型肝炎的宫内感染。目的:研究妊娠合并乙型肝炎胎盘P-gp/MDR1和ABCG2/BCRP的表达,及其与乙肝感染的相关性。
方法:
1.采用免疫组化技术,检测胎盘组织中P-gp/MDR1和ABCG2/BCRP的表达和定位。
2.采用荧光定量PCR技术检测人胎盘组织中P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达。比较乙肝感染胎盘与正常孕妇胎盘中P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达的差异。
3.采用Westernblot技术检测人胎盘组织中P-gp/MDR1和ABCG2/BCRP蛋白质水平的表达。比较乙肝感染胎盘与正常孕妇胎盘中P-gp/MDR1和ABCG2/BCRP蛋白表达的差异。
4.用ELISA检测胎盘组织中IL-1β、IL-6和TNF-α的含量。分析人胎盘组织中P-gp/MDR1和ABCG2/BCRP的表达与IL-1β、IL-6和TNF-α的相关性。
5.体外培养人胎盘滋养细胞BeWo和JEG-3,分别加IL-1β、IL-6干预后,用荧光定量PCR检测细胞P-gp/MDR1和ABCG2/BCRP表达的变化。进一步探讨人胎盘滋养细胞P-gp/MDR1、ABCG2/BCRP的表达与IL-1β和IL-6的相关性。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预后,用HPLC检测3TC在滋养细胞的滤过率的变化。
结果:
1.免疫组化结果显示,乙肝感染胎盘与正常孕妇胎盘中滋养细胞母体面胞膜均有P-gp/MDR1和ABCG2/BCRP表达。P-gp/MDR1和ABCG2/BCRP在足月妊娠胎盘组织中主要表达于胎盘合体滋养细胞母体面胞膜。与以往报道相似,部分合体滋养层细胞高表达,也有部分合体滋养层细胞不表达。
2.荧光定量PCR显示,乙肝感染胎盘P-gp/MDR1 mRNA和ABCG2/BCRPmRNA的表达均高于正常组,差别有显著性。
3.Westernblot显示,乙肝感染胎盘P-gp/MDR1和ABCG2/BCRP蛋白的表达均高于正常组,差别有显著性。HBV感染胎盘组织P-gp/MDR1和ABCG2/BCRP蛋白质水平表达分别是正常足月胎盘表达量的2.43和3.76倍。与相对应的Real Time PCR结果的相关系数为0.980、0.963。
4.ELISA检测胎盘组织中IL-1β、IL-6和TNF-α的含量,乙肝组均高于正常组,乙肝组胎盘IL-1β、IL-6和TNF-α表达量分别是正常组的4.36、1.91、5.12倍。IL-1β、IL-6和TNF-α与P-gp/MDR1和ABCG2/BCRP的表达均有相关性。
5.体外培养人胎盘滋养细胞BeWo和JEG3,分别加IL-1β、IL-6干预12、24、48、72小时后,用荧光定量PCR检测细胞P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达均升高,差别有显著性。P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达随着IL-1β和IL-6作用时间的延长逐渐升高。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预48、72小时后,3TC在细胞的滤过率显著降低。
结论:
1.乙肝感染胎盘与正常孕妇胎盘中滋养细胞膜均有P-gp/MDR1和ABCG2/BCRP表达。
2.乙肝感染胎盘P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达均高于正常组。
3.乙肝感染胎盘P-gp/MDR1和ABCG2/BCRP蛋白的表达均高于正常组。P-gp/MDR1和ABCG2/BCRP蛋白表达升高可能与P-gp/MDR1 mRNA和ABCG2/BCRP mRNA转录增强有关。
4.胎盘组织中IL-1β、IL-6和TNF-α的含量,乙肝组均高于正常组,IL-1β、IL-6和TNF-α与P-gp/MDR1、ABCG2/BCRP的表达均有相关性。提示IL-1β、IL-6和TNF-α可能诱导P-gp/MDR1 mRNA和ABCG2/BCRP mRNA转录增强以及P-gp/MDR1和ABCG2/BCRP蛋白表达。
5.体外培养人胎盘滋养细胞BeWo和JEG3,分别加IL-1β、IL-6干预12、24、48、72小时后,P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达均升高。P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达随着IL-1β和IL-6作用时间的延长逐渐升高。提示IL-1β和IL-6可能在转录水平上调P-gp/MDR1mRNA和ABCG2/BCRP mRNA的表达。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预后,3TC在细胞的滤过率显著降低。可能与IL-1β、IL-6诱导的P-gp/MDR1和ABCG2/BCRP表达上调有关。
Background
P-gp/MDR1 and ABCG2/BCRP are members of the group of transporters in the ATP binding cassette(ABC) superfamily,which are localized in the syncytiotrophoblast of the human placenta.These proteins actively extrudes a wide variety of drugs and toxins from cells,including antiretroviral HBV drugs,such as zidovudine and lamivudine.Regulation of P-gp/MDR1 and ABCG2/BCRP expression may improve access of the antiretroviral drugs to the fetus,so as to prevent materal-fetal transmission of HBV during pregnance.
Objective
To determine the expression of P-gp/MDR1 and ABCG2/BCRP in full-term placentas with HBV infection,and evaluate the relation between pro-inflammatory cytokines with P-gp/MDR1 and ABCG2/BCRP expression.
Study design
1.P-gp/MDR1 and ABCG2/BCRP in full-term placentas were localized by immunohistochemistry.
2.P-gp/MDR1mRNA and ABCG2/BCRP mRNA transcripts were quantified by real-time PCR using relative determinations.The levels of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA in placentas with HBV infection were compared with those of the uninfected groups.
3.The profile of P-gp/MDR1 and ABCG2/BCRP protein levels were quantified using Western blot analysis.P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection were compared with those of the uninfected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas were determined using Enzyme-linked immunosorbent assay(ELISA).The relation between pro-inflammatory cytokines with P-gp/MDR1 and ABCG2/BCRP expression were evaluated.
5.BeWo and JEG-3 cells were treated with IL-1βor IL-6 for different time periods(12 h,24 h,48 h,72h),then the P-gp/MDR1mRNA and ABCG2/BCRP mRNA transcripts were quantified by real-time PCR using relative determinations.
6.The concentrations of lamivudine acrossed the cells were determined using HPLC in BeWo cells treated with IL-1βor IL-6.
Results
1.Immunohistochemically P-gp/MDR1 and ABCG2/BCRP proteins were localized on the maternal side membrance of syncytiotrophoblast in full-term placentas with or without HBV infection.
2.The levels of P-gp/MDR1mRNA and ABCG2/BCRP mRNA in placentas with HBV infection were statistically significantly higher than those of the, non-infected groups using real-time quantitative PCR.
3.The Western blot analysis showed a statistically significant increase in P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection compared with those of the non-infected groups.P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection were 2.43 and 3.76 times higher than those of the non-infected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas with HBV infection were statistically significantly higher than those of the non-infected groups determined by Enzyme-linked immunosorbent assay.Concentrations of IL-1β,IL-6 and TNF-αin placentas with HBV infection were 4.36,1.91,5.12 times higher than those of the non-infected groups.And the increase of IL-1β, IL-6 and TNF-αwere related to the expression of P-gp/MDR1 and ABCG2/BCRP.
5.Relative quantitative PCR showed a statistically significant increase in P-gp/MDR1 mRNA and ABCG2/BCRP mRNA transcription in BeWo and JEG-3 cells treated with IL-1βor IL-6 at 12h,24h,48h and 72h later.And the transcription level was increased remarkably with correspondence to time.
6.The concentrations of lamivudine acrossed the cells were significantly decreased in BeWo cells treated with IL-1βor IL-6 determined by HPLC.
Conclusion
1.P-gp/MDR1 and ABCG2/BCRP proteins express in full-term placentas of both HBV infected and non-infected groups,and localized to the maternal side membrance of syncytiotrophoblast.
2.The levels of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA in placentas with HBV infection are significantly higher than those of the non-infected groups.
3.The P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection are significantly higher compared with those of the non-infected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas with HBV infection are significantly higher than those of the non-infected groups.And the increase of IL-1β,IL-6 and TNF-αare related to the expression of P-gp/MDR1 and ABCG2/BCRP.
5.P-gp/MDR1 mRNA and ABCG2/BCRP mRNA transcription are significantly increased in BeWo and JEG-3 cells after treated with IL-1βor IL-6.IL-1βand IL-6 may activate the transcription of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA.
6.The infiltration rates of lamivudine acrossed the cells are significantly decreased in BeWo cells after treated with IL-1βor IL-6,which maybe related to the up regulation of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA.
方法:
1.采用免疫组化技术,检测胎盘组织中P-gp/MDR1和ABCG2/BCRP的表达和定位。
2.采用荧光定量PCR技术检测人胎盘组织中P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达。比较乙肝感染胎盘与正常孕妇胎盘中P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达的差异。
3.采用Westernblot技术检测人胎盘组织中P-gp/MDR1和ABCG2/BCRP蛋白质水平的表达。比较乙肝感染胎盘与正常孕妇胎盘中P-gp/MDR1和ABCG2/BCRP蛋白表达的差异。
4.用ELISA检测胎盘组织中IL-1β、IL-6和TNF-α的含量。分析人胎盘组织中P-gp/MDR1和ABCG2/BCRP的表达与IL-1β、IL-6和TNF-α的相关性。
5.体外培养人胎盘滋养细胞BeWo和JEG-3,分别加IL-1β、IL-6干预后,用荧光定量PCR检测细胞P-gp/MDR1和ABCG2/BCRP表达的变化。进一步探讨人胎盘滋养细胞P-gp/MDR1、ABCG2/BCRP的表达与IL-1β和IL-6的相关性。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预后,用HPLC检测3TC在滋养细胞的滤过率的变化。
结果:
1.免疫组化结果显示,乙肝感染胎盘与正常孕妇胎盘中滋养细胞母体面胞膜均有P-gp/MDR1和ABCG2/BCRP表达。P-gp/MDR1和ABCG2/BCRP在足月妊娠胎盘组织中主要表达于胎盘合体滋养细胞母体面胞膜。与以往报道相似,部分合体滋养层细胞高表达,也有部分合体滋养层细胞不表达。
2.荧光定量PCR显示,乙肝感染胎盘P-gp/MDR1 mRNA和ABCG2/BCRPmRNA的表达均高于正常组,差别有显著性。
3.Westernblot显示,乙肝感染胎盘P-gp/MDR1和ABCG2/BCRP蛋白的表达均高于正常组,差别有显著性。HBV感染胎盘组织P-gp/MDR1和ABCG2/BCRP蛋白质水平表达分别是正常足月胎盘表达量的2.43和3.76倍。与相对应的Real Time PCR结果的相关系数为0.980、0.963。
4.ELISA检测胎盘组织中IL-1β、IL-6和TNF-α的含量,乙肝组均高于正常组,乙肝组胎盘IL-1β、IL-6和TNF-α表达量分别是正常组的4.36、1.91、5.12倍。IL-1β、IL-6和TNF-α与P-gp/MDR1和ABCG2/BCRP的表达均有相关性。
5.体外培养人胎盘滋养细胞BeWo和JEG3,分别加IL-1β、IL-6干预12、24、48、72小时后,用荧光定量PCR检测细胞P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达均升高,差别有显著性。P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达随着IL-1β和IL-6作用时间的延长逐渐升高。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预48、72小时后,3TC在细胞的滤过率显著降低。
结论:
1.乙肝感染胎盘与正常孕妇胎盘中滋养细胞膜均有P-gp/MDR1和ABCG2/BCRP表达。
2.乙肝感染胎盘P-gp/MDR1 mRNA和ABCG2/BCRP mRNA的表达均高于正常组。
3.乙肝感染胎盘P-gp/MDR1和ABCG2/BCRP蛋白的表达均高于正常组。P-gp/MDR1和ABCG2/BCRP蛋白表达升高可能与P-gp/MDR1 mRNA和ABCG2/BCRP mRNA转录增强有关。
4.胎盘组织中IL-1β、IL-6和TNF-α的含量,乙肝组均高于正常组,IL-1β、IL-6和TNF-α与P-gp/MDR1、ABCG2/BCRP的表达均有相关性。提示IL-1β、IL-6和TNF-α可能诱导P-gp/MDR1 mRNA和ABCG2/BCRP mRNA转录增强以及P-gp/MDR1和ABCG2/BCRP蛋白表达。
5.体外培养人胎盘滋养细胞BeWo和JEG3,分别加IL-1β、IL-6干预12、24、48、72小时后,P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达均升高。P-gp/MDR1 mRNA和ABCG2/BCRP mRNA表达随着IL-1β和IL-6作用时间的延长逐渐升高。提示IL-1β和IL-6可能在转录水平上调P-gp/MDR1mRNA和ABCG2/BCRP mRNA的表达。
6.体外培养人胎盘滋养细胞BeWo,分别加IL-1β、IL-6干预后,3TC在细胞的滤过率显著降低。可能与IL-1β、IL-6诱导的P-gp/MDR1和ABCG2/BCRP表达上调有关。
Background
P-gp/MDR1 and ABCG2/BCRP are members of the group of transporters in the ATP binding cassette(ABC) superfamily,which are localized in the syncytiotrophoblast of the human placenta.These proteins actively extrudes a wide variety of drugs and toxins from cells,including antiretroviral HBV drugs,such as zidovudine and lamivudine.Regulation of P-gp/MDR1 and ABCG2/BCRP expression may improve access of the antiretroviral drugs to the fetus,so as to prevent materal-fetal transmission of HBV during pregnance.
Objective
To determine the expression of P-gp/MDR1 and ABCG2/BCRP in full-term placentas with HBV infection,and evaluate the relation between pro-inflammatory cytokines with P-gp/MDR1 and ABCG2/BCRP expression.
Study design
1.P-gp/MDR1 and ABCG2/BCRP in full-term placentas were localized by immunohistochemistry.
2.P-gp/MDR1mRNA and ABCG2/BCRP mRNA transcripts were quantified by real-time PCR using relative determinations.The levels of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA in placentas with HBV infection were compared with those of the uninfected groups.
3.The profile of P-gp/MDR1 and ABCG2/BCRP protein levels were quantified using Western blot analysis.P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection were compared with those of the uninfected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas were determined using Enzyme-linked immunosorbent assay(ELISA).The relation between pro-inflammatory cytokines with P-gp/MDR1 and ABCG2/BCRP expression were evaluated.
5.BeWo and JEG-3 cells were treated with IL-1βor IL-6 for different time periods(12 h,24 h,48 h,72h),then the P-gp/MDR1mRNA and ABCG2/BCRP mRNA transcripts were quantified by real-time PCR using relative determinations.
6.The concentrations of lamivudine acrossed the cells were determined using HPLC in BeWo cells treated with IL-1βor IL-6.
Results
1.Immunohistochemically P-gp/MDR1 and ABCG2/BCRP proteins were localized on the maternal side membrance of syncytiotrophoblast in full-term placentas with or without HBV infection.
2.The levels of P-gp/MDR1mRNA and ABCG2/BCRP mRNA in placentas with HBV infection were statistically significantly higher than those of the, non-infected groups using real-time quantitative PCR.
3.The Western blot analysis showed a statistically significant increase in P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection compared with those of the non-infected groups.P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection were 2.43 and 3.76 times higher than those of the non-infected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas with HBV infection were statistically significantly higher than those of the non-infected groups determined by Enzyme-linked immunosorbent assay.Concentrations of IL-1β,IL-6 and TNF-αin placentas with HBV infection were 4.36,1.91,5.12 times higher than those of the non-infected groups.And the increase of IL-1β, IL-6 and TNF-αwere related to the expression of P-gp/MDR1 and ABCG2/BCRP.
5.Relative quantitative PCR showed a statistically significant increase in P-gp/MDR1 mRNA and ABCG2/BCRP mRNA transcription in BeWo and JEG-3 cells treated with IL-1βor IL-6 at 12h,24h,48h and 72h later.And the transcription level was increased remarkably with correspondence to time.
6.The concentrations of lamivudine acrossed the cells were significantly decreased in BeWo cells treated with IL-1βor IL-6 determined by HPLC.
Conclusion
1.P-gp/MDR1 and ABCG2/BCRP proteins express in full-term placentas of both HBV infected and non-infected groups,and localized to the maternal side membrance of syncytiotrophoblast.
2.The levels of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA in placentas with HBV infection are significantly higher than those of the non-infected groups.
3.The P-gp/MDR1 and ABCG2/BCRP protein levels in placentas with HBV infection are significantly higher compared with those of the non-infected groups.
4.Concentrations of IL-1β,IL-6 and TNF-αproteins in placentas with HBV infection are significantly higher than those of the non-infected groups.And the increase of IL-1β,IL-6 and TNF-αare related to the expression of P-gp/MDR1 and ABCG2/BCRP.
5.P-gp/MDR1 mRNA and ABCG2/BCRP mRNA transcription are significantly increased in BeWo and JEG-3 cells after treated with IL-1βor IL-6.IL-1βand IL-6 may activate the transcription of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA.
6.The infiltration rates of lamivudine acrossed the cells are significantly decreased in BeWo cells after treated with IL-1βor IL-6,which maybe related to the up regulation of P-gp/MDR1 mRNA and ABCG2/BCRP mRNA.
引文
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