重型乙型肝炎免疫细胞差异表达基因研究
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摘要
目的 NK细胞的细胞毒作用在乙型肝炎病毒(HBV)诱导的肝损伤中起了重要作用，而某些宿主细胞因子和受体基因在这些免疫细胞上的异常表达，可能增强NK等效应细胞对病毒感染细胞的细胞毒作用而参与重型乙肝(FHB)的发病机制。宿主基因表达在免疫应答和炎症反应的发生发展和转归方面所起的重要作用也越来越引起重视，但宿主免疫细胞基因表达在重型肝炎发生中的作用仍不清楚。本实验旨在筛选重型乙肝PBMC差异表达基因，研究其在NK等免疫细胞中的表达及与肝病严重性的关系，从免疫细胞受体基因表达及调节角度探讨乙型肝炎重型化致病机理，为FHB防治开辟新思路和新的前景。
方法 首先应用表达谱基因芯片与抽提自10例FHB和10例无症状表面抗原携带者(ASC)的PBMC中标记的cDNA杂交以检测基因表达，应用GenePix 4000B扫描芯片，ImaGene3.0软件分析数据并筛选差异表达基因。然后应用RT-PCR及免疫组化S-P法，检测所筛基因在FHB、慢性肝炎(CHB)、ASC及正常对照人群的PBMC及肝脏中的差异表达。同时应用流式细胞术(FCM)分选经诱导培养的NK细胞，检测所选基因在FHB、ASC和正常人群中的差异表达。以HepG2、K562、CNE细胞株作为靶细胞，应用MTT比色法分析所选基因对NK细胞细胞毒作用的影响及特异性抗体对细胞毒作用的阻断效应。最后，应用基因重组技术构建含所筛目的基因的真核表达质粒，经双酶切、PCR和DNA测序证实该重组真核表达质粒含有所需目的基因。然后利用脂质体介导将重组真核表达质粒转染CHO细胞，经含G418选择性培养基筛选获得稳定转染重组真核表达质粒的CHO细胞，经RT-PCR和Western Bloting证实转染的CHO细胞中有目的基因表达，然后应用MTT比色法分析含目的基因的重组真核表达质粒对NK细胞的细胞毒
性作用的影响。
结果
1、表达谱芯片筛选结果：应用基因芯片技术建立了重型乙肝PBMC
基因表达数据库。在 8192个基因中，筛选出则B和 ASC差异表达 2
倍以上的基因 249（占 3们，上调基因 SI个，下调 168个，其中细胞
信号和传递，细胞周期和代谢，凋亡及炎症相关类基因占79儿
2、NKGZD InRNA表达：FHB、CHB、对照组及 ASC的 PBMC中 NKGZD
mRNA表达呈依次递减趋势。FHB组 NKGZD mRNA表达最强，与其它各组
卜较，差异有显著性（P＜0．of）。
3、免疫组化结果：在肝脏汇管区表达NKGZD的阳性细胞百分率
不同，差异有显著性um．01人 FHB组表达 NKGZD的阳性细胞百分
率最高，分别与其它3组比较，差异有显著性uo．01人CHB①3，G4）
组与正常对照组比较，差异有显著性u刃．of人但mB①，GZ）组和
正常对照组比较，差异无显著性u川．05）
4、FCM和 FACS分析结果：应用荧光激活细胞分类仪和流式细胞
术分析，分选后能与抗CD16化D56 InAb结合的NK细胞纯度高达99％
以上。FHB组NK细胞NKGZD蛋白表达最强，分别与ASC及对照组比
较，差异有显著性（P＜0．of）。ASC组 NK细胞 NKGZD 表达最弱，分别
与其它2组比较，差异有显著性u的．of人
5、MTT卜色法结果：NK细胞对 3种肿瘤细胞K562、HePGZ和 CNE
均有较强的细胞毒效应。NK细胞的杀伤活性分别为68兄65．7％和
55．l％。力入抗 NKGZD PAb（0．sing／L）后，可显著抑本 NK细胞对 K562、
HePGZ细胞的细胞毒效应。NK细胞对两种靶细胞的杀伤活性分别下降
了 82．9％和 75．6％倍。但抗 NKGZD PAb不能显著抑策 NK细胞对 CNE细
胞的细胞毒效应。
厂二
6、人NKGZD基因在m 细胞中的表达及鉴定：获得的NKGZD的
cDNA序列与GeneBank登陆的cDNA序列一致。成功地构建了稳定表
达的重组真核表达质粒PCDNA3．lw。转染＊ 细胞后，获得可
稳定表达NKGZD的基因工程细胞株。NK细胞与转染了NKGZD的CHO
细胞共培养后，对K562细胞的杀伤率为88见大于与转染空白质粒的
CHO细胞共培养后的杀伤率伤9卿。其杀伤效应能被NKGZD多克隆抗
体所阻断。
结论
1、经表达谱基因芯片证实，HBV感染机体导致重型肝炎过程中，
明显地改变了宿主细胞内基因表达，FHB和 ASC差异表达基因占 3％
（249／8 192）。
2、乙型肝炎外周血PBMC、NK细胞表面NKGZD 过度表达与MB的
发生相关。
3、肝组织内免疫细胞NKGZD过度表达与FHB的发生有关。
4、NKGZD介导的细胞毒作用在激活NK细胞对K562、HepGZ细胞
的杀伤中起主导作用。
5、抗 NKGZD PAb可通过封闭训 细胞表面的 NKGZD 分子而抑制其
对肿瘤细胞吃HepGZ、K562）的细胞毒效应。
6、成功地构建了重组表达载体pCDNA3．1十，并实现了在CHO
细胞中的稳定表达。
Objective It was known that NK cells played an important role in liver damage caused by hepatitis B virus(HBV). Some cytokine and receptor genes expressed abnormally on immunocytes may increase cytotoxicity of NK cells on cells with viral infections and may be associated with the pathogenesis of fulminant hepatitis B(FHB). In recent years the effect of gene expression in host cells have been thought playing a major role in the development and result of inflamation and immune response,and attract more and more attention.So far,little is known about the relation between genes expression on immunocytes and the development of fulminant hepatic failure.In this study,we investigated the relationship between the gene expression on immunocytes (e.g.NK cells)and the development of severe hepatic damage through the screening of genes differential expression on PBMC in patients with FHB. The aim of our experiment was to study the immunopathogenesis of FHB by investigating gene expression and regulation of cell receptor
on immunocytes,that will open a new idea and bright prospects in the prevention and treatment of FFTB.
Methods At first,high-density microarrays consisting of 8192 human cDNAs were applied to analyze gene expression using labeled cDNAs hybridization of PBMC which prepared from 10 patients with
fulminant hepatitis B(FHF) and 10 patients with asymtomatic HBsAg
carries(ASC).Relative expression ratios of each genes were obtained by comparing hybridization of Cy5-labeled cDNA from FHF and Cy3-labeled cDNA from ASC and scanning on a GenePix 4000B,and then by quantitative analysis of genes differential expression using ImaGeneS.O software.The differential expression of target gene were determined in the PBMCs and liver issues obtained from FHB > CHB >, ASC patients and normal peoples by RT-PCR and histochemistry S-P method.Meanwhile, NK cells were sorted after those were cultured and stimulated by PHA and IL-2,and differential expression of target gene were quantitatively determined in patients with FHB> ASC and normal peoples as control by Flow cytometry and immunofluorescent techniques. NK cytolysis on target cells of HepG2, K562 and CNE cells and after NK cells were blocked with specific antibody were analyzed by MTT colorimetry.Finally,an eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique,and was confirmed by double restriction enzymes digestion > PCR and DNA sequence analysis. Then the recombinant plasmid with lipofectamine encapsuled was transfected into CHO cells, and screening by G418 selective culture medium. Finally the CHO cells transfected with recombinant eukaryotic expression plasmid stably were obtained,and the transfected CHO cells containing expressive target gene were confirmed by RT-PCR and Western Blotting techniques.Then the effect of eukaryotic expression
plasmid containing target gene inducing NK cytolysis was analyzed by MTT colorimetry. Results
1. Results of high-densty microarrays analysis Out of 8192 genes,249(3%) cellular genes in the FHB were identified as differentially expressed that 2-fold more than in the ASC.lt included 81 up-regulated genes and 168 down-regulated genes.Among 249 genes,total 79% have been found related with the cell signaling and transduction,cell cycle and metabolism, apoptosis and inflammatory response and immunity-related genes.
2. Expression of NKG2D mRNA In PBMC the level of expression of NKG2D mRNA among the four groups was gradually down from the group of FHB > then the groups of CHEk normal control to ASC in order of level.In group FHB the level of expression of NKG2D mRNA in PBMC was significantly higher than the other three groups(P<0.01).
3. Histochemistry analysis The level of expression of NKG2D gene on immunocytes of hepatic portal areas was significantly higher in the group FHB than in the group CHB and normal control (P<0.01),and in the group CHB(G3/G4) the level of expression of NKG2D was higher than in the group control(P<0.01).But,there
方法 首先应用表达谱基因芯片与抽提自10例FHB和10例无症状表面抗原携带者(ASC)的PBMC中标记的cDNA杂交以检测基因表达，应用GenePix 4000B扫描芯片，ImaGene3.0软件分析数据并筛选差异表达基因。然后应用RT-PCR及免疫组化S-P法，检测所筛基因在FHB、慢性肝炎(CHB)、ASC及正常对照人群的PBMC及肝脏中的差异表达。同时应用流式细胞术(FCM)分选经诱导培养的NK细胞，检测所选基因在FHB、ASC和正常人群中的差异表达。以HepG2、K562、CNE细胞株作为靶细胞，应用MTT比色法分析所选基因对NK细胞细胞毒作用的影响及特异性抗体对细胞毒作用的阻断效应。最后，应用基因重组技术构建含所筛目的基因的真核表达质粒，经双酶切、PCR和DNA测序证实该重组真核表达质粒含有所需目的基因。然后利用脂质体介导将重组真核表达质粒转染CHO细胞，经含G418选择性培养基筛选获得稳定转染重组真核表达质粒的CHO细胞，经RT-PCR和Western Bloting证实转染的CHO细胞中有目的基因表达，然后应用MTT比色法分析含目的基因的重组真核表达质粒对NK细胞的细胞毒
性作用的影响。
结果
1、表达谱芯片筛选结果：应用基因芯片技术建立了重型乙肝PBMC
基因表达数据库。在 8192个基因中，筛选出则B和 ASC差异表达 2
倍以上的基因 249（占 3们，上调基因 SI个，下调 168个，其中细胞
信号和传递，细胞周期和代谢，凋亡及炎症相关类基因占79儿
2、NKGZD InRNA表达：FHB、CHB、对照组及 ASC的 PBMC中 NKGZD
mRNA表达呈依次递减趋势。FHB组 NKGZD mRNA表达最强，与其它各组
卜较，差异有显著性（P＜0．of）。
3、免疫组化结果：在肝脏汇管区表达NKGZD的阳性细胞百分率
不同，差异有显著性um．01人 FHB组表达 NKGZD的阳性细胞百分
率最高，分别与其它3组比较，差异有显著性uo．01人CHB①3，G4）
组与正常对照组比较，差异有显著性u刃．of人但mB①，GZ）组和
正常对照组比较，差异无显著性u川．05）
4、FCM和 FACS分析结果：应用荧光激活细胞分类仪和流式细胞
术分析，分选后能与抗CD16化D56 InAb结合的NK细胞纯度高达99％
以上。FHB组NK细胞NKGZD蛋白表达最强，分别与ASC及对照组比
较，差异有显著性（P＜0．of）。ASC组 NK细胞 NKGZD 表达最弱，分别
与其它2组比较，差异有显著性u的．of人
5、MTT卜色法结果：NK细胞对 3种肿瘤细胞K562、HePGZ和 CNE
均有较强的细胞毒效应。NK细胞的杀伤活性分别为68兄65．7％和
55．l％。力入抗 NKGZD PAb（0．sing／L）后，可显著抑本 NK细胞对 K562、
HePGZ细胞的细胞毒效应。NK细胞对两种靶细胞的杀伤活性分别下降
了 82．9％和 75．6％倍。但抗 NKGZD PAb不能显著抑策 NK细胞对 CNE细
胞的细胞毒效应。
厂二
6、人NKGZD基因在m 细胞中的表达及鉴定：获得的NKGZD的
cDNA序列与GeneBank登陆的cDNA序列一致。成功地构建了稳定表
达的重组真核表达质粒PCDNA3．lw。转染＊ 细胞后，获得可
稳定表达NKGZD的基因工程细胞株。NK细胞与转染了NKGZD的CHO
细胞共培养后，对K562细胞的杀伤率为88见大于与转染空白质粒的
CHO细胞共培养后的杀伤率伤9卿。其杀伤效应能被NKGZD多克隆抗
体所阻断。
结论
1、经表达谱基因芯片证实，HBV感染机体导致重型肝炎过程中，
明显地改变了宿主细胞内基因表达，FHB和 ASC差异表达基因占 3％
（249／8 192）。
2、乙型肝炎外周血PBMC、NK细胞表面NKGZD 过度表达与MB的
发生相关。
3、肝组织内免疫细胞NKGZD过度表达与FHB的发生有关。
4、NKGZD介导的细胞毒作用在激活NK细胞对K562、HepGZ细胞
的杀伤中起主导作用。
5、抗 NKGZD PAb可通过封闭训 细胞表面的 NKGZD 分子而抑制其
对肿瘤细胞吃HepGZ、K562）的细胞毒效应。
6、成功地构建了重组表达载体pCDNA3．1十，并实现了在CHO
细胞中的稳定表达。
Objective It was known that NK cells played an important role in liver damage caused by hepatitis B virus(HBV). Some cytokine and receptor genes expressed abnormally on immunocytes may increase cytotoxicity of NK cells on cells with viral infections and may be associated with the pathogenesis of fulminant hepatitis B(FHB). In recent years the effect of gene expression in host cells have been thought playing a major role in the development and result of inflamation and immune response,and attract more and more attention.So far,little is known about the relation between genes expression on immunocytes and the development of fulminant hepatic failure.In this study,we investigated the relationship between the gene expression on immunocytes (e.g.NK cells)and the development of severe hepatic damage through the screening of genes differential expression on PBMC in patients with FHB. The aim of our experiment was to study the immunopathogenesis of FHB by investigating gene expression and regulation of cell receptor
on immunocytes,that will open a new idea and bright prospects in the prevention and treatment of FFTB.
Methods At first,high-density microarrays consisting of 8192 human cDNAs were applied to analyze gene expression using labeled cDNAs hybridization of PBMC which prepared from 10 patients with
fulminant hepatitis B(FHF) and 10 patients with asymtomatic HBsAg
carries(ASC).Relative expression ratios of each genes were obtained by comparing hybridization of Cy5-labeled cDNA from FHF and Cy3-labeled cDNA from ASC and scanning on a GenePix 4000B,and then by quantitative analysis of genes differential expression using ImaGeneS.O software.The differential expression of target gene were determined in the PBMCs and liver issues obtained from FHB > CHB >, ASC patients and normal peoples by RT-PCR and histochemistry S-P method.Meanwhile, NK cells were sorted after those were cultured and stimulated by PHA and IL-2,and differential expression of target gene were quantitatively determined in patients with FHB> ASC and normal peoples as control by Flow cytometry and immunofluorescent techniques. NK cytolysis on target cells of HepG2, K562 and CNE cells and after NK cells were blocked with specific antibody were analyzed by MTT colorimetry.Finally,an eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique,and was confirmed by double restriction enzymes digestion > PCR and DNA sequence analysis. Then the recombinant plasmid with lipofectamine encapsuled was transfected into CHO cells, and screening by G418 selective culture medium. Finally the CHO cells transfected with recombinant eukaryotic expression plasmid stably were obtained,and the transfected CHO cells containing expressive target gene were confirmed by RT-PCR and Western Blotting techniques.Then the effect of eukaryotic expression
plasmid containing target gene inducing NK cytolysis was analyzed by MTT colorimetry. Results
1. Results of high-densty microarrays analysis Out of 8192 genes,249(3%) cellular genes in the FHB were identified as differentially expressed that 2-fold more than in the ASC.lt included 81 up-regulated genes and 168 down-regulated genes.Among 249 genes,total 79% have been found related with the cell signaling and transduction,cell cycle and metabolism, apoptosis and inflammatory response and immunity-related genes.
2. Expression of NKG2D mRNA In PBMC the level of expression of NKG2D mRNA among the four groups was gradually down from the group of FHB > then the groups of CHEk normal control to ASC in order of level.In group FHB the level of expression of NKG2D mRNA in PBMC was significantly higher than the other three groups(P<0.01).
3. Histochemistry analysis The level of expression of NKG2D gene on immunocytes of hepatic portal areas was significantly higher in the group FHB than in the group CHB and normal control (P<0.01),and in the group CHB(G3/G4) the level of expression of NKG2D was higher than in the group control(P<0.01).But,there
引文
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[56] Bauer S, Groh V, Wu J, et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science, 1999; 285(5428): 727-729
[57] Jinushi M, Takehara T, Kanto T, et al. Critical Role of MHC Class I-Related Chain A and B Expression on IFN-alpha-Stimulated Dendritic Cells in NK Cell Activation: Impairment in Chronic Hepatitis C Virus Infection. J Immunol,2003; 170(3): 1249-1256
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Biol, 1999 Sep; 19(9):2207-2213
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