摘要
慢性乙型肝炎(hepatitis B virus,HBV)感染后呈现多样的疾病谱,目前对于慢乙肝感染后出现不同进展的原因尚无一致意见。但研究表明在HBV感染后的病毒清除过程中细胞免疫反应起重要作用,而免疫清除病毒的同时也对肝组织造成了免疫损伤,其中以T淋巴细胞为主要效应细胞的抗病毒免疫反应与肝脏炎症密切相关。而免疫反应程度在不同宿主间存在差异,因此宿主抗病毒免疫能力的遗传背景差异是影响HBV感染后疾病进展的重要原因。目前研究表明拷贝数变异(copy number variations,CNVs)可通过基因的剂量效应及结构变化对基因表达产生影响,从而影响疾病的易感性及进展等。研究与T淋巴细胞功能相关的免疫因子的基因拷贝数变异在不同慢乙肝感染转归组的差异有助于进一步明确个体免疫遗传背景在慢乙肝感染过程中所起的作用。
研究内容
检测88例正常健康对照组、慢乙肝携带组、慢性乙肝重型肝炎组(重肝组)、原发性肝癌组等慢性HBV感染不同疾病转归组患者基因PDCD1、CD274、BCL2L11、IL7R基因拷贝数,Kruskal-Waills H检验和秩变换分析研究四组间四个基因的拷贝数差异,探讨宿主遗传背景差异与慢乙肝进展的关系。
病例与方法
1.研究对象
慢性HBV携带者、慢性乙型重型肝炎组,原发性肝癌患者主要来源于2006年6月—2008年12月广州市第八人民医院肝病科住院病区和专科门诊,正常健康对照组主要来源于广州市第八人民医院在职职工。纳入年龄为20-60岁。诊断符合2005年12月10日《慢性乙型肝炎防治指南》和2001年9月《原发性肝癌的诊断和分期标准》。
2.研究方法
(1)、利用Taqman real-time PCR技术测定测定正常健康对照组、慢性HBV携带者、慢性乙型重型肝炎组,原发性肝癌组的PDCD1、CD274、BCl2L11、IL7R的基因拷贝数。
(2)、Kruskal-Waills H检验和秩变换分析研究四组间四个基因的拷贝数差异是否存在差异。
结果
1、四组之间PDCD1基因拷贝数存在明显差异(P<0.05[Kruskal-Waills H])。肝癌组PDCD1拷贝数明显低于另三组(P<0.0167[秩变换分析]),携带组和重肝组PDCD1拷贝数高于正常对照组,但差异无显著性(P>0.0167[秩变换分析])。携带组和重肝组间PDCD1的拷贝数差异无显著性(P>0.0167[秩变换分析])。
2、四组之间CD274基因拷贝数也存在明显差异(P<0.05[Kruskal-Waills H])。其中肝癌组CD274拷贝数明显低于携带组和重肝组(P<0.0167[秩变换分析]),肝癌组CD274拷贝数低于正常对照组,但差异无显著性(P>0.0167[秩变换分析]),携带组和重肝组CD274拷贝数明显高于对照组(P<0.0167[秩变换分析]),携带组和重肝组间CD274的拷贝数无显著差异(P>0.0167[秩变换分析])。
3、四组之间BCL2L11基因拷贝数也存在明显差异(P<0.05[Kruskal-Waills H])。肝癌组BCL2L11拷贝数明显低于另三组(P<0.0167[秩变换分析]),携带组明显高于另三组(P<0.0167[秩变换分析]);重肝与对照组间无显著差异(P>0.0167[秩变换分析]),重肝组拷贝较携带组低(P=0.02[秩变换分析])。
4、四组之间IL7R基因拷贝数无明显差异(P>0.05[Kruskal-Waills H])。
结论
1、免疫功能相关的PD-1、PD-L1、Bim的基因在健康对照组、慢性HBV携带组、慢性乙型重型肝炎组、原发性肝癌组间存在基因拷贝数变异(CNVs)。
2、携带组和重肝组PD-1的基因PDCD1的拷贝数与正常对照组相比无显著差异;携带组和重肝组PD-L1的基因CD274的拷贝数较正常对照组高;携带组Bim基因BCL2L11的拷贝数较正常对照组高。
3、不同慢乙肝感染转归组间CD127的基因拷贝数未见差异。
4、PDCD1、CD274、BCL2L11的基因拷贝数在肝癌组均低于正常对照组,与其既往表达研究的结果不一致。有待对肝癌细胞基因组作进一步研究。
5、免疫相关因子的基因拷贝数变异趋势与基因表达趋势不完全一致,特别在携带组和重肝组间未发现显著的CNVs存在。因此,对基因多态性的关联研究,以及联合基因转录水平和蛋白表达水平进行深入研究更有助于全面了解影响慢乙肝转归的免疫遗传因素。
Backgroud
Chronic hepatitis B infection presents a variety of disease spectrum. There is no consensus about the reason for individual being different progression after chronic hepatitis B infection. But research shows that immune response plays an important role in the process of virus removal of HBV infection and immune clearance of the virus also causes immune damage of liver tissue, in which the antiviral immune responses of T lymphocyte cells is closely related to the liver inflammation. The level of immune response is different in the hosts, so the genetic background of host antiviral immunity is an important impact on the disease progression of HBV infection. Present study shows that copy number variation (CNVs) can affect gene expression by gene dosage effect and structural changes, thus affecting the progress and susceptibility of the disease and so on. To study the gene copy number variation (CNVs) of T lymphocyte function–related immune factors in different groups of chronic HBV infection of different progression will help further clarify the role of the genetic background in chronic hepatitis B infection .
Objectives
To study gene copy number of PDCD1, CD274, BCL2L11, IL7R of 88 cases of control group, chronic HBV carriers group, chronic severe hepatitis group, HCC group,using Kruskal-Waills H test and Rank transform analysis to analyze the differences of gene copy number of four genes between four groups,so as to study the relationship between the host genetic background and the chronic hepatitis B progression.
Materials and Methods
Object
88 cases of chronic HBV carriers group, chronic severe hepatitis group and HCC group were admitted patients or outpatients of the 8th People's Hospital of Guangzhou from June 2006 to December 2008 and the control group was main from the employment of Guangzhou 8th People's Hospitalincluding, the age of 20-60 years. All the cases were confirmed as the chronic hepatitis B according to guideline.
Methods
(1). Using Taqman real-time PCR measured to determine gene copy number of PDCD1, CD274, BCl2L11, IL7R of control group, chronic HBV carriers group, chronic severe hepatitis group and HCC group.
(2). Using Kruskal-Waills H test and Rank transform to analysis the differences of gene copy number of four genes between four groups.
Results
1. The gene copy number PDCD1 among the four groups were significantly different (P<0.05 [Kruskal-Waills H test ]). HCC group was significantly lower than the other three groups (P<0.0167 [Rank transformation analysis]), chronic HBV carriers group is higher than the control group, but the difference was not significant (P>0.0167 [Rank transform analysis]). The difference between the carriers group and chronic severe hepatitis group was not significant (P>0.0167 [Rank transformation analysis]).
2. The gene copy number CD274 among the four groups were significantly different (P<0.05 [Kruskal-Waills H test ]). HCC group was significantly lower than the carriers group and chronic severe hepatitis group (P<0.0167 [Rank transformation analysis]).HCC group was lower than the control group, but the difference was not significant (P>0.0167 [Rank transform analysis]).The carriers group and chronic severe hepatitis group was significantly higher than the control group (P<0.0167 [Rank transform analysis]). The difference between the carriers group and chronic severe hepatitis group was not significant (P> 0.0167 [Rank transform analysis]).
3. The gene copy number BCL2l11 among the four groups were significantly different (P<0.05 [Kruskal-Waills H test]). HCC group was significantly lower than the other three groups (P<0.0167 [Rank transformation analysis]). The carriers group was significantly higher than the other three groups (P<0.0167 [Rank transformation analysis]).There is no significant differences between the chronic severe hepatitis group and the control group (P>0.0167 [Rank transformation analysis]). chronic severe hepatitis group was lower than the carriers group (P = 0.02 [Rank transformation analysis]).
4. There is no significant differences among the four groups of IL7R (P<0.05 [Kruskal-Waills H test]).
Conclusions
1.PD-1, PD-L1, Bim ,the immune function related factors,whose gene copy number variation existed among the control group, chronic HBV carriers group, chronic severe hepatitis group and HCC goup.
2.There was no significant difference between chronic HBV carriers group, chronic severe hepatitis group and control group of gene copy number of PDCD1.The gene copy number of CD274 in chronic HBV carriers group and chronic severe hepatitis group were higher than the control group. The gene copy number of BCL2L11 in chronic HBV carriers group was lower than the control group.
3.Ther was no difference among the chronic hepatitis B infection groups of different progression of gene copy number IL7R.
4.The gene copy number of PDCD1, CD274, BCL2L11 in HCC group were lower than the control group, which is inconsistent with the previous findings of its expression. So further study should be put in the genome of hepatoma cells.
5.The gene copy number variation of immune-related factors and was not fully consistent with the trend of the gene expression, and there was no significant CNVs between the chronic HBV carriers group and chronic severe hepatitis group. Therefore, the study of gene polymorphism being combined with gene transcription and protein expression can help to fully understand the impact of immune and genetic factors on the prognosis of chronic hepatitis.
引文
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