p21~(HBsAg/HBsAg)转基因小鼠肝脏病理学研究

[目的]观察p21HBsAg/HBsAg转基因小鼠肝脏病理学改变。[方法]分别选取2,6,12,18,24月龄的SPF级p21HBsAg/HBsAg转基因小鼠和p21+/+野生型小鼠,剖检进行大体观察,取肝脏及肝脏肿瘤组织,进行组织学HE染色及电镜超微结构观察。[结果]p21HBsAg/HBsAg转基因小鼠肝脏大体、光镜和电镜下均有的明显病理改变。随着月龄的增加,肝脏色暗质硬,表面有结节和肿瘤形成;光镜下,肝细胞浊肿,炎症细胞浸润,脂肪变性,点状、灶状和碎屑状坏死,非典型增生,肝细胞癌。癌细胞分化良好,类似肝细胞,形成索状和腺泡状结构。癌细胞核深染,具核分裂像。电镜下,癌细胞核变形,核膜曲折凹陷,线粒体肿胀,数目增多,脊减少。4例18月龄转基因小鼠发生肝细胞癌(4/10),6例24月龄的转基因小鼠发生肝细胞癌(6/10),其中2例发现远处转移;[结论]p21HBsAg/HBsAg转基因小鼠肝脏出现明显病理损害,18月龄小鼠开始发展成高分化的肝细胞癌,高龄小鼠形成的肝细胞癌能够转移。     

p21HBsAg/HBsAg转基因小鼠肝癌发生过程中PCNA的表达及意义

[目的]探讨乙型肝炎病毒表面抗原定位整合(p21HBsAg/HbsAg)转基因小鼠肝癌发生过程中PCNA的表达及意义。[方法]分别选取2,6,12,18,24月龄的SPF级转基因小鼠,取肝脏及肝脏肿瘤组织,进行免疫组织化学S-P法染色。[结果]①阳性肝细胞核内可见棕黄色反应颗粒;②2,6月龄转基因小鼠肝脏有少量散在分布的肝细胞呈PCNA阳性表达,阳性率分别为2.5%,3%;12月龄转基因小鼠肝脏PCNA阳性表达主要出现在非典型增生的肝细胞,阳性率23.65%;18月龄转基因小鼠发生的肝细胞癌PCNA阳性率61.68%;24月龄的转基因小鼠发生的肝细胞癌PCNA阳性率63.56%。12月龄转基因小鼠PCNA阳性率高于2,6月龄转基因小鼠(p<0.01),18月龄和24月龄的转基因小鼠PCNA阳性率高于12月龄转基因小鼠(p<0.05)。[结论]①PCNA能准确反映乙型肝炎病毒表面抗原定位整合转基因小鼠肝细胞的增殖能力,PCNA与肝细胞癌的发生、发展密切相关;②非典型增生的肝细胞有较高的PCNA阳性表达,是一群具有肿瘤增殖潜能的癌前细胞群。     

透射电镜观察p21HBHBsAg/HBsAg转基因小鼠肝癌细胞凋亡

目的 观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡.方法 用透射电镜观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡的形态学改变.结果 细胞凋亡早期,细胞核染色体发生边集,核形不规整,核膜表面凹凸;凋亡中期,核内染色质凝聚,趋边呈月牙状,核膜孔消失,核膜呈波纹状皱缩;凋亡晚期,核固缩,细胞膜出芽形成小泡,可见凋亡小体.结论 p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡具有典型性病变特征,是研究HBV感染诱发肝癌发病机理的合适动物模型.     

透射电镜观察p21-（HBsAg/HBsAg）转基因小鼠肝癌细胞凋亡

目的观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡。方法用透射电镜观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡的形态学改变。结果细胞凋亡早期,细胞核染色体发生边集,核形不规整,核膜表面凹凸;凋亡中期,核内染色质凝聚,趋边呈月牙状,核膜孔消失,核膜呈波纹状皱缩;凋亡晚期,核固缩,细胞膜出芽形成小泡,可见凋亡小体。结论p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡具有典型性病变特征,是研究HBV感染诱发肝癌发病机理的合适动物模型。     

p16在HBsAg和HBx转基因小鼠肝脏肿瘤中的表达

目的:探讨p16基因在由乙型肝炎病毒基因整合引起的小鼠肝细胞癌发生发展中的表达变化规律。方法:以乙肝病毒表面抗原基因(HBsAg)及X基因(HBx)定位整合转基因小鼠及对照小鼠的肝脏组织为标本,利用North-ern印迹、Western印迹及免疫组织化学检测p16在乙肝病毒基因定位整合转基因小鼠肝脏正常组织与肿瘤组织中的差异表达。结果:p16主要在小鼠胚胎期的肝脏中表达,在新生小鼠和成年小鼠的肝脏组织中几乎检测不到其表达;在HBsAg转基因小鼠和HBx转基因小鼠的肝脏肿瘤中,p16的表达明显升高。结论:p16基因在HBsAg或HBx诱导的肝细胞癌发生过程中被重新激活,也许发挥重要的作用。     

不同月龄p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠血清酶含量的测定

目的测定p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠纯合型及野生型小鼠的血清酶,探讨各种血清酶在两种纯合型小鼠及野生型小鼠的变化规律。方法采用荷兰半自动生化分析仪Ⅱ对于肝功能相关的7种血清酶进行测定,应用SSPS10统计学软件进行T检验及方差分析比较。结果p2^1HBsAg/HBsAg和p21^HBX/HBX转基因小鼠不同年龄、不同的指标雌雄之间差异显著,并且随着年龄的变化,雌雄之间的变化规律不同。结论p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠的血清酶在不同的年龄具有一定的变化规律,说明p21^HBsAg/HBsAg和p21^HBX/HBX转基因小鼠的血清酶具有一定的特征,为其功能性研究及临床检验具有重要参考价值。     

重组HBsAg疫苗辅以CpG ODN对转基因小鼠的免疫治疗效果

目的利用免疫耐受的乙型肝炎病毒(hepatitis B virus,HBV)转基因(Transgenic mice,Tg)小鼠模型,研究重组HBsAg疫苗辅以CpG ODN的免疫治疗效果,为HBV的临床免疫治疗提供思路和依据.方法重组HBsAg疫苗单独或辅以CpG ODN,同时设干扰素(IFN)药物组和生理盐水(NS)照组,多次免疫治疗HBV转基因小鼠,于免疫前和末次免疫后2周、4周眼球后静脉丛取血,动态观察各组小鼠血清中HBsAg量、HBsAg阴转率、Anti-HBs阳性率和HBVDNA拷贝数的变化.在治疗后应用HE染色观察各组小鼠肝组织病变活动度以及SP组化法观察活肝组织中HBsAg表达量的改变.结果在免疫治疗后2周,HBsAg疫苗组和HBsAg CpG组血清中的HBsAg量较免疫前和同期的IFN组、NS组均有明显降低(P＜0.05),并且到4周时降低作用依然很明显;免疫治疗后2周时两组100%出现Anti-HBs抗体;HBsAg CpG组治疗后2周血清HBsAg有1只转阴,4周时阴转数增加到3只.其他三组中均无阴转;免疫治疗后2周至4周HBsAg CpG组的小鼠HBV DNA的拷贝数可降低1～2个数量级,IFN组2周部分出现轻微降低但到4周时出现回升;HBsAg CpG组肝组织中HBsAg量的表达均出现不同程度的降低;病理学检测显示HBsAg CpG组肝组织中浸润大量淋巴细胞,可见恢复期的肝小叶.肝组织病变活动度情况为HBsAg CpG组＞HBsAg组＞IFN组、NS组.结论CpG ODN增强重组HBsAg疫苗对HBV转基因小鼠的免疫治疗效果.重组HBsAg疫苗辅以CpG ODN可作为临床上免疫治疗慢性HBV感染的可行性途经.     

HBsAg阳性转基因小鼠肝脏中昼夜节律基因dbp转录水平变化的研究

目的 验证HBsAg阳性转基因小鼠肝脏中昼夜节律表达基因dbp (coding for the D site albumin promoter binding protein)转录水平的变化。 方法 用实时荧光定量聚合酶链式反应方法对比研究雌、雄及个体HBsAg阳性转基因小鼠及对照鼠肝脏dbp转录水平的变化。研究不同时间点HBsAg阳性转基因小鼠及对照鼠肝脏dbp转录水平的变化。 结果 #59 及#10 品系组HBsAg阳性转基因小鼠肝组织中dbp均较对照组动物上调, 但个体间差别较大。初步结果显示雌性正常小鼠dbp转录水平较雄性为高。而在雌性HBsAg阳性转基因小鼠中dbp的上调进一步增强。与此相反,雄性转基因小鼠dbp的上调则较同性对照鼠更弱。转基因小鼠dbp转录,在8：00时及14：00时平均水平均高于对照鼠,然而在20：00时及2：00时则与对照鼠相当。结论 本研究首次报道HBsAg阳性转基因小鼠肝脏中dbp表达的上调,提供了乙型肝炎病毒蛋白表达与昼夜节律基因变化有所联系的证据。至于本研究在转基因鼠中的发现是否在乙型肝炎患者中存在及其意义还有待在患者中作进一步临床验证与研究。     

佐剂CpG-ODN与重组HBsAg疫苗联合免疫乙型肝炎病毒转基因小鼠的免疫应答研究

目的 以CpG-ODN为佐剂与重组HBsAg(rHBsAg)疫苗合用,研究其对乙型肝炎病毒转基因(HBV Tg)小鼠模型的免疫应答效果.方法 40只HBV Tg小鼠随机分为4组,每组小鼠分别注射rHBsAg疫苗(单用rHBsAg组)、rHBsAg疫苗+CpG-ODN(试验组)、rIFNα-2b(IFN组)、生理盐水(对照组).经多次免疫HBV转基因小鼠,于免疫前、后不同时间采血,动态观察各组小鼠血清中HBsAg量、抗-HBs阳性率和HBV DNA的变化,检测肝组织中HBsAg的表达.检测免疫小鼠的外周血T淋巴细胞亚群和白细胞介素2(IL-2)、IL-12(p70)以及γ干扰素(IFN-γ)的含量,分别检测免疫小鼠的脾细胞增殖和细胞毒性T淋巴细胞(CTL)杀伤功能并计算各组小鼠肝组织活性指数(HAI).结果 rHBsAg组和rHBsAg+CpG组在免疫小鼠后2周100%诱导抗-HBs;rHBsAg+CpG组能显著降低血清中的HBsAg量或使HBsAg转阴,rHBsAg+CpG组肝组织中HBsAg的表达量与血清中一样降低,并降低血清中HBV DNA的拷贝数.rHBsAg组的CD3+、CD4+、CD8+细胞在T细胞中所占百分比,IL-2、IL-12(p70)和IFN-γ的含量以及淋巴细胞特异性增殖和杀伤效应均明显高于对照组(P＜0.05).rHBsAg+CpG组与rHBsAg组比较,免疫小鼠产生更强的HBV特异性细胞应答(P＜0.05),且以Th1型细胞免疫应答为主.在rHBsAg+CpG组肝组织中出现大量淋巴细胞,肝脏的HAI在4个组中最高.结论 CpG-ODN作为佐剂可以增强重组HBsAg疫苗诱导HBV转基因小鼠产生抗病毒免疫应答,重组HBsAg疫苗辅以CpG ODN可作为免疫治疗慢性HBV感染的可行性途经.     

LPS/D-GalN诱发NF-κB转基因小鼠急性致死性肝损伤模型的建立

目的以NF-κB转基因BALB/c小鼠建立一个LPS/D-GalN诱发的急性致死性肝损伤模型。方法采取腹腔注射高剂量的LPS/D-GalN建立急性致死性肝损伤小鼠模型,观察模型小鼠的促炎症细胞因子水平和NF-κB的活性改变,以及肝脏功能和病理改变情况。结果模型组小鼠生存时间为8～10 h,模型建立后小鼠血清TNF-α、IL-6和MCP-1水平显著升高,在2～4h达到高峰;肝脏外观出现瘀血和出血,肝脏小叶被严重破坏,肝细胞严重坏死和出血;血清ALT/AST水平在模型诱发后持续迅速上升;整体成像显示NF-κB的活性在4～6 h达到高峰。正常对照组小鼠以上指标无显著变化。结论成功建立LPS/D-GalN诱发的NF-κB转基因小鼠的急性致死性肝损伤模型。     

Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and alpha-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.     

大剂量HBsAg对HBV转基因小鼠T细胞免疫调节的研究

目的：研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法：用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果：HBsAg组免疫后脾细胞产生的Th1类细胞因子（IFN-γ、IL-2）、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加（P〈0.05）。结论：大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

Expression of HBsAg and HBcAg in liver tissue: correlation with disease activity.

The patterns of Hepatitis B surface antigen (HBsAg) and Hepatitis B core antigen (HBcAg) expression were studied in liver biopsies taken from 41 patients with chronic HBV disease. Immunohistochemical methods were used on deparaffinized sections for the identification of HBsAg and HBcAg in liver tissue. Twenty-one of the 41 cases (51.2%) were classified as inactive liver disease and 20 (48.8%) as active liver disease. In liver biopsies with inactive disease, HBsAg demonstrated varying types of cytoplasmic expression in a rather high number of hepatocytes distributed mainly in clusters, while HBcAg was rarely expressed in liver nuclei. On the other hand, in liver biopsies with active disease HBsAg was characterized by a diffuse cytoplasmic expression in a few discrete hepatocytes, while HBcAg was expressed in the nuclei of the hepatocytes in 70% of the cases and in half of the positive cases it was also detected in the cytoplasm. In conclusion, HBsAg expression in a few scattered hepatocytes correlates with active liver disease and positive HBcAg, while varying HBsAg cytoplasmic expression in a rather high number of clustered hepatocytes is related to chronic inactive liver disease and negative expression of HBcAg.     

CpG Oligodeoxynucleotides with Hepatitis B Surface Antigen (HBsAg) for Vaccination in HBsAg-Transgenic Mice

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.