【作者】 曹峻；

【导师】 温浩；

【作者基本信息】 新疆医科大学 ， 内科学， 2010， 博士

【摘要】 目的：在建立稳定的HBV转基因小鼠模型的基础上,应用真菌植物松茸提取物、α-干扰素化学性预防治疗HBV转基因小鼠,初步评价真菌植物松茸提取物与α-干扰素预防HBV转基因小鼠肝脏癌变作用的效果,研究真菌植物松茸提取物等药物可能的对HBV转基因小鼠组织、生化、免疫等方面的影响,进一步说明上述药物抑制HBV转基因小鼠肝脏癌变的作用机制。方法：按照标准随机选择56只HBV转基因小鼠C57BL/6J-TgN,随机分为真菌松茸提取物(AMH)组、α-干扰素(INF-α)组、α-干扰素联合AMH组、阴性对照组。每组14只,根据不同的给药方式预防性治疗12个月,其中,第11月经眼球内眦静脉取血1次,测血清AST。第12月末,每只小鼠称取体重,经摘眼球取血处死动物,待测血清标本。采用双抗体夹心法酶联免疫吸附试验(ELISA法)测定血清中肿瘤坏死因子(TNF-α)、白细胞介素-2(IL-2)、白细胞介素-10(IL-10)；切除小鼠肝脏,称取肝脏的重量,观察肝脏的形态及有无异常结节性改变,并观察肺、脾、肾和淋巴结有无肿瘤及转移灶,一部分肝脏组织保存于10%福尔马林溶液中,另一部分肝脏组织立即放入冻存管中保存于液态氮中存储。制作标本蜡块和病理切片,光镜下观察不同组肝组织结构变化,包括非癌变组织和癌变组织的变化。确定每组小鼠中发生肝组织癌变的具体数量,计算不同组中转基因小鼠肝组织癌变的百分数。肝组织做成切片进行免疫组化检查,显微镜下观察乙肝表面抗原(HBsAg)、转化生长因子β1(TGF-β1)、转化生长因子β1受体Ⅱ(TβRⅡ)、磷酸化Smad2/3 (P-Smad2/3)、Smad4、Smad7在肝脏组织中的表达。以出现棕黄色为阳性,进行结果分析。选取液态氮中存储的肝脏组织,行实时荧光定量PCR检测(Real-time fluorescent quantitative PCR, RT-PCR)结果：1)肝脏组织形态改变：大体形态肝脏增大、肿胀,肝脏肿瘤结节为灰白色的单个或多个结节。光镜下可见：肝细胞弥漫性肿胀,细胞胞质伊红均染(毛玻璃样变),核内可见嗜酸性小体,肝细胞坏死,伴淋巴细胞浸润,散在单个肝细胞质固缩深染伴核固缩深染。肿瘤内可见明显异常核分裂像,癌细胞向肝组织周围浸润。AMH组预防性用药后,上述表现程度上稍轻,癌变发生的百分数稍低,但不能完全防止全部转基因小鼠肝脏炎性改变及癌变的发生；2)血清学指标：经治疗干预后,AMH组(294±51)、α-干扰素组(231±59)和α-干扰素联合AMH组(215±64)的AST值均低于阴性对照组(322±45)(P<0.05)。α-干扰素组、α-干扰素联合AMH组AST值分别低于AMH组(P<0.05)。而α-干扰素组和α-干扰素联合AMH组比较,AST值差别无统计学意义(P>0.05)；α-干扰素加AMH组(58.43±14.42)、AMH组(25.37±8.36)、α-干扰素组(35.84±12.36)与阴性对照组(15.75±8.45)相比,血清IL-2有增高(P<0.05)；α-干扰素加AMH组与AMH组和α-干扰素组相比IL-2升高(P<0.05)；a-干扰素加AMH组(12.03±6.54)与阴性对照组(45.97±14.03)、AMH组(21.58±11.20)、α-干扰素组(18.74±9.36)相比,血清TNF-α有明显降低(P<0.01)；α-干扰素加AMH组(160.25±70.03)、AMH组(289.58±110.20)、α-干扰素组(240.69±95.36)与阴性对照组相比(375.03±120.48),血清IL-10值降低(P<0.05)；α-干扰素加AMH组与AMH组、α-干扰素组相比,血清IL-10值也有降低(P<0.05)；α-干扰素加AMH组中血清AST的水平与血清TNF-α水平呈负相关(F值6.529,P<0.05)；3)免疫组化与RT-PCR指标：经治疗干预后,AMH组(4.92±1.20)、α-干扰素组(4.57±1.09)和α-干扰素联合AMH组(2.86±0.94)肝组织中的TGF-β1的表达均低于阴性对照组(6.53±1.78)(P<0.05)。联合AMH组分别与AMH组、α-干扰素组比较,肝组织中TGF-β表达的变化均有统计学意义(P<0.05)；经治疗后,AMH组(3.51±1.78)、α-干扰素组(4.63±1.44)和α-干扰素联合AMH组(5.72±1.55)肝组织中Smad4的表达均高于阴性对照组(3.06±0.87)(P<0.05)。α-干扰素联合AMH组肝组织中Smad4的表达高于AMH组有统计学意义(P<0.05)；经治疗干预后,AMH组、α-干扰素组和α-干扰素联合AMH组肝组织中P-Smad2/3的表达均高于阴性对照组(P<0.05)。α-干扰素联合AMH组肝组织中Smad4的表达分别高于AMH组、α-干扰素组有统计学意义(P<0.05);经治疗干预后,AMH组(4.10±0.75)、α-干扰素组(4.57±0.93)和α-干扰素联合AMH组(5.38±1.25)肝组织中TβRⅡ的表达均明显高于阴性对照组(3.36±0.62)(P<0.01)；经治疗后,AMH组、α-干扰素组和α-干扰素联合AMH组肝组织中P-Smad2/3的表达均低于阴性对照组(P<0.05)。阴性对照组(5.38±2.21)、AMH组(5.19±1.91)、α-干扰素组(4.86±1.82)肝组织中Smad7的表达均高于α-干扰素联合AMH组(2.17±1.64)(P<0.05)。α-干扰素联合AMH组肝组织中TGF-β1mRNA的表达低于阴性对照组(P<0.05)。α-干扰素联合AMH组肝组织中TβRⅡmRNA的表达高于阴性对照组(P<0.05)。AMH组、α-干扰素组和α-干扰素联合AMH组肝组织中Smad2mRNA的表达均高于阴性对照组(P<0.05)。结论：1)HBV转基因C57BL/6J-TgN小鼠可以从HBV慢性感染状态发展到肝癌形成的过程,可以部分模拟人类的该病转归；2)应用HBV转基因C57BL/6J-TgN小鼠观察不同药物化学预防性治疗后,AMH可以明显改善肝脏功能,降低血清转氨酶水平,减缓癌变的发生；3)AMH可以诱导HBV转基因小鼠Thl类细胞因子作用增强,Th2类细胞因子作用降低,抑制恶性肿瘤的生成；4)AMH可能通过影响TGF-β/Smad信号通路的传导,降低肝癌的发生；5)AMH和α-干扰素的联用在预防肝癌发生中效果可能更强。更多还原

【Abstract】 Objective:Based on the established HBV transgenic mouse model with pine mushroom extract (AMH), and a-interferon were applicated for the preventional chemotherapy using HBV transgenic mice in different groups, which aimed at evaluating the effects of AMH andα-interferon for the prevention of HBV transgenic mice oncogenesis in liver tissues. The influence of histology, biochemical, immunological changes, and other implications were explored in the HBV transgenic mice with oral AMH anda-interferon. Further explanation of the possible mechanism in liver oncogenesis with those drugs was investigated by inhibiting the HBV transgenic mice models with HCC. Method:Forty-six HBV transgenic C57BL/6J-TgN mice were randomly divided into four groups as follows; 1) AMHgroup; 2) a-interferon group; 3) a-interferon combined with AMH group, and; 4) control groups. There were 14 mice for each group, according to the different methods for preventive purpose. The blood was taken from the angular vein of an eye in the 11th month. The serum samples were measured for aspartate aminotransferase (AST). At the end of 12 months, each mouse model was weighed, and blood collected for further analysis. After then, all the models were sacrefied, and the liver tissues were stored in -80℃cryopreservation. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was tested for those of serum tumor necrosis factor (TNF-a), interleukin -2 (IL-2), Interleukin -10 (IL-10). The liver tissue from each mouse was taken and weighted. And then, the morphological changed from abnormal liver nodules, lung, spleen, kidney and lymph node tumors and metastases were observed under the. Part of the liver tissue was preserved in 10% formalin, and the remained part of the liver tissue immediately kept into the freezing tube in liquid nitrogen storage. Hepatic tissue Structural changes of liver tissues were microscopily observed in the above four groups, including non-cancerous tissue and oncogenesis tissue. It was necessary to identify the each occurrence of liver cancer in those mouse models in a specific amount and calculate in different groups. Several important immunohistochemistry tests were made in those liver tissues for analysis such as:1) Hepatitis B surface antigen (HbsAg); 2) transforming growth factorβ1 (TGF-(31); 3) transforming growth factorβ1 receptorⅡ(TβRⅡ); 4) phosphorylated Smad2 (P-Smad2); 5) Smad4, and; 6) Smad7. The result were observed and diagnosed by the microscope. Selected liver tissue were analyzed by the real-time fluorescence quantitative PCR detection (RT-PCR) such as:1) TGF-β1mRNA; 2) TβRⅡmRNA; 3) Smad2 mRNA. Results:1) Morphological changes in liver tissue:increased general form of the liver, swelling, liver tumor nodules as the white single or multiple nodules. Light microscope:diffuse swelling of liver cells, both cytoplasmic eosin staining (ground glass-like change), the nuclear bodies seen in eosinophils, liver cell necrosis, with infiltration of lymphocytes, scattered in the cytoplasm of a single liver stained with pyknotic deeply stained nuclear condensation. Obvious abnormalities were seen in the tumor mitotic cancer cells infiltrating to the surrounding liver tissues. After a little light on the performance level AMH plus a-interferon group had slightly lower percentage of cancer occurrence. However, it may not completely prevented from hepatic inflammatory changes and the incidence of cancer; 2) serum analysis. AST values in a-interferon group plus AMH group (215±64), a-interferon group (231±59) and AMH (294±51) group were lower than that in negative control group (322±45) (P< 0.05). AST values inα-interferon group, a-interferon plus AMH group AST values were lower than that in AMH group (P< 0.05); Serum IL-2 levels in a-interferon plus AMH group (58.43±14.42), AMH alone group (25.37±8.36), a-interferon group (35.84±12.36) were higher than that in negative control group (15.75±8.45) (P<0.05), in addition, IL-2 levels in a-interferon plus AMH group, a-interferon was significantly higher than the AMH group (P<0.05); compared with a-interferon group, IL-2 levels in a-interferon was increased (P<0.05); TNF-a level in a-interferon plus AMH group (12.03±6.54), AMH group (21.58±11.20), and a-interferon group (18.74±9.36) was significantly decreased than that in negative control group (45.97±14.03) (P<0.01); Serum IL-10 levels in a-interferon plus AMH group (160.25±70.03), AMH group (289.58±110.20), a-interferon group (240.69±95.36) compared with the negative control group (375.03±120.48) had decreased (P<0.05); serum IL-10 levels in a-interferon plus AMH group was lower than that in AMH group, a-interferon group (P<0.05); 3) Immunohistochemistry and RT-PCR analysis. AMH group (4.92±1.20), a-interferon group (4.57±1.09) and a-interferon plus AMH group (2.86±0.94) of liver tissue expression of TGF-β1 were lower than that in the negative control group (6.53±1.78) (P<0.05). In AMH, AMH group plus a-interferon group, and a-interferon group, the liver tissue expression of TGF-β1 showed significant changes (P<0.05); AMH group (3.51±1.78), a-interferon group (4.63±1.44) and AMH plus a-interferon group (5.72±1.55) in combination with Smad4 expression in liver tissues were higher than that in the negative control group (3.06±0.87) (P<0.05). AMH plus a-interferon group in combination with Smad4 expression in liver tissue was significantly higher than that in the AMH group (P< 0.05); AMH group (4.10±0.75), a-interferon group (4.57±0.93) and a-interferon plus AMH (5.38±1.25) in liver tissue P-Smad 2/3 expression was higher than that in the negative control group (3.36±0.62) (P<0.05). AMH plus a-interferon group in combination with P-Smad2/3 expression in liver tissues were higher than that in the AMH group, a-interferon group was statistically significant (P<0.05); AMH group (5.20±1.68), a-interferon group (5.49±1.85) and the a-interference plus AMH group (8.13±2.46) liver tissue expression of TβRⅡwere significantly higher than that in the negative control group (4.97±1.52) (P<0.01); AMH group, a-interferon group and a-interferon plus AMH group of liver tissue P-Smad2/3 were significantly lower than that in the negative control group (P<0.05). AMH group (5.19±1.91), a-interferon group (4.86±1.82) and negative control group (5.38±2.21) of liver tissue expression of Smad7 were higher than that in the a-interferon plus AMH group (2.17±1.64) (P< 0.05). The expression of TGF-β1mRNA in a-interferon plus AMH group was lower than that in the negative control group (P<0.05). AMH group plus a-interferon group with TβRⅡmRNA expression was higher than that in the negative control group (P< 0.05). AMH group, a-interferon group and a-interferon plus AMH group Smad2 expression were higher than that in the negative control group (P<0.05). Conclusion: 1) The process of HBV transgenic mice developing from chronic infection status to hepatocellular carcinoma formation can be in part to simulate that in humans; 2)AMH group in treatment of hepatitis B preventive C57BL/6J-TgNtransgenic mice could improve liver function and reduce levels of serum AST. It may inhibit the HBV virus detention in the liver cells, reduce the degeneration and necrosis of liver cells and slow the occurrence of oncogenesis; 3) AMH may increase the Thl-type cytokines levels and decreas the Th2 type cytokine levels in order to inhibit tumor formation; 4) AMH may reduce the incidence of hepatic cancer through influencing the TGF-β/Smad signal pathway transmission; 5) AMH plus a-interferon group showed better effective for the prevention of liver cancer in those animal models.更多还原