1、 521 CHINESE JOURNAL OF ANATOMY Vol.45 No.6 2022 解剖学杂志 2022 年第 45 卷第 6 期doi：10.3969/j.issn.1001-1633.2022.06.006论 著富马酸替诺福韦二吡呋酯联合聚乙二醇化干扰素对肝硬化大鼠 肝细胞凋亡、免疫功能及内毒素水平的影响*闪海霞 卢瑞杰 颜成果 王 英 和青森 范崇桂（南阳市中心医院感染性疾病科，南阳 473000）摘要 目的：目的：研究富马酸替诺福韦二吡呋酯（TDF）联合聚乙二醇化干扰素-2b（PEG-IFN-2b）对肝硬化（代偿期）大鼠肝细胞凋亡、免疫功能及内毒素水平的影响。方法：方法：
2、大鼠随机分为对照组（健康大鼠常规饲养）、模型组（肝硬化模型组）、TDF治疗组（肝硬化模型+TDF）、干扰素治疗组（肝硬化模型+PEG-IFN-1b）、联合治疗组（肝硬化模型+TDF+PEG-IFN-2b）。H-E染色观察肠、肝病理形态。脱氧核苷酸末端转移酶介导缺口末端标记法测细胞凋亡指数（AI）。流式细胞术测免疫功能。鲨试剂显色基质法测内毒素。免疫组织化学检测大鼠肝组织Bax、Bcl-2蛋白表达。结果：结果：模型组大鼠肝小叶结构破坏，肝细胞排列紊乱，可见片状坏死灶，有明显炎性细胞浸润。与模型组肝组织病变相比，TDF治疗组、干扰素治疗组与联合治疗组肝组织结构较为完整，肝细胞坏死减少，汇管区炎性细
3、胞浸润也较模型组减轻，且以联合治疗组效果较为显著。模型组大鼠回肠黏膜上皮细胞变性、坏死，大量绒毛脱落，上皮下囊状间隙扩大，中央乳糜管扩张，固有层裸露，并伴有毛细血管扩张、充血；与模型组相比，TDF治疗组、干扰素治疗组、联合治疗组均有所改善，且以联合治疗组效果明显。与对照组相比，模型组大鼠内毒素、肝细胞凋亡、CD8+T淋巴细胞数、Bax升高，Bcl-2、CD3+T淋巴细胞数、CD4+T淋巴细胞数、CD4+/CD8+降低。与联合治疗组相比，TDF治疗组与干扰素治疗组内毒素、肝细胞凋亡、CD8+T淋巴细胞数、Bax降低，Bcl-2、CD3+T淋巴细胞数、CD4+T淋巴细胞数、CD4+/CD8+升高。
4、与干扰素治疗组相比，联合治疗组内毒素、肝细胞凋亡、CD8+T淋巴细胞数、Bax降低，Bcl-2、CD3+T淋巴细胞数、CD4+T淋巴细胞数、CD4+/CD8+升高。结论：结论：TDF联合IFN-1b通过提高肝硬化（代偿期）大鼠免疫功能，降低内毒素水平以及减少肝细胞凋亡，改善肝硬化程度。关键词 富马酸替诺福韦二吡呋酯；聚乙二醇化干扰素；肝硬化；肝细胞；免疫功能；内毒素Effects of tenofovir dipyoproxil fumarate combined with pegylated interferon on hepatocyte apoptosis,immune function
5、 and endotoxin levels in cirrhotic rats*Shan Haixia，Lu Ruijie，Yan Chengguo，Wang Ying，He Qingsen，Fan Chonggui（Infectious Diseases Department，Nanyang Central Hospital，Nanyang 473000，China）Abstract Objective：To study the effects of tenofovir disoproxil fumarate（TDF）combined with pegylated IFN-2b（PEG-IF
6、N-2b）on liver cell apoptosis，immune function，and endotoxin levels in cirrhotic rats.Methods：Rats were randomly divided into a control group（normal feeding），a model group（liver cirrhosis model group），a TDF treatment group（liver cirrhosis model+TDF），a interferon treatment group（liver cirrhosis model+P
7、EG-IFN-1b），and a combination treatment group（liver cirrhosis model+TDF+PEG-IFN-2b）.H-E staining was used to observe the morphology of intestinal and liver diseases.Apoptosis was measured by deoxynucleotide terminal transferase mediated Nick end labeling.Immune function was measured by flow cytometry
8、.Detection of endotoxin by shark reagent chromogenic matrix.Bax and Bcl-2 were detected by immunohistochemistry.Results：In the model group，the hepatic lobule structure of rats was damaged，the arrangement of hepatocytes was disordered，there were patellar necrosis foci and obvious inflammatory cell in
9、filtration.Compared with the model group，the liver tissue structure of the TDF treatment group，interferon treatment group and combination treatment group was more complete，the necrosis of hepatocytes was reduced，and the infiltration of inflammatory cells in the portal area was also reduced，and the e
10、ffect of the combination treatment group was more significant.In the model group，the ileum*河南省卫健委医学科技公关计划联合共建项目(LHGJ20200905)第 1 作者 E-mail：收稿日期：2021-07-06；修回日期：2022-04-22mucosal epithelial cells were denaturated and necrotic，a large number of villi were shed，the subepithelial cystic space was expand
11、ed，the central chylus duct 522 was dilated，the lamina propria was exposed，accompanied by telangiectasia and congestion.Compared with the model group，the TDF treatment group，interferon treatment group and combination treatment group were improved，and the combination treatment group had a significant
12、effect.Compared with the control group，the levels of endotoxin，hepatocyte apoptosis，CD8+T lymphocyte and Bax were increased in the model group，while the levels of Bcl-2，CD3+T lymphocyte，CD4+T lymphocyte and CD4+/CD8+were decreased.Compared with the combined treatment group，endotoxin，hepatocyte apopt
13、osis，CD8+and Bax were decreased，while Bcl-2，CD3+T lymphocyte，CD4+T lymphocyte and CD4+/CD8+were increased in the TDF treatment group and interferon treatment group.Compared with interferon treatment group，endotoxin，hepatocyte apoptosis，CD8+T lymphocyte and Bax were decreased，while Bcl-2，CD3+T lympho
14、cyte，CD4+T lymphocyte and CD4+/CD8+were increased in combination treatment group.Conclusion：TDF combined with IFN-2b can improve the degree of liver cirrhosis by improving immune function，reducing endotoxin levels，and reducing hepatocyte apoptosis.Key words tenofovir disoproxil fumarate；pegylated in
15、terferon；liver cirrhosis；liver cells；immune function；endotoxin在国内，肝硬化以乙型肝炎病毒为主1。该病早期无特异性特征，不易发现，一经确诊，多数已为晚期，治疗难度大，并伴有多种并发症产生2-3。肝硬化产生后会使肠道内的细菌过度生长，导致内毒素增加，加之肠黏膜机械屏障、免疫及生物屏障等功能破坏，内毒素吸收增多，肝对内毒素清除能力下降，从而加剧肝损伤4。因此改善肠黏膜损伤对阻止肝硬化发展具有一定的作用。目前，干扰素被广泛应用于慢性乙肝的治疗中，且效果较为显著，它具有抗病毒和免疫调节的双重作用，但其疗效有限，众多学者认为多种有效的抗病毒药
16、的有效组合，是治疗慢性乙型肝炎的重要方案5。富马酸替诺福韦二吡呋酯（tenofovir disoproxil fumarate，TDF）是抗病毒药物的一种，常用于治疗慢性乙肝病毒6。因此采用TDF联合聚乙二醇化干扰素-2b（PEG-IFN-2b）对肝硬化（代偿期）大鼠肝细胞凋亡、免疫功能及内毒素水平的影响进行研究。1 材料和方法1.1 实验动物与分组1.1 实验动物与分组SPF级健康SD大鼠55只，体质量220 300 g，由长沙天勤公司提供生产许可证号：SCXK（湘）-2019-0001，常规饲养，自由取食、水。随机分为对照组（健康大鼠常规饲养）、模型组（肝硬化模型组）、TDF治疗组（肝硬化模型+TDF）、干扰素治疗组（肝硬化模型+PEG-IFN-2b）、联合治疗组（肝硬化模型+TDF+PEG-IFN-2b），每组11只。1.2 实验试剂1.2 实验试剂TDF（成都倍特公司）；PEG-IFN-2b（厦门特宝生物工程股份有限公司）；核苷酸混合缓冲液（珠海泛海公司）；无热源肝素钠抗凝真空采血管（苏州灵岩公司）；流式细胞仪（常州必达公司）；枸橼酸修复液（南京巴傲得公司）；乌拉坦（武汉梦奇公