PDF下载 ( 2677 KB)
扶正化瘀方通过改变急性肝损伤小鼠模型肝脏CD8+T淋巴细胞表型功能预防肝纤维化的价值分析
DOI: 10.3969/j.issn.1001-5256.2022.02.017
Value of Fuzheng Huayu prescription in preventing liver fibrosis by altering the phenotypic function of CD8+ T lymphocytes in the liver of mice with acute liver injury
-
摘要:
目的 观察应用扶正化瘀方后急性肝损伤小鼠模型肝脏CD8+ T淋巴细胞对共培养的肝星状细胞的影响，探讨扶正化瘀方预防肝纤维化的作用机制。 方法 18只SPF级雄性C57BL/6NCrl Vr小鼠随机分为正常组、模型组、扶正化瘀方组，每组6只。扶正化瘀方组提前给予扶正化瘀方灌胃5 d。实验前12 h以2 mL/kg体质量的剂量腹腔注射10%的CCl4，腹主静脉取血，留取血清并检测ALT、AST，留取部分肝脏做病理观察；应用流式细胞术分选小鼠肝脏CD8+ T淋巴细胞，预先小鼠体内用药，再与小鼠肝星状细胞株(JS 1)以2∶ 1比例共培养于96孔板中24 h、48 h，使用qPCR方法检测各组Col.Ⅰ mRNA和α-SMA mRNA表达变化。符合正态分布的计量资料多组间比较采用方差分析，进一步两两比较采用SNK-q或LSD-t检验。 结果 模型组的ALT和AST水平均显著高于正常组(P值均＜0.000 1)。扶正化瘀方组小鼠的ALT升高的幅度明显小于模型组(P＜0.001)。HE染色显示，扶正化瘀方组小鼠肝细胞变性、坏死程度较模型组有所减轻。与正常组比较, 模型组总淋巴细胞、CD45、CD4-CD8+T、CD8+CD28-T显著上升, 而扶正化瘀方组与模型组相比较, 上述淋巴细胞比例显著降低(P值均＜0.001)。自各组小鼠肝脏分离的CD8+ T淋巴细胞与JS 1共培养48 h，模型组与对照组(JS 1单培养)和正常组相比α-SMA mRNA的表达明显增加(P值均＜0.01)；Col.Ⅰ mRNA表达也明显高于对照组和正常组(正常小鼠肝脏CD8+ T淋巴细胞与JS 1共培养)(P值均＜0.001)。扶正化瘀方组α-SMA mRNA和Col.Ⅰ mRNA表达显著低于模型组(P值均＜0.01)。 结论 扶正化瘀方能通过改变小鼠肝脏CD8+ T淋巴细胞功能表型间接抑制活化的肝星状细胞。 -
关键词:
- 肝硬化 /
- 扶正化瘀方 /
- 肝星状细胞 /
- CD8阳性T淋巴细胞
Abstract:Objective To investigate the effect of liver CD8+ T lymphocytes on co-cultured hepatic stellate cells (HSCs) after the application of Fuzheng Huayu prescription in a moues model of acute liver injury, as well as the mechanism of action of Fuzheng Huayu prescription in preventing liver fibrosis. Methods A total of 18 specific pathogen-free male C57BL/6NCrl Vr mice were randomly divided into normal group, model group, and Fuzheng Huayu prescription group, with 6 mice in each group. The mice in the Fuzheng Huayu prescription group were given Fuzheng Huayu prescription for 5 days in advance. At 12 hours before the experiment, 10% CCl4 was injected intraperitoneally at a dose of 2 mL/kg body weight. Blood was collected from the main abdominal vein, and the serum was separated to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Part of the liver was used for pathological observation. After the mice were pretreated with medication in vivo, flow cytometry was used for the sorting of mouse liver CD8+ T lymphocytes, which were then co-cultured with the mouse HSC cell line (JS 1) in a 96-well plate at a ratio of 2∶ 1, and after co-culture for 24 and 48 hours, qPCR was used to measure the changes in the mRNA expression of Col.I and α-SMA. An analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK-q test or the least significant difference t-test was used for further comparison between two groups. Results The model group had significantly higher activities of ALT and AST than the normal group (both P < 0.000 1), and compared with the model group, the Fuzheng Huayu prescription group had a significantly lower degree of increase in ALT activity (P < 0.001). HE staining showed that the Fuzheng Huayu prescription group had a significantly lower degree of hepatocyte degeneration and necrosis compared with the model group. Compared with the normal group, the total lymphocytes, CD45, CD4-CD8+ T and CD8 + CD28-T in the model group increased significantly, while the proportion of the above lymphocytes in the Fuzheng Huayu formula group decreased significantly compared with the model group (P < 0.001). CD8+ T lymphocytes isolated from the liver of mice in each group were co-cultured with JS 1 for 48 hours, and compared with the control group (JS 1 cultured alone) and the normal group, the model group had a significant increase in the mRNA expression of α-SMA (both P < 0.01) and significantly higher mRNA expression of Col.I than the control group and the normal group (normal mouse liver CD8+ T lymphocytes co-cultured with JS 1) (both P < 0.001). The Fuzheng Huayu prescription group had significantly lower mRNA expression levels of α-SMA and Col.I than the model group (both P < 0.01). Conclusion Fuzheng Huayu prescription can indirectly inhibit activated HSCs by altering the functional phenotype of CD8+ T lymphocytes in mouse liver. -
临床上，胰腺恶性肿瘤大部分为原发肿瘤，胰腺转移癌少见[1]。胰腺转移癌与胰腺原发肿瘤鉴别困难，极易造成误诊，延误治疗。目前，胰腺转移癌国内外尚无统一治疗标准，原发肿瘤的生物学特性和针对原发肿瘤的综合治疗是决定其预后的主要因素，个体化差异较大。近期，笔者接诊1例宫颈鳞癌胰腺转移患者，现将病例资料及经验总结报告如下。
1. 病例资料
患者女性，63岁，因“皮肤、巩膜黄染20余天”于2021年3月27日入本院。起初黄染程度不重，后进行性加重，无腹痛腹胀，无阴道流血等其他不适。曾于2013年9月6日因阴道异常流血于外院行宫颈活检，病理学诊断为宫颈中分化鳞癌ⅢB期，予局部放疗联合TP方案(多西他赛+奥沙利铂)化疗，于2015年结束治疗后复查腹盆腔脏器、腹腔、盆腔均未见肿瘤转移。出院后患者再未复查，亦无特殊不适。入院查体：皮肤巩膜明显黄染，腹平坦，腹部无压痛，腹部未触及肿块。辅助检查：TBil 282.99 μmol/L，DBil 203.80 μmol/L，CA19-9正常；妇科彩超示：老年性子宫；全腹盆腔增强CT示：肝内外胆管扩张，主胰管可见(图 1a)，胆囊明显增大，胆总管下段狭窄，动脉期见胰腺钩突区低密度肿块(图 1b)，静脉期肿块轻度强化(图 1c)，子宫显示不明显，腹膜后可见多发淋巴结肿大；磁共振胰胆管造影示：胰头区异常信号肿块，肝内外胆管扩张，胆汁淤积；超声内镜示：胰头钩突区占位，直径约3 cm。入院后予以护肝利胆治疗，患者黄疸进行性加重，建议患者行穿刺活检、PET-CT检查，患方拒绝。2021年4月4日行剖腹探查：术中探查发现肿瘤位于胰头钩突区，质地硬，侵犯十二指肠壁和胆总管，腹膜后见多发淋巴结肿大，行胰头十二指肠切除术。术后病理学检查结果：胰腺钩突肿块大小3.0 cm×2.0 cm×1.5 cm，癌组织侵犯十二指肠及胆总管，胰腺切缘未见癌。癌组织呈团块状或条索状癌巢(图 2a)，癌细胞及核大小、形态不一，分布不规则，核分裂像多见，癌组织侵犯淋巴管及血管，脉管内见癌栓(图 2b)，周围淋巴结可见癌转移(2/2)。免疫组化：P16(弥漫性+)，P63(+)，P40(+)，CK7(+)，CK8(+)，CK19(+)，ER(+)(图 2c、d)。诊断考虑胰腺钩突区中分化鳞状细胞癌，结合临床病史及免疫组化结果，考虑宫颈鳞癌转移。目前，该患者于本院进一步针对原发肿瘤行化疗等综合治疗，日常生活行为已恢复正常。
图 2 术后病理学检查结果注：a，低倍镜下见癌细胞成团块状或条索状不规则排列(HE染色，×100)；b，高倍镜下见脉管侵犯(HE染色，×400)；c，P16弥漫性阳性表达(免疫组化SP法，×400)；d，P40表达阳性(免疫组化SP法，×400)。2. 讨论
据统计，宫颈癌的患病/病死率在女性恶性肿瘤中居第4位，鳞状细胞癌是最常见的病理类型，占比约80%，P16、P40等表达阳性对宫颈鳞癌诊断有重要意义。宫颈鳞癌早期往往无特殊表现，晚期可出现异常阴道流血、腰痛、盆腔痛、性交痛、贫血等表现，诊断时大多发生转移，多转移至直肠、膀胱、盆腔、肺、骨、肝等。临床上，胰腺癌多为原发肿瘤，胰腺转移癌罕见，仅占胰腺恶性肿瘤总数的2%~3%[2]。在一项纳入973例胰腺肿瘤手术标本病理资料的研究[3]中，共38例为胰腺转移瘤，主要包括淋巴瘤11例，胃癌7例，肾癌6例，肺癌2例，肝癌、前列腺癌、卵巢癌、子宫癌各1例，默克尔细胞癌1例，另有3例胃肠道恶性间质瘤和1例腹膜后平滑肌肉瘤；81例转移性胰腺肿瘤尸检报告显示，主要来源肺癌34例，胃肠道20例，肾脏4例，乳腺3例，肝脏2例，卵巢和膀胱各1例，另有6例来自造血系统，黑色素瘤、肉瘤和间皮瘤各2例。来源于宫颈的胰腺转移癌并不多见，笔者在国内外文献检索系统中，共检出各类型宫颈癌胰腺转移病例报告10例[4-7]，其中鳞癌3例，神经内分泌癌5例，腺癌2例；转移灶位于胰头3例，位于颈部1例，位于胰腺体部3例，位于尾部、体尾部和胰头及体尾部各1例。转移灶位于胰头的3例患者中，1例为鳞癌，2例为神经内分泌癌，胰腺转移部位与宫颈癌病理类型无明显关联。本例患者在宫颈鳞癌同步放化疗后再未复查，6年后转移至胰腺钩突，临床表现为梗阻性黄疸，行胰头十二指肠切除术，术后病理学检查考虑鳞癌，结合免疫组化考虑为宫颈癌来源。目前，国内尚未见类似报道，国外也鲜有相关报道。
原发性胰腺癌与胰腺转移癌鉴别需依赖血清CA19-9水平、CT、MRI、磁共振胰胆管造影、内镜逆行胰胆管造影、超声内镜等检查手段。本例患者CT和MRI特点较原发性胰腺癌无特异表现，鉴别困难。血清CA19-9水平升高常见于胆道、胰腺恶性肿瘤，对诊断原发性胰腺癌有较高的敏感度和特异度[8]。鉴别困难时，可采用通过超声、CT及内镜引导下穿刺活检获取胰腺组织学、细胞学标本进行确诊，但需要评估穿刺活检的出血风险[9]。一般认为，超声内镜经十二指肠细针穿刺抽吸活检可提高胰腺疾病诊断的准确性，且相对安全[10-11]。本例患者术前影像学检查提示胰头钩突区占位，CA19-9水平不高，且有宫颈鳞癌病史，建议进一步行穿刺活检、PET-CT检查。但患者存在梗阻性黄疸，肝功能持续恶化，有手术指征，且患者家属不同意术前行穿刺活检、PET-CT等进一步检查，因此术前未能获得病理学诊断。
转移性胰腺癌临床表现因胰腺转移部位不同而异。在笔者检出的10例报告中，仅有1例(鳞癌)出现梗阻性黄疸，提示宫颈癌胰腺转移鲜有梗阻性黄疸症状，部分病例可因肿瘤压迫出现胆胰管扩张。转移性胰腺癌出现胰胆管梗阻较少见，可能与原发肿瘤主要经过淋巴及血行途径转移侵犯胰腺，不侵犯胆胰管有关[12]。本例患者胰腺转移性肿瘤致梗阻性黄疸，目前国内尚未见类似报道，国外仅有1例类似报道，但未分析相关转移机制。本例患者影像学检查示肝、胃、肠道等器官均未见肿瘤，但有腹膜后淋巴结肿大，病理学检查阳性；在既往10例报告中，3例胰腺转移性宫颈鳞癌有2例伴腹膜后淋巴结肿大，因此笔者推测宫颈鳞癌胰腺转移可能与腹膜后淋巴结侵犯相关。
关于转移性胰腺癌是否需要手术，意见尚未统一。以急腹症、进行性黄疸、出血为临床表现患者，应行急诊手术治疗，解除症状同时，切除病灶送检，根据病理学检查结果制订综合治疗方案。研究[13-14]表明，转移性胰腺癌行手术切除相较于保守治疗可延长生存期，且根治性手术相较于姑息性手术，预后更好。术后根据原发病灶辅以化疗等综合治疗，可提高治疗效果。对无法接受手术的转移性胰腺癌患者，应根据原发肿瘤的生物学特性制订以放疗为主的个体化综合治疗方案[15]。本例患者影像学检查提示存在胆道梗阻，并见胰头钩突区占位；胆红素水平持续升高，手术指征明确，手术切除胰腺钩突病灶、解除胆道梗阻后，胆红素水平明显下降，目前患者恢复良好，进一步接受化疗等综合治疗，延长生存期。
笔者经验总结：(1)对于诊断不明确的胰腺肿瘤，术前条件允许情况下需行穿刺活检明确性质，以指导下一步治疗方案；(2)胰腺转移癌在具备手术条件情况下，应积极采取以根治性手术为主的治疗方案，姑息性切除方案亦可行。胰腺转移癌为晚期癌症，术后需早期予以放化疗、靶向、免疫治疗等综合治疗；(3)对于不具备手术条件的患者，需根据原发肿瘤予以抗肿瘤综合治疗，包括放疗、化疗以及免疫治疗、靶向治疗等[16-17]。
-
表 1 qPCR引物列表
Cat.No. TM值 名称 上游引物 下游引物 Tnt0338 85.1 GAPDH 5′AAA TGG TGA AGG TCG GTG TG 3′ 5′AGG TCA ATG AAG GGG TCG TT 3′ Tnt1328 81.1 α-SMA 5′CAC ACA CCT TTT AGA ACC CA 3′ 5′GTT TTC CCC CTT CCT CTT ACT 3′ Tnt1680a 85.9 Col.Ⅰ 5′TGA CTG GAA GAG CGG AGA GTA 3′ 5′GAC GGC TGA GTA GGG AAC AC 3′ 
下载: 导出CSV
表 2 各组肝脏相关细胞占总淋巴细胞比例
分组 动物数(只) 总淋巴细胞(个/mL) CD45(%) CD4-CD8+T(%) CD8+CD28-T(%) 正常组 6 8.62×106±0.425×106 40.20±1.31 3.96±0.152 3.73±0.251 模型组 6 2.53×107±0.157×107 58.80±1.90 1.37±0.152 1.27±0.153 扶正化瘀方组 6 1.69×107±0.447×107 29.60±1.43 1.63±0.115 1.67±0.152 F值 316.846 265.604 260.143 143.394 P值 ＜0.001 ＜0.001 ＜0.001 ＜0.001
下载: 导出CSV
-
[1] DING BS, CAO Z, LIS R, et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis[J]. Nature, 2014, 505(7481): 97-102. DOI: 10.1038/nature12681. [2] CAMPANA L, IREDALE JP. Regression of liver fibrosis[J]. Semin Liver Dis, 2017, 37(1): 1-10. DOI: 10.1055/s-0036-1597816. [3] IRSHAD M, GUPTA P, IRSHAD K. Immunopathogenesis of liver injury during hepatitis C virus infection[J]. Viral Immunol, 2019, 32(3): 112-120. DOI: 10.1089/vim.2018.0124. [4] XING YM. Current status and prevention of chronic hepatitis B infection[J]. J World Latest Med Inf, 2017, 69: 174-175. DOI: 10.19613/j.cnki.1671-3141.2017.69.130.邢亚明. 慢性乙型肝炎感染现状及预防[J]. 世界最新医学信息文摘, 2017, 69: 174-175. DOI: 10.19613/j.cnki.1671-3141.2017.69.130. [5] LIU CH, LIU P, LIU C, et al. Discussion on serum pharmacological methods of effective Chinese herbal compound prescriptions for anti-liver fibrosis[J]. Chin J Exp Med Formul, 1998, 2: 16-19. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX802.006.htm刘成海, 刘平, 刘成, 等. 抗肝纤维化有效中药复方血清药理学方法探讨[J]. 中国实验方剂学杂志, 1998, 2: 16-19. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX802.006.htm [6] KOYAMA Y, BRENNER DA. Liver inflammation and fibrosis[J]. J Clin Invest, 2017, 127(1): 55-64. DOI: 10.1172/JCI88881. [7] NOTAS G, KISSELEVA T, BRENNER D. NK and NKT cells in liver injury and fibrosis[J]. Clin Immunol, 2009, 130(1): 16-26. DOI: 10.1016/j.clim.2008.08.008. [8] SCHUSTER S, CABRERA D, ARRESE M, et al. Triggering and resolution of inflammation in NASH[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(6): 349-364. DOI: 10.1038/s41575-018-0009-6. [9] ARRESE M, CABRERA D, KALERGIS AM, et al. Innate immunity and inflammation in NAFLD/NASH[J]. Dig Dis Sci, 2016, 61(5): 1294-1303. DOI: 10.1007/s10620-016-4049-x. [10] JENNE CN, KUBES P. Immune surveillance by the liver[J]. Nat Immunol, 2013, 14(10): 996-1006. DOI: 10.1038/ni.2691. [11] CHEN Y, OU Y, DONG J, et al. Osteopontin promotes collagen I synthesis in hepatic stellate cells by miRNA-129-5p inhibition[J]. Exp Cell Res, 2018, 362(2): 343-348. DOI: 10.1016/j.yexcr.2017.11.035. [12] WU P, TAN XY. The effect of Fuzheng Huayu capsule on immine regulation and anti-fibrosis in hepatic C virus infected patients with decompensated cirrhosis[J]. Chin J Integr Trad West Med Dig, 2016, 24(9): 658-660. DOI: 10.3969/j.issn.1671-038X.2016.09.02.吴平, 谭小燕. 扶正化瘀胶囊对丙型肝炎失代偿期肝硬化患者免疫调节以及抗纤维化作用研究[J]. 中国中西医结合消化杂志, 2016, 24(9): 658-660. DOI: 10.3969/j.issn.1671-038X.2016.09.02. [13] CHEN H, YANG BW, YUAN M, et al. Prevention and therapeutic effects of Fuzheng Huayu capsule on liver fibrosis and expression of connective tissue growth factor in rats[J]. J Sichuan Univ (Med Sci Edi), 2016, 47(2): 197-202. https://www.cnki.com.cn/Article/CJFDTOTAL-HXYK201602012.htm陈晗, 杨碧伟, 袁满, 等. 扶正化瘀胶囊对肝纤维化大鼠的防治作用及对结缔组织生长因子表达的影响[J]. 四川大学学报(医学版), 2016, 47(2): 197-202. https://www.cnki.com.cn/Article/CJFDTOTAL-HXYK201602012.htm [14] WANG J. The effect of Fuzheng Huayu capsule assisting western medicine on intestinal mucosal barrier function, immune function and liver function in patients with hepatitis B liver cirrhosis[J]. Mod J Integr Tradit Chin West Med, 2017, 26(27): 3051-3053. DOI: 10.3969/j.issn.1008-8849.2017.27.031.王军. 扶正化瘀胶囊辅助西医对乙肝肝硬化患者肠黏膜屏障功能、免疫功能及肝功能状态的影响[J]. 现代中西医结合杂志, 2017, 26(27): 3051-3053. DOI: 10.3969/j.issn.1008-8849.2017.27.031. [15] XU HY, YUN CX, WANG YX. The effects of PSP on the subset of T cell and apoptosis of the tumo-bearing mice[J]. J Qiqihar Med Coll, 2004, 25(5): 485-487. DOI: 10.3969/j.issn.1002-1256.2004.05.003.许惠玉, 运晨霞, 王雅贤. 桃仁总蛋白对荷瘤鼠T淋巴细胞亚群及细胞凋亡的影响[J]. 齐齐哈尔医学院学报, 2004, 25(5): 485-487. DOI: 10.3969/j.issn.1002-1256.2004.05.003. [16] MA YL. Study on the immune regulation mechanism of Fuzheng Huayu recipe against liver fibrosis based on the changes of CD8+ T lymphocyte subsets[D]. ShangHai: ShangHai University of Traditional Chinese Medicine, 2017.马亚丽. 基于CD8+T淋巴细胞亚群的变化探讨扶正化瘀方抗肝纤维化的免疫调节机制[D]. 上海: 上海中医药大学, 2017. [17] SPAHN J, PIERCE RH, CRISPE IN. Ineffective CD8+ T-cell immunity to adeno-associated virus can result in prolonged liver injury and fibrogenesis[J]. Am J Pathol, 2011, 179(5): 2370-2381. DOI: 10.1016/j.ajpath.2011.08.004. [18] XIE M, JIANG Q, ZHAO S, et al. Prognostic value of tissue-infiltrating immune cells in tumor microenvironment of follicular lymphoma: A Meta-analysis[J]. Int Immunopharmacol, 2020, 85: 106684. DOI: 10.1016/j.intimp.2020.106684. [19] KLAMETHA, NEUBAUER A, KELLER C, et al. Aberrant CD3-positive, CD8-low, CD7-negative lymphocytes may appear during viral infections and mimic peripheral T-cell lymphoma[J]. Diagnostics, 2020, 10(4): 204. DOI: 10.3390/diagnostics10040204. [20] ZHANG PX, BIAN PY, YE CT, et al. Changes in senescence-and function-related parameters of CD8+T cells after direct-acting antiviral treatment in patients with hepatitis C virus infection[J]. J Clin Hepatol, 2020, 36(7): 1496-1501. DOI: 10.3969/j.issn.1001-5256.2020.07.011.张沛欣, 边培育, 叶传涛, 等. HCV感染者直接抗病毒药物治疗前后CD8+T淋巴细胞衰老和功能相关指标的变化[J]. 临床肝胆病杂志, 2020, 36(7): 1496-1501. DOI: 10.3969/j.issn.1001-5256.2020.07.011. 期刊类型引用(1)
1. 李帅，王娟，夏仕雪，顾鹏. 子宫颈混合性腺神经内分泌癌胰腺转移1例. 中国医学影像学杂志. 2022(12): 1283-1284 . 
百度学术其他类型引用(0)
-
下载:
百度学术
