2型糖尿病胰腺癌裸鼠模型的建立及活体成像观察
DOI: 10.12449/JCH240625
Establishment and in vivo imaging observation of a nude mouse model of type 2 diabetes mellitus and pancreatic cancer
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摘要:
目的 建立可动态观察成瘤过程并进行体内研究的2型糖尿病(T2DM)胰腺癌裸鼠模型。 方法 首先,通过慢病毒载体GV260转染人胰腺癌细胞(PANC-1细胞)构建能稳定表达萤火虫荧光素酶的胰腺癌细胞株(PANC-1-Luc细胞)。然后,将36只SPF级裸鼠随机分为对照组(n=12,血糖正常的胰腺癌裸鼠)和模型组(n=24,T2DM胰腺癌裸鼠)。对照组:先给予繁殖饲料喂养,之后将PANC-1-Luc细胞异位种植于裸鼠皮下;模型组:先给予高脂饲料喂养联合腹腔注射1% STZ,之后将PANC-1-Luc细胞异位种植于裸鼠皮下。用荧光活体成像系统和人工测量法同步动态监测2组裸鼠胰腺癌生长情况,绘制肿瘤生长曲线、分析荧光值与肿瘤体积的关系。显微镜下观察裸鼠皮下肿瘤及胰岛,验证造模是否成功;同时,通过免疫组化检测肿瘤组织Ki-67的表达来分析高血糖对裸鼠胰腺癌生长的影响。正态分布计量资料组间比较采用成组t检验,非正态分布计量资料组间比较采用Mann-Whitney U检验。 结果 确定PANC-1细胞慢病毒载体稳定转染的最佳病毒滴度为5×107 TU/mL,用嘌呤霉素筛选的最佳浓度为20 μg/mL、最佳筛选时间为9天;PANC-1-Luc细胞的荧光值与细胞数量呈线性正相关,线性方程为y=42.56x-42 504(r=0.977,P=0.004)。T2DM裸鼠模型血糖值为23.05(19.25~26.40)mmol/L,且每只裸鼠的血糖均高于11.1 mmol/L,与对照组裸鼠血糖值[6.15(5.20~7.30)mmol/L]相比,差异有统计学意义(Z=-8.45,P<0.001)。与对照组相比,模型组胰腺组织内胰岛数量减少、体积减小、形状不规则、边界模糊,同时移植瘤病理学检查确认镜下为胰腺癌组织,可判定T2DM裸鼠胰腺癌模型造模成功。模型组皮下肿瘤大小与荧光值呈线性正相关,线性方程为y=232 348 691x-8 258 608(r=0.911,P=0.031);模型组移植瘤Ki-67免疫组化阳性率显著高于对照组[(50.333±7.808)% vs (15.917±4.055)%,t=13.55,P<0.001],说明模型组肿瘤增殖较快。 结论 本研究所构建的T2DM裸鼠胰腺癌模型可模拟T2DM背景下胰腺癌发生、发展的病理过程,动态观察高血糖对体内胰腺癌细胞生长的影响,从而为T2DM背景下胰腺癌发生、发展的体内研究提供新的实验载体。 Abstract:Objective To establish a nude mouse model of type 2 diabetes mellitus (T2DM) and pancreatic cancer that allows dynamic observation of tumor formation process and facilitates in vivo research. Methods At first, human pancreatic cancer PANC-1 cells were transfected with lentiviral vector GV260 to construct the pancreatic cancer cell line PANC-1-Luc with stable expression of firefly luciferase. Then, 36 specific pathogen-free nude mice were randomly divided into control group with 12 mice and model group with 24 mice (nude mice with T2DM and pancreatic cancer). The mice in the control group were fed with breeding diet and were then given ectopic subcutaneous implantation of PANC-1-Luc cells, and those in the model group were first given high-fat diet and intraperitoneal injection of 1% STZ, followed by ectopic subcutaneous implantation of PANC-1-Luc cells. The fluorescence in vivo imaging system and the manual measurement method were used for simultaneous and dynamic monitoring of the growth of pancreatic cancer in nude mice in the two groups, and the tumor growth curve was plotted to investigate the correlation between fluorescence value and tumor volume. Subcutaneous tumors and pancreatic islets were observed under a microscope to verify whether the model was successfully established, and immunohistochemistry was used to measure the expression of Ki-67 in tumor tissue to investigate the influence of hyperglycemia on the growth of pancreatic cancer in nude mice. The independent-samples t test was used for comparison of normally distributed continuous data between groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups. Results The optimal virus titer was determined as 5×107 TU/mL for the stable transfection of lentiviral vector in PANC-1 cells, and the optimal concentration selected with puromycin was 20 μg/mL, with an optimal selection time of 9 days. The fluorescence value of PANC-1-Luc cells was linearly and positively correlated with the number of cells, with the linear equation of y=42.56x-42 504 (r=0.977, P=0.004). The blood glucose value of T2DM nude mice was 23.05 (19.25 — 26.40) mmol/L, with a blood glucose level of >11.1 mmol/L in each nude mouse, and there was a significant difference in blood glucose value between the T2DM nude mice and the control nude [6.15 (5.20 — 7.30) mmol/L] (Z=-8.45, P<0.001). Compared with the control group, the model group had reductions in the number and volume of pancreatic islets, with irregular shapes and unclear boundaries, and pathological examination confirmed that the xenograft tumor was pancreatic cancer tissue, which showed that the model was established successfully. In the model group, there was a linear positive correlation between subcutaneous tumor size and fluorescence values, with the linear equation of y=232 348 691x-8 258 608 (r=0.911, P=0.031). The model group had a significantly higher positive rate of Ki-67 than the control group (50.333%± 7.808% vs 15.917%±4.055%, t=13.55, P<0.001), suggesting rapid tumor proliferation in the model group. Conclusion The T2DM nude mouse model of pancreatic cancer established in this study can simulate the pathological process of the development and progression of pancreatic cancer in the context of T2DM and dynamically observe the influence of hyperglycemia on the growth of pancreatic cancer cells in vivo, thereby providing a new experimental vector for the in vivo study of the development and progression of pancreatic cancer in the context of T2DM. -
Key words:
- Diabetes Mellitus, Type 2 /
- Pancreatic Neoplasms /
- Disease Models, Animal
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甲状腺功能亢进症是一种常见的内分泌疾病,其治疗方法主要有药物、手术和同位素治疗,其中药物治疗是目前最常用、最主要的治疗方法。抗甲状腺药物(anti-thyroid drug,ATD)主要包括甲巯咪唑(MMI)和丙硫氧嘧啶(PTU)。MMI和PTU疗效确切,口服用药方便,总体安全性较高,但少数患者使用后可出现粒细胞减少、皮疹、肝功能损害以及关节疼痛等不良反应,尤其以药物性肝损伤(DILI)较为严重,起病急,病情重,预后差,可致死亡[1]。本文就ATD所致肝损伤(下文简称ATD肝损伤)的诊断和治疗原则进行阐述,指导临床医师规范用药。
1. 临床表现
ATD致肝损伤无特异性表现,急性起病,与其他各种急、慢性肝病类似。轻者可无任何症状,重者可出现黄疸,伴或不伴不同程度的乏力、食欲减退、肝区胀痛及上腹部不适等。胆汁淤积明显者可出现黄疸、大便颜色变浅和瘙痒等表现[2]。
过去我国将急性DILI的严重程度分为1~5级(轻度、中度、重度、急性肝衰竭、致命)[3],但分级不统一,不便于制订规范的治疗方案。随着循证证据的累积和进展,目前我国将急性DILI按严重程度不同分为4级,分别为1级轻度、2级中度、3级重度和4级致命[4]。
PTU较MMI更容易引发肝损伤。PTU治疗后14.3%的患者出现ALT无症状性升高,提示亚临床肝损伤,其中严重肝损伤发生率约为0.1%[5]。PTU诱导的肝损伤大多为肝细胞型,病理可见实质性坏死伴出血、小叶结构塌陷和门静脉前混合炎症浸润[6],起病急且进展迅速,呈非剂量依赖性,肝功能和肝酶检测对于评估肝功能损伤情况无明确的预测作用[7]。MMI引起的肝损伤主要是胆汁淤积型[8],通常在药物使用的最初几周发生,发生率为0.1%~0.2%,表现为肝细胞和/或胆小管内胆汁淤积,肝活检显示门静脉水肿和炎症,并伴有小管内胆汁淤积和中度微血管脂肪变性,其不良反应呈剂量依赖性,停药后多数患者能恢复正常,但所需时间较长。
2. ATD肝损伤的机制
目前ATD肝损伤的发生机制尚不十分明确,可能与药物代谢动力学及免疫学机制、年龄、性别、药物相互作用以及潜在疾病(如Graves病)等因素有关[9]。在药物代谢方面,MMI在人体内主要由CYP450酶和FMO酶代谢,MMI的主要代谢产物N-甲基硫脲可能是导致肝损伤的主要因素,N-甲基硫脲使氧自由基和过氧化脂质体形成增多,损伤肝细胞,同时,减少了还原型谷胱甘肽的储存量,造成肝细胞进一步损伤[10]。PTU在人体内主要在肝脏被葡萄糖醛酸化代谢,在髓过氧化物酶(MPO)介导下生成的代谢活性代谢产物与白蛋白共价结合,抑制谷胱甘肽转移酶和谷胱甘肽过氧化物酶,导致肝细胞抗氧化作用明显减弱。此外,PTU活性代谢产物可损伤细胞线粒体,出现线粒体形状异常,可见巨大线粒体。同时,线粒体功能异常,可出现线粒体内外膜破碎和基质溶解,从而诱导细胞损伤[11]。在免疫方面,部分接受ATD治疗的患者体内存在细胞因子和自身抗体,激活免疫反应造成肝细胞损伤。此外,ATD改变了肠道微生物群的结构,通过脂多糖相关信号通路,引起肝损伤[12]。
3. 诊断与鉴别诊断
目前虽有多个DILI的特异性生物学标志物(如miR-122、GLDH、HMGB1等)被报道用于协助诊断DILI[13-14],但尚未进行严格的临床验证,无法推广使用。当前诊断DILI仍是基于病史采集、临床症状、血清生化、影像学及组织学等排他性诊断[4],诊断标准主要有:(1)用药、停药与肝脏生化改变有明确合理的时效关系;(2)肝损伤的临床或病理表现与使用药物已知的肝毒性一致;(3)停药或减量后肝损伤明显改善或恢复正常;(4)再次用药后肝损伤再次出现:(5)排除肝损伤其他病因且无法用其他原发疾病或治疗手段解释。
在ATD肝损伤的诊断与鉴别诊断流程中,推荐采用先分型,再排除,后病因的诊断原则。关于ATD肝损伤的诊断与鉴别诊断流程可见图1。
ATD肝损伤诊断有困难时可行肝穿刺活检,适应证为:(1)其他竞争性病因无法排除,尤其是自身免疫性肝炎仍无法排除;(2)停用MMI或PTU后,肝酶仍持续升高者;或肝细胞损伤型(主要为使用PTU)患者的ALT峰值在发病后的30~60天,胆汁淤积型(主要为使用MMI)患者的ALP峰值在180天内,未下降>50%者;(3)持续肝酶升高超过180天,怀疑存在慢性肝病者。药物再暴露,因其具有潜在的严重后果,可能会导致快速、更严重的再次肝损伤甚至急性肝衰竭,且除ATD外还可使用131I等方法治疗,因此不建议对ATD肝损伤患者进行再暴露,且避免患者再次暴露于相似的可疑药物。因此,在诊断ATD肝损伤时,应遵循宁可活检不再暴露的原则。
4. 治疗原则
目前,ATD肝损伤的治疗方法和流程并不统一[15]。常规的ATD肝损伤防治措施应包括:及时停药,促进肝损伤尽早恢复,防止肝损伤重症化及慢性化,降低死亡风险。
4.1 及时停药
只要怀疑ATD肝损伤,首先应做到及时停药,尽量避免再次使用同类药物,停药是对因治疗中最重要的措施,也是最基本的治疗原则。及时停药后约95%患者可自行改善甚至痊愈,少数发展为慢性,极少数进展为急性/亚急性肝衰竭。有报道[16]肝细胞损伤型恢复时间约(3.3±3.1)周,胆汁淤积型约(6.6±4.2)周。对于轻度亚临床肝损伤时,可减少ATD剂量,密切监测肝功能,如肝损伤显著,则立即停药。若出现下列情况之一应考虑停药:(1)血清ALT或AST>8倍正常值上限;(2)ALT或AST>5倍正常值上限,持续2周;(3)ALT或AST>3倍正常值上限,且TBil>2倍正常值上限或INR>1.5;(4)ALT或AST>3倍正常值上限,伴逐渐加重的疲劳、恶心、呕吐、右上腹疼痛或压痛、发热、皮疹和/或嗜酸性粒细胞增多(>5%)[17]。及时停药,不再暴露是治疗ATD肝损伤的基本原则。
4.2 药物治疗
在大多数ATD引起的肝损伤病例中,除了及时停药与监测肝功能外,应结合循证医学证据,合理选择护肝治疗药物。ATD肝损伤治疗中常用药物包括:
4.2.1 糖皮质激素
糖皮质激素常用于甲亢危象的治疗[18],可抑制免疫或变态反应,减少外周T4向T3的转换,抑制甲状腺素的合成,也有利于改善患者黄疸。糖皮质激素可减轻MMI引起的肝炎[19],泼尼松龙可改善ATD肝损伤黄疸患者病情[20]。糖皮质激素联合甘草素治疗DILI可同时改善肝生化和组织学,且安全性良好。糖皮质激素使用的适应证,主要有重症DILI患者,合并甲亢危象和有过敏性皮疹的DILI患者;慢性DILI患者伴转氨酶升高超过10倍,或者超过5倍伴有TBil超过2倍,或者病理提示肝炎活动明显,可以考虑糖皮质激素治疗[21]。一些研究也指出,糖皮质激素可能增加不良事件的发生。糖皮质激素的使用并非ATD肝损伤的常规治疗方案,需严格掌握适应证,充分权衡获益与风险。
4.2.2 N-乙酰半胱氨酸(NAC)/谷胱甘肽(GSH)
NAC是由L-半胱氨酸和乙酰基形成,是细胞内还原型谷胱甘肽的前体,可促进GSH的生成;同时含有活性巯基,可对抗不同原因所致组织氧化损伤。NAC可用于预防MMI诱导的肝损伤[22];静脉注射NAC可显著提高Ⅰ~Ⅱ级早期昏迷患者的无移植生存率[23]。GSH属于解毒类药物,提供活性的巯基,参与肝细胞代谢过程,减轻组织损伤,促进修复,促进有毒物的转化和排泄。GSH对MMI所致肝损伤具有较好的疗效[24]。目前,NAC静脉注射被普遍用于药物急性肝衰竭的成人患者,且应尽早使用,每天50~150 mg/kg。同时,使用还原型谷胱甘肽,1.8 g,静脉滴注,每天1次。
4.2.3 异甘草酸镁/甘草酸二胺/双环醇
异甘草酸镁/甘草酸二胺可抑制炎症因子、调节肝细胞免疫功能、抑制氧化应激、保护干细胞膜等,可有效降低急性DILI患者的ALT和AST水平,促进ALT和AST及肝损伤恢复[25]。异甘草酸镁可显著改善MMI致DILI患者的肝功能,并促进机体自身免疫调节功能[26]。双环醇具有抗细胞凋亡、清除氧自由基、抗脂质过氧化等功能,可降低ALT和AST,尤其是降低ALT。双环醇治疗DILI,可以显著提高ALT正常化比率[27]。双环醇治疗急性肝损伤,50 mg,每天3次,效果优于25 mg,每天3次[28]。异甘草酸镁0.2 g,静脉滴注,每天1次。使用异甘草酸镁/甘草酸二胺时,注意复查电解质,关注血钾水平。
4.2.4 S-腺苷蛋氨酸/熊去氧胆酸
S-腺苷蛋氨酸退黄作用较好,适用于胆汁代谢障碍、ALP升高为主的胆汁淤积型DILI。熊去氧胆酸可以促进内源性胆汁酸的代谢,抑制其重吸收,增加胆汁排泄。腺苷蛋氨酸1 g,口服,每天3次。熊去氧胆酸胶囊,0.2 g,口服,每天2次。
4.2.5 多烯磷脂酰胆碱
进入肝细胞后,以完整的分子与肝细胞膜及细胞器膜结合,增加膜的完整性、稳定性和流动性,促进肝细胞再生。多烯磷脂酰胆碱注射液,465~930 mg,每天1次,静脉注射。
在ATD肝损伤的护肝治疗中,不宜同时使用多种同一类型的护肝药,应根据导致肝损伤的药物和损伤程度选择恰当的护肝药物,做到有的放矢,精准治疗。
4.3 血浆置换和人工肝治疗
治疗性血浆置换可以有效清除胆红素、胆汁酸和高水平的甲状腺激素等,减少甲状腺素对肝脏的直接损伤,并可以暂时部分替代肝脏的解毒功能,稳定机体内环境,为肝细胞再生及进一步治疗赢得宝贵时间。血浆置换可以明显降低血中T3和T4水平[29]。推荐ATD所致3级或4级肝损伤、普通护肝治疗措施效果不佳等情况下,及时行血浆置换。推荐使用新鲜冷冻血浆作为置换液,以提高甲状腺结合球蛋白水平,置换容积应为总血浆体积的1~1.5倍,根据病情每天或每2~3天进行1次血浆置换,持续置换到临床症状和肝功能指标显著改善。血浆置换是手术或131I治疗之前有效和安全的治疗选择。分子吸附再循环人工肝是一种将白蛋白透析和常规透析结合的技术,可以减少置换的次数,减少甲状腺激素的反弹,缩短肝功能恢复时间,在普通血浆置换效果欠佳时可选择使用[30]。
4.4 131I治疗
在DILI得到一定程度控制后,及时、足量的131I治疗,可以降低ATD相关肝损伤的死亡[31]。因此ATD肝损伤后,应及时停用ATD,综合护肝治疗,β-受体阻滞剂改善高代谢症状,适时予131I治疗。
5. 总结与展望
ATD的使用已有近70年历史,这类药物在治疗甲亢方面迄今仍占据着极其重要的地位。由于其所引起的肝损伤等不良反应并非罕见,有些甚至为致命性损伤[32]。因此,在使用ATD时应遵循充分评估,识别风险,监测肝功能,权衡利弊,及时停药,综合治疗的基本原则,最大限度降低ATD肝损伤的发生或进展。
除此之外,医药专业人员和公众可利用LiverTox和HepaTox网络平台,了解各类肝毒性药物的信息并增加对DILI的认知。临床药师也应加入治疗决策团队,通过审核药物配伍、提醒潜在的药物相互作用,以及必要时的血药浓度监测等,降低DILI风险。同时,加强针对公众的健康和合理用药教育,指导患者按药品说明书用药,纠正错误的服药习惯。
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表 1 模型组及对照组裸鼠皮下肿瘤大小变化(cm3)
Table 1. Changes in subcutaneous tumor size of nude mice in the model group and the control group (cm3)
时间 对照组(n=12) 模型组(n=24) t值 P值 第1周 0.013±0.007 0.036±0.021 3.535 0.004 第2周 0.020±0.012 0.046±0.022 3.490 0.002 第3周 0.027±0.015 0.051±0.023 3.490 0.002 第4周 0.035±0.019 0.061±0.034 2.285 0.035 第5周 0.039±0.019 0.070±0.037 2.549 0.018 表 2 模型组及对照组裸鼠皮下肿瘤荧光值变化(p⋅s-1⋅cm-2⋅sr-1)
Table 2. Changes in subcutaneous tumor values of nude mice in the model group and the control group (p·s-1·cm-2·sr-1)
时间 对照组(n=12) 模型组(n=24) t值 P值 第1周 6 460.83±4 200.33 985 416.67±97 565.32 34.726 <0.001 第2周 28 169.17±20 112.81 2 029 250.00±612 538.25 11.311 <0.001 第3周 131 601.67±101 868.71 3 251 666.67±981 546.40 10.953 <0.001 第4周 174 560.00±133 203.77 3 897 500.00±1 248 631.07 10.270 <0.001 第5周 359 734.17±236 685.20 9 650 833.33±4 384 569.45 7.330 <0.001 -
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