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2型糖尿病自发胰腺癌KPC小鼠新型共病模型的构建及评价
DOI: 10.12449/JCH260420
Construction and evaluation of a novel KPC mouse model of type 2 diabetes mellitus comorbid with spontaneous pancreatic cancer
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摘要:
目的 通过基因编辑-代谢干预双驱动策略构建KPC小鼠新型2型糖尿病（T2DM）自发胰腺癌共病模型，并结合传统模型进行对比和评价。 方法 将14只雄性KPC小鼠随机分为新型模型组（T2DM-KPC组，n=7）和对照组（KPC组，n=7）；另将14只雄性BALB-c/nu裸鼠随机分为传统模型组（T2DM-胰腺癌组，n=7）和对照组（单纯胰腺癌组，n=7）。KPC组及单纯胰腺癌组采用普通饲料喂养，T2DM-KPC组和T2DM-胰腺癌组采用高脂饲料喂养。4周后，T2DM-KPC组和T2DM-胰腺癌组腹腔注射链脲佐菌素。随后，T2DM-KPC组和KPC组随着时间的推移自发形成胰腺原发肿瘤；T2DM-胰腺癌组和单纯胰腺癌组待血糖稳定2周后皮下接种肿瘤，形成传统皮下移植瘤。观察4组小鼠成瘤率、成瘤时间、体重及血糖变化；对KPC组和T2DM-KPC组进行测序并分析分子分型；利用苏木精-伊红染色、Masson染色和免疫组织化学染色评价T2DM-KPC组胰腺肿瘤组织病理学和病理学微环境特征，并与T2DM-胰腺癌组进行比较。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。计数资料多组间比较采用Fisher确切概率法。 结果 T2DM-KPC组成瘤率为85.71%，成瘤时间为（104.40±2.87）d；T2DM-胰腺癌组成瘤率为71.43%，成瘤时间为（95.20±9.47）d，2组成瘤率、成瘤时间、体重和血糖水平比较，差异均无统计学意义（P值均>0.05）。分子分型提示，KPC组模型与人类胰腺导管腺癌的胰祖细胞型高度相似，T2DM-KPC组模型与人类胰腺导管腺癌的免疫原性型高度相似。苏木精-伊红染色显示，T2DM-KPC组肿瘤细胞排列成形状各异的腺管样结构，细胞异型性明显，能真实反映原发胰腺癌的病理学特征，且肿瘤较KPC组侵袭性更强。免疫组织化学和Masson染色结果显示，T2DM-KPC组肿瘤增殖（Ki-67为评价指标）程度和纤维化（α-平滑肌肌动蛋白和Masson为评价指标）程度均较T2DM-胰腺癌组明显升高（P值均<0.05），说明T2DM-KPC组小鼠模型可以更好地复现胰腺癌的高增殖和高纤维化特征。 结论 本研究成功构建了T2DM自发胰腺癌KPC小鼠新型共病模型。该模型能准确模拟T2DM合并胰腺癌的组织病理学形态和特有的基质环境，实现T2DM背景下胰腺组织从上皮内瘤变到浸润癌再到转移的全过程模拟，还可支持免疫治疗转化研究，为T2DM自发胰腺癌的体内研究提供了新的实验载体。 Abstract:Objective To construct a novel KPC mouse model of type 2 diabetes mellitus （T2DM） comorbid with spontaneous pancreatic cancer based on the gene editing-metabolic intervention dual-driven strategy， and to compare it with traditional models. Methods A total of 14 male KPC mice were randomly divided into novel model group （T2DM-KPC group with 7 mice） and control group （KPC group with 7 mice）， and 14 male BALB/c-nu nude mice were randomly divided into traditional model group （T2DM-pancreatic cancer group with 7 mice） and control group （pancreatic cancer group with 7 mice）. The mice in the KPC group and the pancreatic cancer group were fed with normal diet， and those in the T2DM-KPC group and the T2DM-pancreatic cancer group were fed with a high-fat diet. After 4 weeks， the mice in the T2DM-KPC group and the T2DM-pancreatic cancer group were given intraperitoneal injection of streptozotocin. Subsequently， the mice in the KPC group and the T2DM-KPC group developed primary pancreatic tumor spontaneously over time， while those in the T2DM-pancreatic cancer group and the pancreatic cancer group were inoculated with tumor cells to form subcutaneous tumor xenograft at 2 weeks after stabilization of blood glucose. The 4 groups were observed in terms of tumor formation rate， tumor formation time， body weight， and the change in blood glucose； RNA sequencing was performed for tumors from the KPC group and the T2DM-KPC group， and then molecular subtyping was performed； HE staining， Masson staining， and immunohistochemical staining were used to assess the histopathological features and tumor microenvironment of pancreatic tumor from the T2DM-KPC group， which were compared with those of the T2DM-pancreatic cancer group. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups； the Fisher’s exact test was used for comparison of categorical data between multiple groups. Results The T2DM-KPC group had a tumor formation rate of 85.71% and a tumor formation time of 104.40±2.87 days， while the T2DM-pancreatic cancer group had a tumor formation rate of 71.43% and a tumor formation time of 95.20±9.47 days， and there were no significant differences between the two groups in tumor formation rate， tumor formation time， body weight， and blood glucose （all P>0.05）. Molecular subtyping showed that the model in the KPC group highly resembled the pancreatic progenitor subtype of human pancreatic ductal adenocarcinoma （PDAC）， and the model in the T2DM-KPC group highly resembled the immunogenic subtype of PDAC. HE staining showed that tumor cells in the T2DM-KPC group were arranged into glandular tubular structures of varying shapes， exhibiting significant cellular atypia， and this model faithfully recapitulated the pathological features of primary pancreatic cancer and showed greater invasiveness than the KPC group. Immunohistochemical staining and Masson staining showed that compared with the T2DM-pancreatic cancer group， the T2DM-KPC group had significantly higher degrees of tumor proliferation （assessed by Ki-67 expression） and fibrosis （assessed by α-SMA and Masson） （all P<0.05）， suggesting that the mouse model in the T2DM-KPC group could better recapitulate the features of hyperproliferation and pronounced desmoplasia in human pancreatic cancer. Conclusion A novel KPC mouse model of T2DM comorbid with spontaneous pancreatic cancer is successfully constructed in this study. This model can accurately mimic the histopathological architecture and stromal microenvironment of T2DM comorbid with pancreatic cancer， realize the longitudinal simulation of the progression of pancreatic tissue from intraepithelial neoplasia to invasive carcinoma and metastasis in the presence of T2DM， and support the translational research on immunotherapy， thereby providing a novel experimental carrier for in vivo studies on spontaneous pancreatic cancer in T2DM. -
Key words:
- Pancreatic Neoplasms /
- Diabetes Mellitus， Type 2 /
- Comorbidity /
- Disease Models， Animal /
- KPC Mice
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图 1 KPC小鼠构建策略与作用机制
Figure 1. Construction strategy and mechanism of action of KPC mice
注： a，Krasem4（LSL-G12D） 杂合小鼠基因型鉴定电泳结果（部分示例）；b，Pdx1-cre阳性小鼠基因型鉴定电泳结果（部分示例）；c，p53点突变小鼠终点分析鉴定结果（部分示例）。HO，突变型纯合子；HE，杂合子；WT，野生型纯合子；Negative，阴性对照；Unknown，未分类/信号模糊；N/A，无数据/无效。
图 2 KPC小鼠基因型鉴定结果
Figure 2. Genotype identification results of KPC mice
注： a，KPC组；b，T2DM-KPC组。
图 3 KPC组和T2DM-KPC组小鼠成瘤情况
Figure 3. Tumor formation in mice of the KPC group and the T2DM-KPC group
注： a，单纯胰腺癌组成瘤情况；b，两组皮下移植瘤模型成瘤情况对比；c，T2DM-胰腺癌组成瘤情况。
图 4 单纯胰腺癌组和T2DM-胰腺癌组裸鼠成瘤情况
Figure 4. Tumor formation in nude mice of the simple pancreatic cancer group and the T2DM-pancreatic cancer group
注： a，4组体重变化比较；b，4组血糖变化比较；c，4组末周体重比较；d，4组末周血糖比较。*P<0.05。
图 6 4组体重和血糖的比较
Figure 6. Comparison of weight and blood glucose among the four groups
注： a，KPC组；b，T2DM-KPC组。
图 7 KPC组和T2DM-KPC组胰腺肿瘤及周围脾脏组织大体形态
Figure 7. Gross morphology of pancreatic tumor and surrounding spleen tissue in the KPC group and the T2DM-KPC group
注： a，KPC组；b，T2DM-KPC组。
图 8 KPC组和T2DM-KPC组胰腺肿瘤组织苏木精-伊红染色结果（×200）
Figure 8. Hematoxylin and eosin staining results of tumor tissues in the KPC group and the T2DM-KPC group （×200）
注： 1，单纯胰腺癌组；2，T2DM-胰腺癌组。
图 9 单纯胰腺癌组和T2DM-胰腺癌组皮下移植瘤大体形态
Figure 9. Gross morphology of subcutaneous transplanted tumors in the simple pancreatic cancer group and the T2DM- pancreatic cancer group
注： a，单纯胰腺癌组；b，T2DM-胰腺癌组。
图 10 单纯胰腺癌组和T2DM-胰腺癌组皮下移植瘤组织苏木精-伊红染色结果（×200）
Figure 10. Hematoxylin and eosin staining results of subcutaneous transplanted tumor tissues in the simple pancreatic cancer group and the T2DM-pancreatic cancer group （×200）
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