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汤晓娟, 任媛, 贺敏, 丁宁, 吕元, 李勇敏, 赵亮, 何永恒.结直肠癌3D细胞肿瘤球的构建及天马颗粒抗肿瘤作用的初探[J].湖南中医药大学学报,2026,46(3):499-508[点击复制] |
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| 结直肠癌3D细胞肿瘤球的构建及天马颗粒抗肿瘤作用的初探 |
| 汤晓娟,任媛,贺敏,丁宁,吕元,李勇敏,赵亮,何永恒 |
| (湖南省中西医结合医院(湖南省中医药研究院附属医院), 湖南 长沙 410006;湖南中医药大学, 湖南 长沙 410208;湖南大学生命医学交叉研究院, 湖南 长沙 410082) |
| 摘要: |
| 目的 建立一种包含肠癌细胞（HCT-116）与内皮细胞（HUVEC）的3D细胞肿瘤球模型，并在该模型上验证天马颗粒对3D细胞肿瘤球生长和血管新生的影响。方法 不同比例HCT-116与HUVEC混合液培养于超低吸附96孔板，通过检测3D细胞肿瘤球体积、细胞数量、ATP含量确定最佳培养比例。将HCT-116和HUVEC以2∶1比例混合，分为空白对照组（15%空白血清）、盐酸阿霉素组（15%空白血清+100 ng/mL盐酸阿霉素）、天马颗粒含药血清低剂量组（5%含药血清+10%空白血清）、天马颗粒含药血清中剂量组（10%含药血清+5%空白血清）、天马颗粒含药血清高剂量组（15%含药血清+0%空白血清）；检测3D细胞肿瘤球体积、细胞数量、ATP含量。将2D细胞和3D细胞肿瘤球分为2D组（15%空白血清）、3D组（15%空白血清）、盐酸阿霉素组（15%空白血清+100 ng/mL盐酸阿霉素）、天马颗粒含药血清低剂量组（5%含药血清+10%空白血清）、天马颗粒含药血清中剂量组（10%含药血清+5%空白血清）、天马颗粒含药血清高剂量组（15%含药血清+0%空白血清）；进行RNA测序分析，RT-qPCR检测丙酮酸激酶M1（PKM1）、丙酮酸激酶M2（PKM2）、葡萄糖转运蛋白1（Glut1）mRNA表达，Western blot检测CD31、P21、P53蛋白表达，TSA多重免疫荧光检测CD31、增殖细胞核抗原（PCNA）表达。结果 培养第9天，细胞比例为2∶1时，3D细胞肿瘤球体积最大、细胞数量最多、ATP含量最高（P<0.05，P<0.01）。与空白对照组相比，各给药组3D细胞肿瘤球体积均缩小、细胞数量均减少、ATP含量均降低（P<0.01）。与盐酸阿霉素组相比，天马颗粒含药血清低剂量组3D细胞肿瘤球体积增大、细胞数量增加、ATP含量升高（P＜0.05，P＜0.01）；天马颗粒含药血清中剂量组3D细胞肿瘤球体积缩小（P<0.01）；天马颗粒含药血清高剂量组3D细胞肿瘤球体积缩小、细胞数量减少、ATP含量降低（P＜0.01）。与天马颗粒含药血清低剂量组相比，天马颗粒含药血清中、高剂量组3D细胞肿瘤球体积均缩小、细胞数量均减少、ATP含量均降低（P＜0.05，P＜0.01）。与天马颗粒含药血清中剂量组相比，天马颗粒含药血清高剂量组3D细胞肿瘤球体积缩小、细胞数量减少、ATP含量降低（P＜0.05，P＜0.01）。与2D组相比，3D组糖酵解通路上调；与3D组相比，天马颗粒含药血清高剂量组糖酵解通路下调。与2D组相比，3D组PKM1、PKM2、Glut1 mRNA表达水平及CD31蛋白表达水平均升高（P<0.01），P53、P21蛋白表达水平均降低（P＜0.05，P＜0.01）。与3D组相比，各给药组PKM1和Glut1 mRNA表达水平均降低（P＜0.05，P＜0.01），P53、P21蛋白表达水平均升高（P＜0.05，P＜0.01）；盐酸阿霉素组、天马颗粒含药血清中、高剂量组PKM2 mRNA表达水平及CD31蛋白表达水平均降低（P＜0.05，P＜0.01）。与盐酸阿霉素组相比，天马颗粒含药血清低剂量组PKM1和PKM2 mRNA表达水平均升高（P＜0.05，P＜0.01），P21蛋白表达水平降低（P＜0.05）；天马颗粒含药血清中剂量组PKM2 mRNA表达水平及P21、CD31蛋白表达水平均降低（P＜0.05，P＜0.01）；天马颗粒含药血清高剂量组PKM1、PKM2和Glut1 mRNA表达水平及P53、P21、CD31蛋白表达水平均降低（P＜0.05，P＜0.01）。与天马颗粒含药血清低剂量组比较，天马颗粒含药血清中剂量组PKM1、PKM2 mRNA表达水平及CD31蛋白表达水平均降低（P＜0.05，P＜0.01）；天马颗粒含药血清高剂量组PKM1、PKM2和Glut1 mRNA表达水平及P53、P21和CD31蛋白表达水平均降低（P＜0.05，P＜0.01）。与天马颗粒含药血清中剂量组比较，天马颗粒含药血清高剂量组Glut1 mRNA表达水平及P53、P21蛋白表达水平均降低（P＜0.05，P＜0.01）。与空白对照组相比，盐酸阿霉素组及天马颗粒含药血清高剂量组PCNA、CD31阳性细胞数量均减少（P＜0.05，P＜0.01）；与盐酸阿霉素组比较，天马颗粒含药血清高剂量组CD31阳性细胞数量减少（P＜0.01）。结论 肠癌细胞与内皮细胞比例为2∶1时可构建较稳定的3D细胞肿瘤球模型，天马颗粒含药血清可抑制肿瘤球生长和血管新生。 |
| 关键词: 结直肠癌 天马颗粒 3D细胞肿瘤球 糖酵解 血管新生 |
| DOI：10.3969/j.issn.1674-070X.2026.03.009 |
| 投稿时间：2025-10-29 |
| 基金项目:国家自然科学基金项目（82374462）；湖南省自然科学基金项目（2024JJ6286）；长沙市自然科学基金项目（kq2403131）；湖南省卫生健康委员会科研项目（W20243256）；湖南省中医药管理局科研项目（B2024031）。 |
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| Construction of 3D colorectal cancer cell tumorspheres and preliminary exploration of the anti-tumor effects of Tianma Granules |
| TANG Xiaojuan, REN Yuan, HE Min, DING Ning, LYU Yuan, LI Yongmin, ZHAO Liang, HE Yongheng |
| (Hunan Provincial Hospital of Integrated Traditional Chinese and Western Medicine (The Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine), Changsha, Hunan 410006, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;School of Biomedical Sciences, Hunan University, Changsha, Hunan 410082, China) |
| Abstract: |
| Objective To establish a 3D cell tumorsphere model incorporating colorectal cancer cells (HCT-116) and endothelial cells (HUVEC), and validate the effects of Tianma Granules on the growth and angiogenesis of the 3D cell tumorspheres within this model. Methods HCT-116 and HUVEC were mixed in varying ratios and cultured in ultra-low attachment 96-well plates. The optimal culture ratio was determined by measuring the volume, cell count, and ATP content of the 3D cell tumorspheres. HCT-116 and HUVEC were mixed at a 2∶1 ratio and divided into five groups: blank control group (15% blank serum), doxorubicin hydrochloride (15% blank serum + 100 ng/mL doxorubicin), and low-, medium-, and high-dose Tianma Granules-containing serum (TGCS) groups (5%, 10%, and 15% drug-containing serum respectively, supplemented with blank serum to a total of 15%). The volume, cell count, and ATP content of the 3D cell tumorspheres were then measured. Additionally, 2D cells and 3D cell tumorspheres were divided into six groups: 2D control (15% blank serum), 3D control (15% blank serum), doxorubicin group (15% blank serum + 100 ng/mL doxorubicin hydrochloride), and low-, medium-, and high-dose TGCS groups (5%, 10%, and 15% drug-containing serum respectively, supplemented with blank serum to a total of 15%). RNA sequencing analysis was performed, and RT-qPCR was used to measure the mRNA expressions of pyruvate kinase M1 (PKM1), pyruvate kinase M2 (PKM2), and glucose transporter 1 (Glut1). Western blot was conducted to check the protein expressions of CD31, P21 and P53. TSA multiple immunofluorescence was employed to examine the expressions of CD31 and proliferating cellnuclear antigen (PCNA). Results On the 9th day of culture, when the cell ratio was 2∶1, the 3D cell tumorspheres exhibited the maximum volume, the highest cell count, and the highest ATP content (P<0.05, P<0.01). Compared with the blank control group, the 3D cell tumorspheres in all drug-administered groups showed reductions in the volume, cell count, and ATP content (P<0.05). Compared with the doxorubicin hydrochloride group, the low-dose TGCS group showed increases in the volume, cell count, and ATP content of 3D cell tumorspheres (P<0.05, P<0.01); the medium-dose TGCS group showed reductions in the volume of 3D cell tumorspheres (P<0.01); the high-dose TGCS group exhibited reductions in the volume, cell count, and ATP content of the 3D cell tumorspheres (P<0.01). Compared with the low-dose TGCS group, the medium- and high-dose TGCS groups exhibited reductions in the volume, cell count, and ATP content of the 3D cell tumorspheres (P<0.05, P<0.01). Compared with the medium-dose TGCS group, the high-dose TGCS group exhibited reductions in the volume, cell count, and ATP content of the 3D cell tumorspheres (P<0.05, P<0.01). Compared with the 2D group, the glycolytic pathway was upregulated in the 3D group; conversely, compared with the 3D group, the glycolytic pathway was downregulated in the high-dose TGCS group. Compared with the 2D group, the mRNA expression levels of PKM1, PKM2 and Glut1, as well as the protein expression level of CD31, increased in the 3D group (P<0.01), while the protein expression levels of P53 and P21 decreased (P<0.05, P<0.01). Compared with the 3D group, the mRNA expression levels of PKM1 and Glut1 reduced in all drug-administered groups (P<0.05, P<0.01), while the protein expression levels of P53 and P21 increased (P<0.05, P<0.01). The mRNA expression level of PKM2 and the protein expression level of CD31 decreased in the doxorubicin hydrochloride group, as well as the medium- and high-dose TGCS groups (P<0.05, P<0.01). Compared with the doxorubicin hydrochloride group, the low-dose TGCS group exhibited elevated mRNA expression levels of PKM1 and PKM2 (P<0.05, P<0.01) and reduced protein expression level of P21 (P<0.05). The medium-dose TGCS group showed decreased mRNA expression level of PKM2 and protein expression levels of P21 and CD31 (P<0.05, P<0.01). The high-dose TGCS group demonstrated reduced mRNA expression levels of PKM1, PKM2 and Glut1, as well as protein expression levels of P53, P21 and CD31 (P<0.05, P<0.01). Compared with the low-dose TGCS group, the medium-dose group exhibited decreased mRNA expression levels of PKM1 and PKM2, as well as protein expression level of CD31 (P<0.05, P<0.01); while the high-dose group showed reduced mRNA expression levels of PKM1, PKM2 and Glut1, as well as protein expression levels of P53, P21 and CD31 (P<0.05, P<0.01). Compared with the medium-dose TGCS group, the high-dose group exhibited reduced mRNA expression level of Glut1 and protein expression levels of P53 and P21 (P<0.05, P<0.01). Compared with the blank control group, both the doxorubicin hydrochloride group and the high-dose TGCS group showed a decrease in the number of PCNA- and CD31-positive cells (P<0.05, P<0.01). Compared with the doxorubicin hydrochloride group, the high-dose TGCS group demonstrated a reduction in the number of CD31-positive cells (P<0.01). Conclusion A relatively stable 3D cell tumorsphere model can be established when the ratio of colorectal cancer cells to endothelial cells is 2∶1. TGCS can inhibit tumorsphere growth and angiogenesis. |
| Key words: colorectal cancer Tianma Granules 3D tumorsphere glycolysis angiogenesis |
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