Lentivirus-mediated human interleukin-12 fusion gene-transfected rat bone marrow mesenchymal stem cells inhibit CT26 tumor growth

doi:10.3969/j.issn.2095-4344.0953

Abstract

Abstract:

BACKGROUND: Interleukin-12 as the most potent anti-tumor factor has been a hot topic in research, but little is reported on its use in the treatment of colorectal cancer.
OBJECTIVE: To construct rat bone marrow mesenchymal stem cell lines stably expressing human interleukin-12 (hIL-12-BMSCs), and to observe its effect on colorectal cancer (CT26).
METHODS: Primers were designed and synthesized, and purified p40 and p35 gene fragments were amplified by PCR. The single-chain double-subunit fusion gene, hIL-12, was obtained by Overlap-ping PCR ligation, and 293T cells were co-transfected with lentiviral packaging system. Recombinant lenovirus overexpressing hIL-12 (LV-IL-12) was constructed to transfect rat BMSCs. The stable strain was then cultured via drug screening. Thirty-two nude mice were subcutaneously injected with CT26 cells to make animal models, and then randomly divided into four groups to receive peritumoral injection of hIL-12-BMSCs (0.2 mL; hIL-12-BMSCs group), 0.2 mL of PBS (PBS group), 0.2 mL of BMSCs (BMSCs group), or 0.2 mL of LV-IL-12 (LV-IL-12 group). Tumor growth was then statistically analyzed in each group.
RESULTS AND CONCLUSION: There was no significant difference in tumor volume between the PBS and BMSCs groups (P > 0.05), indicating that BMSCs cannot promote or inhibit the growth of CT26 tumors. However, from the 7th day after injection, the tumor volume showed a significant difference between hIL-12-BMSCs and PBS groups as well as between PBS and LV-IL-12 groups (P < 0.01), indicating that Hil-12 has a significant inhibitory effect on CT26 tumor growth. From the 10th day after injection, a significant difference in the tumor volume was found between the hIL-12-BMSCs and LV-IL-12 groups (P < 0.05), indicating that hIL-12-BMSCs are more effective than LV-IL-12 in inhibiting the growth of CT26. To conclude, hIL-12-BMSCs considerably inhibit the growth of CT26 tumor.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Colorectal Neoplasms, Interleukin-12, Bone Marrow, Mesenchymal Stem Cells, Lentivirus Infections, Tissue Engineering

CLC Number:

R394.2

He Yang, Lin Chen, Gao Qin, Song Jing-xiang, Tu Xiao-huang. Lentivirus-mediated human interleukin-12 fusion gene-transfected rat bone marrow mesenchymal stem cells inhibit CT26 tumor growth[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3944-3949.

Figures/Tables 2

2.1 BMSCs形态及鉴定结果培养24 h后细胞呈圆形，少量贴壁；72 h后大部分贴壁，呈梭形；第3代细胞形态已较均一，绘制细胞生长曲线(图1)。流式细胞仪检测细胞表面标志物CD29、CD90阳性表达率为91.10%，93.50%，而CD34、CD45阴性表达率为99.18%，97.78%，表明BMSCs培养成功(图2)。 2.2 PCR扩增产物电泳图及测序结果产物行1.5%琼脂糖凝胶电泳(图3，4)，IL-12p35(648 bp)、IL-12p40 (1 008 bp)、IL-12(1 620 bp)的测序结果及片段大小与预期一致，表明构建IL-12融合基因成功，待用。 2.3 构建慢病毒/IL-12过表达体慢病毒转染293T细胞72 h后，实验组、对照组均见大量绿色荧光(图5)，且荧光表达稳定，表明过表达体构建成功。 2.4 过表达体转染BMSCs 过表达体转染BMSCs 24 h后，即见荧光，且强度随时间增强，72 h后绝大多数细胞呈现绿色(图6)。流式细胞仪测定在MOI=10，30，50的转染率，实验组分别为35.4%，83.1%，97.3%，对照组分别为31.8%，79.9%，91.2%。MOI=50时，荧光最强且细胞形态无明显变化，表明慢病毒可将hIL-12基因转染至BMSCs，且转染率超过85%，满足实验需求。 2.5 hIL-12-BMSCs形态及鉴定结果过表达体转染BMSCs 24 h后，即见荧光，强度随时间增强且形态未见明显改变(图7)。ELISA法检测实验组12，24，48，72，96 h上清液中hIL-12蛋白量分别为(5.08±0.99)，(57.68±3.73)，(102.16±6.26)，(146.64±7.46)，(189.47±3.80) ng/106细胞，对照组未检测到hIL-12蛋白的表达，表明hIL-12-BMSCs构建成功(图8)。 2.6 各组裸鼠生存及肿瘤生长情况所有裸鼠注射CT26细胞悬液7 d后均见长径≥5 mm瘤体，造模成功率100%，观察期内所有裸鼠均存活。各组各时间段瘤体体积数据及肿瘤生长曲线见表1和图9。4组瘤体体积在前4 d差异无显著性意义(P > 0.05)，观察期内PBS组与BMSCs组肿瘤体积差异均无显著性意义(P > 0.05)，表明BMSCs本身对CT26瘤体生长无明显促进或抑制作用；7 d后hIL-12-BMSCs组与PBS组、PBS组与LV-IL-12组间肿瘤体积差异均有显著性意义(P < 0.01)，表明IL-12对CT26瘤体生长有明显抑制作用；10 d后hIL-12-BMSCs组与LV-IL-12组间肿瘤体积差异有显著性意义(P < 0.05)，表明hIL-12-BMSCs抑制CT26生长优于LV-IL-12。"

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