a Medical Oncology, Changzhi people's Hospital Affiliated to Shanxi Medical University , Changzhi , Shanxi Province , China.
b Department of Pathophysiology , Medical School of Zhengzhou University , Zhengzhou , Henan Province , China.
Hum Vaccin Immunother. 2018 May 4;14(5):1294-1304. doi: 10.1080/21645515.2018.1427532. Epub 2018 Feb 22.
The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice.
Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3 and CD8 T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice.
The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates.
Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.
本研究使用小鼠脑微血管内皮细胞系 bEnd.3 开发疫苗，并观察其在免疫小鼠中的抗肺转移作用。
体外培养小鼠 bEnd.3 细胞，然后用戊二醛固定，用于免疫小鼠；用转移性癌细胞系 U14 对小鼠进行攻击，通过苏木精和伊红染色观察转移性癌组织的变化。用羧基荧光素琥珀酰亚胺基酯（CSFE）和碘化丙啶（PI）检测脾 T 淋巴细胞的细胞毒性活性；用流式细胞术测定 CD3 和 CD8 T 细胞亚群的比例。用酶联免疫吸附试验（ELISA）、免疫细胞化学和免疫印迹检测免疫小鼠血清的特异性反应。
bEnd.3 和人脐静脉内皮细胞（HUVEC）疫苗组的转移结节数少于 NIH3T3 疫苗组和磷酸盐缓冲盐水（PBS）对照组。bEnd.3 诱导和 HUVEC 诱导的细胞毒性 T 淋巴细胞（CTL）对 bEnd.3 和 HUVEC 靶细胞表现出明显的溶解活性，而 bEnd.3 和 HUVEC 疫苗组小鼠的血清对膜蛋白表现出特异性免疫反应，并抑制体外靶细胞增殖。免疫印迹结果显示 bEnd.3 和 HUVEC 裂解物中分别有 180KD 和 220KD 以及 130KD 和 220KD 的特异性条带。
同种异体 bEnd.3 疫苗诱导针对肿瘤血管内皮细胞的主动和特异性免疫反应，导致产生针对增殖抗原 VEGF-R II、整合素、内等的抗体。用这种疫苗免疫抑制了宫颈癌 U14 细胞的肺转移，并延长了这些小鼠的生存时间。
