负载MAGE-A3抗原的树突状细胞与细胞因子诱导杀伤细胞共培养对子宫内膜癌肿瘤干细胞及恶性进展的影响

目的：探究负载黑色素瘤相关抗原基因A3（MAGE-A3）的树突状细胞（DC）与细胞因子诱导杀伤细胞（CIK）对子宫内膜癌肿瘤干细胞及恶性进展的影响。方法：采集人外周血分离单个核细胞，利用细胞因子分别诱导生成DC和CIK；MAGE-A3孵育DC后与CIK共培养，流式细胞仪检测DC-CIK、MAGE-A3-DC-CIK的表型；流式细胞仪分选子宫内膜癌细胞系Ishikawa的CD133+干细胞，以子宫内膜癌干细胞作为靶细胞，分别以CIK、DC-CIK及MAGE-A3-DC-CIK作为效应细胞，MTT法检测效靶比为10∶1、20∶1、40∶1的细胞杀伤活性，ELISA法检测联合培养细胞上清液中IFN-γ、IL-2、IL-12、IL-17水平，Annexin V-FITC/PI双染法检测子宫内膜癌干细胞凋亡；建立子宫内膜癌干细胞移植瘤裸鼠模型，尾静脉注射DC-CIK或MAGE-A3-DC-CIK，观察裸鼠肿瘤生长情况，每隔2 d测量瘤体大小，21 d后取肿瘤组织，电子天平称重，HE染色观察肿瘤组织病理形态学变化，免疫组织化学染色检测肿瘤组织内Ki-67表达。结果：分离获得细胞表面CD80、CD86、HLA-DR表达分别达到88%、86%和90%的DC；MAGE-A3-DC-CIK组细胞表面CD8+CD3+、CD56+CD3+比例均高于DC-CIK组（P<0.01）；经磁珠分选后获得CD133+细胞比例高达90.23%的子宫内膜癌干细胞；与CIK组和DC-CIK组比较，MAGE-A3-DC-CIK组在不同效靶比下对子宫内膜癌干细胞的杀伤能力升高，细胞上清中IFN-γ、IL-2、IL-12及IL-17的分泌水平升高，培养液上清处理的子宫内膜癌干细胞凋亡率增加，差异均具有统计学意义（P<0.01）；同时，与对照组和DC-CIK组比较，MAGE-A3-DC-CIK组移植瘤裸鼠的肿瘤体积小，肿瘤重量轻，肿瘤组织内细胞稀疏，Ki-67阳性细胞率减小，差异均具有统计学意义（P<0.01）。 结论：负载MAGE-A3的DC与CIK联合培养能促进CIK成熟，提高对子宫内膜癌干细胞的杀伤性，并抑制移植瘤裸鼠的恶性进展。

Effects of co-culture of dendritic cells loaded with MAGE-A3 antigen and cytokine-induced killer cells on tumor stem cells and malignant progression of endometrial cancer

AIM: To explore the effects of dendritic cells (DC) and cytokine-induced killer cells (CIK) carrying melanoma-associated antigen gene A3 (MAGE-A3) on endometrial cancer tumor stem cells and malignant progression. METHODS: Human peripheral blood was collected to separate mononuclear cells, and DC and CIK cells were induced by cytokines, respectively. DCs were incubated with MAGE-A3 and then co-cultured with CIK, and the phenotypes of DC-CIK and MAGE-A3-DC-CIK were detected by flow cytometry; The CD133+ stem cells of the endometrial cancer cell line Ishikawa were sorted by flow cytometry, the endometrial cancer stem cells were used as target cells, and CIK, DC-CIK and MAGE-A3-DC-CIK were used as effector cells, respectively, MTT method was used to detect the cytotoxic activity of effector-target ratios of 10∶1, 20∶1 and 40∶1, ELISA method was used to detect IFN-γ, IL-2, IL-12 and IL-17 in the supernatant of the combined cultured cells, Annexin V-FITC/PI double staining was used to detect the apoptosis of endometrial cancer stem cells; A nude mouse model of endometrial cancer stem cell transplanted tumor was established, DC-CIK or MAGE-A3-DC-CIK was injected into the tail vein, and the tumor growth in nude mice was observed. The tumor size was measured every 2 days, after 21 days, the tumor tissue was taken and weighed on an electronic balance. HE staining was used to detect e the pathological changes of tumor tissues, and immunohistochemical staining was used to detect Ki-67 expression in tumor tissues. RESULTS: The expressions of CD80, CD86 and HLA-DR on the isolated DC surface were 88%, 86% and 90%, respectively. The ratios of CD8+CD3+ and CD56+CD3+ on the surface of MAGE-A3-DC-CIK group were higher than those of DC-CIK group (P<0.01); After sorting by magnetic beads, endometrial cancer stem cells with a proportion of CD133+ cells as high as 90.23% were obtained; Compared with CIK group and DC-CIK group, MAGE-A3-DC-CIK group has higher killing ability on endometrial cancer stem cells under different target ratios, the secretion level of IFN-γ, IL-2, IL-12, IFN-γ and IL-17 in the cell supernatant were increased, and the apoptotic rate of endometrial cancer stem cells treated with the culture medium supernatant was increased, the difference were statistically significant (P<0.01); At the same time, compared with the control group and DC-CIK group, the tumor volume of the transplanted nude mice in the MAGE-A3-DC-CIK group was smaller, the tumor weight was lighter, the cells in the tumor tissue were sparse, and the rate of Ki-67 positive cells was reduced, the difference were statistically significant (P<0.01). CONCLUSION: The co-culture of DC loaded with MAGE-A3 and CIK can promote the maturation of CIK, improve the lethality of endometrial cancer stem cells, and inhibit the malignant progression of transplanted tumor in nude mice.