肿瘤干细胞样细胞RNA致敏树突状细胞治疗大鼠9L脑肿瘤

doi:10.3969/j.issn.1671-167X.2015.04.023

北京大学学报(医学版) ›› 2015, Vol. 47 ›› Issue (4): 661-666. doi: 10.3969/j.issn.1671-167X.2015.04.023

肿瘤干细胞样细胞RNA致敏树突状细胞治疗大鼠9L脑肿瘤

肖宗宇1△，陈晓娟2，杨艺3，徐如祥3

（1.青海大学附属医院神经外科，西宁810000； 2. 青海省人民医院神经内科，西宁810000；3. 北京军区总医院附属八一脑科医院，北京100700）

出版日期:2015-08-18 发布日期:2015-08-18
通讯作者: 肖宗宇 E-mail:xiaozongyu@hotmail.com
基金资助:
青海大学中青年科研基金（2014-QYY-6）资助

Experimental study of dendritic cells transfected with cancer stem like cells RNA against 9L brain tumors

XIAO Zong-yu1△, CHEN Xiao-juan2, YANG Yi3, XU Ru-xiang3

(1. Department of neurosurgery, Affiliated Hospital of Qinghai University, Xining 810000, China; 2. Department of Neurology, People’s Hospital of Qinghai Province, Xining 810000，China; 3. Affiliated Bayi Brain Hospital of Military General Hospital of Beijing PLA, Beijing 100700，China)

Online:2015-08-18 Published:2015-08-18
Contact: XIAO Zong-yu E-mail:xiaozongyu@hotmail.com
Supported by:
Supported by Youth Foundation of Qinghai University (2014-QYY-6)

摘要/Abstract

摘要：

目的：利用大鼠9L肿瘤干细胞样细胞（cancer stem like cells，CSLCs）总RNA致敏树突状细胞（dendritic cells，DC）治疗9L/F344大鼠颅内胶质瘤，观察DC疫苗对脑胶质瘤的治疗作用，探讨其免疫反应的机制，为DC疫苗的临床应用提供实验基础。方法：40只颅内荷瘤F344大鼠随机分成4组，每组10只，作为实验组：（1）树突状细胞组（DC-9L）：注射转染贴壁9L细胞RNA的DC组；（2）DC-9LTS组：注射转染9L肿瘤球RNA的DC组；（3）DC组：注射未经转染RNA；（4）磷酸盐缓冲液（phosphate buffered saline，PBS）组：注射100 μL PBS。非荷瘤F344大鼠10只设为对照组，仅在右侧尾状核注射10 μL DMEM/F12培养基。利用大鼠9L肿瘤干细胞样细胞的总RNA致敏DC，制备DC-9LTS,采用皮下注射的方式，对9L/F344大鼠颅内胶质瘤模型进行免疫治疗，观察荷瘤大鼠生存期，同时检测血清干扰素-γ（interferon-gamma，IFN-γ）的浓度，免疫组织化学染色对各治疗组肿瘤组织CD4、CD8淋巴细胞浸润情况进行分析。结果：各实验组大鼠的中位生存期分别为：PBS组21 d，DC组为21 d，DC-9L组为31 d，DC9LTS组为36 d。DC-9LTS组大鼠的中位生存期与其余各组比较，差异有统计学意义（P＜0.01）；DC-9L组与DC组、PBS组比较，差异有统计学意义（P＜0.01）；而DC组与PBS组之间差异无统计学意义(χ2＝0.071，P＝0.789)，DC-9LTS组的IFNγ浓度为（157.08±7.25） ng/L，高于其余各组（P＜0.05），且大鼠肿瘤组织内部及瘤周组织均有大量CD8淋巴细胞浸润。DC-9L组中，在肿瘤组织内部及瘤周也可见CD8淋巴细胞浸润， DC-9LTS组CD8的平均光密度值（D）明显高于DC-9L组（P＜0.001），DC-9LTS组及DC-9L组均未见CD4淋巴细胞的表达。在DC组和PBS组中，肿瘤组织中未见CD4或CD8淋巴细胞浸润。结论：利用大鼠9L肿瘤干细胞样细胞总RNA体外致敏树突状细胞，制备树突状细胞疫苗，回输治疗9L/F344大鼠颅内胶质瘤，能明显延长荷瘤大鼠生存期，为靶向性杀伤脑肿瘤干细胞的胶质瘤免疫治疗提供了实验基础及理论依据。

关键词: 脑肿瘤, 肿瘤干细胞, 树突细胞, 免疫疗法, 神经胶质瘤

Abstract:

Objective：To investigate the anti-tumor efficacy of dendritic cells (DC) vaccination transfected with total RNA of cancer stem like cells and to discuss the mechanism of immune response, so as to provide experimental basis for clinical application.Methods: Dendritic cells were isolated from F344 bone marrow cells, then these dendritic cells were transfected with total RNA of 9L cancer stem cells or 9L monolayer cells. F344 rats bearing with 9L brain tumors were treated by subcutaneous injection of either PBS, unpulsed DC, DC transfected with 9L monolayer cells RNA (DC-9LTS) or DC transfected with 9L tumor spheres RNA (DC-9L)3, 10, 17. And 21 days after tumor implantation, the brains and sera were obtained from the different groups, the lymphocytes infiltration was detected by immunohistochemistry, and the concentration of interferon-γ(IFN-γ) was tested by ELISA. The survival time was observed and determined using the method of Kaplan Meier analysis.Results: The rats vaccinated with DC transfected with 9L tumor spheres RNA (DC9LTS) and the monolayer cell RNA (DC-9L) expired with median survival time of 36 and 31 days, respectively. The animals bearing intracranial 9L gliosarcoma were vaccinated with unpulsed DC vaccine, all expired with a median survival time of 21 days. The Kaplan-Meier survival curve showed that the rats treated with DC-9LTS had longer survival than the other groups (P＜0.01). There was significant difference among DC-9L group, DC group, and PBS group (P＜0.01). There was no significant difference between DC group and PBS group (χ2＝0.071, P=0.789).The concentration of IFNgamma of DC-9LTS group［（157.08±7.25) ng/L］was much higher than those of the other groups (P＜0.05). DC-9LTS could effectively enhance T-cell infiltration, a large number of CD8+ cells were detected in and around the tumor in DC-9LTS group, compared with DC-9L group, DC and PBS group (P＜0.001). The expression of CD8+ cell was not detected in DC group and PBS group. However no expression of CD4+ cells was observed in all the groups. Conclusion: Immunotherapy using DC transfected with 9L CSLCs total RNA was more effective for the treatment of 9L brain gliomas, and the strategy prolonged the survival of 9L glioma-bearing rats significantly, which provides a scientific foundation for further investigation of this approach to eradicate gliomas.

Key words: Brain neoplasms, Neoplastic stem cells, Dendritic cells, Immunotherapy, Glioma

中图分类号:

R739.4

肖宗宇, 陈晓娟, 杨艺, 徐如祥. 肿瘤干细胞样细胞RNA致敏树突状细胞治疗大鼠9L脑肿瘤[J]. 北京大学学报(医学版), 2015, 47(4): 661-666.

XIAO Zong-Yu, CHEN Xiao-Juan, YANG Yi, XU Ru-Xiang. Experimental study of dendritic cells transfected with cancer stem like cells RNA against 9L brain tumors[J]. Journal of Peking University(Health Sciences), 2015, 47(4): 661-666.

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肿瘤干细胞样细胞RNA致敏树突状细胞治疗大鼠9L脑肿瘤

Experimental study of dendritic cells transfected with cancer stem like cells RNA against 9L brain tumors

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