雷帕霉素联合调节性T细胞对受体小鼠移植心脏及抗肿瘤免疫的影响
发布时间：2018-08-15 19:09
【摘要】：目的研究短期使用雷帕霉素联合调节性T细胞(regulatory T cell,Treg)在诱导同种异体小鼠心脏移植物长期存活,同时探讨此方案对移植受体肿瘤免疫的影响。方法免疫磁珠法分离得到受体小鼠脾脏Treg,经CD3/CD28单克隆抗体磁珠及2000 U/mL重组小鼠白介素2(recombinant murine IL-2,rmIL-2)体外扩增后,流式细胞术检测纯度;建立小鼠腹腔同种异体心脏移植模型(H-2~b到H-2~d),分为对照组(单纯移植)、雷帕霉素组、雷帕霉素联合Treg组。雷帕霉素组连续14 d经腹腔注射雷帕霉素1mg/(kg·d),雷帕霉素联合Treg组注射相同剂量的雷帕霉素,并于移植当天经尾静脉过继输注扩增后的Treg(1×107/只),同时设同基因移植组(H-2~d到H-2~d),每日观察移植心脏搏动并在相应时点进行组织学评价。在受体肿瘤免疫实验中设三组同种异体心脏移植(H-2~b到H-2~d),同时经尾静脉过继输注B16-F10细胞(H-2~b)(供体来源),另三组同种异体心脏移植(H-2~d到H-2~b)小鼠经尾静脉过继输注B16-F10细胞(H-2~b)(受体来源),两周后比较肺部肿瘤结节数量。结果CD4~+CD25~+Foxp3~+Treg扩增前纯度为86.68%±0.02%(n=5),体外加入扩增2周后Treg数量达到了初始数量的30~40倍,流式细胞术检测CD4+CD25+Foxp3+Treg纯度为84.58%±0.03%,与扩增前相比差异无统计学意义(P0.05)。对照组移植心脏中位生存时间(MST)为7 d,雷帕霉素组为15 d,雷帕霉素联合Treg过继输注能够显著延长移植心脏MST至93 d,组织学显示长期存活心脏淋巴细胞浸润及慢性血管病变;对于供体来源肿瘤,对照组未见肿瘤结节,雷帕霉素组为(15±8)个,雷帕霉素联合Treg组小鼠肺部肿瘤结节为(14±7)个,后2组类似无显著性差异;对于受体来源肿瘤,对照组肿瘤结节为(70±12)个、雷帕霉素组为(28±9)个、雷帕霉素联合Treg组小鼠肺部肿瘤结节为(146±12)个,与对照组、雷帕霉素组相比均显著增加。结论短期使用低剂量雷帕霉素联合Treg能够显著延长小鼠移植心脏的存活时间,但仍不能抑制受体肿瘤的发生。
[Abstract]:Objective to study the effect of rapamycin combined with regulatory T cell (regulatory T cell Treg on the long-term survival of cardiac allografts in mice, and to explore the effect of rapamycin combined with regulatory T cell (regulatory T cell Treg on tumor immunity. Methods the spleen of recipient mice was isolated by immunomagnetic bead method and amplified by CD3/CD28 monoclonal antibody magnetic beads and 2000 U/mL recombinant mouse interleukin 2 (recombinant murine IL-2rmIL-2. Flow cytometry was used to detect the purity. A model of intraperitoneal allograft heart transplantation (H-2B to H-2d) was established and divided into three groups: control group, rapamycin combined with Treg group. Rapamycin group was intraperitoneally injected with rapamycin 1mg/ (kg d), and rapamycin combined with Treg group for 14 days, and the same dose of rapamycin was injected into the group. The Treg (1 脳 107 / mouse) was injected through the tail vein on the day of transplantation, and the homologous gene transplantation group (H-2D to H-2D) was set up. The heart beating was observed daily and the histological evaluation was made at the corresponding time point. Three groups of allogeneic heart transplantation (H-2B to H-2d) were established in the tumor immunological test of the recipient, and B16-F10 cells (H-2B) (donor source) were injected via caudal vein, and B16-F10 cells were infused through caudal vein in the other three groups of allograft heart transplantation (H-2D-H-2B) mice. (h-2 B) (receptor source), comparing the number of pulmonary tumor nodules two weeks later. Results the purity of CD4 ~ CD25 ~ Foxp3- Treg was 86.68% 卤0.02% (nd5). After 2 weeks of amplification, the number of Treg reached 3040 times of the initial number, and the purity of CD4 CD25 Foxp3 Treg detected by flow cytometry was 84.58% 卤0.03. There was no significant difference compared with that before amplification (P0.05). The median survival time (MST) was 7 days in the control group and 15 days in the rapamycin group. Rapamycin combined with Treg adoptive infusion could significantly prolong the MST to 93 days. For donor-derived tumors, no tumor nodules were found in the control group (15 卤8) in rapamycin group and (14 卤7) in rapamycin combined with Treg group, but there was no significant difference between the latter two groups. There were (70 卤12) nodules in control group, (28 卤9) in rapamycin group and (146 卤12) in rapamycin combined with Treg group, which were significantly higher than those in control group and rapamycin group. Conclusion Short-term administration of rapamycin combined with Treg can significantly prolong the survival time of transplanted heart in mice, but still can not inhibit the occurrence of recipient tumor.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R617
本文编号：2185139
[Abstract]:Objective to study the effect of rapamycin combined with regulatory T cell (regulatory T cell Treg on the long-term survival of cardiac allografts in mice, and to explore the effect of rapamycin combined with regulatory T cell (regulatory T cell Treg on tumor immunity. Methods the spleen of recipient mice was isolated by immunomagnetic bead method and amplified by CD3/CD28 monoclonal antibody magnetic beads and 2000 U/mL recombinant mouse interleukin 2 (recombinant murine IL-2rmIL-2. Flow cytometry was used to detect the purity. A model of intraperitoneal allograft heart transplantation (H-2B to H-2d) was established and divided into three groups: control group, rapamycin combined with Treg group. Rapamycin group was intraperitoneally injected with rapamycin 1mg/ (kg d), and rapamycin combined with Treg group for 14 days, and the same dose of rapamycin was injected into the group. The Treg (1 脳 107 / mouse) was injected through the tail vein on the day of transplantation, and the homologous gene transplantation group (H-2D to H-2D) was set up. The heart beating was observed daily and the histological evaluation was made at the corresponding time point. Three groups of allogeneic heart transplantation (H-2B to H-2d) were established in the tumor immunological test of the recipient, and B16-F10 cells (H-2B) (donor source) were injected via caudal vein, and B16-F10 cells were infused through caudal vein in the other three groups of allograft heart transplantation (H-2D-H-2B) mice. (h-2 B) (receptor source), comparing the number of pulmonary tumor nodules two weeks later. Results the purity of CD4 ~ CD25 ~ Foxp3- Treg was 86.68% 卤0.02% (nd5). After 2 weeks of amplification, the number of Treg reached 3040 times of the initial number, and the purity of CD4 CD25 Foxp3 Treg detected by flow cytometry was 84.58% 卤0.03. There was no significant difference compared with that before amplification (P0.05). The median survival time (MST) was 7 days in the control group and 15 days in the rapamycin group. Rapamycin combined with Treg adoptive infusion could significantly prolong the MST to 93 days. For donor-derived tumors, no tumor nodules were found in the control group (15 卤8) in rapamycin group and (14 卤7) in rapamycin combined with Treg group, but there was no significant difference between the latter two groups. There were (70 卤12) nodules in control group, (28 卤9) in rapamycin group and (146 卤12) in rapamycin combined with Treg group, which were significantly higher than those in control group and rapamycin group. Conclusion Short-term administration of rapamycin combined with Treg can significantly prolong the survival time of transplanted heart in mice, but still can not inhibit the occurrence of recipient tumor.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R617
【参考文献】
相关期刊论文 前2条
1 韩洪瀛;温盛霖;;肝移植患者术后生存质量及心理干预[J];器官移植;2013年03期
2 孙伟;;肺部恶性肿瘤患者CD4~+ CD25~+ Treg等指标的临床测定及意义[J];细胞与分子免疫学杂志;2011年09期
，本文编号：2185139
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