雷帕霉素聯(lián)合調(diào)節(jié)性T細(xì)胞對(duì)受體小鼠移植心臟及抗腫瘤免疫的影響
發(fā)布時(shí)間：2018-08-15 19:09
【摘要】：目的研究短期使用雷帕霉素聯(lián)合調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)在誘導(dǎo)同種異體小鼠心臟移植物長期存活,同時(shí)探討此方案對(duì)移植受體腫瘤免疫的影響。方法免疫磁珠法分離得到受體小鼠脾臟Treg,經(jīng)CD3/CD28單克隆抗體磁珠及2000 U/mL重組小鼠白介素2(recombinant murine IL-2,rmIL-2)體外擴(kuò)增后,流式細(xì)胞術(shù)檢測純度;建立小鼠腹腔同種異體心臟移植模型(H-2~b到H-2~d),分為對(duì)照組(單純移植)、雷帕霉素組、雷帕霉素聯(lián)合Treg組。雷帕霉素組連續(xù)14 d經(jīng)腹腔注射雷帕霉素1mg/(kg·d),雷帕霉素聯(lián)合Treg組注射相同劑量的雷帕霉素,并于移植當(dāng)天經(jīng)尾靜脈過繼輸注擴(kuò)增后的Treg(1×107/只),同時(shí)設(shè)同基因移植組(H-2~d到H-2~d),每日觀察移植心臟搏動(dòng)并在相應(yīng)時(shí)點(diǎn)進(jìn)行組織學(xué)評(píng)價(jià)。在受體腫瘤免疫實(shí)驗(yàn)中設(shè)三組同種異體心臟移植(H-2~b到H-2~d),同時(shí)經(jīng)尾靜脈過繼輸注B16-F10細(xì)胞(H-2~b)(供體來源),另三組同種異體心臟移植(H-2~d到H-2~b)小鼠經(jīng)尾靜脈過繼輸注B16-F10細(xì)胞(H-2~b)(受體來源),兩周后比較肺部腫瘤結(jié)節(jié)數(shù)量。結(jié)果CD4~+CD25~+Foxp3~+Treg擴(kuò)增前純度為86.68%±0.02%(n=5),體外加入擴(kuò)增2周后Treg數(shù)量達(dá)到了初始數(shù)量的30~40倍,流式細(xì)胞術(shù)檢測CD4+CD25+Foxp3+Treg純度為84.58%±0.03%,與擴(kuò)增前相比差異無統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)照組移植心臟中位生存時(shí)間(MST)為7 d,雷帕霉素組為15 d,雷帕霉素聯(lián)合Treg過繼輸注能夠顯著延長移植心臟MST至93 d,組織學(xué)顯示長期存活心臟淋巴細(xì)胞浸潤及慢性血管病變;對(duì)于供體來源腫瘤,對(duì)照組未見腫瘤結(jié)節(jié),雷帕霉素組為(15±8)個(gè),雷帕霉素聯(lián)合Treg組小鼠肺部腫瘤結(jié)節(jié)為(14±7)個(gè),后2組類似無顯著性差異;對(duì)于受體來源腫瘤,對(duì)照組腫瘤結(jié)節(jié)為(70±12)個(gè)、雷帕霉素組為(28±9)個(gè)、雷帕霉素聯(lián)合Treg組小鼠肺部腫瘤結(jié)節(jié)為(146±12)個(gè),與對(duì)照組、雷帕霉素組相比均顯著增加。結(jié)論短期使用低劑量雷帕霉素聯(lián)合Treg能夠顯著延長小鼠移植心臟的存活時(shí)間,但仍不能抑制受體腫瘤的發(fā)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% 鹵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% 鹵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 鹵8) in rapamycin group and (14 鹵7) in rapamycin combined with Treg group, but there was no significant difference between the latter two groups. There were (70 鹵12) nodules in control group, (28 鹵9) in rapamycin group and (146 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R617
本文編號(hào)：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% 鹵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% 鹵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 鹵8) in rapamycin group and (14 鹵7) in rapamycin combined with Treg group, but there was no significant difference between the latter two groups. There were (70 鹵12) nodules in control group, (28 鹵9) in rapamycin group and (146 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R617
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 韓洪瀛;溫盛霖;;肝移植患者術(shù)后生存質(zhì)量及心理干預(yù)[J];器官移植;2013年03期
2 孫偉;;肺部惡性腫瘤患者CD4~+ CD25~+ Treg等指標(biāo)的臨床測定及意義[J];細(xì)胞與分子免疫學(xué)雜志;2011年09期
，本文編號(hào)：2185139
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