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三级职业资格证书淋巴腺内脏器官(TLO)诱导性建立还有在抗肉瘤抗体中的性能钻研

陈红 胡翔 张惠媛 胡洪波

陈红, 胡翔, 张惠媛, 等. 三级淋巴器官(TLO)诱导形成及其在抗肿瘤免疫中的功能研究[J]. koko体育app 学报(医学版), 2022, 53(1): 35-42. doi: 10.12182/20220160102
引用本文: 陈红, 胡翔, 张惠媛, 等. 三级淋巴器官(TLO)诱导形成及其在抗肿瘤免疫中的功能研究[J]. koko体育app 学报(医学版), 2022, 53(1): 35-42. doi:
CHEN Hong, HU Xiang, ZHANG Hui-yuan, et al. Induction and Anti-Tumor Function of Tertiary Lymphoid Organs[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2022, 53(1): 35-42. doi: 10.12182/20220160102
Citation: CHEN Hong, HU Xiang, ZHANG Hui-yuan, et al. Induction and Anti-Tumor Function of Tertiary Lymphoid Organs[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2022, 53(1): 35-42. doi:

三级淋巴器官(TLO)诱导形成及其在抗肿瘤免疫中的功能研究

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基金项目: 国家自然科学基金(No.82025002、No.81871232)和科技部重点研发专项(No. 2019YFA0110200)资助
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    E-mail: hongbohu@dikai.net.cn

Induction and Anti-Tumor Function of Tertiary Lymphoid Organs

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  • 摘要:   目的   在体外诱导形成三级淋巴器官(tertiary lymphoid organs, TLO),并评价其在抗肿瘤免疫中的功能。   方法   利用慢病毒系统在NIH3T3细胞上过表达淋巴毒素-β受体(lymphotoxin-beta receptor, LTβR),并检测LTβR-NIH3T3细胞中LTβR的过表达效率;通过免疫印迹实验和qPCR探究过表达LTβR的NIH3T3细胞内非经典核因子(nuclear factor, NF)-κB信号通路的情况。构建B16-OVA黑色素瘤小鼠荷瘤模型,探究LTβR-NIH3T3细胞诱导TLO形成的能力及抗肿瘤效果。  结果   利用慢病毒感染在NIH3T3细胞中过表达LTβR,流式检测发现GFP+细胞比例达99%。过表达LTβR能在NIH3T3细胞内激活非经典NF-κB信号通路。小鼠肿瘤模型结果表明,注射LTβR-NIH3T3细胞能在肿瘤附近诱导形成淋巴样组织,并促进了T细胞和MHCⅡ+巨噬细胞的浸润,明显抑制荷瘤小鼠的肿瘤生长,并延长小鼠生存期。  结论   LTβR-NIH3T3细胞通过诱导TLO的形成,促进抗肿瘤免疫,为肿瘤免疫治疗提供新的思路。
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    图  1  检测LTβR-NIH3T3细胞中LTβR的过表达效率

    Figure  1.  🌟 Examination of LTβR overexpression in LTβR-NIH3T3 cells

    A: Flow cytometry analysis of the expression of LTβR in Vector-NIH3T3 and LTβR-NIH3T3 cells; B: qPCR analysis of the expression of Ltbr in Vector-NIH3T3 and LTβR-NIH3T3 cells including before and after sorting. ****P<0.000 1, n=3.

    图  2  🐓 Western blot检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活

    Figure  2.  🎀 Western blot was used to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells.

    The relative molecular mass goes from top to bottom: 100×103, 44×103, 52×103, 70×103, 68×103.

    图  3  qPCR检测非经典NF-κB通路在LTβR-NIH3T3细胞中的激活

    Figure  3.  ඣ qPCR was done to examine the activation of non-canonical NF-κB pathway in LTβR-NIH3T3 cells

    *P<0.05,**P<0.01, ***P<0.001. Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells. n=3.

    图  4  LTβR-NIH3T3细胞的抗肿瘤效果评价

    Figure  4.  🐠 Evaluation of antitumor function of LTβR-NIH3T3 cells

    A: Measurement of tumor size (Vec: n=11; LTβR: n=15); B: The tumor was weighted at 21 d after tumor stripping (Vec: n=11; LTβR: n=15); C: The tumor growth curves (Vec: n=11; LTβR: n=15); D: HE staining of tumor microenvironment (white triangles show lymphoid-like tissue). Vec: Vector-NIH3T3 cells; LTβR: LTβR-NIH3T3 cells; ** P<0.01, Vec vs. LTβR.

    图  5  LTβR-NIH3T3细胞影响肿瘤浸润免疫细胞类型

    Figure  5.  ൩ Composition of tumor infiltrating immune cells affected by LTβR-NIH3T3 cells

    A: Flow cytometry analysis of tumor infiltrating immune cells; B: Proportion of live cells; C: Total cell number/mm3 tumor. *P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).

    图  6  LTβR-NIH3T3细胞影响肿瘤巨噬细胞类型

    Figure  6.  💜 The effect of LTβR-NIH3T3 cells on macrophages in tumors

    A: Flow cytometry analysis of MHCII+F4/80 macrophages and MHCⅡF4/80+ macrophages; Ba, Bb, Bc, Bd: Proportion and total cell number/mm3 tumor of MHCⅡ+F4/80 macrophages and MHCⅡF4/80+ macrophages; C: Ratio of MHCⅡ+/(F4/80+). * P<0.05, **P<0.01. Vec: Vector-NIH3T3 cells (n=11); LTβR: LTβR-NIH3T3 cells (n=15).

    表  1  qPCR引物序列及长度

    Table  1.   Sequences and length of qPCR primers

    GeneForward (5′→3′)Reverse (5′→3′)Length/bp
    Ltbr CATGCTAGCATGCGCCTGCCCCGGGCCTC TGAGCGGCCGCTCAGAGGTCTTGGCATCCTAGTG 212
    Ccl19 GAAAGCCTTCCGCTACCTTC GAGGTGCACAGAGCTGATAG 92
    Ccl21 TCCGAGGCTATAGGAAGCAA CTTCCTCAGGGTTTGCACAT 108
    Madcam1 GAGCAAGAAGAGGAGATACAAGAG TGGTGACCTGGCAGTGAAG 117
    Icam1 GTGCTTTGAGAACTGTGGCA GGTCCTTGCCTACTTGCTG 119
    Vcam1 GGAAGCTGGAACGAAGTATCC AAACACTTGACCGTGACCG 109
    18S ACAGGGAGAAAGCGCAAAAC TGTGGCCTTGTGGTGAAGAG 237
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出版历程
  • 收稿日期:  2022-09-26
  • 修回日期:  2022-12-09
  • 网络出版日期:  2023-01-24
  • 刊出日期:  2023-01-24

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