【作者】 刘雪明；

【导师】 王立新；

【作者基本信息】 东南大学 ， 免疫学， 2022， 硕士

【摘要】 胸腺肽α-1(Thymosinα-1,Tα-1)是一种小分子活性肽,临床上已与化疗联合应用于肿瘤患者的治疗,但分子机制尚未明了。我们前期研究发现:多种化疗药物(如:紫杉醇、顺铂、硼替佐米等)能诱导肿瘤细胞凋亡,巨噬细胞在胞葬凋亡细胞过程中激活TBK1信号通路、分泌IL-10并导致M2型极化,而Tα-1能与凋亡细胞表面磷脂酰丝氨酸(PS)结合,通过TLR7-SHIP1通路逆转胞葬巨噬细胞的M2型极化、增强肿瘤浸润淋巴细胞(TILs)活性,从而提高化疗的抗肿瘤效果。凋亡肿瘤细胞会产生凋亡小体(ABs),由于凋亡小体表面含有大量PS分子,往往导致树突状细胞(DC)摄取凋亡小体后的抗原提呈功能受到抑制。因此,Tα-1能否与凋亡小体表面PS分子结合,负载Tα-1凋亡小体能否提高DC的抗原提呈功能、促进肿瘤特异性T细胞应答并提高肿瘤微环境TILs的数量,有待进行深入研究。目的:探讨Tα-1是否调节DC摄取肿瘤细胞来源凋亡小体后的抗原提呈功能及其诱导的T细胞应答,旨在进一步探索Tα-1增强T细胞应答及其抗肿瘤作用的机制。方法:1.Tα-1调节吞噬凋亡小体DC的功能及其机制1.1 Tα-1调节吞噬凋亡小体DC的功能用100n M化疗药硼替佐米诱导4T1细胞凋亡,差速离心法提取凋亡小体,用流式细胞术及透射电子显微镜鉴定凋亡小体。诱导小鼠骨髓来源的树突状细胞(BMDC),将Tα-1和凋亡小体共孵育(ABs/Tα-1),用Tα-1(100μg/ml)、ABs(100μg/ml)和ABs/Tα-1(100μg/ml)与BMDC共培养24 h,收获细胞培养上清液,用ELISA法检测IL-12p70表达量;收集细胞,流式细胞术检测DC表面MHCⅡ和CD86分子的表达。1.2 Tα-1调节吞噬凋亡小体DC功能的机制Tα-1和ABs共孵育,用Annexin V-FITC标记ABs表面的PS分子,Tα-1抗体标记Tα-1分子;流式细胞术检测ABs与Tα-1结合的比例。与CY5标记的Tα-1(Tα-1-CY5)和ABs共孵育(ABs/Tα-1-CY5),Annexin V-FITC标记ABs表面的PS分子,DC2.4与ABs/Tα-1-CY5共孵育6 h,Lyso-Tracker Red标记DC溶酶体,用流式细胞术检测ABs和Tα-1-CY5被DC吞噬的比例,用激光共聚焦显微镜观察DC吞噬ABs/Tα-1-CY5后的情况。2.Tα-1调节DC提呈凋亡小体抗原的功能及其抗肿瘤作用2.1 Tα-1调节DC提呈凋亡小体抗原的功能将OVA蛋白和纳米铝偶联(α-Al2O3-OVA)后,多次皮下注射免疫小鼠。分离小鼠脾细胞并用不同浓度的OVA蛋白刺激,ELISA检测细胞培养上清中IFN-γ的含量。OVA+B16F10来源的ABs、Tα-1预处理的OVA+B16F10来源ABs以及Tα-1与BMDC共培养24h;收获上述处理的BMDC分别与OVA蛋白特异性应答小鼠的脾细胞混合培养24 h,用流式细胞术检测IFN-γ+CD3+CD8-及IFN-γ+CD3+CD8+细胞的比例,ELISA检测细胞培养上清中IFN-γ的含量。2.2 Tα-1增强化疗的抗肿瘤作用及机制小鼠皮下接种OVA+B16F10细胞,将小鼠分为四组,分别接受生理盐水(NS)、Tα-1、顺铂(DDP)或DDP联合Tα-1的治疗一组只用Tα-1。观察并记录肿瘤大小并绘制成肿瘤生长曲线。末次治疗第二天安乐死小鼠,流式细胞术检测小鼠淋巴结中DC和CD8α+DC的比例及其CD86和MHCⅡ类分子表达情况、淋巴结和脾脏中IFN-γ+CD3+CD8-及IFN-γ+CD3+CD8+细胞的比例,ELISA法检测肿瘤组织中浸润T淋巴细胞(Tumor infiltrating lymphocyte,TIL)分泌IFN-γ的水平。结果:1.Tα-1调节吞噬凋亡小体DC的功能及其机制1.1 Tα-1调节吞噬凋亡小体DC的功能硼替佐米诱导4T1细胞凋亡再用差速离心法可提取出纯度超过90%的ABs。Tα-1对BMDC分泌IL-12p70以及表达CD86和MHCⅡ类分子都没有直接调节作用;BMDC吞噬ABs,IL-12p70的分泌以及CD86和MHCⅡ类分子的表达水平都下调,而Tα-1可以上调其分泌IL-12p70分泌以及表达CD86和MHCⅡ类分子。1.2 Tα-1调节吞噬凋亡小体DC功能的机制流式细胞术结果显示Tα-1可以通过PS分子和ABs特异性结合,结合能力高达90%。流式细胞术结果显示DC2.4可以同时吞噬ABs和结合在其表面的Tα-1;激光共聚焦显微镜观察到ABs/Tα-1被DC2.4吞噬后定位于吞噬溶酶体。2.Tα-1调节DC提呈凋亡小体抗原的功能及其抗肿瘤作用2.1 Tα-1调节DC提呈凋亡小体抗原的功能ELISA结果证实OVA蛋白可以特异性刺激α-Al2O3-OVA免疫小鼠的脾细胞分泌IFN-γ并且有浓度依赖性,OVA蛋白特异性应答T细胞诱导成功。流式细胞术和ELISA结果显示BMDC吞噬ABs后抗原提呈能力被抑制:小鼠脾细胞IFN-γ分泌量以及IFN-γ+CD3+CD8-及IFN-γ+CD3+CD8+细胞的比例下降;而Tα-1可以上调吞噬ABs的BMDC的抗原提呈能力:小鼠脾细胞IFN-γ分泌量及IFN-γ+CD3+CD8-及IFN-γ+CD3+CD8+细胞的比例增多。2.2 Tα-1增强化疗的抗肿瘤作用及机制单独使用Tα-1不能缓解小鼠黑色素瘤的生长,低剂量的DDP能缓解肿瘤生长,但低剂量DDP联合Tα-1治疗具有协同效应。与其他三组相比,联合治疗组小鼠淋巴结中DC和CD8α+DC比例最高,且DC表面抗原提呈相关分子CD86和MHC II类分子表达水平也更高;小鼠脾细胞和淋巴结细胞中IFN-γ分泌量及IFN-γ+CD3+CD8-及IFN-γ+CD3+CD8+细胞的比例更高;同时TILs分泌IFN-γ分泌水平最高。结论:1.Tα-1与凋亡小体表面的PS分子结合并被树突状细胞吞噬,上调吞噬凋亡小体的树突状细胞分泌IL-12以及表达CD86和MHC II类分子;2.Tα-1上调吞噬DC提呈凋亡小体抗原的功能及T细胞应答,Tα-1增加DDP的抗肿瘤作用;更多还原

【Abstract】 Thymosin alpha 1(Thymosin α-1,Tα-1)is a small peptides molecules.Tα-1 is often used in combination with chemotherapy in the treatment of tumor patients.However,the mechanisms have not been fully elucidated.In our previous study we found that apoptotic tumor cells in large amounts are induced by chemotherapeutic drugs(Paclitaxel,Cisplatin,Bortezomib,ect).Macrophages activate TBK1 signaling pathway,secrete IL-10 and lead to M2 polarization during the efferocytosis.Tα-1binds with surface PS on apoptotic cells,reverses M2 polarization by TLR7-SHIP1 signaling pathway and enhances the activity of tumor infiltrating lymphocytes(TILs)to upregrate anti-tumor effects of chemotherapy.apoptotic bodies(ABs)are breakages of apoptotic tumor cells.ABs also have large amounts of surface PS.Antigen presentation in dendritic cells(DC)is depressed after apoptotic bodies engulfment.Therefore,one question still to be studied is whether Tα-1binds with ABs,upregulates the apoptotic bodies antigen presentation in dendritic cells,or enhances tumor specific T cell response and amounts of TILs.Objectives:To study whether Tα-1 regulates the tumor cells-drived apoptotic bodies antigen presentation in dendritic cells and its mechanism.To study the mechanism of antitumor immunity induced by Tα-1.Methods:1.Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment and and its mechanism1.1 Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment4T1 were induced apoptosis by 100 n M Bortezomib.Apoptotic bodies were extracted by differential centrifugation.Flow cytometry and transmission electron microscopy were used to identify apoptotic bodies.Bone Marrow-Derived Dendritic Cells(BMDC)were generated and co-cultured with apoptotic bodies incubated with Tα-1(ABs/Tα-1,100μg/ml),Tα-1(100μg/ml),and ABs(100μg/ml)for 24 hours.IL-12p70 in supernatants was detected by ELISA,and CD86 and MHC Ⅱ expression in BMDC were detected by flow cytometry.1.2 The mechanism of Tα-1 regulating the function of dendritic cells after apoptotic bodies engulfmentApoptotic bodies were incubated with Tα-1.And then the PS of ABs were labeled with Annexin V-FITC.The propotion of apoptotic bodies binded with Tα-1 was detected by flow cytometry.Apoptotic bodies were incubated with Tα-1 linked with CY5(Tα-1-CY5).Then ABs/Tα-1-CY5 were labeled with Annexin V-FITC and co-cultured with DC for 6 hours.Next,DC lysosomes were labeled with Lyso-Tracker Red.Finally,the proportion of apoptotic bodies binded with Tα-1-CY5 were detected by flow cytometry.ABs/Tα-1-CY5 were detected by confocal scanning laser microscopy.2.Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells2.1 Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells in vitroMice were immunized subcutaneously with alpha-alumina nanoparticles coupled with OVA(α-Al2O3-OVA).Mice spleen cells were stimulated with different concentrations of OVA.IFN-γ in supernatants were detected by ELISA.Apoptotic bodies from OVA+B16F10(OVA+ABs),OVA+/ABs incubated with Tα-1(OVA+ABs/ Tα-1)and Tα-1 were co-cultured with BMDC for 24 hours.Then those prepared BMDC were co-cultured with spleen cells from OVA specific response mice for 24 hours.Finally,the proportion of IFN-γ+ CD3+ CD8-and IFN-γ+ CD3+ CD8+ cells was detected by flow cytometry.IFN-γ in supernatants was detected by ELISA.2.2 Thymosin α-1 enhances the chemotherapy efficacy in the treatment of melanomaMice were immunized subcutaneously with OVA+B16F10.Then,those mice were divided into 4 groups: only Tα-1 group,only DDP group,Tα-1 and DDP group,Saline group(NS);The tumour growth curve were made on the basis of tumour size;In the next day after the last therapy,proportion of DC and CD8α+ DC cells and their CD86,MHC Ⅱ expression in lymph nose cells were detected by flow cytometry;proportion of IFN-γ+ CD3+ CD8-and IFN-γ+CD3+CD8+cells in spleen cells and Lymph node cells were detected by flow cytometry;Secretion IFN-γ of tumor infiltrating lymphocyte(TILs)was detected by ELISA.Results:1.Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfment and and its mechanism1.1 Thymosin α-1 regulates the function of dendritic cells after apoptotic bodies engulfmentOver 90% purity apoptotic bodies were extracted by differential centrifugation from apoptotic 4T1.Tα-1 have no modulation of IL-12p70 secretion,MHC Ⅱ and CD86 expression of dendritic cells.Dendritic cells after apoptotic bodies engulfment showed a downregulation of IL-12p70 secretion,MHC Ⅱ and CD86 expression.Tα-1 can enhance the IL-12p70 secretion,MHC Ⅱ and CD86 expression of dendritic cells after apoptotic bodies engulfment.1.2 The mechanism of Tα-1 regulating the function of dendritic cells after apoptotic bodies engulfmentMore the 90% apopototic bodies were binded with Tα-1 by the surface PS.DC2.4 engulfed apopototic bodies and Tα-1 binded with apoptotic bodies;ABs/Tα-1 were associated with DC2.4 phagosome-lysosome.2.Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells2.1 Thymosin α-1 regulates the apoptotic bodies antigen presentation in dendritic cells in vitroSpleen cells of the mouse had a dose-dependent up-dependent secretion of IFN-γ.OVA specific response T cells had been induced.Dendritic cells after apoptotic bodies engulfment showed a downregulated antigen presentation: decreased proportion of IFN-γ+CD3+CD8-and IFN-γ+CD3+CD8+ cells in spleen cells.Tα-1 upregulated antigen presentation in DC: increased proportion of IFN-γ+CD3+CD8-and IFN-γ+CD3+ CD8+ cells in spleen cells and secretion of IFN-γ.2.2 Thymosin α-1 enhances the chemotherapy efficacy in the treatment of melanomaOnly Tα-1 cannot depress the tumour growth.But Low-Dose DDP can depress the growth.However,Low-Dose DDP and Tα-1 have a significant depression on tomour growth.Compared with other three groups,DDP and Tα-1 group showed the highest proportion of DC and CD8α+DC cells in lymph node cells;the highest expression of CD86 and MHC Ⅱ;the highest proportion of IFN-γ+ CD3+ CD8-and IFN-γ+ CD3+ CD8+ cells in spleens cells and lymph nodes;the highest secretion of IFN-γ from TILs.Conclusion:1.Thymosin α-1 bind with surface PS on apoptotic bodies is engulfed by dendritic cells.Thymosin α-1 enhances the IL-12 secretion,CD86 and MHCII expression of dendritic cells after apoprtotic bodies engulfment;2.Thymosin α-1 upregulates the antigen presentation in dendritic cells after apoprtotic bodies engulfment,and has anti-tumour effects with DDP;更多还原