[關(guān)鍵詞]
[摘要]
目的 通過體外實驗探究四君子湯含藥血清(SJZDCS)聯(lián)合PD-1抗體對肺癌腫瘤浸潤CD4+T淋巴細(xì)胞免疫功能的影響和機制。方法 取雄性SD大鼠,連續(xù)ig四君子湯6 d,麻醉取血,制備含藥血清。取雄性C57BL/6J小鼠,建立Lewis肺癌荷瘤小鼠模型,飼養(yǎng)第18天后處死小鼠,提取腫瘤并分離腫瘤浸潤淋巴細(xì)胞,磁珠分選腫瘤浸潤CD4+T淋巴細(xì)胞。建立Lewis細(xì)胞與腫瘤浸潤CD4+T淋巴細(xì)胞共培養(yǎng)體系,CCK-8法檢測Lewis細(xì)胞的增殖,篩選SJZDCS的最佳作用濃度和時間。建立Lewis細(xì)胞與腫瘤浸潤CD4+T淋巴細(xì)胞間接共培養(yǎng)體系,分為對照組、PD-1抗體(20 μg·mL-1)組、SJZDCS(20%)組、SJZDCS(20%)+PD-1抗體(20 μg·mL-1)組,干預(yù)72 h;酶聯(lián)免疫吸附實驗(ELISA)檢測共培養(yǎng)細(xì)胞上清中免疫功能相關(guān)細(xì)胞因子乳酸脫氫酶(LDH)、轉(zhuǎn)化生長因子-β1(TGF-β1)、腫瘤壞死因子-α(TNF-α)、干擾素-γ(IFN-γ)、白細(xì)胞介素(IL)-2、IL-4、IL-6和IL-10表達(dá)水平,Western blotting檢測腫瘤浸潤CD4+T淋巴細(xì)胞中PD-1和Ras-MEK-ERK通路蛋白表達(dá)水平。結(jié)果 20% SJZDCS干預(yù)腫瘤浸潤CD4+T淋巴細(xì)胞72 h對抑制共培養(yǎng)Lewis細(xì)胞的增殖效果最佳。與對照組相比,PD-1抗體組TNF-α、IFN-γ、IL-2和TGF-β1水平顯著升高(P<0.01),IL-4、IL-6、IL-10和LDH水平顯著降低(P<0.05);SJZDCS組IL-2水平顯著升高(P<0.01),IL-4、IL-6、LDH和TGF-β1水平顯著降低(P<0.05、0.01);SJZDCS+PD-1抗體組TNF-α、IFN-γ和IL-2水平顯著升高(P<0.01),IL-4、IL-6、LDH和TGF-β1水平降低(P<0.01)。與PD-1抗體組相比,SJZDCS+PD-1抗體組TNF-α、IFN-γ、IL-2升高水平和IL-4、IL-6和TGF-β1的降低水平更顯著(P<0.01)。與SJZDCS組相比,SJZDCS+PD-1抗體組TNF-α、IFN-γ、IL-2和IL-6的水平更高(P<0.01)。與對照組相比,PD-1抗體組、SJZDCS組、SJZDCS+PD-1抗體組CD4+T淋巴細(xì)胞中PD-1蛋白表達(dá)顯著降低(P<0.01),p-MEK1/2和MRK1/2蛋白表達(dá)顯著升高(P<0.05、0.01);PD-1抗體組、SJZDCS+PD-1抗體組Ras和P-ERK1/2蛋白表達(dá)顯著升高(P<0.01)。與PD-1抗體組相比,SJZDCS+PD-1抗體組ERK1/2、p-MEK1/2和p-ERK1/2蛋白表達(dá)顯著升高(P<0.01),Ras蛋白表達(dá)顯著降低(P<0.01)。與SJZDCS組相比,SJZDCS+PD-1抗體組Ras、p-MEK1/2、MEK1/2和p-ERK1/2蛋白表達(dá)顯著升高(P<0.05)。結(jié)論 SJZDCS單用或與PD-1抗體聯(lián)用,可在體外調(diào)節(jié)肺癌腫瘤浸潤CD4+T淋巴細(xì)胞免疫功能,其機制與調(diào)控PD-1及下游Ras-MEK-ERK通路表達(dá)有關(guān)。
[Key word]
[Abstract]
Objective The objective of this study was to investigate the impact and underlying mechanisms of Sijunzi Decoction containing serum (SJZDCS) in combination with a PD-1 antibody on the immune function of CD4+T lymphocytes infiltrating lung cancer tumors through in vitro experiments. Methods Male SD rats were administered Sijunzi Decoction via continuous gavage for 6 d, after which blood was collected under anesthesia to prepare serum containing the medication. Lewis lung cancer tumor-bearing mouse model was established using male C57BL/6J mice. After 18 d, the mice were sacrificed, tumors were excised, and tumorinfiltrating lymphocytes were isolated. CD4+T lymphocytes were sorted using magnetic beads, and a co-culture system with Lewis cells was established, and divided into control group, PD-1 antibody (20 μg·mL-1) group, SJZDCS (20%) group, SJZDCS (20%)+ PD-1 antibody (20 μg·mL-1) group, intervention for 72 h. The proliferation of Lewis cells was assessed using the CCK-8 assay to determine the optimal concentration and exposure time of the medicated serum. Immune function-related cytokines, including lactate dehydrogenase (LDH), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, and IL-10, were measured in the supernatant of the co-cultured cells using an enzyme-linked immunosorbent assay (ELISA). The expression levels of PD-1 and proteins involved in the Ras-MEK-ERK pathway in CD4+T lymphocytes were evaluated using Western blotting. Results The intervention of 20% SJZDCS on tumor infiltrating CD4+T lymphocytes for 72 h showed the best inhibitory effect on the proliferation of co cultured Lewis cells. Compared with the control group, the levels of TNF-α, IFN-γ, IL-2 and TGF-β1 were significantly increased (P < 0.01) and the levels of IL-4, IL-6, IL-10 and LDH were significantly decreased (P < 0.05) in the PD-1 antibody group. The level of IL-2 was significantly increased (P < 0.01) and the levels of IL-4, IL-6, LDH and TGF-β1 were significantly decreased (P < 0.05, 0.01) in the SJZDCS group. The levels of TNF-α, IFN-γ and IL-2 were significantly increased (P < 0.01) and the levels of IL-4, IL-6, LDH and TGF-β1 were decreased (P < 0.01) in the SJZDCS+PD-1 antibody group. Compared with the PD-1 antibody group, the increase in the levels of TNF-α, IFN-γ, IL-2 and the decrease in the levels of IL-4, IL-6 and TGF-β1 were more significant (P < 0.01). Compared with the SJZDCS group, the levels of TNF-α, IFN-γ, IL-2 and IL-6 were higher (P < 0.01) in the SJZDCS+PD-1 antibody group. Compared with the control group, the expression of PD-1 protein in CD4+T lymphocytes was significantly decreased (P < 0.01) in the PD-1 antibody group, SJZDCS group and SJZDCS+ PD-1 antibody group, and the expression of p-MEK1/2 and MRK1/2 proteins was significantly increased (P < 0.05, 0.01). The expression of ERK1/2, p-MEK1/2 and p-ERK1/2 proteins was significantly increased (P < 0.01) in the PD-1 antibody group and SJZDCS+PD-1 antibody group, and the expression of Ras protein was significantly decreased (P < 0.01). Compared with the PD-1 antibody group, the expression of ERK1/2, p-MEK1/2, and p-ERK1/2 proteins was significantly increased (P < 0.01) in the SJZDCS+PD-1 antibody group, while the expression of Ras protein was significantly decreased (P < 0.01). Compared with the SJZDCS group, the expression of Ras, p-MEK1/2, MEK1/2, and p-ERK1/2 proteins was significantly increased (P < 0.05) in the SJZDCS+PD-1 antibody group. Conclusions Sijunzi Decoction medicated serum, whether used alone or in combination with PD-1 antibodies, can modulate the immune function of CD4+T lymphocytes infiltrating lung cancer tumors in vitro. This effect is associated with the regulation of PD-1 expression and the Ras-MEK-ERK signaling pathway.
[中圖分類號]
R285.5
[基金項目]
國家自然科學(xué)基金面上項目(82074405)