目的 通過采用腫瘤新抗原疫苗對(duì)產(chǎn)生表皮細(xì)胞生長因子受體-酪酸激酶抑制劑（EGFR-TKI）耐藥性的肺癌大鼠模型進(jìn)行治療，并進(jìn)一步探究其機(jī)制。方法 采用胸部X射線照射，劑量為3 Gy，每周3次，構(gòu)建大鼠肺癌模型，對(duì)EGFR-TKI治療2周后，大鼠腫瘤大小不再減小，視為耐藥性大鼠肺癌模型建立成功。采用標(biāo)準(zhǔn)的固相合成肽化學(xué)和反相高效液相色譜法合成和純化多肽制備特異性新抗原，將大鼠肺癌EGFR-TKI耐藥模型隨機(jī)分為模型組、傳統(tǒng)化療組[采用傳統(tǒng)一線聯(lián)合化療方式，先ip培美曲塞10 mg/（kg·d），再ip順鉑0.6 mg/（kg∙d），連續(xù)用藥10 d]，新抗原組（采用sc特異性新抗原進(jìn)行化療，劑量3 mL/只，連續(xù)用藥2次）。PET-CT檢測(cè)各組大鼠的腫瘤大小，蘇木素-伊紅（HE）染色觀察組織病變情況，流式細(xì)胞術(shù)檢測(cè)各組大鼠外周血淋巴細(xì)胞CD4⁺、CD8⁺、CD127⁺表達(dá)，酶聯(lián)免疫吸附實(shí)驗(yàn)檢測(cè)外周血γ干擾素（IFN-γ）、白細(xì)胞介素（IL）-2、IL-5水平。結(jié)果 新抗原組腫瘤質(zhì)量顯著低于模型組和傳統(tǒng)化療組（P<0.05），同時(shí)PET-CT掃描新抗原組腫瘤病灶出現(xiàn)縮小趨勢(shì)；HE染色中，相比于模型組和傳統(tǒng)化療組，新抗原組肺組織中炎性浸潤現(xiàn)象呈現(xiàn)顯著下降趨勢(shì)，同時(shí)新生毛細(xì)血管的增加量和肺泡腔的孔徑呈現(xiàn)上升趨勢(shì)；相比于模型組和傳統(tǒng)化療組，新抗原組大鼠外周血淋巴細(xì)胞中CD4⁺、CD4⁺/CD127⁺、CD8⁺/CD127⁺的表達(dá)情況呈現(xiàn)顯著升高，CD8⁺的表達(dá)則顯著降低（P<0.05）；相比于模型組和傳統(tǒng)化療組，新抗原組大鼠外周血中IL-2、IFN-γ、IL-5水平顯著下降（P<0.05）。結(jié)論 采用腫瘤新抗原疫苗對(duì)EGFR-TKI耐藥后肺癌大鼠模型進(jìn)行干預(yù)，能夠有效地縮減腫瘤質(zhì)量，對(duì)肺部組織炎性病變程度起到一定的緩解作用，能夠減輕炎癥浸潤情況，通過升高外周血淋巴細(xì)胞內(nèi)CD4⁺、CD4⁺/CD127⁺、CD8⁺/CD127⁺，減少CD8⁺因子的表達(dá)，起到免疫抑制作用。另外，新抗原疫苗能夠降低外周血IFN-γ、IL-2、IL-5炎性因子水平，減輕肺部炎性程度。

Objective To further explore the mechanism of tumor neoantigen vaccine to treat lung cancer rat models with epidermal growth factor receptor-tyrosate kinase inhibitor (EGFR-TKI) resistance. Methods The rat lung cancer model was established by chest X-ray irradiation at a dose of 3 Gy, 3 times weekly. After EGFR-TKI treatment for 2 weeks, the tumor size of the rats did not decrease, which was regarded as the successful establishment of drug-resistant rat lung cancer model. Specific neoantigens are prepared by synthesizing and purifying peptides using standard solid-phase synthetic peptide chemistry and reversed-phase high performance liquid chromatography. The EGFR-TKI resistant rat lung cancer model was randomly divided into model group, conventional chemotherapy group[Traditional first-line combined chemotherapy, peritoneally injected pemetrexed 10 mg/(kg·d), followed by cisplatin 0.6 mg/(kg·d) for 10 consecutive days], neoantigen group (Specific neoantigen was injected subcutaneously for 2 times at a dose of 3 mL). The tumor size of rats in each group was detected by PET-CT, the histological changes were observed by hematoxylin-eosin (HE)

staining, the expression of CD4+, CD8+, and CD127+ in peripheral blood lymphocytes of rats in each group was detected by flow cytometry, and the levels of interferon (IFN)-γ, interleukin (IL)-2, and IL-5 in peripheral blood were detected by enzyme linked immunosorbent assay. Results The tumor quality in the neoantigen group was significantly lower than that in the model group and traditional chemotherapy group (P < 0.05), and the tumor lesions in the PET-CT scanning neoantigen group showed a trend of shrinkage. In HE staining, compared with the model group and traditional chemotherapy group, the inflammatory infiltration in the lung tissues of the neoantigen group showed a significant decreasing trend, while the increase of new capillaries and the aperture of alveolar cavity showed an increasing trend. Compared with model group and conventional chemotherapy group, the expression of CD4+, CD4+/CD127+, and CD8+/CD127+ in peripheral blood lymphocytes of neoantigen group was significantly increased, while the expression of CD8+ was significantly decreased (P < 0.05). Compared with model group and conventional chemotherapy group, the levels of IL-2, IFN-γ, and IL-5 in peripheral blood of neoantigen group were significantly decreased (P < 0.05). Conclusion The intervention of tumor neoantigen vaccine on EGFR-TKI resistant lung cancer rat model can effectively reduce the tumor quality, alleviate the degree of inflammatory lesions in lung tissue to a certain extent, and reduce inflammatory infiltration. By increasing CD4+, CD4+/CD127+, and CD8+/CD127+ in peripheral blood lymphocytes, and reducing the expression of CD8+ factors, play an immunosuppressive role. In addition, neoantigen vaccine can reduce the levels of IFN-γ, IL-2, IL-5 inflammatory factors in peripheral blood, and reduce the degree of lung inflammation.

R979.1

天津市北辰區(qū)科技計(jì)劃項(xiàng)目（SHGY-2020006）