文雪
摘要:目的:PDL1廣泛表達(dá)于腫瘤組織,因此,PDL1/PD1通路可能在腫瘤免疫逃逸中起到一定作用。本研究通過檢測(cè)化療藥物作用前后PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)情況,為進(jìn)一步研究其在膠質(zhì)瘤靶向治療中的作用提供實(shí)驗(yàn)依據(jù)。方法 利用流式細(xì)胞儀檢測(cè)化療藥物作用前后PDL1在膠質(zhì)瘤細(xì)胞株的表達(dá)。結(jié)果:膠質(zhì)瘤細(xì)胞株高表達(dá)PDL1,化療藥物作用后PDL1的表達(dá)顯著升高。結(jié)論 PDL1可以作為膠質(zhì)瘤治療的潛在靶點(diǎn)。
關(guān)鍵詞:PDL1;膠質(zhì)瘤;化療藥物
Abstract: Objective PDL1 is widely expressed in tumor tissues. Therefore, the PDL1/PD1 pathway may play a role in tumor immune escape. This study detected the expression of PDL1 in glioma cells before and after the action of chemotherapeutics, and provided experimental evidence for further research on its role in targeted therapy of glioma. Methods Flow cytometry was used to detect the expression of PDL1 in glioma cell lines before and after the action of chemotherapy drugs. Results The glioma cell line highly expressed PDL1, and the expression of PDL1 was significantly increased after chemotherapeutic drugs. Conclusion PDL1 can be used as a potential target for the treatment of glioma.
Keywords: PDL1; glioma; chemotherapy drugs
程序性死亡配體-1 (PDL1) 是一種主要的免疫檢查點(diǎn)配體分子,它在腫瘤和不同類型癌癥的腫瘤微環(huán)境中上調(diào)表達(dá)[1]。臨床研究證實(shí),抑制PDL1與程序性細(xì)胞死亡受體 (PD1)在各種類型的免疫細(xì)胞(包括抗原呈遞細(xì)胞、效應(yīng) T 細(xì)胞、自然殺傷細(xì)胞 (NK)、胸腺細(xì)胞、骨髓細(xì)胞)上表達(dá),阻斷其相互作用,能夠誘導(dǎo)免疫系統(tǒng)對(duì)腫瘤的持久控制和抑制不同類型的癌癥 [2-4]。本研究通過檢測(cè)PDL1在不同膠質(zhì)瘤細(xì)胞株的表達(dá)情況,為研究負(fù)性調(diào)節(jié)分子PDL1的表達(dá)與膠質(zhì)瘤細(xì)胞對(duì)化療藥物抵抗的相關(guān)性及免疫逃逸機(jī)制提供實(shí)驗(yàn)依據(jù)。
1 材料及方法
1.1細(xì)胞及試劑 U251 細(xì)胞系為本實(shí)驗(yàn)室凍存;替莫唑胺(TMZ)購(gòu)自先靈葆雅公司;DMEM培養(yǎng)基購(gòu)自Gibco公司;胰酶購(gòu)自Sigma公司;抗PDL1抗體購(gòu)自eBioscience公司。
1.2 細(xì)胞培養(yǎng) 將凍存的U251細(xì)胞復(fù)蘇后,以每孔200個(gè)細(xì)胞培養(yǎng)在含有15%胎牛血清DMEM培養(yǎng)基的六孔板中,置于37℃、5%CO2條件下培養(yǎng)。24小時(shí)后,將培養(yǎng)基更換為包含TMZ(0、25、50和100μmol/L),共同孵育72小時(shí)。
1.3流式細(xì)胞術(shù)檢測(cè)PDL1的表達(dá) 胰酶消化并收集上述各組腫瘤細(xì)胞,然后用冰冷的PBS洗滌,按照試劑說明加入抗PDL1抗體,采用流式細(xì)胞術(shù)檢測(cè)并分析結(jié)果。
1.4統(tǒng)計(jì)分析 使用Origin Pro 軟件進(jìn)行統(tǒng)計(jì)分析,統(tǒng)計(jì)顯著性由t 檢驗(yàn)確定,并且在 p ≤ 0.05 時(shí)被認(rèn)為是顯著的。值表示為平均值±標(biāo)準(zhǔn)差。
2 結(jié)果
TMZ濃度為0時(shí),PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)為35%±2.1;TMZ濃度為25μmol/L時(shí),PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)為40%±3.6;TMZ濃度為50μmol/L時(shí),PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)為53%±6.4;TMZ濃度為100μmol/L時(shí),PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)為68%±7.8。結(jié)果表明,PDL1高表達(dá)于膠質(zhì)瘤細(xì)胞的表達(dá),化療藥物TMZ可以誘導(dǎo)PDL1在膠質(zhì)瘤細(xì)胞的表達(dá),其表達(dá)程度與TMZ濃度呈正相關(guān)。
3 討論
膠質(zhì)瘤是 21 世紀(jì)最具破壞性和無法治愈的疾病;它的特點(diǎn)是組織異質(zhì)性高,從低度 I-II 級(jí)向高度 III-IV 級(jí)惡性腫瘤快速轉(zhuǎn)變。90% 的高級(jí)別膠質(zhì)瘤患者盡管進(jìn)行了最大程度的手術(shù)腫瘤切除、放療和化療,但仍會(huì)出現(xiàn)腫瘤復(fù)發(fā) [5,6]?;颊呖偵嫫诘闹形粩?shù)為 15-18 個(gè)月,五年后只有約 10% 的患者存活。尋找新的治療靶點(diǎn)發(fā)揮抗腫瘤免疫,可能會(huì)顯著提高膠質(zhì)瘤患者的存活率。研究證明,PD-L1表達(dá)于惡性腫瘤組織,放療及化療均可誘導(dǎo)腫瘤PDL1的表達(dá)增加。本研究檢測(cè)了不同濃度化療藥物TMZ作用前后PDL1在膠質(zhì)瘤細(xì)胞的表達(dá)情況。結(jié)果顯示,膠質(zhì)瘤細(xì)胞本身高表達(dá)PDL1,當(dāng)膠質(zhì)瘤細(xì)胞與TMZ混合培養(yǎng)后,PDL1的表達(dá)顯著增加,且其增加程度與TMZ的濃度成正比,說明化療藥物可以誘導(dǎo)PDL1在膠質(zhì)瘤細(xì)胞的表達(dá),阻斷PDL1/PD1 相互作用,可能是膠質(zhì)瘤治療的潛在策略。
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基金項(xiàng)目:2020年度湖北省教育廳科學(xué)研究計(jì)劃指導(dǎo)性項(xiàng)目(B2020389)