張雯雯 劉璐瑤 曹娟
[摘要] 目的 研究雙硫侖(DSF)對口腔鱗狀細胞癌(OSCC)腫瘤細胞上清液誘導腫瘤相關(guān)巨噬細胞(TAMs)分泌的細胞因子基因表達的影響。 方法 將RAW264.7細胞隨機分為對照組和研究組,研究使用兩種誘導方法,第一種誘導方式為對照組用CAL27腫瘤細胞上清誘導RAW264.7細胞48 h,研究組在CAL27腫瘤細胞上清液誘導RAW264.7細胞的同時加入DSF/葡萄糖酸銅(DSF/Cu)2 μmol/L干預48 h;第二種誘導方式為對照組用CAL27腫瘤細胞上清誘導RAW264.7細胞48 h后,繼續(xù)誘導48 h,研究組CAL27腫瘤細胞上清誘導RAW264.7細胞48 h后,用DSF/Cu 2 μmol/L干預48 h。檢測兩種誘導方式下,一氧化氮合酶(iNOS)、白細胞介素12(IL-12)、腫瘤壞死因子-α(TNF-α)、精氨酸酶1(Arg-1)、IL-10的基因表達情況。 結(jié)果 誘導過程中加入DSF/Cu,研究組iNOS mRNA水平高于對照組,Arg-1 mRNA水平低于對照組,差異均有高度統(tǒng)計學意義(均P < 0.01)。兩組IL-12、TNF-α、IL-10 mRNA水平,差異無統(tǒng)計學意義(P > 0.05)。誘導48 h后加入DSF/Cu,研究組iNOS mRNA、IL-12 mRNA、TNF-α mRNA、IL-10 mRNA水平高于對照組,Arg-1 mRNA水平低于對照組,差異有統(tǒng)計學意義(P < 0.05或P < 0.01)。 結(jié)論 DSF可調(diào)節(jié)TAMs分泌細胞因子的基因表達,有助于進一步闡明DSF抗OSCC的作用機制,可能為將來口腔腫瘤的診治提供實驗依據(jù)和治療策略。
[關(guān)鍵詞] 雙硫侖;葡萄糖酸銅;腫瘤相關(guān)巨噬細胞;口腔鱗狀細胞癌
[中圖分類號] R739.8 ? ? ? ? ?[文獻標識碼] A ? ? ? ? ?[文章編號] 1673-7210(2020)08(b)-0012-04
[Abstract] Objective To study the effect of Disulfiram (DSF) on the genes expression of cytokine secreted by tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) tumor cell supernatant. Methods RAW264.7 cells were randomly divided into control group and research group. Two induction methods were used, the first inducement was that CAL27 tumor cell supernatant was used to induce RAW264.7 cells for 48 h in control group; and DSF and copper gluconate (DSF/Cu) 2 μmol/L were added to the CAL27 tumor cell supernatant to induce RAW264.7 cells for 48 h in research group. The second inducement was that RAW264.7 cells were induced with CAL27 tumor cell supernatant for 48 h, and then continued to be induced for 48 h in control group; in research group, after CAL27 tumor cell supernatant induced RAW264.7 cells for 48 h, DSF/Cu 2 μmol/L was used to intervene for 48 h. The gene expression levels of nitric oxide synthase (iNOS), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), argininase 1 (Arg-1) and IL-10 were measured. Results DSF/Cu was added in the induction process, iNOS mRNA level of research group was higher than that of control group, Arg-1 mRNA level was lower than that of control group, and the differences were highly statistically significant (all P < 0.01). There were no significant differences in IL-12, TNF-α and IL-10 mRNA levels between two groups (P > 0.05). DSF/Cu was added 48 h after induction, iNOS mRNA, IL-12 mRNA, TNF-α mRNA, IL-10 mRNA levels in research group were higher than those in control group, and Arg-1 mRNA level was lower than that in control group, with statistically significant differences (P < 0.05 or P < 0.01). Conclusion DSF can regulate the gene expression of cytokines secreted by TAMs, which helps to further clarify the mechanism of disulfiram against OSCC. It may provide experimental basis and treatment strategies for the diagnosis and treatment of oral tumors in the future.
[Key words] Disulfiram; Copper gluconate; Tumor-associated macrophages; Oral squamous cell carcinoma
口腔鱗狀細胞癌(oral squamous cell carcinoma,OSCC)是口腔中常見的癌癥,占頭頸癌的90%以上[1]。OSCC具有侵襲性強、轉(zhuǎn)移率高和預后差的特點,5年生存率不足50%[2]。近年來,OSCC的腫瘤微環(huán)境(TME)受到廣泛關(guān)注,成為研究熱點之一。腫瘤相關(guān)巨噬細胞(TAMs)是TME的重要免疫細胞群,參與腫瘤的發(fā)生發(fā)展[3]。TAMs在腫瘤進展期間可處于不同的極化狀態(tài),主要為M1型和M2型。M1型分泌細胞因子白細胞介素12(IL-12)、腫瘤壞死因子-α(TNF-α)、IL-6和硝酸氧化物發(fā)揮抗腫瘤作用[4]。M2型分泌IL-10、精氨酸酶1(Arg-1)等因子促進腫瘤的發(fā)展[5]。雙硫侖(DSF)作為安全抗酒精藥物[6],近年來,主要作為治療癌癥的潛在研究藥物,其可損害血管生成,抑制腫瘤侵襲轉(zhuǎn)移,進而加速腫瘤衰退[7-9]。研究顯示,培養(yǎng)基中的銅能增強DSF的抗癌活性,解決DSF容易失去活性的問題[10-11]。有研究顯示[12],將腫瘤細胞上清液與巨噬細胞共同孵育將其誘導為TAMs,可以更好地模擬腫瘤微環(huán)境。因此,本研究通過觀察DSF/葡萄糖酸銅(DSF/Cu)及OSCC腫瘤細胞上清液對RAW264.7細胞的兩種作用方式,誘導TAMs分泌細胞因子基因表達的影響,探討DSF/Cu抗OSCC作用機制,為OSCC的干預提供理論依據(jù)以及治療策略。
1 材料與方法
1.1 細胞株
人類舌鱗狀細胞癌細胞系CAL27細胞,小鼠RAW264.7巨噬細胞購于武漢大學細胞實驗室。
1.2 藥品與試劑
特級胎牛血清(批號:04-001-1A)、PBS(批號:02-024-1ACS)購自美國BI公司;DMEM(美國gibco公司,批號:06-1055-57-1ACS);二甲基亞砜(DMSO,美國sigma公司,批號:D2650);CCK-8(美國APExBIO公司,批號:K1018);RNA提取試劑盒(批號:9767)、反轉(zhuǎn)錄試劑盒(批號:RR047A)、PCR試劑盒(批號:RR820A)購自TKR公司;DSF(大連美侖生物公司,批號:MB1892);葡萄糖酸銅(中國麥克林公司,批號:C832356)。
1.3 方法
1.3.1 試驗藥物的配制 ?使用0.1 mg稱量精度的天平(美國奧豪斯,型號:PX224ZH)分別量取0.1 g DSF和葡萄糖酸銅,DMSO溶解DSF,PBS稀釋配比得DSF稀釋液。去離子水稀釋溶解0.1 g葡萄糖酸銅。
1.3.2 腫瘤細胞上清液的收集及DSF/Cu和CAL27細胞上清混合液的制備 ?取對數(shù)生長期狀態(tài)良好的CAL27細胞接種至培養(yǎng)瓶中,生長密度約為80%時,換新完全培養(yǎng)基培養(yǎng),24 h后收集培養(yǎng)基,1000 r/min,離心5 min,收集上清液,按7∶3比例將上清液與新完全培養(yǎng)基制備為腫瘤細胞上清液。將DSF/Cu溶于腫瘤細胞上清液中形成DSF/Cu和CAL27細胞上清混合液。
1.3.3 CCK-8法測定DSF/Cu對RAW264.7細胞活力 ?選對數(shù)生長期RAW264.7細胞接種在96孔板(2000個/孔),貼壁后,換腫瘤細胞上清100 μL加不同濃度DSF/Cu,每組設(shè)6個副孔。DSF為對照組(0 μmol/L)、研究組(0.5、1、2、4、8 μmol/L),葡萄糖酸銅為對照組(0 μmol/L)、研究組(0.5、1、2、4、8 μmol/L)作用48 h后,每孔加CCK-8 10 μL,孵育4 h。酶標儀檢測450 nm吸光度(OD)值。計算細胞生長活性:存活率(%)=(加藥組OD/對照組OD)×100%。
1.3.4 實時熒光定量聚合酶鏈式反應(PCR)檢測mRNA表達 ?選對數(shù)生長期RAW264.7細胞種于六孔板,隨機分對照組和研究組,每組3個副孔,用CAL27腫瘤細胞上清液誘導RAW264.7細胞。按照TKR說明書,提取并純化RNA,Nano drop 2000C儀器檢測RNA的純度及濃度,取合格總RNA為模板,按TKR逆轉(zhuǎn)錄試劑盒逆轉(zhuǎn)錄為cDNA。以β肌動蛋白為內(nèi)參,實時熒光定量PCR兩步法進行擴增:95℃預變性30 s,95℃ 5 s,60℃ 20 s 40個循環(huán)。應用2-ΔΔCT法分析mRNA相對表達量。序列如下:β-actin:5′-GTGCT-ATGTTGCTCTAGACTTCG-3′,5′-ATGCCACAGGAT-TCCATACC-3′;IL-10:5′-CTGCTATGCTGCCTGCTC-TTACTG-3′,5′-ATGTGGCTCTGGCCGACTGG-3′;IL-12:5′-ACGAGAGTTGCCTGGCTACTAGAG-3′,5′-TCTG-AAGTGCTGCGTTGATGGC-3′;TNF-α:5′-CTCATG-CACCACCATCAAGGACTC-3′,5′-AGACAGAGGCA-ACCTGACCACTC-3′;Arg-1:5′-TGCTCACACTGAC-ATCAACACTCC-3′,5′-GGTCTACGTCTCGCAAGCC-AATG-3′;iNOS:5′-TGCCACGGACGAGACGGATAG-3′,5′-CTC-TTCAAGCACCTCCAGGAACG-3′。
1.4 觀察指標
①采用CAL27細胞上清液孵育RAW264.7誘導形成TAMs,觀察組在TAMs誘導過程中加入DSF/Cu作用48 h,檢測兩組iNOS、IL-12、TNF-α、Arg-1、IL-10的mRNA水平;②CAL27腫瘤細胞上清液誘導RAW264.7細胞誘導形成TAMs,在誘導48 h后觀察組加入DSF/Cu再作用48 h,對照組繼續(xù)誘導48 h,檢測兩組iNOS、IL-12、TNF-α、Arg-1、IL-10的mRNA水平。
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(收稿日期:2020-02-18)