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      雙硫侖對(duì)腫瘤相關(guān)巨噬細(xì)胞分泌細(xì)胞因子基因表達(dá)的影響

      2020-10-09 10:32張?chǎng)?/span>劉璐瑤曹娟
      關(guān)鍵詞:雙硫侖

      張?chǎng)? 劉璐瑤 曹娟

      [摘要] 目的 研究雙硫侖(DSF)對(duì)口腔鱗狀細(xì)胞癌(OSCC)腫瘤細(xì)胞上清液誘導(dǎo)腫瘤相關(guān)巨噬細(xì)胞(TAMs)分泌的細(xì)胞因子基因表達(dá)的影響。 方法 將RAW264.7細(xì)胞隨機(jī)分為對(duì)照組和研究組,研究使用兩種誘導(dǎo)方法,第一種誘導(dǎo)方式為對(duì)照組用CAL27腫瘤細(xì)胞上清誘導(dǎo)RAW264.7細(xì)胞48 h,研究組在CAL27腫瘤細(xì)胞上清液誘導(dǎo)RAW264.7細(xì)胞的同時(shí)加入DSF/葡萄糖酸銅(DSF/Cu)2 μmol/L干預(yù)48 h;第二種誘導(dǎo)方式為對(duì)照組用CAL27腫瘤細(xì)胞上清誘導(dǎo)RAW264.7細(xì)胞48 h后,繼續(xù)誘導(dǎo)48 h,研究組CAL27腫瘤細(xì)胞上清誘導(dǎo)RAW264.7細(xì)胞48 h后,用DSF/Cu 2 μmol/L干預(yù)48 h。檢測(cè)兩種誘導(dǎo)方式下,一氧化氮合酶(iNOS)、白細(xì)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(dá)情況。 結(jié)果 誘導(dǎo)過(guò)程中加入DSF/Cu,研究組iNOS mRNA水平高于對(duì)照組,Arg-1 mRNA水平低于對(duì)照組,差異均有高度統(tǒng)計(jì)學(xué)意義(均P < 0.01)。兩組IL-12、TNF-α、IL-10 mRNA水平,差異無(wú)統(tǒng)計(jì)學(xué)意義(P > 0.05)。誘導(dǎo)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(duì)照組,Arg-1 mRNA水平低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P < 0.05或P < 0.01)。 結(jié)論 DSF可調(diào)節(jié)TAMs分泌細(xì)胞因子的基因表達(dá),有助于進(jìn)一步闡明DSF抗OSCC的作用機(jī)制,可能為將來(lái)口腔腫瘤的診治提供實(shí)驗(yàn)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(xì)胞;口腔鱗狀細(xì)胞癌

      [中圖分類號(hào)] R739.8 ? ? ? ? ?[文獻(xiàn)標(biāo)識(shí)碼] A ? ? ? ? ?[文章編號(hào)] 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(xì)胞癌(oral squamous cell carcinoma,OSCC)是口腔中常見(jiàn)的癌癥,占頭頸癌的90%以上[1]。OSCC具有侵襲性強(qiáng)、轉(zhuǎn)移率高和預(yù)后差的特點(diǎn),5年生存率不足50%[2]。近年來(lái),OSCC的腫瘤微環(huán)境(TME)受到廣泛關(guān)注,成為研究熱點(diǎn)之一。腫瘤相關(guān)巨噬細(xì)胞(TAMs)是TME的重要免疫細(xì)胞群,參與腫瘤的發(fā)生發(fā)展[3]。TAMs在腫瘤進(jìn)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(xì)胞因子白細(xì)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(jìn)腫瘤的發(fā)展[5]。雙硫侖(DSF)作為安全抗酒精藥物[6],近年來(lái),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(jìn)而加速腫瘤衰退[7-9]。研究顯示,培養(yǎng)基中的銅能增強(qiáng)DSF的抗癌活性,解決DSF容易失去活性的問(wèn)題[10-11]。有研究顯示[12],將腫瘤細(xì)胞上清液與巨噬細(xì)胞共同孵育將其誘導(dǎo)為TAMs,可以更好地模擬腫瘤微環(huán)境。因此,本研究通過(guò)觀察DSF/葡萄糖酸銅(DSF/Cu)及OSCC腫瘤細(xì)胞上清液對(duì)RAW264.7細(xì)胞的兩種作用方式,誘導(dǎo)TAMs分泌細(xì)胞因子基因表達(dá)的影響,探討DSF/Cu抗OSCC作用機(jī)制,為OSCC的干預(yù)提供理論依據(jù)以及治療策略。

      1 材料與方法

      1.1 細(xì)胞株

      人類舌鱗狀細(xì)胞癌細(xì)胞系CAL27細(xì)胞,小鼠RAW264.7巨噬細(xì)胞購(gòu)于武漢大學(xué)細(xì)胞實(shí)驗(yàn)室。

      1.2 藥品與試劑

      特級(jí)胎牛血清(批號(hào):04-001-1A)、PBS(批號(hào):02-024-1ACS)購(gòu)自美國(guó)BI公司;DMEM(美國(guó)gibco公司,批號(hào):06-1055-57-1ACS);二甲基亞砜(DMSO,美國(guó)sigma公司,批號(hào):D2650);CCK-8(美國(guó)APExBIO公司,批號(hào):K1018);RNA提取試劑盒(批號(hào):9767)、反轉(zhuǎn)錄試劑盒(批號(hào):RR047A)、PCR試劑盒(批號(hào):RR820A)購(gòu)自TKR公司;DSF(大連美侖生物公司,批號(hào):MB1892);葡萄糖酸銅(中國(guó)麥克林公司,批號(hào):C832356)。

      1.3 方法

      1.3.1 試驗(yàn)藥物的配制 ?使用0.1 mg稱量精度的天平(美國(guó)奧豪斯,型號(hào):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(xì)胞上清液的收集及DSF/Cu和CAL27細(xì)胞上清混合液的制備 ?取對(duì)數(shù)生長(zhǎng)期狀態(tài)良好的CAL27細(xì)胞接種至培養(yǎng)瓶中,生長(zhǎng)密度約為80%時(shí),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(xì)胞上清液。將DSF/Cu溶于腫瘤細(xì)胞上清液中形成DSF/Cu和CAL27細(xì)胞上清混合液。

      1.3.3 CCK-8法測(cè)定DSF/Cu對(duì)RAW264.7細(xì)胞活力 ?選對(duì)數(shù)生長(zhǎng)期RAW264.7細(xì)胞接種在96孔板(2000個(gè)/孔),貼壁后,換腫瘤細(xì)胞上清100 μL加不同濃度DSF/Cu,每組設(shè)6個(gè)副孔。DSF為對(duì)照組(0 μmol/L)、研究組(0.5、1、2、4、8 μmol/L),葡萄糖酸銅為對(duì)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(biāo)儀檢測(cè)450 nm吸光度(OD)值。計(jì)算細(xì)胞生長(zhǎng)活性:存活率(%)=(加藥組OD/對(duì)照組OD)×100%。

      1.3.4 實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(PCR)檢測(cè)mRNA表達(dá) ?選對(duì)數(shù)生長(zhǎng)期RAW264.7細(xì)胞種于六孔板,隨機(jī)分對(duì)照組和研究組,每組3個(gè)副孔,用CAL27腫瘤細(xì)胞上清液誘導(dǎo)RAW264.7細(xì)胞。按照TKR說(shuō)明書(shū),提取并純化RNA,Nano drop 2000C儀器檢測(cè)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(dòng)蛋白為內(nèi)參,實(shí)時(shí)熒光定量PCR兩步法進(jìn)行擴(kuò)增:95℃預(yù)變性30 s,95℃ 5 s,60℃ 20 s 40個(gè)循環(huán)。應(yīng)用2-ΔΔCT法分析mRNA相對(duì)表達(dá)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(biāo)

      ①采用CAL27細(xì)胞上清液孵育RAW264.7誘導(dǎo)形成TAMs,觀察組在TAMs誘導(dǎo)過(guò)程中加入DSF/Cu作用48 h,檢測(cè)兩組iNOS、IL-12、TNF-α、Arg-1、IL-10的mRNA水平;②CAL27腫瘤細(xì)胞上清液誘導(dǎo)RAW264.7細(xì)胞誘導(dǎo)形成TAMs,在誘導(dǎo)48 h后觀察組加入DSF/Cu再作用48 h,對(duì)照組繼續(xù)誘導(dǎo)48 h,檢測(cè)兩組iNOS、IL-12、TNF-α、Arg-1、IL-10的mRNA水平。

      [4] ?Jeannin P,Paolini L,Adam C,et al. The roles of CSFs on the functional polarization of tumor-associated macrophages [J]. FEBS J,2018,285(4):680-699.

      [5] ?Griess B,Mir S,Datta K,et al. Scavenging reactive oxygen species selectively inhibits M2 macrophage polarization and their pro-tumorigenic function in part,via Stat3 suppression [J]. Free Radic Biol Med,2020,147:48-60.

      [6] ?Banerjee P,Geng T,Mahanty A,et al. Integrating the drug,disulfiram into the vitamin E-TPGS-modified PEGylated nanostructured lipid carriers to synergize its repurposing for anti-cancer therapy of solid tumors [J]. Int J Pharm,2019,557:374-389.

      [7] ?Yang Q,Yao Y,Li K,et al. An Updated Review of Disulfiram:Molecular Targets and Strategies for Cancer Treatment [J]. Curr Pharm Des,2019,25(30):3248-3256.

      [8] ?Wu X,Xue X,Wang LH,et al. Suppressing autophagy enhances disulfiram/copper-induced apoptosis in non-small cell lung cancer [J]. Eur J Pharmacol,2018,827:1-12.

      [9] ?Zhong Y,Sun R,Geng Y,et al. N-Oxide polymer-cupric ion nanogels potentiate disulfiram for cancer therapy [J]. Biomater Sci,2020,8(6):1726-1733.

      [10] ?Majera D,Skrott Z,Bouchal J,et al. Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors,vorinostat and disulfiram [J]. Prostate,2019,79(4):352-362.

      [11] ?McMahon A,Chen W,Li F. Old wine in new bottles:Advanced drug delivery systems for disulfiram-based cancer therapy [J]. J Control Release,2020,319:352-359.

      [12] ?Zhang Y,Du W,Chen Z,et al. Upregulation of PD-L1 by SPP1 mediates macrophage polarization and facilitates immune escape in lung adenocarcinoma [J]. Exp Cell Res,2017,359(2):449-457.

      [13] ?Kwon IS,Yim JH,Lee HK,et al. Lobaric Acid Inhibits VCAM-1 Expression in TNF-alpha-Stimulated Vascular Smooth Muscle Cells via Modulation of NF-kappa B and MAPK Signaling Pathways [J]. Biomol Ther(seoul),2016, 24(1):25-32.

      [14] ?Kanemaru H,Yamane F,F(xiàn)ukushima K,et al. Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages [J]. Proc Natl Acad Sci U S A,2017,114(35):E7331-E7340.

      [15] ?Goswami KK,Ghosh T,Ghosh S,et al. Tumor promoting role of anti-tumor macrophages in tumor microenvironment [J]. Cell Immunol,2017,316:1-10.

      [16] ?Berti FCB,de Oliveira KB. IL-10 in cancer:Just a classical immunosuppressive factor or also an immunostimulating one [J]. AIMS Allergy Immunol,2018,2(2):88-97.

      [17] ?Martinenaite E,Ahmad SM,Bendtsen SK,et al. Arginase-1-based vaccination against the tumor microenvironment:the identification of an optimal T-cell epitope [J]. Cancer Immunol Immunother,2019,68(11):1901-1907.

      [18] ?Liu B,Wang X,Chen TZ,et al. Polarization of M1 tumor associated macrophage promoted by the activation of TLR3 signal pathway [J]. Asian Pac J Trop Med,2016,9(5):484-488.

      [19] ?Chiang CF,Chao TT,Su YF,et al. Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-kappa B signaling [J]. Oncotarget,2017,8(13):20706-20718.

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      (收稿日期:2020-02-18)

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