2021, Vol. 48扩展功能
文章信息
- 其力格尔, Sadia Nawab, 范殊璇, 曹博, 李雨昕, 田明振, 张商浩, 马伟, 邓子新
- BAO Qelger, Sadia Nawab, FAN Shuxuan, CAO Bo, LI Yuxin, TIAN Mingzhen, ZHANG Shanghao, MA Wei, DENG Zixin
- ω-3多不饱和脂肪酸抗肿瘤新途径——抑制具核梭杆菌黏附宿主细胞
- New anti-tumor mechanism of ω-3 polyunsaturated fatty acids—inhibiting Fusobacterium nucleatum adherence to host cells
- 微生物学通报, 2021, 48(3): 820-829
- Microbiology China, 2021, 48(3): 820-829
- DOI: 10.13344/j.microbiol.china.200337
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文章历史
- 收稿日期: 2020-04-03
- 接受日期: 2020-05-22
- 网络首发日期: 2020-09-07
2. 上海交通大学致远学院 上海 200240;
3. 上海市民办平和双语学校 上海 200000
2. Zhiyuan College, Shanghai Jiao Tong University, Shanghai 200240, China;
3. Shanghai Pinghe School, Shanghai 200000, China
许多体内外以及临床研究表明，二十二碳六烯酸(Docosahexaenoic Acid，DHA)、二十碳五烯酸(Eicosapentaenoic Acid，EPA)等ω-3多不饱和脂肪酸具有显著的预防及治疗肿瘤发生和发展的作用[1-4]。如喂食富含DHA食物的神经母细胞瘤移植瘤大鼠肿瘤生长速度显著减慢[5]；补充DHA的乳腺癌化疗患者中，血浆、红细胞中DHA水平高的患者相对于含量低的患者，肿瘤进展延迟和总生存期延长[6]。结直肠癌[7]、胃肠癌[8]、乳腺癌[9]等多种肿瘤研究均表明ω-3多不饱和脂肪酸通过增加细胞膜流动性[10]、抗炎活性[11]、减少促进致癌物前列腺素E2的合成[7]、增加细胞内ROS水平[12]等途径抑制肿瘤细胞生长[13]、诱导肿瘤细胞凋亡[13]。然而，Hanson等2020年针对108 194人的研究结果显示，补充DHA、EPA、ALA对肿瘤发生以及肿瘤患者死亡风险几乎没有影响[14]。如此矛盾的相关研究报道揭示不饱和脂肪酸对肿瘤发生、发展作用的复杂性，暗示可能存在未知途径与作用。
Bray等2018年的研究报道显示，全球结直肠癌发病率和死亡率分别位于恶性肿瘤的第3位与第2位[15]。近年来肠道微生物菌群功能或结构失调与结直肠癌之间存在密切关联的研究报道越来越多[16-20]。其中，Chen等发现相较于健康志愿者，结直肠癌患者的组织样本中Fusobacterium、Porphyromonas、Peptostreptococcus、Gemella、Mogibacterium和Klebsiella等属菌的丰度显著增加，同时，Feacalibacterium、Blautia、Lachnospira、Bifidobacterium和Anaerostipes等属菌丰度显著减少[21]。
近年来，对革兰氏阴性专性厌氧的具核梭杆菌(F. nucleatum)与结直肠癌(Colorectal Cancer，CRC)等肿瘤发生、发展[22]、化疗耐药等密切相关[23-25]，引起了国内外专家的广泛关注。研究表明，F. nucleatum菌体[26-29]、表面蛋白[30-37]及脂多糖[38]等代谢物分别具有促进肿瘤细胞侵袭、诱发炎症反应、促进细胞生长分裂、免疫细胞招募等多样、全局的作用[39]。
F. nucleatum表面蛋白FadA与结直肠癌细胞表面的E-钙黏蛋白结合能激活β-Catenin信号通路，促进肿瘤细胞的增殖[22]。Fap2作为F. nucleatum与人体细胞结合的另一个关键表面蛋白，通过与CRC细胞过表达的Gal-GalNAc结合，导致F. nucleatum在CRC病灶部位富集[35]。Fap2蛋白同时还能与T淋巴细胞和NK细胞表面的抑制性受体TIGIT作用，导致NK细胞毒性和淋巴细胞活性的抑制[40]，最终保护CRC细胞免受自然杀伤细胞(Natural Killer Cell，NK)毒性、肿瘤浸润淋巴细胞和T细胞攻击。RadD作为F. nucleatum的另一个重要黏附素通过与Streptococcus mutans[41][41]、S. mutans、白色念珠菌 [42]的结合，促进口腔细菌聚集及生物膜的形成。由此可见，F. nucleatum很可能是人体异常微生物群落形成的先锋物种。
基于上述研究报道，尤其是DHA与EPA能够下调F. nucleatum FadA表达[43]的研究报道，本研究计划通过系统分析DHA、EPA、ALA对具核梭杆菌、结直肠正常与肿瘤细胞的生长以及对具核梭杆菌与人体细胞互作的影响，探究多不饱和脂肪酸、F. nucleatum、结直肠肿瘤三者的相互关系，为明确多不饱和脂肪酸抑制肿瘤机制提供信息。
1 材料与方法 1.1 材料 1.1.1 细胞株与细胞培养细胞株：人结直肠腺癌细胞Caco-2和正常结肠上皮细胞NCM460由本实验室保存。
细胞培养：Caco-2细胞采用含20%胎牛血清的DMEM高糖培养基培养；NCM460细胞采用含10%胎牛血清的DMEM高糖培养基培养；2种细胞于37 ℃、5% CO2、饱和湿度的细胞培养箱中培养。细胞传代、冻存等操作按常规方法进行。
1.1.2 实验菌株与培养实验菌株：F. nucleatum ATCC 25586由本实验室保存。
菌株培养：F. nucleatum采用BHI培养基在85% N2、5% CO2、10% H2、37 ℃厌氧培养。
1.1.3 主要试剂和仪器DMEM高糖培养基，HyClone公司；胎牛血清，BI公司；DHA、EPA、ALA，上海阿拉丁生化科技股份有限公司；MTT细胞增殖及细胞毒性检测试剂盒，南京凯基生物科技发展有限公司；细菌总RNA提取试剂盒，Magen生物公司；FastQuant cDNA第一链合成试剂盒，天根生化科技(北京)有限公司；Hieff UNICON® qPCR SYBR Green Master Mix，上海翊圣生物科技有限公司。多功能酶标仪，Tecan公司；荧光定量PCR仪，Thermo Fisher公司；荧光正置显微镜，蔡司公司。
1.2 方法 1.2.1 DHA、EPA、ALA的细胞毒性检测采用MTT方法检测了DHA、EPA、ALA的细胞毒性。将Caco-2细胞、NCM460细胞培养至80%覆盖时，胰酶消化，96孔板每孔接种5 000个细胞。待细胞过夜贴壁后，加入DHA、EPA、ALA使终浓度分别为0、30、60、120 µg/mL，处理24、48、72 h后，采用MTT细胞增殖及细胞毒性检测试剂盒进行细胞活性检测。
1.2.2 具核梭杆菌对结直肠腺癌细胞的黏附试验F. nucleatum用ω-3多不饱和脂肪酸预处理组：将培养至指数末期的F. nucleatum按1%比例分别接种于含30 μg/mL DHA、EPA、ALA的BHI培养基继续培养12 h后，将菌悬液4 000 r/min离心10 min。用DMEM培养基悬浮细菌，调整菌浓度至1×108 CFU/mL。
在加入细胞培养级盖玻片的24孔细胞培养板中铺1×105个Caco-2细胞，置37 ℃、5% CO2培养箱中培养至80%覆盖度时，用PBS洗3遍，每孔分别加入经DHA、EPA、ALA预处理或未处理的500 μL 1×108 CFU/mL具核梭杆菌。
共培养组：此时未处理组中加入终浓度为30 µg/mL的DHA、EPA、ALA。对照组与各实验组均设置3个复孔。两组分别继续于37 ℃、5% CO2条件下培养2 h后，吸去培养液，用PBS冲洗3遍，洗去未结合的具核梭杆菌，自然干燥，冰甲醇固定10 min，革兰氏染色，100倍油镜观察、拍照、计单细胞黏附的细菌数。
1.2.3 总RNA提取、反转录、定量PCR检测待F. nucleatum长到对数生长期，即OD600为0.5左右时分别用30 µg/mL的DHA、EPA、ALA处理3 h，随后采用细菌总RNA提取试剂盒提取具核梭杆菌总RNA，具体操作按试剂盒说明书进行。反转录根据FastQuant cDNA第一链合成试剂盒进行反转录，总RNA使用量为1 µg。定量PCR采用Hieff UNICON® qPCR SYBR Green Master Mix配制20 µL的反应体系进行实验，操作按说明书进行。rpoB等定量PCR引物采用Primer 3网站设计，由捷瑞公司合成，引物具体序列见表 1。定量PCR条件为：95 ℃ 15 min；95 ℃ 10 s，60 ℃ 20 s，72 ℃ 20 s，共40个循环，最后进行溶解曲线分析。基因表达相对定量采用2–ΔΔCt方法计算，内参基因为rpoB。
| 基因gene | 引物primers | 序列Sequences (5′→3′) |
| Fap2 | 正向Forward | CAGTTGCTACAGATGGTGTGGTAGG |
| 反向Reverse | TGAATTTGCTTCTCCATACATTCCTG | |
| FadA | 正向Forward | GCAGCAAGTTTAGTAGGTGAATTACAA |
| 反向Reverse | GTCTAGCAGCGTCAGCTTGTG | |
| RadD | 正向Forward | GATATGAGCAACAACAATGCGAAAG |
| 反向Reverse | GTGCCTTGACCATCAGAAGAAACTT | |
| rpoB | 正向Forward | ATCTTCCACCATCAAGTTGAACCAT |
| 反向Reverse | GCTTCTGGAAATAGACCTGAATGGA |
实验结果组间差异采用SPSS 25.0软件进行ANOVA分析，当P≤0.05时认为差异具有统计学意义。
2 结果与分析 2.1 DHA、EPA、ALA对Caco-2、NCM460的细胞毒性作用用不同浓度的DHA、EPA、ALA分别处理正常结肠上皮细胞NCM460 24、48和72 h，结果如图 1所示，30 μg/mL DHA处理的各时间处理组NCM460相对存活率分别为60.19%、72.00%和74.35%；60 μg/mL DHA处理组分别是3.52%、3.81%和2.76%；120 μg/mL DHA处理组分别是6.24%、3.16%和2.76%。30 μg/mL EPA处理组分别是67.22%、85.41%和70.94%；60 μg/mL EPA处理组分别是9.04%、4.35%和4.42%；120 μg/mL EPA处理组分别是6.65%、3.55%和2.25%。30 μg/mL ALA处理组分别是65.73%、75.84%和71.09%；60 μg/mL ALA处理组分别是32.77%、38.24%和37.93%；120 μg/mL ALA处理组分别是3.97%、1.87%和1.42%。30、60、120 μg/mL各浓度的不同时间处理组之间差异不显著。
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| 图 1 DHA (A)、EPA (B)、ALA (C)对Caco-2、NCM460细胞的毒性作用 Figure 1 Cytotoxicity evaluation of DHA (A), EPA (B) and ALA (C) on Caco-2, NCM460 cells |
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对于结直肠肿瘤细胞Caco-2，30 μg/mL DHA处理的各时间处理组相对存活率分别是87.71%、93.25%和91.76%；60 µg/mL DHA处理组分别是69.62%、80.75%和74.51%；120 µg/mL DHA处理组分别是11.04%、17.20%和5.51%。30 µg/mL EPA处理组分别是89.87%、95.74%和99.54%；60 µg/mL EPA处理组分别是72.83%、69.78%和67.18%；120 µg/mL EPA处理组分别是13.81%、19.49%和9.34%。30 µg/mL ALA处理组分别是89.91%、94.04%和93.49%；60 µg/mL ALA处理组分别是82.39%、92.07%和80.54%；120 µg/mL ALA处理组分别是33.16%、28.21%和17.83%。30、60、120 µg/mL各浓度的不同时间处理组之间差异不显著(图 1)。
2.2 DHA、EPA、ALA对F. nucleatum黏附能力的影响我们前期分别用30、60、120 μg/mL等不同浓度的DHA、EPA、ALA处理F. nucleatum 0−24 h，经具核梭杆菌生长检测发现，前14 h DHA、EPA、ALA抑制率较强，最高抑制率分别是71.61%、77.38%、81.91%；但随着培养时间的延长，抑制作用逐渐减弱，处理24 h后最高抑制率分别下降至16.83%、14.99%、17.24% (结果未展示)。该结果与文献[43]报道一致ADDIN EN.CITE.DATA。
综合考虑DHA、EPA、ALA对Caco-2、NCM460和F. nucleatum的生长抑制作用，后续选择30 μg/mL浓度的DHA、EPA、ALA处理F. nucleatum并检测其黏附Caco-2能力的变化。
如图 2所示，经30 μg/mL DHA、EPA、ALA处理12 h的F. nucleatum，Caco-2细胞上黏附的细菌数量分别比对照组减少了81.04% (P=0)、93.63% (P=0)和68.63% (P=0)。
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| 图 2 30 μg/mL DHA、EPA、ALA预处理抑制F. nucleatum黏附Caco-2细胞 Figure 2 30 μg/mL DHA, EPA and ALA pretreatment suppress the adhesive ability of F. nucleatum to Caco-2 cells 注：A：单个细胞上F. nucleatum平均黏附数柱状图；B：F. nucleatum黏附照片。***：P < 0.001 Note: A: Column chart of the average number of F. nucleatum adhere to single cell; B: Pictures of F. nucleatum adhesion. ***: P < 0.001 |
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当Caco-2与F. nucleatum共培养时加30 μg/mL DHA、EPA、ALA，如图 3所示，Caco-2细胞上黏附的细菌数量未发生显著变化。
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| 图 3 30 μg/mL DHA、EPA、ALA直接作用对F. nucleatum黏附Caco-2细胞没有影响 Figure 3 Treating with 30 μg/mL DHA, EPA and ALA directly have no influence on the adhesive ability of F. nucleatum to Caco-2 cells 注：A：单个细胞上F. nucleatum平均黏附数柱状图；B：F. nucleatum黏附照片 Note: A: Column chart of the average number of F. nucleatum adhere to single cell; B: Pictures of F. nucleatum adhesion |
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检测30 μg/mL的DHA、EPA、ALA处理3 h的具核梭杆菌FadA、Fap2等黏附相关基因、定殖有关基因RadD的表达量发现，DHA处理组的Fap2基因表达比对照组下调了10.22% (P=0.027)，与对照组有显著性差异(P < 0.05)。EPA处理组的FadA、Fap2基因表达分别显著下调了23.49% (P=0)、15.09% (P=0.003)。ALA处理组的FadA基因表达比对照组显著下调了26.75% (P=0.012)，详见图 4。
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| 图 4 30 μg/mL DHA、EPA、ALA对Fap2、FadA、RadD表达量的影响 Figure 4 Effects of 30 μg/mL DHA, EPA and ALA on the expression of Fap2, FadA, RadD Note: *: P < 0.05; ***: P < 0.001 |
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许多动物实验、流行病学等临床研究表明ω-3多不饱和脂肪酸具有降低结直肠癌风险的作用[7, 13, 44]，但也有无效的报道[14]。这种研究结果不一致甚至矛盾的现象，反映了ω-3多不饱和脂肪酸抗肿瘤作用机制的复杂性。如表 2所示，不同细胞对不同种类的ω-3多不饱和脂肪酸敏感性各不相同。如：66 μg/mL DHA处理24 h时，人牙龈成纤维细胞(hGFs)活性下降约50%，而人牙周膜细胞(hPDLCs)仅下降约30%；同时，60 μg/mL的EPA对此2种牙周细胞活性基本没有影响[43]。然而即使浓度高达197 μg/mL的DHA处理24 h，大鼠胶质母细胞瘤细胞C6细胞活性仍保持80%左右[45]。然而仅用20 μg/mL和13 μg/mL DHA处理人胃癌细胞GT-38[46]与人膀胱癌细胞EJ-138[47]48 h，就能使2种细胞活性下降约50%。
| 细胞种类 Cell lines or kinds |
处理方法 Experimental method |
细胞活性 cell viability (%) |
参考文献 References |
| 人牙龈成纤维细胞 Human gingival fibroblasts (hGFs) |
66 μg/mL DHA 24 h 60 μg/mL EPA 24 h |
About 50% 细胞毒性不明显 No obvious cytotoxicity |
[43] |
| 人牙周膜细胞 Human periodontal ligament cells (hPDLCs) |
66 μg/mL DHA 24 h 60 μg/mL EPA 24 h |
About 70% 细胞毒性不明显 No obvious cytotoxicity |
[43] |
| 大鼠胶质母细胞瘤细胞C6 Rat glioblastoma cell line C6 |
197 μg/mL DHA 24 h | About 80% | [45] |
| 人胃癌细胞GT-38 Epstein-Barr virus-associated gastric carcinoma GT38 |
20 μg/mL DHA 48 h | About 50% | [46] |
| 人膀胱癌细胞EJ-138与HTB-9 Human bladder cancer cell lines EJ-138 and HTB-9 |
13 μg/mL DHA 48 h | About 50% | [47] |
| 大鼠胶质细胞C6G Rat glioma cells C6G |
33 μg/mL DHA 24 h | 45.2% | [48] |
| 人神经母细胞瘤细胞SH-SY5Y Neuroblastoma cell SH-SY5Y |
33 μg/mL DHA 24 h | 45.9% | [48] |
| 星形胶质细胞Astrocytes | 33 μg/mL DHA 24 h | 100% | [48] |
Toit-Kohn等采用低血清培养基培养Caco-2、NCM460细胞时，16.4 µg/mL DHA能够显著降低Caco-2细胞活力达80%，而正常结肠细胞NCM460的活力不受影响[49-50]。本研究中，采用正常培养条件培养Caco-2、NCM460细胞时，30 µg/mL的DHA、EPA、ALA处理Caco-2细胞活性仅下降10%左右，而NCM460细胞活性则下降30%左右。即使是抑制作用最弱的ALA，仍呈现抑制正常细胞(NCM460)作用强于肿瘤细胞(Caco-2)的类似现象，详见图 1。同时，研究结果显示，DHA、EPA、ALA对Caco-2、NCM460的生长抑制作用仅具有显著的剂量依赖作用，无时间依赖作用。
鉴于上述实验结果，我们认为DHA、EPA、ALA的抗肿瘤作用不仅仅是由于直接抑制肿瘤细胞生长，而是通过其他间接途径实现。同时，文献报道与本研究预实验发现DHA、EPA、ALA仅能在14 h内短暂抑制F. nucleatum生长[43, 51-52]，之后抑制作用基本消失。该实验结果表明，DHA、EPA、ALA也不是通过杀死或完全抑制F. nucleatum的生长发挥抗肿瘤作用。
进一步检测DHA、EPA、ALA对FadA、Fap2、RadD等具核梭杆菌黏附、侵袭肠上皮细胞过程关键基因[39]表达水平的影响以及对F. nucleatum黏附宿主细胞能力的影响等实验发现，30 μg/mL DHA、EPA、ALA预处理能够显著降低Caco-2细胞上附着的具核梭杆菌数量，降幅分别达到81.04% (P=0)、93.63% (P=0)和68.63% (P=0)。该结果与文献报道的DHA和EPA抗肿瘤活性强于ALA报道一致[53]。但F. nucleatum与Caco-2共培养时再加入相同浓度的DHA、EPA、ALA对F. nucleatum黏附能力没有显著影响，该实验结果进一步表明DHA、EPA、ALA并不直接影响F. nucleatum对肿瘤细胞的黏附能力。
根据DHA、EPA、ALA处理分别显著下调F. nucleatum的Fap2、FadA表达的实验结果，我们认为，抑制FadA、Fap2等黏附相关基因表达减弱了F. nucleatum黏附宿主细胞的能力，是ω-3多不饱和脂肪酸抗肿瘤活性的关键组成部分，相关分子机制值得深入开展研究。
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