Progress on Treatment of Bone and Soft Tissue Tumors
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摘要:
骨与软组织肿瘤发病率低,属于罕见病范畴,每年针对该类肿瘤进行的研究,不管是国际还是国内,都屈指可数。本文以近年开展的骨与软组织肿瘤的研究为基础,对骨肉瘤、尤文肉瘤、软组织肉瘤、肉瘤随访、靶向治疗及免疫治疗这些核心内容进行述评,并分析我国该领域目前存在的问题,提出解决方案和建议,为该领域临床医师进行该类疾病的诊治过程中提供参考。
Abstract:Bone and soft tissue tumor is a rare disease and its incidence is low. There are few studies on this kind of tumors in the world every year. Based on the research of bone and soft tissue tumor in recent years, this paper reviews the core contents of osteosarcoma, Ewing's sarcoma, soft tissue sarcoma, sarcomas follow-up, targeted therapy and immunotherapy. At the same time, we analyze the unsolved problems and put forward the solutions and suggestions to provide references for clinicians in this field.
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Key words:
- Bone tumor /
- Soft tissue tumor /
- Malignant /
- Progress
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0 引言
结直肠癌(colorectal cancer, CRC)是全球发病率排名第三的恶性肿瘤,亦是较常见的消化道肿瘤之一,严重威胁着人类健康。研究[1-2]证实,结直肠癌的发生与肠道菌群失调密切相关,肠道菌群在结直肠癌的发生中扮演了重要角色。
正常人体肠道内含有不同类型、不同含量的细菌,它们具有刺激免疫功能、维护肠道屏障功能、抑制病原体增殖、促进物质代谢与吸收等作用[3-4]。而肠道菌群结构失衡可造成肠黏膜损伤,引起炎性反应或癌变等的发生。研究发现,受损的肠道上皮通透性增加,致使病原体穿过肠上皮的概率变大,持续的抗原刺激过度激活肠道免疫系统、补体系统、内分泌系统等,加剧肠道损伤,累积形成炎症性肠癌[5]。特定肠道共生菌可以改变肿瘤微环境中各种细胞的分布与活性,产生各种不同的细胞因子调控炎性反应,但不同种细菌或同种不同株细菌的功能不一。
课题组前期研究[6]发现肠道微生态的改变可以调控Wnt/β-catenin信号通路,改善固有层免疫细胞的作用方式,抑制大肠癌的发生。此外,已有研究报道[7]阿克曼菌(Akkermansia muciniphila, AKK)通过调控固有层免疫细胞的方式维持肠道稳态,但具体机制尚不明确。为探讨阿克曼菌是否通过调节肠道菌群改善肠道炎性反应进而防治CRC,本研究利用氧化偶氮甲烷(azoxymethane, AOM)和葡聚糖硫酸钠(dextran sulfate sodium, DSS)建立炎症相关性CRC小鼠模型,将阿克曼菌和肠道干细胞联系起来,探索他们在炎症性肠癌中的作用。
1 材料与方法
1.1 实验动物
C57BL/6J雌性小鼠,5~6周龄,体质量(20±2)g,无特定病原体(SPF)级动物,购自上海斯莱克实验动物有限责任公司。动物生产许可证号:SCXK(沪)2019-0018,在上海中医药大学动物实验中心饲养。饲养室温(25±1)℃,湿度(50±5)%,12 h光照与黑暗交替循环,自由进食灭菌饲料和饮用水。动物实验伦理号:PZSHUTCM191129007。
1.2 主要试剂
氧化偶氮甲烷AOM(货号A5486)购自美国Sigma-Aldrich公司;葡萄糖硫酸钠盐DSS(货号116570400)购自美国MP Biomedicals公司。Ki-67(货号:12202)购自美国CST公司,Lgr5(货号:RAY8180926,RayBiotech)。阿克曼菌菌株(BAA 835)购于美国ATCC。
1.3 AKK菌液的制备及保存
将冻干菌粉接种于添加了0.02%黏蛋白的脑心浸液(BHI)平板中活化,将细菌置于含有10%H2、10%CO2、80%N2的37℃厌氧培养箱中培养,根据细菌在600 nm时的OD值绘制生长曲线,并结合细菌平板计数确定细菌浓度,4℃ 500×g离心5 min,PBS重悬,将浓度调整至1.0×109 CFU/ml保存于4℃冰箱,此过程在厌氧操作台中进行。
1.4 模型建立及分组
首先给予抗生素混合物(Abx)饮水方式,主要包括氨苄西林(1 mg/ml)、新霉素(1 mg/ml)、甲硝唑(1 mg/ml)、万古霉素(0.5 mg/ml),喂养小鼠4周,模拟伪无菌小鼠。随后建立AOM/DSS炎症相关性结直肠癌小鼠模型,具体为:小鼠均于腹腔注射12.5 mg/kg AOM,7天后饲喂2.5%DSS饮用水,连续一周,第2~4周给予常规饮用水,此为1个循环。连续3个循环结束后恢复常规饮用水。使用阿克曼菌处理小鼠时,将保存的菌群液4℃离心5 min,弃去上清液,无菌PBS洗涤3次,后重悬于无菌PBS中,37℃水浴,最后灌胃于小鼠。细菌离开厌氧瓶至灌入小鼠体内需在1 h内完成。通过随机数字表法分组,每组8只,包括模型组(Model):Abx+AOM/DSS+PBS灌胃;阿克曼菌组(AKK):Abx+AOM/DSS+AKK灌胃;阳性药物对照组(阿司匹林,Aspirin):Abx+AOM/DSS+Aspirin(1.56 g/kg)灌胃。
1.5 方法
1.5.1 观察指标及处理方法
在实验过程中观察各组小鼠的饮食、活动、精神,毛发情况及小鼠的粪便性状,每周称量小鼠的体质量并记录。小鼠处死前禁食8 h,质量分数为5%水合氯醛麻醉,剖开小鼠胸腔及腹腔,自盲肠末端与结肠分离,直至肛门处,将小鼠结直肠分离,将结肠内容物取出保存。PBS漂洗后,量取结直肠长度并记录,若有肿瘤,观察小鼠成瘤情况并记录位置、大小和数目。肿瘤负荷=每组肿瘤总直径之和/每组初始总数量。
1.5.2 免疫组织化学观察小鼠结肠组织中Ki-67和Lgr5表达
将肿瘤组织置于4%甲醛中浸泡24 h固定,制成石蜡切片标本。石蜡切片常规脱蜡,3%H2O2溶液消除组织内源性过氧化物酶活性,0.01 mol/L柠檬酸盐缓冲液微波法进行抗原修复,10%山羊血清封闭液封闭后,1:100稀释的Ki-67抗体进行染色。每张切片选取6个代表性视野观察,统一条件下拍照。Image-Pro Plus v6.2图像分析软件,计算和分析肿瘤组织中Ki-67的表达。
1.5.3 Western blot检测Lgr5的表达
取各组小鼠的肠肿瘤组织研磨后,加入0.25%(含EDTA)胰蛋白酶消化细胞,收集细胞悬液,4℃ 500×g离心5 min。弃上清液,每管加入1 ml冷PBS重悬细胞,4℃ 500×g离心5 min,清洗细胞。加入适量RIPA裂解液(含蛋白酶抑制剂),超声破碎仪超声3个循环,充分裂解细胞。冰上裂解20 min后,4℃ 12 000 r/min离心15 min,获得细胞总蛋白。BCA法蛋白定量后,取等量总蛋白加入1×上样缓冲液,100℃加热8 min。经电泳、转膜和封闭后,加入抗Lgr5一抗(1:1 000稀释),4℃孵育过夜;TBST洗膜3次后加入二抗(1:2 000稀释),室温孵育2 h;TBST洗膜3次后,ECL曝光。
1.5.4 qRT-PCR检测干细胞相关基因
TRIzol试剂提取结肠组织总RNA。PrimeScriptTM RT Master Mix(RR036A, Takara)试剂盒反转录。SYBR FAST qPCR Kit(RR420A, TaKaRa)进行PCR扩增。数据通过2-ΔΔCt法进行分析。引物序列如下:Lgr5(正向:5’-GTGGCAGCAAGTATGGCG-3’,反向:5’- AGCAAAGGGAATTGAGCAAG-3’)、CD133(正向:5’-CTGGGGCTGCTGT TTATTATTCTG-3’,反向:5’-ACGCCTTGTCCTTGGTAGTGTTG-3’)、Nanog(正向:5’-GTCTGCTACTGATGCTCT-3’,反向:5’-ATCTGCTGGAGGCTGAGGTA-3’)和ALDH1(正向:5’-GTCTGCTACTGATGCTCT-3’,反向:5’-ATCTGCTGGAGGCTGAGGTA-3’);GAPDH(正向:5’-TGAAGGTCGGTGTGAACGGATTTG-3’反向:5’-TCTCGCTCCTGGAAGATGGTGATG-3’)。
1.6 统计学方法
采用SPSS13.0统计软件包处理数据。各组数据用(x±s)表示,两独立样本均数比较采用Independent-Samples t-Test,多样本均数比较采用单因素方差分析,均数间两两比较用SNK-q检验。P < 0.05为差异有统计学意义。
2 结果
2.1 AKK对AOM/DSS小鼠生活状态及生存率的影响
造模后,模型组小鼠出现活动度减小、毛发枯燥无光泽、体重下降、便溏、便血、脱肛等现象。三组小鼠体质量整体呈上升趋势,在每次给予DSS饮水后,小鼠体质量均出现下降趋势,停止给予DSS饮水后小鼠体质量逐渐恢复,见表 1,阿克曼活菌组小鼠体重增长最为明显,其次为阿司匹林组,模型组小鼠体重总体增长最小。实验结果表明,AOM/DSS会引起小鼠体质量下降,并与AKK干预组存在差异。结合患病动物的体重下降百分率(体重不变为0,1~5为1分,5~10为2分,10~15为3分,> 15为4分)、大便黏稠度(正常为0,松散大便为2分,腹泻为4分)和大便出血(正常0分,隐血阳性为2分,显性出血为4分)三种情况进行综合评分,将3项结果的总分除以3即得到DAI值[6, 8]。
表 1 实验结束时各组AOM/DSS小鼠DAI评分Table 1 DAI score in AOM/DSS mice at the end of experiment2.2 AKK对AOM/DSS小鼠结直肠长度及肿瘤生长的影响
与模型组相比,AKK组和Aspirin组小鼠腺瘤总数量明显减少(P=0.003, P=0.008);腺瘤直径大于5 mm时,AKK组和Aspirin组小鼠腺瘤数量明显减少(P < 0.001, P < 0.001),直径 < 5 mm时,AKK组小鼠腺瘤数量有下降趋势。提示AKK可能通过调节肠道菌群抑制了AOM/DSS小鼠肠道腺瘤的生长,降低了肠道腺瘤的数量,见图 1、2。
2.3 AKK对AOM/DSS小鼠结直肠Ki67蛋白表达的影响
免疫组织化学结果显示,Ki67在模型组肿瘤组织中呈强阳性表达,核呈棕黄色颗粒;在AKK组中表达明显减弱,提示AKK延缓肿瘤的发展过程与抑制细胞增殖有关。与模型组相比,阿克曼菌干预后,小鼠肠道肿瘤中Lgr5蛋白表达显著降低,提示AKK抑制炎症性结直肠癌的发生可能与靶向抑制结直肠癌干细胞Lgr5+的表达相关,见图 3、4。
2.4 AKK对AOM/DSS诱导大肠癌小鼠的肠道肿瘤干性相关基因的调控作用
三组均出现了大肠肿瘤,但在AKK干预后,小鼠肠道肿瘤发生率和发展速度得到了减缓,表现出了明显的预防作用。本实验采用qRT-PCR法发现,AKK抑制了Lgr5(P=0.005)、ALDH1(P=0.007)、CD133(P=0.123)、Nanog(P=0.002)基因的表达(P < 0.05),见图 5。
2.5 AKK对AOM/DSS小鼠结直肠细胞干性标记Lgr5蛋白表达的影响
与模型组相比,Lgr5的表达在AKK组明显降低。实验结果提示,AKK对AOM/DSS诱导的小鼠结肠细胞的干性因子Lgr5表达具有一定抑制作用,差异有统计学意义(P=0.006),见图 6。
3 讨论
嗜黏蛋白阿克曼菌是一种定植在肠上皮黏液层的椭圆形革兰氏阴性菌,广泛分布于人体与动物的消化道[9]。阿克曼菌是专性厌氧、不能运动、无芽孢形成的椭圆形真细菌,隶属于疣微菌门。有报道表示[10],肠道菌群与结直肠癌疗效及预后相关联。最近一份报道显示,阿克曼菌的丰度与炎性反应发生还有随年龄增长下降的结肠黏液厚度相关[11]。虽然对阿克曼菌的了解随着研究的进展不断增加,但我们对阿克曼菌如何介导上述反应仍然知之甚少,尤其是大肠癌炎癌转化中的作用机制尚需进一步明确。
AOM/DSS模型能模拟炎性肠病诱发结直肠癌的全过程,而被广泛用于研究结肠炎相关性结直肠癌的癌变机制以及结直肠癌的化学预防研究,病理形态与人的结肠癌前病变相似,是一种较为理想的动物模型[12]。本研究结果表明AOM/DSS诱发小鼠发生了结直肠肿瘤,使小鼠质量增加,阿克曼菌干预后能够明显抑制结直肠的缩短,减小肿瘤体积,减少肿瘤区域。Ki67是一种用来表达增殖的细胞核抗原,灵敏、特异的增殖期细胞的高低反映了细胞的增殖指数[13]。临床中该蛋白的升高往往与肿瘤的发生发展、转移及预后密切相关。本研究证实模型组小鼠结直肠组织中Ki67蛋白阳性表达增多,阿克曼菌能减少Ki67的阳性表达,表明阿克曼菌抑制结直肠癌的发生。与健康人群相比,CRC患者的微小微单胞菌、核梭杆菌、口腔消化链球菌和嗜黏性阿克曼氏菌明显增多,提示阿克曼氏菌具有显著的预防和治疗CRC的作用[14]。也有研究[15]发现AKK可以通过加重结肠炎早期的炎性反应,促进肠上皮细胞的增殖,从而促进小鼠结肠炎症相关性结直肠癌的发生。
近年来,有研究[16]表明Lgr5与β-catenin通路的异常激活可能促进肿瘤的恶化。Lgr5+肠道干细胞(ISC)是一类特异性表达Lgr5、具有持续增殖分化功能的细胞,Lgr5+ ISC功能缺失引起肠上皮修复障碍诱发炎症性肠病,过度激活则可能诱发结直肠肿瘤[17]。醛脱氢酶(ALDH)是一组依赖烟酰腺嘌呤核苷酸磷酸(NADP)作为辅酶的催化酶,其生理功能是将内源性和外源性醛类物质氧化为相应的羧酸类物质。ALDH1被认为是一个有前景的CSC标志[18],它的存在可能是干细胞长期存活的基础。CD133和Nanog作为常用的CSC标志物,愈发受到重视[19]。研究[20]证实CD133+细胞具有明显的体外成球性、致瘤性、细胞分化及化疗耐药等特性。Nanog是一种在内细胞团、原始生殖细胞及胚胎干细胞表达的新转录因子[21]。该基因能在多种上皮性恶性肿瘤中高表达,与更差的预后相关,能感知、整合并将细胞微环境信号转导到膜相关的细胞骨架蛋白或细胞核中,调控细胞行为,使其作为典型的CSC表面标志物。Western blot结果显示,AKK能抑制AOM/DSS小鼠肿瘤的发生和发展,同时影响Lgr5蛋白的肠道隐窝部位定位,从而抑制了肿瘤干细胞增殖,以实现防治结肠癌的作用,提示AKK可作为抗结肠癌预防的新方法。
综上所述,AKK可以抑制AOM/DSS诱导的大肠癌,在一定程度上改善肠道微环境,并减缓AOM/DSS造成的肠道干细胞恶性病变。隐窝增殖细胞可能参与了这一过程。对于临床预防大肠癌发生而言,AKK可能是一种化学预防结肠癌的潜在益生菌。
作者贡献牛晓辉:文献调研、整理及论文撰写 -
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