【作者】 范莎莎；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学（专业学位）， 2014， 博士

【摘要】 1.背景与目的：肝脏是维持机体内环境稳定和机体健康的最重要的器官之一,脂肪肝、肝炎、肝硬化和原发性肝癌是肝内最常见的四大严重疾病,也是危害人类健康的重要原因之一。事实上,每年大约有一百万人死于肝炎病毒感染及其导致的肝硬化和肝癌。特别需要指出的是,作为肝脏最常见的原发恶性肿瘤—原发性肝细胞性肝癌(Hepatocellular carcinoma HCC)因为发病率较高、发现困难、治疗方法少等原因已经在导致人类死亡的各种疾病中位居前列。目前,原发性肝细胞性肝癌在全球范围内肿瘤致死率方面位列第五位,而在一些非洲和亚洲国家,原发性肝细胞性肝癌在肿瘤性死因中已经位居首位。肝癌所导致的人力、财力、物力和医疗资源的大量消耗在我国更加明显,主要原因在于世界上几乎一半的新发病例出现我国,以2000年为例,全世界新发病例总数62.4万人,发生在我国的大约占其中得55%,这个数字实在有点触目惊心。据我国卫生部统计,从20世纪90年代起原发性肝癌(PHC)已经演变为第二位的癌症杀手,在城市仅次于肺癌,在农村仅次于胃癌。而与此相对应的现实是：肝癌每年新发病例已达564000例,而死亡病例也有549000例之多。由于没有切实有效的早期诊断方法,发现时已处于中晚期；加上没有合理有效的根治方案,手术效果较差,化疗和放疗的效果也不明显,造成肝癌病人的5年生存率很低,复发率和死亡率很高。曾有报道指出肝癌根治切除术后5年复发率为51.6%,其中小肝癌的复发率就高达43.6%。虽然除手术根治术以外,还有一些新的治疗方法进入临床(如射频消融治疗、微波治疗、冷冻消融治疗、无水酒精瘤内注射、肝动脉介入栓塞化疗等局部治疗手段),但这些方法对于提高肝癌的总的生存质量效果收效甚微。早发现才能早治疗,因此,肝癌的早期诊断早已经成为人们关注的焦点,但遗憾的是实际有效的方案仍然欠缺,早期发现肝癌仍然是巨大的挑战。在医学科学日益发展的今天,虽然在诊断领域的投入已经越来越多,但有效的标志物的成果还是寥寥无几。事实上,很多我们眼中可能是早期肝癌的病人,在治疗的过程中却发现其实肿瘤细胞已经长期存在并发生了转移,患者已是处于肿瘤的中、晚期,各种现象都表明目前的检测技术仍然不能及时发现和有效检测早期阶段的肝癌。这也是有很大一部分所谓的“早期”患者在实施完手术后,却很快发生了复发。所以,对于肿瘤标志物的寻找仍然是一个重要方向,也是肿瘤早期发现的重要方法。由于血清检测的灵敏度高,操作便捷、不仅对判断肿瘤的发生、转移、复发有重要价值,而且对指导临床治疗,改善患者预后有重要意义,因此血清学标志物的研究是目前研究的一个热点,也是难点和重点。肝细胞癌是常见的恶性程度极高的肿瘤,其发病存在一定的区域分布特性,其中,在肝炎病毒流行的亚洲和非洲,发病率相对较高。在肝癌的普查上,常规的肝癌易感个体的普查可以明显延长很多病人的生存期,但是对于许多晚期肝癌病人来说,效果仍然不容乐观。对于一部分晚期肝癌患者,虽然只有很小的肿瘤,外科手术也完全切除,但是术后长期生存质量的改善却很不明显,而术后肝内或肝外转移及复发就是肝癌病人预后极差的主要原因。事实上,目前术后复发和转移的估计常常不足或过度,往往会造成术后治疗不当,对于病人来说无疑又是一个不利因素。许多研究证实炎症、免疫、肿瘤三者有着密切的关系。目前认为炎症反应在肿瘤的发生、进展、恶性转化、侵袭和转移中都扮演了重要角色。同时在机体炎症过程和免疫应答两者之间又有着密切的关系,已有学者提出有1/5的癌症是与慢性感染相关。炎症和肿瘤是一对矛盾体,Dvorak HF等将肿瘤称为“不能愈合的伤口”,Schrader J提出肿瘤一方面利用促炎的微环境来进行组织重塑、血管生成和发生侵犯和转移;另一方面通过促进抑制性巨噬细胞生成、聚集Treg细胞来抑CTLs的作用,从而阻止免疫系统攻击肿瘤细胞。肿瘤作为对机体的一种损伤,在其发生发展的过程中,机体也会出现相应的炎症反应,特别是对于恶性肿瘤,因为是破坏性比较强,激起病人的自我防御机制也会相对明显一些。鉴于炎症与肿瘤的密切联系,对于肿瘤发生过程中炎症相关因素的参与的研究也就成为了一个重要的方向。HTATIP2\TIP30(HIV-I Tat interactive protein2)又名CC3,是人类免疫缺陷病毒在体外转录时发现的一种分子量30kd的结合蛋白(Tat interactive protain, TIP30),其作为HIV转录的辅助因子可以和Tat相互结合特异性提高HIV增殖。以往的研究认为HTATIP2/TIP30是一种被广泛表达于人体组织和一些肿瘤组织中抑癌基因。HTATIP2/TIP30能够与已知的酪氨酸激酶抑制剂,血管生成抑制剂共同参与细胞周期和细胞凋亡相互作用。XiaoHua等研究发现,在杂合背景下(50%来源于C57BJ6小鼠和Svj129来源于svJ129小鼠),Tip30基因敲除可导致约30%的小鼠发生原发性肝癌,在人原发性肝癌的免疫组化检查中发现33%的原发性肝细胞癌TIP30蛋白表达低于正常肝组织。Zhao等的研究发现TIP30高表达可抑制肝癌的转移,同时TIP30可通过多种机制抑制肝细胞的凋亡。HTATIP2/TIP30可通过与DNA结合、跟RNA多聚酶II形成复合物等调节细胞增殖、凋亡及血管生成相关基因而达到抑制肿瘤转移的作用。Budhu A等利用肝癌癌旁组织基因的17个细胞因子表达谱与其他临床预后指标相比,发现他们可以作为独立预后因子,Thl/Th2细胞因子失衡对于肿瘤预后预测有重要的意义。他们认为不同转移潜能肝脏微环境中有着截然不同的优势细胞因子表达谱,微环境由抗炎状态向免疫抑制,这些研究也揭示了细胞因子改变与肿瘤复发转移的关系。TNF-α和IL-1β在原发性肝癌中表达高于正常人,另外癌旁这两个因子也增高,MIF等另外的血管生成相关基因都与肝癌肝内转移有关。TNF-α作为一种具有抗肿瘤细胞因子,在抑制肿瘤侵袭和转移过程中发挥重要作用。IL-6和TNF-α是促进肝癌发生发展的两种重要炎症介质。致癌剂DEN可诱导小鼠肝癌发生,IL-6基因敲除小鼠中DEN诱导肝癌的发生率、肿瘤数量和大小显著降低,IL-6和TNF-α可分别激活肿瘤细胞内NF-kβ和STAT3信号通路,导致肿瘤增殖、抗凋亡、血管生成、侵袭和转移相关基因的表达,使肝癌得以发生发展。Th17分泌的细胞因子除了IL-17(IL-17A)外,还包括IL-17F,以及IL-21、IL-22、IL-6、TNF-α等细胞因子,Th17细胞被认为是一群重要的介导炎症反应的重要细胞还能激活树突状细胞和T淋巴辅助细胞,释放细胞因子维持慢性炎症状态和致癌变环境。IL-17是一种重要的炎症介质,它可以通过诱导其他炎症细胞因子如IL-6, TNF-α,及趋化因子如MCP-1、MIP-2等的表达,介导炎症细胞到局部的浸润及组织损伤。白细胞介素27(Interleukine27,IL-27)是2002年发现并命名的一种新的细胞因子,它与IL-12以及另一种新发现的细胞因子IL-23共同组成IL-12分子家族,具有复杂的生物学功能,在抗感染免疫,抗肿瘤免疫以及自身免疫病中发挥重要作用。基于已有的研究对HTATIP2/TIP30及炎症细胞因子与肿瘤及肝癌的各种相关报道,HTATIP2/TIP30基因的下调可能与原发性肝癌的发生有关。因此,该蛋白是否能够通过血清血检测,其表达水平与肝癌的发生、发展是否相关方面的研究显得非常必要和关键。此外,结合已有的AFP的临床价值,对HTATIP2/TIP30和AFP的肝癌的临床资料的相关性进行评估也显得很有意义,这样更有利于系统地分析和检验二者的检验效能,本研究拟通过运用Luminex xMAP技术检测,一次性对52例肝癌患者血清中的IL-1β、IL-6、IL-10、IL-17、 IL-27、TNF-α等细胞因子进行检测,进一步探讨这些炎症细胞因子预测肝癌转移可能。以便更好地对其进行利用,这也是本研究的目的所在。2.实验方法：2.1患者选择选择2013年9月和2013年12月来医院就诊的肝癌病人和健康体检者,肝癌患者条件为：未经过任何治疗经病理为肝细胞癌的患者。共有84名受试者参与,并分别分为对照组：共包括32名健康体检人(无肝癌或其他肿瘤及明确诊断的疾病),男18例,女14例。在对照组中年龄范围为21-72岁(平均年龄43±15岁)；肝癌组：共52例,男45例,女7例,肝癌患者年龄范围为27至80岁(平均年龄54±12岁),HBV感染率(76.9%),平均肿瘤大小为5.6±4.9厘米,各入组人员在入院后进行相应治疗之前完成相应的CT、超声、血生化常规、凝血功能、血清学乙肝病毒定性和定量检测。2.2标本收集入院后征得患者知情同意,嘱患者晨起空腹,实验人员应用一次性真空采血管采外周静脉血约2ml,4摄氏度下4,000rpm离心10分钟,得到血清,共留取84例外周静脉血清。将血清置于-80摄氏度超低温冰箱中冷冻保存,备用,直至生化测定。2.3酶联免疫吸附实验(Enzyme-linked immunosorbent assay)根据HTATIP2\TIP30ELISA试剂盒(Cusabio Biotech.)说明书方法,检测肝癌组与对照组血清中HTATIP2\TIP30的浓度,最低检出量0.16ng/ml。批内精密度(化验内精度)为CV%<8%,批间精密度(检测精度之间)的CV%<10%。2.4Roche电化学发光法测定HBeAg状态所有入组实验对象通过电化学发光法测定HBeAg,按照罗氏化学发光法检测HBeAg的SOP流程进行样本处理、上机、Elecsys软件分析判定该标本HBeAg阴、阳性。2.5荧光定量PCR测定血清HBV DNA水平所有入组实验对象按HBV荧光定量检测试剂盒说明书进行操作,将实验对象血清与PCR反应液充分混匀,与HBV定量标准品共同进行PCR扩增,待扩增结束后通过ABI7100进行数据分析。2.6Luminex xMAP检测肝癌患者血清中细胞因子Luminex xMAP技术(multi-anlyte profiling beads)eBioscience的ProcartaPlex试剂盒,细胞因子标准品(包括IL-1β,IL-10, IL-6, IL-17, TNF-α, IL-27)共6种细胞因子的标准品及样品稀释液。数据结果通过分析软件Luminex200分析,通过检测待测样品捕捉图像,继而经过分析图像,绘制标准曲线,计算得出待测样品的数据。2.7统计学方法SPSS17.0计算机程序软件包进行统计计算。结果以均数±标准差(SD)。单因素方差分析,组间多重比较选择LSD和SNK两两之间的多重比较分析对照组和肝癌组,及肝癌I-Ⅱ,Ⅲ-Ⅳ期均值。(Operating Characteristic curve) ROC受试者诊断特征曲线分析各指标对肝癌与正常组,肝癌转移组和无转移组、早期肝癌与正常组的诊断效能。Pearson相关分析(正态分布资料),Spearman(非正态分布资料)临床检测指标与细胞因子及生化指标的相关性。二元logistic回归分析和建立联合指标的诊断模型,Hosrner-Lemeshow进行logistic回归拟合优度检验,P值精确到小数点后3位,以P<0.05为有统计学差异。Medcalc绘制ROC曲线,Graphpade Prism5绘制柱状图及相关线图。3结果：3.1肝癌组与正常组及各期肝癌组血清HTATIP2/TIP30的水平HTATIP2/TIP30在肝癌组中浓度明显低于正常组分别为4.51±2.64和9.50±2.04(P<0.001),且Ⅰ-Ⅱ和Ⅲ-Ⅳ期浓度分别为4.22±1.98和3.02±1.80,结果显示Ⅲ-Ⅳ期较Ⅰ-Ⅱ期也降低,且差异有统计学意义(P<0.001)。3.2ROC曲线分析HTATIP2/TIP30与AFP诊断肝癌通过受试者诊断特征曲线(Receive Operating Characteristic)ROC曲线分析,HTATIP2/TIP30和AFP诊断肝癌和正常对照组的诊断效能,两指标的曲线下面积分别为0.928(P<0.001)和0.884(P<0.001),认为两指标诊断肝癌均有统计学意义。HTATIP2/TIP30截断点为7.271ng/ml时,诊断肝癌的敏感度84.6%,特异度93.7%,约登指数最大。3.3ROC曲线分析HTATIP2/TIP30与AFP诊断早期肝癌ROC分析HTATIP2/TIP30与AFP预测肝癌早期患者与正常对照组。HTATIP2/TIP30曲线面积AUC为0.789(0.789±0.069,P<0.001),敏感度88.2%,特异度50%；AFP曲线面积AUC为0.858(0.858±0.055,P<0.001),敏感度94.1%,特异度59.4%,两者诊断肝癌均有统计学意义,AFP对于早期肝癌的诊断效能稍优于HTATIP2/TIP30。3.4ROC曲线分析HTATIP2/TIP30与AFP诊断肝癌转移ROC分析HTATIP2/TIP30和AFP诊断肝癌转移,其对应的曲线下面积分别为(0.804,P<0.001)和(0.513,P=0.581),HTATIP2/TIP30作为诊断肝癌转移有统计学意义。HTATIP2/TIP30约登指数最大时的截断点为5.367ng/ml,其对应的预测肝癌转移的敏感度为91.7%,特异度为57.1%。而AFP(P=0.081)对于预测肝癌组转移无统计学意义。HTATIP2/TIP30对预测肝癌转移的诊断效能高于AFP, HTATIP2/TIP30可能成为肝癌转移的标志物。3.5HTATIP2/TIP30水平与临床指标的Spearman相关性分析HTATIP2/TIP30与乙肝病毒DNA和Child-Pugh评分呈负相关,相关系数和P值分别为(r=-0.364, P=0.002;)(r=-0.419, P=0.008;),相关有统计学意义,但是与乙肝病毒DNA相关不密切。与肿瘤最大径和AFP、粒细胞和淋巴细胞比值(NLR)在本研究中相关没有统计学意义。3.6HTATIP2/TIP30、HBvDNA水平、AFP三指标的Logistic逐步回归模型通过HTATIP2/TIP30、HBvDNA水平、AFP浓度3个因素做logistic向后逐步回归分析,选择进入变量的概率标准为0.10,剔除变量的概率标准为0.15,应变量“0”为正常对照,“1”为肝癌,得出回归方程：P=1[1+e-(4.449-0.717×1+0.042×2)]其中x1代表HTATIP2/TIP30, x2代表AFP, p为logistic模型预测概率,取值范围在0-1之间,e为自然对数(e=2.718),Odd Ratio (OR)值分别为：0.488和1.043；95%CI(95%可信区间)分别为(0.331-0.791)和(0.998-1.089)。回归方程提示HTATIP2/TIP30与AFP都是肝癌发生的独立影响因素,HTATIP2/TIP30为保护性因素,即HTATIP2/TIP30血清水平高的患者其患肝癌的风险为水平低的患者的0.488倍。AFP为危险因素,即AFP血清水平高的患者其患肝癌的风险为水平低的患者的1.043倍。两者回归模型PRE预测肝癌发生的曲线下面积(AUC)为0.963±0.020(P<0.001),其约登指数最大时对应的敏感度90.6%,特异度90.4%,均较两指单独标诊断时显著增高。3.7肝癌组血清多个细胞因子的检测运用Luminex xMAP方法检测肝癌转移与无转移组中血清标本中(IL-1β, IL-6, IL-10, IL-17A, TNF-α,IL-27)6个细胞因子的含量,结果显示无转移组中的各细胞因子含量分别为(IL-1β,3.93±2.07; IL-6,6.61±4.63; IL-10,7.77±5.49; IL-17A,2.67±2.53; IL-27,255.49±94.33),转移组的各组细胞因子含量为(IL-1β,4.11±1.95; IL-6,6.30±5.39; IL-10,8.70±4.77; IL-17A,5.56±2.85; IL-27,353.19±177.55),其中IL-17A, IL-27两组细胞因子在转移组浓度明显增高,P值分别为(0.015,P<0.001),差异有统计学意义。3.8HTATIP2ATIP30水平与血清各细胞因子Pearson相关性分析Pearson相关性分析HTATIP2/TIP30、IL-1β、IL-6、IL-10、IL-17、IL-27、 TNF-α等细胞因子相关性发现,IL-17与TIP30存在负相关(r=-0.601,P<0.001),两者相关较为密切,且相关有统计学意义。3.9ROC分析IL-17和IL-27预测肝癌转移能力ROC分析IL-17与IL-27两指标对肝癌的转移与无转移组,曲线下面积为分别为(0.818,P=0.044)和(0.664,P<0.001),两者作为指标预测肝癌转移均有统计学意义。IL-27预测肝癌转移的效能较IL-17更好。以IL-27为238.95pg/mL临界值时,敏感度为：95.8%,特异度为：64.3%,约登指数最大。IL-17选择临界值为2.005pg/ml,约登指数最大,其敏感度为83.3%,特异度35.7%。3.10logistic回归模型预测肝癌转移选择HTATIP2/TIP30、IL-27做logistic向后逐步回归分析,因变量选择“0”为无转移,“1”为转移。选择进入变量的概率标准为0.10,剔除变量的概率标准为0.15,得出回归方程：P=1[1+e-(-1.264-0.642×1+0.013x2)]其中x1代表HTATIP2/TIP30,×2代表IL-27,P为logistic模型预测概率,取值范围在0-1之间,e为自然对数(e=2.718), Odd Ratio (OR)值分别为：0.526和1.013；95%CI(95%可信区间)分别为(0.346-0.799)和(1.003-1.026)。这一回归方程提示HTATIP2/TIP30与IL-27都是肝癌转移的独立影响因素,HTATIP2/TIP30为保护性因素,HTATIP2/TIP30血清水平高的患者其转移的风险为水平低的患者的转移风险的0.526倍。IL-27为危险因素,IL-27血清水平高的患者其转移的风险为水平低的患者转移风险的1.013倍。两者回归模型PRE预测肝癌转移的曲线下面积(AUC)为0.903±0.043,P<0.001,敏感度87.5%,特异度82.1%,较两指标单独预测时明显增高.4结论：4.1血清中肝癌组HTATIP2/TIP30的水平显著低于正常对照组,且Ⅲ-Ⅳ期水平较Ⅰ-Ⅱ更加降低,差异有统计意义。4.2ROC分析HTATIP2/TIP30与AFP诊断各期肝癌组与对照组时,曲线下面积(AUC)较AFP高,敏感度和特异度均较AFP高。诊断早期肝癌时,其曲线下面积(AUC)稍小于AFP。对转移与无转移组的预测时,HTATIP2/TIP30明显优于AFP。提示HTATIP2/TIP30不仅是个可以诊断肝癌的指标,同时也是一个较好的预测肝癌转移的指标。4.3Spearman相关分析发现HTATIP2/TIP30水平与乙肝病毒DNA浓度和Child-Pugh分数呈负相关,有统计学意义,但与乙肝病毒DNA浓度相关不太密切。4.4通过Logistic逐步回归得出的HTATIP2/TIP30和AFP回归模型,回归方程提示HTATIP2/TIP30与AFP都是肝癌发生的独立影响因素HTATIP2/TIP30为保护性因素,即HTATIP2/TIP30血清水平高的患者其转移的风险为水平低的患者转移风险的0.488倍。AFP为危险因素,即AFP血清水平高的患者其转移的风险为水平低的患者转移风险的1.043倍。两者回归模型PRE预测肝癌发生的曲线下面积(AUC)为0.963±0.020(P<0.001)、其约登指数最大时对应的特异度90.6%,特异度90.4%均较两指单独标诊断时显著增高。4.5肝癌组转移与无转移组中的IL-17和IL-27较转移组明显增高,通过Pearson相关分析发现IL-17与HTATIP2/TIP30呈负相关,相关较为密切,相关有统计学意义。4.6ROC分析IL-27和IL-17预测肝癌转移,曲线下面积(AUC)较IL-17高,敏感度和特异度均较IL-17高。我们发现了新的指标IL-27能较好的预测肝癌转移。4.7通过Logistic逐步回归得出的HTATIP2/TIP30和IL-27回归模型,回归方程提示HTATIP2/TIP30与IL-27都是肝癌转移的独立影响因素,HTATIP2/TIP30为保护性因素,即HTATIP2/TIP30血清水平高的患者其转移的风险为水平低的患者转移风险的0.526倍,IL-27为危险因素,即IL-27血清水平高的患者其转移的风险为水平低的患者转移风险的1.013倍。两者回归模型PRE预测肝癌转移的曲线下面积(AUC)、敏感度和特异度均较两指标单独预测时明显增高。创新之处：1.本研究首次在肝癌患者的血清中检HTATIP2/TIP30水平,并发现其能成为诊断肝癌和预测转移的指标。2.首次通过液相芯片发现IL-17和IL-27与肝癌转移相关,两指标可联合用于肝癌转移的预测。更多还原

【Abstract】 1. Background and objectives:The liver is one of most important organs in the internal environment to maintain the stability and health of the body. Fatty liver, hepatitis, cirrhosis and primary liver cancer are the important reasons for the four most common serious liver diseases, but also endanger human health. In fact, there are nearly one million people died of HCC leaded by hepatitis virus infection and cirrhosis. Particularly, as the most common primary malignant liver primary hepatocellular carcinoma (Hepatocellular carcinoma HCC) because of the higher incidence found difficult, less treatment reasons has led to the death of a variety of human diseases in the forefront. Currently, primary hepatocellular carcinoma ranked fifth worldwide cancer death rate, while in some African and Asian countries, primary hepatocellular carcinoma in tumor death has been ranked.Consume caused by human hepatocellular carcinoma, financial, material and medical resources more obvious in our country, mainly because almost half of the new cases of the world. For example, in2000years the total number of cases worldwide emerging624000people, it is a bit shocking which have accounted for about55%of this figure occurred in our country. According to Ministry of Health statistics, from the1990s hepatocellular carcinoma (PHC) has evolved into the second cancer killer just behind lung cancer in the city, and gastric cancer in the countryside. This corresponds with the reality is:the HCC cases each year has reached564,000cases, while there are549,000cases of deaths as much. Since There is no effective method for early diagnosis, much of the cases found in the middle and late stage, combined with the absence of reasonable and effective solution to cure, most of the HCC had poor surgical results, the effect of radiotherapy and chemotherapy were not evident, resulted5-year survival rate of patients with HCC is very low. The recurrence rate and mortality rate is very high. There have been reports that5years after radical resection of HCC recurrence rate was51.6%, of which a small hepatocellular carcinoma recurrence rate as high as43.6%. There have been reports that5years after radical resection of HCC recurrence rate was51.6%, of which a small hepatocellular carcinoma recurrence rate as high as43.6%. Although surgical resection besides, there are some new treatments into clinical (such as radiofrequency ablation, microwave therapy, cryoablation, the intratumoral injection of ethanol,hepatic artery chemoembolization local treatment intervention, etc.) but these methods have little effect for improving the overall quality of life of HCC.Early detection and early treatment, therefore, early diagnosis of liver cancer had already become the focus of attention, actually effective treatments still lacks, early detection of liver cancer remains a huge challenge. Fast growing in medical science today, although investment in the field of diagnosis has been increasing, but a few markers were effective. In fact, many of early stage of HCC may has occurred long-standing and metastasis, the patient has a tumor in the middle and late, various phenomena indicate that current testing technology is still not timely detection and effective detection of early-stage of HCC. So, looking for tumor markers remains an important direction and it is also an important method for the early detection of the cancer.Hepatocellular carcinoma is a common highly malignant tumor, the regional distribution of certain characteristics of its incidence exist where hepatitis virus is endemic in Asia and Africa, the incidence is relatively high. In the census of liver cancer, liver cancer susceptible individuals routine screening can significantly prolong the survival of many patients, but for many patients with advanced hepatocellular carcinoma, the effect is still not optimistic. For the part of the patients with advanced hepatocellular carcinoma, although only a small tumor, surgery is completely removed, but to improve the quality of long-term survival after surgery is still unknown.For the part of the patients with advanced hepatocellular carcinoma, although only a small tumor, surgery is completely removed, but much of the cancer can not removed by the surgery, to improve the quality of long-term survival after surgery is very important. Many intrahepatic or extrahepatic metastasis and recurrence is poor prognosis of patients with liver cancer the main reason. In fact, the current estimates of survival are often inadequate or excessive, often caused by improper postoperative treatment, for patients these reasons undoubtedly were negative factors.Many studies have confirmed that inflammation, immunity, cancer three closely related. Now that the inflammation in tumor incidence, progression, malignant transformation, invasion and metastasis have played an important role. We also understand very clearly between the inflammatory process and the immune response and has a close relationship between the two, there has been some scholars have proposed cancer1/5is associated with chronic infection. Inflammation and cancer is a contradiction, Dvorak HF, such as tumor called a "non-healing wounds" Schrader J, infered proinflammatory made of the tumor microenvironment for tissue remodeling, angiogenesis, invasion and metastasis occurs in one hand. to promote the inhibition of CTLs by the inhibition of macrophage production, aggregation Treg cells, thereby preventing immunization system to attack tumor cells in the other hand. As a cancer on the body damage, occurred in the course of its development, inflammatory response, especially for cancer, because it is destructive relatively strong, sparking the patient’s self-defense mechanisms will be relatively obvious. Given the close links between inflammation and cancer, and for participating in the process of inflammation-related factors in tumorigenesis has become an important research direction.It was known that there has been very significantly progress in the development of anti-cancer agents, including tyrosine kinase inhibitors, angiogenesis inhibitors, and agents that interact with the cell cycle and cell death (apoptosis). HTATIP2/TIP30was an evolutionarily conserved gene that is expressed ubiquitously in human tissues and some tumor tissues. The protein displayed a serinethreonine kinase activity that could phosphorylate the carboxyl terminal domain of RNA polymerase II in a TAT-dependent manner. So, it was necessary to evaluate its potential value. It was evidenced that HTATIP2/TIP30and EGFR had a close link in signaling pathways in liver. As reported, Endothelial Growth Factor Receptor (EGFR) was also central to the promotion of cell growth and had a role in the development of HCC. With the role of VEGF as a major antigenic factor in HCC, preventing EGFR activity was an attractive method for anti-HCC. Zhang, C, et al found that HTATIP2/TIP30banding with Endophilin B and ACSL4, form a complex in the cytoplasm, then participate in the transport of EGFR. In HTATIP2/TIP30gene knockout studies, after HTATIP2/TIP30knocked out, EGF-induced EGFR degradation were inhibited. The reason was that HTATIP2/TIP30, CSL4, Endophilin B1(EndoB1) formed a complex protein, which promoted EGFR from early endosomes into the liposomal sorting, and then could accelerate the degradation of EGFR in liver cells in mice and mammary cells. HTATIP2/TIP30gene knockout cells could not make the access of early endosomes for EGFR to the liposomal degradation, resulted in the activation of EGFR and its downstream signaling and ultimately promoted cell proliferation.The immunosuppressive microenvironment by the inflammatory state revealed the relationship between cytokine changes and tumor recurrence and metastasis TNF-a and IL-1β in primary liver cancer patients were higher than normal, the other side of these two factors have increased cancer, in addition to the blood vessels, such as generating MIF gene are associated with liver cancer liver metastasis TNF-a as a has anti-tumor cytokine, Intracellular NF-kβ and STAT3signaling pathway, leading to tumor proliferation, anti-apoptosis, angiogenesis, invasion and metastasis related genes to the development of liver cancer. Apart Th17cytokines secreted IL-17(IL-17A), but also includes IL-17F, and IL-21, IL-22, IL-6, TNF-a and other cytokines, Thl7cells are considered an important group important mediated inflammation can activate dendritic cells and T cells in lymphoid helper cells, release of cytokines to maintain the state of chronic inflammation and carcinogenesis environmentIL-17is an important mediator of inflammation, it can be induced by other inflammatory cytokines such as IL-6, the expression of TNF-a, and chemokines such as MCP-1, MIP-2, etc., to a local inflammatory cell mediated infiltration and tissue damage. Interleukin27(interleukine27, IL-27) was discovered in2002and named a new cytokine, IL-12, and it is with another newly discovered cytokine IL-23together constitute the IL-12family of molecules with complex biological functions in the anti-infection immunity, anti-tumor immunity and autoimmunity plays an important role. Based on existing research on HTATIP2/TIP30and inflammatory mediators and cancer and liver cancer in a variety of relevant reports, down HTATIP2/TIP30gene may be associated with the incidence of primary liver cancer, and therefore, the protein is detected through blood serum, determine the expression levels of normal liver cancer occurrence and development of research-related aspects are very necessary and critical. In addition, combined with the existing AFP clinical value, relevance and evaluating HTATIP2/TIP30, AFP in HCC clinical data also appears to make sense, This is more conducive to systematically analyze and test the performance of the two tests, we detected through the use of Luminex xMAP technology, high efficiency, high sensitivity,52cases of hepatocellular carcinoma in patients with hepatocellular carcinoma in patients with serum IL-1β,IL-6, IL-10, IL-17, IL-27, TNF-α levels were detected, and further explore these inflammatory factors may predict liver cancer metastasis. To be better utilized, and this is where the purpose of the present study.2Methods:2.1PatientsA total of84subjects, which included52patients diagnosed on the basis of National Comprehensive Cancer Network (NCCN) guidelines and32healthy individuals were recruited. All the subjects were from Hunan Provincial People’s Hospital between September2013and December2013. Before therapy, CT scan, ultrasound, biochemical, and serological parameters were performed to determine the presence of tumors and asides. According to our routine clinical approach,2ml of blood were taken from each patient. This study was approved by the Local Human Ethics Committee of the Ministry of Health. The clinical characteristics of the study population are shown in Table1. Table (1-1) Clinical datas in each group.2.2Blood samplingBlood samples were collected from these patients and healthy individuals and put them in biochemical tubes and then immediately taken to the biochemistry laboratory, where they were centrifuged at4,000rpm for10minutes at4℃.The serum were kept at-80℃until the biochemical measurements for HTATIP2/TIP30and other evaluation were conducted.2.3Biochemical analysesHTATIP2/TIP30concentration was determined using a commercially available ELISA kit (Cusabio Biotech. catalog number:CSB-E14917H ELISA kit). For this kit, the minimum detectable dose of human HTATIP2\TIP30was0.16ng/ml. The sensitivity of this assay, or lower limit of detection (LLD), was defined as the lowest protein concentration that could be differentiated from zero. Intra-assay precision (precision within an assay) was CV%<8%, and inter-assay precision (precision between assays) was CV%<10%.2.4Roche electrochemiluminescence assay HBeAg statusAll subjects enrolled HBeAg determined by electrochemiluminescence detection of HBeAg SOP sample handling processes in accordance with Roche chemiluminescence, aircraft, Elecsys software analysis determined that the specimens HBeAg positive and negative.2.5Quantitative PCR serum HBV DNA levelsAll enrolled subjects by HBV fluorescence quantitative detection kit instructions to operate the experimental subjects serum PCR reaction mixture and mix well with a common standard quantitative HBV PCR amplification, to be amplified by ABI7100after the data analysis2.6Luminex xMAP serum cytokines in patients with liver cancerLuminex xMAP technology (multi-anlyte profiling beads) eBioscience the ProcartaPlex kit standards cytokines (including IL-Iβ, IL-10, IL-6, IL-17, TNF-a, IL-27) cells in a total of6standard and sample dilution factor. Data analyzed by Luminex200results analysis software that captures images by detecting the sample, and then through image analysis, the standard curve, calculated from the data sample to be tested.2.7Statistical analysesSPSS Statistics17computer program package for statistical calculations. The results are expressed as mean±standard deviation (SD). ANOVA and multiple comparison analysis of the healthy control group and two groups between HCC and each stage of HCC.(Operating Characteristic curve) ROC analysis of various indicators of HCC group and the control group, metastatic and nonmetastatic, early diagnosis of HCC and control group. Spearman clinical detection of cytokines and biochemical indices and indicators of relevance. Multivariate logistic regression analysis and the establishment of joint diagnostic model indicators, Hosrner-Lemeshow goodness of fit of the logistic regression test, P values are accurate to three decimal places, with P<0.05as statistically significant. Medcalc draw ROC curve, Graphpade Prism5draw a histogram and associated line graph.3Results:3.1Serum level of HTATIP2/TIP30The HTATIP2/TIP30level was significantly lower in the HCC group (mean,4.505ng/ml; range,0.191-11.07) compared with the healthy group (mean,9.499ng/ml; range,5.759-13.278, unpaired t-test,*P<0.001, P<0.05) For the subgroups, the serum level of HTATIP2/TIP30was significantly lower in the (mean,3.667ng/ml; range,0.191-8.965) than in the SD group (mean,6.089ng/ml; range,2.452-11.07),(unpaired t-test,**P=0.001, P<0.05) for the diagnosis of the subgroups, the PD group or SD group was detected by CT scan and evaluated by response evaluation criteria in solid tumors RECIST,NCI2000.3.2ROC curves of HTATIP2/TIP30and AFPAlthough the level of HTATIP2/TIP30was elevated in majority of the HCC patients, there was considerable overlap between hepatocellular carcinoma patients and the healthy controls. Therefore, ROC analysis was used to detect the optimal cut-off points of HTATIP2/TIP30and AFP for discrimination HCC patients and the healthy individuals The Area under the curves (AUCs) of HTATIP2/TIP30and AFP were0.928(0.926±0.027) and0.888(0.888±0.043) respectively.(P<0.001, P <0.05) ROC analysis was also used to detect the optimal cut-off points of HTATIP2/TIP30and AFP for discrimination for the subgroup. The Area under the curves (AUCs) of HTATIP2/TIP30(0.769±0.066, P=0.001, P<0.05) was greater than that of AFP (versus0.668±0.086, P=0.043, P<0.05).3.3HTATIP2/TIP30and AFP for diagnosisIf combined the HTATIP2/TIP30and AFP to diagnosis of HCC and healthy individuals, with the optical cut-points (7.271ng/ml for HTATIP2/TIP30,11.930ng/ml for AFP), the sensitivity, specificity and Youden index was showed in If combined the HTATIP2/TIP30and AFP to diagnosis of HCC progressed or not, with optical cut-points (6.240ng/ml for HTATIP2/TIP30, and31.190ng/ml for AFP), the sensitivity, specificity and Youden index was showed in.3.4Relationships between clinical characteristics and HTATIP2/TIP30 We analyzed relationships between HTATIP2/TIP30level, tumor characteristics, serological parameters and Child-Pugh score. HTATIP2/TIP30level showed a statistically significantly negative correlation with HBV DNA load (r=-0.364, P=0.002) and Child-Pugh score (r=-0.419, P=0.008), although not associated with tumor size and AFP.3.5Logistic regression model to predict HCCBy HTATIP2/TIP30, HBvDNA level, AFP concentration of three factors do backward stepwise logistic regression analysis, the probability choose to enter the variable standard of0.10, excluding the probability standard variable is0.15, derived regression equation P=1/[1+e-(4.449-0.717×1+0.042×2)]:where×1representatives HTATIP2/TIP30, x2behalf of AFP, Odd Ratio (OR) values were:0.488and1.043;95%CI (95%confidence interval) were (0.331-0.791) and (0.998-1.089). When AUC Logistic regression analysis model predictors PRE ROC curve was0.963, more than HTATIP2/TIP30and the individual indicators AFP, its largest Youden index corresponding sensitivity of90.6%and a specificity of90.4%compared with the two indicators alone diagnosis significantly increased.3.6Detection serum of multiple inflammatory cytokines in HCCUsing Luminex xMAP method for detecting HCC metastasis and without metastasis in serum samples content (IL-1β, IL-6, IL-10, IL-17A, TNFa, IL-27) and the results showed no metastasis group the cytokines levels were (IL-1β,3.93±2.07; IL-6,6.61±4.63; IL-10,7.77±5.49; IL-17A,255.49±94.33; IL-27,2.67±2.53), the transfer group content of each group of cytokines (IL-1β,4.11±1.95; IL-6,6.30±5.39; IL-10,8.70±4.77; IL-17A,5.56±2.85IL-27,353.19±177.55), which IL-17A, IL-27cytokine groups was significantly higher in the transfer group, the difference was statistically significant. P values were (0.015, P<0.001)3.7Correlation analysis HTATIP2/TIP30each serum levels of inflammatory cytokinesPearson correlation analysis HTATIP2/TIP30, IL-1β,IL-6, IL-10, IL-17, IL-27, TNF-a and other cytokines correlation was, IL-17and TIP30negative correlation (r=-0.601, P<0.001),3.8ROC analysis of IL-17A and IL-27predict liver cancer metastasisROC analysis of IL-17and IL-27both indicators of liver metastasis and without metastasis, respectively, the area under the curve (0.818, P=0.044) and (0.664, P <0.001), both as a predictor of liver cancer metastasis statistically significant. To IL-27is238.95pg/mL threshold is, the sensitivity was:95.8%, specificity was:64.3%maximum Youden index. IL-17select critical value2.005pg/ml, the largest Youden index, the sensitivity was83.3%and specificity of35.7%. IL-27and IL-17compared to a better prediction of liver cancer metastasis.3.9Logistic regression model to predict HCC metastasisLogistic regression analysis HTATIP2/TIP30and IL-27, the dependent variable select "0" for the metastasis,"1" for the transfer. Choose to enter the variable probability standard is0.10, excluding the probability standard variable is0.15, derived regression equation:P=1/[1+e-(-1.264-0.642×1+0.013x2)],×1representatives HTATIP2/TIP30, x2behalf of IL-27, P is a logistic model to predict the probability ranges between0-1, e is the natural logarithm (e=2.718), Odd Ratio (OR) values were:0.526and1.013;95%CI (95%confidence interval), respectively (0.346-0.799) and (1.003-1.026). The regression equation prompt independent factors HTATIP2/TIP30and IL-27are HCC metastasis, the risk of high HTATIP2/TIP30serum levels in patients with metastasis was0.526times its low level of patient transfer risks. IL27-risk as a risk factor, high serum levels of IL-27in patients with metastasis was1.013times its low level of patient transfer risk. PRE regression model to predict both the area under the curve of HCC metastasis (AUC) was0.903±0.043, P<0.001, sensitivity87.5%, specificity82.1%, significantly higher than the two indicators predict when alone.Novelty1. This study is the first time to detect the level of HTATIP2/TIP30in the serum of HCC patients, and found it is a powerful indicator in dignose and predict metastasis.2. For the first time by liquid chip found IL-17and IL-27associated with hepatocellular carcinoma metastasis, two indicators may be used to predict HCC metastasis.更多还原