Application of Ultrasound Combined with DSA-guided Single-incision Technique via Axillary Vein Access in Implantation of Totally Implantable Venous Access Port
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摘要:目的
探讨超声联合数字减影血管造影技术（DSA）引导下行单切口经腋静脉（AV）入路植入输液港（TIVAP）在临床应用中的可行性及安全性。
方法回顾性分析在福建省肿瘤医院就诊并接受超声联合DSA引导下行单切口经AV入路TIVAP植入术的240例患者临床资料。记录手术相关信息，包括AV宽度、AV穿刺成功率、植入成功率、超声引导穿刺时间、手术时间及术中、术后并发症等。
结果240例患者均成功植入TIVAP，植入成功率100%。229例患者于超声联合DSA引导下行单切口经AV穿刺植入TIVAP，AV穿刺成功率95.42%（229/240），11例因AV穿刺失败，改超声联合DSA引导下经同侧颈内静脉（IJV）穿刺植入TIVAP。240例患者术前超声探查定位下测得拟穿刺段AV平均宽度为（7.56±1.26）mm，其中AV 1次穿刺成功195例，2次穿刺成功26例，3次穿刺成功8例，成功率分别为81.25%、10.83%、3.34%；超声引导下平均穿刺时间（0.85±0.52）min，手术平均时间（25.9±4.8）min。术中并发症发生率为1.67%（4/240），未发生血胸、血气胸及严重致死性并发症；TIVAP留置期间相关并发症发生率为2.92%（7/240），未见导管相关血流感染、导管相关静脉血栓、导管断裂/移位、夹闭综合征、药物外渗等并发症发生。
结论超声联合DSA引导下行单切口经AV植入TIVAP术是一种可行且安全的植入方式，具有较高的成功率、较短的手术时长和较低的并发症风险，可作为TIVAP植入方式的另一种选择。
Abstract:ObjectiveTo evaluate the technical feasibility and safety of a single-incision technique via axillary vein (AV) for placement of totally implantable venous access port (TIVAP) guided by ultrasound combined with DSA in clinical application.
MethodsWe retrospectively analyzed clinical data of 240 patients who received TIVAP by single incision technique via AV access guided by ultrasound combined with DSA. We observed and recorded operation-related information such as AV width, AV puncture success rate, implantation success rate, ultrasound-guided puncture time, operation time and intraoperative and postoperative complications, etc.
ResultsAll 240 patients were successfully implanted with TIVAP, and the success rate was 100%. In 229 cases, TIVAP was implanted through single-incision AV puncture under the guidance of ultrasound combined with DSA, and the success rate of AV puncture was 95.42% (229/240). In 11 cases, TIVAP was implanted through the ipsilateral internal jugular vein (IJV) under the guidance of ultrasound combined with DSA due to the failure of AV puncture. In the 240 patients, the average width of AV of the intended puncture segment was (7.56±1.26) mm measured by preoperative ultrasound exploration and positioning, in which 195 cases were successfully punctured once, 26 cases were successfully punctured twice, and 8 cases were successfully punctured three times, with the success rate of 81.25%, 10.83% and 3.34%, respectively. The average puncture time under ultrasound guidance was (0.85±0.52) min, and the average operation time was (25.9±4.8) min. The incidence of intraoperative complications was 1.67% (4/240). No hemothorax, hemopneumothorax or serious fatal complications occurred. The incidence of complications during TIVAP retention was 2.92% (7/240). No complication such as catheter-related bloodstream infection, catheter-related venous thrombosis, catheter rupture/displacement, clipping syndrome or drug extravasation was observed.
ConclusionUltrasound combined with DSA guided single-incision technique via AV access in the implantation of TIVAP is a feasible and safe implantation method with high technical success rate, short operation time and low risk of complications. It can be used as another choice of TIVAP implantation method.
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Key words:
- Ultrasound /
- DSA /
- Single-incision /
- Axillary vein /
- Totally implantable venous access port
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0 引言
宫颈癌在全球最常见的女性恶性肿瘤中排名第二，目前仍然是女性癌症相关死亡的主要原因之一。虽然宫颈癌早期可以通过根治手术辅以放化疗得以治愈，但是一些高危因素患者的预后仍然不容乐观[1]。目前的临床数据表明，影响宫颈癌预后的因素包括肿瘤大小、浸润深度、血管的侵犯、子宫旁组织的浸润、盆腔淋巴结转移等，其中盆腔淋巴结的转移被认为是影响术后疗效最重要的危险因素[2]。作为早期诊断及预后评估的分子指标，非编码RNA（non-coding RNA, ncRNAs）已日益得到重视和研究。近年研究表明ncRNAs与宫颈鳞状细胞癌的发生发展有密切关系，参与调控宫颈鳞状细胞癌进程的分子机制和雌孕激素表达[3]。
ncRNAs主要包括microRNAs和lncRNAs[4]。miRNA可通过调控靶基因从而促进宫颈癌细胞凋亡，并能改变肿瘤细胞对药物的敏感度，且能在组织、血清和血浆等标本中稳定存在。miRNA有望成为诊断宫颈癌的新标志物和治疗的新靶点[5]。近年来，lncRNA-miRNAs的调节关系吸引了越来越多学者的注意，lncRNAs和microRNAs的交互调节是目前的研究热点[6]。本研究探讨了LncRNA HCG11和miR-590-3p在宫颈鳞状细胞癌组织中的表达，并且利用qRT-PCR结果和starBase v2.0数据库探讨两者之间的关系，结合患者的临床资料探讨LncRNA HCG11和miR-590-3p与性别、年龄、组织大小、病理分级、淋巴结转移、临床分期以及预后的关系。
1 资料与方法
1.1 临床资料
1.1.1 宫颈鳞状细胞组织样本和临床病理数据收集
2013年7月—2016年3月在南阳市第二人民医院妇科接受根治性子宫切除术和盆腔淋巴结清扫的58例宫颈鳞状细胞癌患者，年龄28~72岁。根据国际妇产科联合会（FIGO，2014）的诊断标准，58例宫颈鳞状细胞癌病例中，Ⅰ~Ⅱ期12例、Ⅲ~Ⅳ46例；20例有淋巴结转移、38例无淋巴结转移；分化良好（G1）13例、中度分化（G2）32例、低分化（G3）13例。所有患者术前均未接受化疗或放疗；所有病例均有病理组织学诊断支持；所有标本直接取自手术切除物，均为配对组织。每对为肿物和癌旁正常组织距离病灶≥5 cm，经病理证实无肿瘤细胞浸润各一份。在手术切除后10 min之内获得。取材后置于无菌冻存管中，储存至-80℃。所有患者均获得随访，末次随访时间为2016年8月，随访时间5~36月。
1.1.2 试剂与仪器
RNA提取试剂盒（天根生物公司，中国），RNA反转录试剂盒（诺维赞生物公司，美国），荧光定量PCR试剂盒（康为世纪生物公司，中国），Biotek Epoch微量核酸定量仪购自美国Biotek公司，7500荧光定量PCR仪购自美国ABI公司。
1.1.3 qRT-PCR检测基因LncRNA HCG11和miR-590-3p的引物序列
通过PCR引物设计软件Primer Premier 5.0设计LncRNA HCG11、GAPDH、has-miR-590-3p和U6的引物序列，具体序列如下：HCG11上游引物：5′-AGGAGTGGTTGCATTTGGGA-3′，下游引物：5′-CCCACCACGCAGTGAATAGT-3′；GAPDH上游引物：5'-GGAAGGACTCATGACCACAGTCC-3'，下游引物：5'-TCGCTGTI'GAAGTCAGAGGAGACC-3'。has-miR-590-3p-5p引物为5′-CCTGGCTTTTCATTCCTATGTGA-3′，U6引物为5′-GCTTCGGC AGCACATATACTAAA-3′，并由上海生工生物公司合成。引物干粉离心后用DEPC水配制成20 µmol/L，置于－20℃冰箱备用。
1.2 方法
1.2.1 总RNA的提取和反转录
从子宫颈鳞状细胞癌组织和癌旁组织中抽提总RNA，按总RNA提取试剂盒说明书进行，采用定量分光光度法在A260/280处对提取RNA进行定量。按照反转录试剂盒（诺维赞公司，R111-01/02）说明书对总RNA进行反转录，反转录反应由1 µg的总RNA，与10 µl 2×RT mix，2 µl mix，1 µl Oligo dNTPs，1 µl Random hexamers，6 µl ddH2O总体系20 µl。25℃ 5 min，50℃ 15 min，85℃ 5 min，经反转录形成cDNA。
1.2.2 qRT-PCR检测LncRNA HCG11和miR-590-3p
根据康为世纪生物公司实时PCR试剂盒说明书进行荧光定量PCR。实时PCR体系为20 µl : 2 µl反向转录产物，2 µl引物，2 µl反向引物，10 µl 2×mix，纯水4 µl。1个循环包括95℃ 30 s、95℃ 5 s和60℃ 30 s，总共40个循环。所有样本重复三次。以2-ΔCt表示基因的相对表达水平。
1.3 统计学方法
采用SPSS 19.0软件进行统计学分析。计量资料以（x±s）表示，计数资料采用构成比（%）表示，58对配对的宫颈鳞状细胞癌和癌旁组织比较采用两配对样本t检验，与临床资料联合分析采用卡方检验，采用Kaplan-Meier法计算生存函数，差异比较采用Log rank检验，采用Cox比例风险模型用于宫颈鳞状细胞癌预后的多因素分析。以P < 0.05为差异有统计学意义。
2 结果
2.1 LncRNA HCG11和miR-590-3p的表达
应用qRT-PCR检测58对配对的宫颈鳞状细胞癌组织及癌旁组织中LncRNA HCG11和miR-590-3p的表达，以癌旁为参考值，通过配对样本t检验分析2-ΔCt值。结果显示宫颈鳞状细胞癌组织中LncRNA HCG11的平均表达量为（8.23±3.77）低于宫颈鳞状细胞癌癌旁组织（9.62±3.93），miR-590-3p平均表达量为（9.61±4.17）高于宫颈鳞状细胞癌癌旁组织（8.18±4.22），差异有统计学意义（t=3.249, P=0.0019; t=3.403, P=0.0012），见图 1。
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图 1 58对配对的宫颈鳞状细胞癌组织及癌旁组织中LncRNA HCG11和miR-590-3p的表达Figure 1 LncRNA HCG11 and miR-590-3p expression in 58 pairs of squamous carcinoma of cervix and adjacent non-tumor tissuesT: squamous carcinoma of cervix(SCC) tissues; N: adjacent non-tumor tissues; A: the expression of LncRNA HCG11 in SCC tissues was inferior to that in non-tumor tissues; B: the expression of miR-590-3p in squamous carcinoma of cervix tissues was superior to that in non-tumor tissues2.2 LncRNA HCG11和miR-590-3p表达相关性分析
通过NONCODE database分析我们知道LncRNA HCG11在宫颈癌细胞系HeLa中的相对表达量为1.874，见图 2A，说明宫颈癌细胞中可以检测到该基因的表达。通过starBase v2.0数据库分析了与LncRNA HCG11可以结合miRNA，发现miR-590-3p与LncRNA HCG11有三个结合位点，见图 2B。通过qRT-PCR结果，分析了宫颈鳞状细胞癌组织中LncRNA HCG11和miR-590-3p核酸水平的相关性，见图 2C，结果呈负相关（r=-0.642, P =0.000）。
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图 2 LncRNA HCG11和miR-590-3p表达相关性分析Figure 2 Correlation analysis between LncRNA HCG11 and miR-590-3p expression1: adipose; 2: adrenal; 3: brain; 4: brain_R; 5: breast; 6: colon; 7: fores kin; 8: heart; 9: hela_R; 10: HLF_1; 11: HLF_2; 12: kidney; 13: liver; 14: liver_R; 15: lung; 16: lymph Node; 17: ovary; 18: placenta_R; 19: prostate; 20: skeltal muscle; 21: testes; 22: testes_R; 23: thyroid; 24: white blood cell. A: NONCODE database showed that the expression of LncRNA HCG11 in HeLa cells; B: starBase v2.0 databases showed that there were three binding sites between miR-590-3p and LncRNA HCG11; C: qPCR indicated that on nucleic acid level, LncRNA HCG11 expression was negatively correlated with miR-590-3p expression in squamous carcinoma of cervix tissues2.3 LncRNA HCG11和miR-590-3p与临床病理特征的相关性
肿瘤组织中LncRNA HCG11和miR-590-3p的表达水平与组织大小、淋巴结转移以及临床分期（P=0.027）有相关性，即癌组织大、有淋巴结转移、临床分期差的患者LncRNA HCG11表达水平越低，而miR-590-3p表达水平越高，见表 1。
表 1 LncRNA HCG11和miR-590-3p的相对表达情况与患者临床病理因素的关系Table 1 Relative expression of LncRNA HCG11 and miR-590-3p and their correlation with patients' clinicopathological factors
2.4 LncRNA HCG11和miR-590-3p与宫颈鳞状细胞癌患者预后的关系
根据qRT-PCR的结果，用Kaplan-Meier方法分析LncRNA HCG11和miR-590-3p在宫颈鳞状细胞癌组织中的表达和患者生存期之间的关系。按照二者在癌组织中表达量中位数把患者分为高表达组和低表达组，其中LncRNA HCG11高表达者24例、低表达者34例。miR-590-3p高表达者35例、低表达者23例。随访时间15~36月，其中复发14例、死亡14例、13例因复发转移死亡、1例因放化疗相关并发症死亡。分析表明，在宫颈鳞状细胞癌患者中，LncRNA HCG11低表达患者的总生存期明显低于高表达患者（P=0.024）；miR-590-3p高表达患者的总生存期明显低于低表达患者（P=0.047），见图 3。Cox单变量分析表明肿瘤大小、淋巴结转移、临床分期和LncRNA HCG11以及miR-590-3p的表达情况与生存相关。Cox比例风险模型多变量分析表明LncRNA HCG11和miR-590-3p表达情况分别是独立的危险因素（P=0.037, 0.042），见表 2。
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图 3 LncRNA HCG11和miR-590-3p与患者的预后的关系Figure 3 Relationship of LncRNA HCG11 and miR-590-3p expression with patients' prognosisA: The overall survival of patients with low LncRNA HCG11 expression was obviously shorter than that with high LncRNA HCG11 expression; B: The overall survival of patients with high miR-590-3p expression was significantly shorter than that with low miR-590-3p expression表 2 58例宫颈鳞状细胞癌患者不同预后因素对OS的单因素和多因素分析Table 2 Univariate and multivariate analyses of different prognostic factors for OS of 58 patients with squamous carcinoma of cervix
2.5 Gene Oncology和KEGG通路分析
为了预测HCG11的功能，首先使用TCGA构建共表达网络识别差异表达的mRNA和HCG11基因之间的相互作用。根据Pearson相关系数建立基因共表达网络。然后使用MAS 3.0（http://bioinfo.capitalbio.com/mas3/project/index）执行GO和通路分析。根据GO分析结果把前1 000个差异表达的基因进行分类。分析显示，这些基因与信号转导、转录、细胞黏附、细胞转化和蛋白质氨基酸磷酸化相关。通路分析显示HCG11的共表达基因主要参与MAPK信号通路、钙信号通路、Jak-STAT信号通路和Wnt信号通路等。
3 讨论
非编码RNA占整个人类基因组的98%左右，包括lncRNAs和microRNAs[7]。miRNA能够结合到目标mRNA的3′UTR，调节mRNA的稳定性和翻译过程，最终导致mRNA的翻译被抑制或被降解[7-8]。miRNA可分为癌基因和抑癌基因，参与细胞的多个生物过程，包括增殖、凋亡、代谢、细胞分化等[9]。越来越多的证据表明，microRNA参与多种癌症的进展过程。与已经非常熟悉的microRNAs相比，LncRNAs方面的研究相对较少。长非编码RNA（LncRNAs）由超过200个核苷酸组成，是ncRNAs中重要的成员，不能被翻译成蛋白质[10]。到目前为止，绝大多数的LncRNAs已被鉴定出来[11-12]。许多研究已经证明，LncRNAs在癌症的发生发展中发挥了重要作用[13-15]。目前LncRNA的研究处于起步阶段，因此大多数LncRNA的功能仍是未知。LncRNA能不能作为肿瘤诊断和预后的生物标志物，以及在临床中的意义，人们还没有一个确切的结论。
目前，很多国家都在致力寻找并建立一些可以有效筛查宫颈鳞状细胞癌的项目，从而降低其发病率和死亡率。这些筛查基本需依赖细胞学巴氏涂片，有些联合检查可以预防多达91%的浸润性宫颈鳞状细胞癌[2]。然而，这些项目花费较高，因此，寻找客观精准而又方便检测的标志物，来提示发病和预后是宫颈鳞状细胞癌目前重要的研究方向。一些与宫颈鳞状细胞癌预后相关的分子虽然已得到初步证实，但是它们的作用机制仍不确定。
目前，国内外已发表的与宫颈鳞状细胞癌致病相关的lncRNA有HOTAIR[16]、MALAT1[17]、H19[18]、EBIC[19]、MEG3[20]、GAS5[21]，XLOC_010588[22]等。这7个LncRNAs在有些文献中的报道是抑癌作用，有些文献中报道是促癌作用，比如TUG1在结肠癌中表现为上调[23]，在非小细胞肺癌中表达下调[24]，与其他文献报道不同，说明LncRNAs的表达存在组织特异性、个体差异性，且在组织中表达差异并不显著，均不足以成为宫颈癌的特异肿瘤标志物。
该研究发现与癌旁组织相比HCG11在宫颈鳞状细胞癌组织中显著下调，且与肿瘤组织大小、淋巴结转移以及临床分期有关。starBase v2.0数据库分析表明miR-590-3p与LncRNA HCG11有三个结合位点。随后研究发现miR-590-3p在宫颈鳞状细胞癌组织中表达明显上调，且与组织大小、淋巴结转移、临床分期以及预后有关，而且发现LncRNA HCG11和miR-590-3p核酸水平呈显著的负相关。为了进一步探讨HCG11的功能，我们进行了GO和KEGG通路分析。结果表明受其表达影响的基因参与包括信号转导、转录调控、细胞黏附、恶性转化和氨基酸磷酸化等过程。HCG11参与MAPK信号通路、钙信号通路、Jak-STAT信号通路和Wnt信号通路的调控。
目前关于LncRNA HCG11与肿瘤研究的报道较少，其中HCG11在乳腺癌与前列腺癌中表达下调的患者预后较差。miR-590-3p与肿瘤的研究比较多，Yang等[25]发现miR-590-3p通过激活PI3K-AKT信号通路下调PTEN，促进AKT1-S473的磷酸化作用，进而发挥促进肝癌细胞增殖。Sun等[26]发现，miR-590-3p通过Hippo通路促进结肠癌细胞的增殖和转移。LncRNA的作用机制之一就是通过海绵样吸附作用，竞争性与microRNA结合，抑制其进一步发挥基因调控的作用。由此推测，LncRNA HCG11有可能通过调控miR-590-3p的表达发挥其抑癌作用。可以通过进一步的体外细胞实验对该调控机制进行验证。
Competing interests: The authors declare that they have no competing interests.作者贡献：刘伟夫：选题设计、试验执行、病例资料及数据收集、文章撰写及修改张孔志：指导选题设计及试验执行余文昌：指导试验执行陈示光：病例资料及数据收集王小珑：病例资料收集 -
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图 1 单切口AV入路植入输液港影像资料
Figure 1 Image of single-incision technique via AV access in implantation of TIVAP
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图 2 AV入路导丝、导管误入非目标血管影像资料
Figure 2 Image of guide wire and catheter mistakenly entering non-target vessels via AV approach
表 2 AV不同穿刺次数成功的一般资料比较(x±s)
Table 2 General information of patients with different times of successful axillary vein puncture (x±s)
下载: 导出CSV
表 3 患者一般资料与腋静脉宽度之间相关性分析
Table 3 Correlation between general date of patients and width of axillary vein
下载: 导出CSV
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