中医药对淋巴管新生与回流功能的作用研究进展＊

淋巴管作为循环系统的重要组成部分之一，具有调节机体体液稳态，协助免疫监视和肠道脂质吸收等重要作用。淋巴管新生是机体生理和病理过程中维持脉管系统结构和功能正常的重要手段，淋巴管新生调控对于防治肿瘤、心血管等诸多疾病有着潜在的临床转化意义；淋巴回流功能则与关节炎症等疾病发病机制关系密切。在循环系统中，相较于中医药调控血管相关疾病的发病机制已取得很大进展，近年来对于淋巴管的研究则明显相对滞后。本文从中医药作用于淋巴管新生及回流功能角度对这一领域的研究进展作一综述，以期为临床上中医药治疗相关疾病提供新的思路与方法。     

结直肠癌淋巴管生成的特点及其临床病理意义

目的探讨结直肠癌中淋巴管的分布特点、增殖状态及其与转移和预后的关系。方法采用淋巴管特异标记podoplanin对96例结直肠癌及其相应正常组织进行免疫组织化学染色检测微淋巴管密度,以CD34标记血管检测微血管密度作为对比,并分别与Ki-67进行双标免疫组织化学染色检测淋巴管和血管增殖活性,结合结直肠癌临床病理参数和预后分析。结果结直肠癌中心及浅表部淋巴管多为闭锁的条索状,边缘区淋巴管多呈管样扩张状。结直肠癌边缘区淋巴管密度(51.2±25.5)及较正常结直肠组织(29.4±9.0)和肿瘤其他区域显著性增高(P<0.01),并且其淋巴管内皮Ki67指数(0.23±0.17)也较其他区域显著性增高(P<0.05)。结直肠癌边缘区微淋巴管密度与淋巴管受累、淋巴结转移、远处器官转移及预后密切相关(P<0.01或P<0.05)。结论结直肠癌组织中存在新生淋巴管,且主要分布于肿瘤边缘区,癌周围淋巴管密度增加与癌细胞转移相关,结直肠癌边缘区微淋巴管密度测定对评估其淋巴结转移和预后判断可能具有意义。     

Akt介导VEGF信号通路在促进胃癌淋巴管新生中的作用

随着研究的深入,肿瘤淋巴管新生已被证实在实体肿瘤淋巴转移途径中起着至关重要的作用,并可作为一个评价肿瘤预后的重要指标.但长期以来由于缺乏对淋巴管内皮特异因子和淋巴管检测技术的认识,对淋巴管转移具体机制与肿瘤淋巴管新生分子途径尚未完全明确.Akt信号通路是细胞内一条重要的信号传导通路,与肿瘤发生、增殖、抗凋亡、转移等密切相关.近年来发现Akt在介导VEGF信号通路与肿瘤淋巴管转移或肿瘤淋巴管新生的也存在着密切关系.本文对Akt信号通路在胃癌淋巴管新生中重要作用与研究进展作一综述.     

A practical application of quantitative vascular image analysis in breast pathology

Aims

Quantitative image analysis of histopathology slides is becoming an important technology in diagnostic pathology. To this end, it is essential to combine a robust image analysis software with the most commonly used immunohistochemical staining methods. In this investigation, we describe a practical application of NIH ImageJ software for quantitative vascular image analysis for diaminobenzene chromogen-based CD34 immunostain in breast cancer. CD34 immunostain is in a unique position to identify lymphangiogenesis and angiogenesis simultaneously in a given tumor tissue. This investigation aims at establishing a practical quantitative vascular image analysis solution for diagnostic pathologists by using ImageJ, and CD34 immunostain.

Methods and results

Tissue microarray slides containing breast cancer tissue were immunostained for CD34 for simultaneous identification of lymphatic endothelial cells (LEC) and blood vessel endothelial cells (BEC). Digital images were analyzed using NIH ImageJ software. A CD34 score was quantified for each tissue core as a percentage (CD34-positive area/area of tissue core). The mean CD34 scores were 0.24%, 0.40%, 1.30%, 2.33%, 2.64%, and 3.44% for normal breast tissue, in stage IIA, IIB, IIIA, IIIB, and IIIC breast cancer tissue cores, respectively (p < 0.0001). The mean CD34 scores were 0.70% and 2.21% for lymph node-negative and lymph node-positive breast cancer patients, respectively (p < 0.0001).

Conclusions

ImageJ software seems to be an attractive quantitative image analysis tool for diagnostic pathology for immunohistochemistry-based applications because of its capabilities, availability, and ease of use with most image formats. Our results show the feasibility, versatility, and ease of use of ImageJ and CD34 immunohistochemistry for vascular image analysis in breast pathology. Given the prospects of novel lymphatic and vascular endothelium-targeting therapeutics in breast oncology, the practical analysis of combined LEC and BEC density described in this report could enable diagnostic pathologists to apply quantitative vascular image analysis easily in their pathology practice and translational research.     

Podoplanin在口腔鳞状细胞癌中的表达及与淋巴结转移的关系

目的:采用淋巴管特异性标志物podoplanin标记口腔鳞癌组织和正常口腔组织中的淋巴管,并计数淋巴管密度(lymphatic vessel density,LVD)值,探讨口腔鳞癌及癌周淋巴管密度与淋巴结转移的相关性及可能机制。方法:采用免疫组织化学方法检测21例正常口腔黏膜组织和88例口腔鳞癌患者组织的podoplanin表达;用podoplanin标记淋巴管,并计数口腔鳞癌和癌周组织的淋巴管密度。采用SPSS13.0软件包对数据进行t检验,分析正常组织和癌组织、淋巴结转移组和未转移组的淋巴管密度(癌周、癌内)。结果:口腔鳞癌及癌周组织的淋巴管密度均高于正常口腔黏膜组织,有显著差异(P<0.05)。癌组织内淋巴管小而闭锁,癌组织周围的淋巴管大而扩张;淋巴结转移组的癌周淋巴管密度(14.270±4.610)显著高于无转移组(9.450±2.411),差异具有显著性(P<0.05),癌组织淋巴管密度在2组间无显著差异。结论:口腔鳞癌患者癌周淋巴管的生成可能是影响区域淋巴结转移的重要因素。     

直肠癌中血管内皮生长因子C和血小板衍生生长因子AA、BB的表达及其与淋巴管生成和淋巴结转移的关系

目的探讨血管内皮生长因子C（VEGF-C）、血小板衍生生长因子（PDGF）-AA、PDGF-BB在直肠癌组织中的表达情况及其与患者淋巴管生成和淋巴结转移之间的关系。 方法采用免疫组织化学SP法检测60例直肠癌组织（直肠癌组）和30例正常组织（对照组）中VEGF-C、PDGF-AA和PDGF-BB的表达。应用D2-40检测直肠癌中微淋巴管密度（LMVD）,并做相关统计分析。 结果VEGF-C、PDGF-AA和PDGF-BB在直肠癌组织中阳性表达率均显著高于对照组(VEGF-C：63.3% vs 3.3%,P<0.05;PDGF-AA：40.0% vs 13.3%,P<0.05;PDGF-BB：55.0% vs 6.7%,P<0.05)。VEGF-C和PDGF-BB在淋巴结阳性组的阳性表达率较淋巴结阴性组均显著升高（VEGF-C：86.4% vs 50.0%,P<0.01;PDGF-BB：77.3% vs 42.1%,P<0.05）,但PDGF-AA表达与淋巴结转移无关（54.5% vs 31.6%,P>0.05）。直肠癌中淋巴结阳性组的LMVD高于淋巴结阴性组[（13.11±2.01）/mm² vs (8.13±2.99）/mm²,P<0.05],VEGF-C、PDGF-AA和PDGF-BB阳性表达组的LMVD高于阴性表达组[VEGF-C：（12.06±2.20） /mm² vs（6.32 ±2.41） /mm²,P<0.05;PDGF-AA：（11.89± 2.46）/mm² vs （8.67±3.67）/mm²,P<0.05;PDGF-BB：（12.19±2.22） /mm² vs（7.33 ±3.02）/mm²,P<0.05]。直肠癌中VEGF-C、PDGF-AA、PDGF-BB三者的表达具有显著相关性（VEGF-C vs PDGF-AA：r_s=0.366,P<0.05;VEGF-C vs PDGF-BB：r_s=0.650,P<0.05;PDGF-AA vs PDGF-BB：r_s=0.320,P<0.05）。 结论淋巴管生成是直肠癌淋巴结转移的重要原因之一,VEGF-C、PDGF-AA和PDGF-BB均参与直肠癌淋巴管生成,进而促进淋巴结转移,且三者可能存在一定的协同作用。针对VEGF-C、PDGF-AA、PDGF-BB的联合用药可能更利于直肠癌淋巴结转移的防治。     

胃癌组织中微淋巴管的生成特点及临床病理意义

目的:探讨胃癌组织中微淋巴管的生成特点及与临床病理特征之间的关系.方法:对胃癌56例及相应正常胃组织12例进行双重免疫组织化学染色,采用微淋巴管特异标记物Podoplanin标记微淋巴管,同时采用Ki-67来检测微淋巴管内皮细胞的增殖活性,并观察微淋巴管分布特点,分析其与临床病理特征之间的关系.结果:胃癌组织中存在微淋巴管,但分布不一致.癌内微淋巴管数目少,并且多呈闭锁条索状,癌周微淋巴管数目多且多扩张,呈管腔样,有时可在其中发现癌栓.胃癌癌周微淋巴管密度(11.89±3.95)与癌内微淋巴管密度(5.83±3.26)相比较,差别有显著统计学意义(P<0.01),与相应正常组织微淋巴管密度(6.93±1.32)相比较,差别亦有统计学意义(P<0.05).胃癌癌周微淋巴管内皮细胞Ki-67指数(0.04±0.02)分别与癌内微淋巴管内皮细胞Ki-67指数(0)及相应正常胃组织微淋巴管内皮细胞Ki-67指数(0)相比较,差别均有统计学意义(P<0.05).胃癌癌周微淋巴管密度与肿瘤的分化程度、淋巴结转移以及淋巴管受累相关(P<0.05).胃癌癌周微淋巴管内皮细胞Ki-67指数仅与淋巴管受累相关(P<0.05).结论:胃癌组织中存在新生淋巴管,且主要存在于癌周,癌周淋巴管是发生淋巴结转移的主要途径.     

Cell transplantation therapy for a rat model of secondary lymphedema

Background

Although lymphedema is a progressive and lifelong condition, substantial advances in therapeutic intervention are limited. The development of a novel therapy for lymphedema is urgent for those patients suffering from it. The aim of this study was to investigate the usefulness of a new cell transplantation therapy in the rat tail model of secondary lymphedema.

Materials and methods

We prepared two cell sources, human dermal microvascular endothelial cells (HDMECs) and lymphatic endothelial cells (LECs), which were collected from the resected normal dermis of patients with breast cancer. After the animal model of secondary lymphedema of the nude rats' tails was established, phosphate-buffered saline, purified LECs, or unpurified HDMECs were injected in the rats' tails five times for more than 14 d. The evaluations were performed by measuring the circumference, fluorescence lymphography, and histologic analysis of the rats' tails between each group.

Results

The isolated cells by the simple immunomagnetic sorting from HDMECs were positive for a pan-endothelial marker (CD31) and lymphatic-specific markers (podoplanin, lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1], and prospero homebox 1 [Prox-1]), and were considered to be LECs. In the cell transplantation group, which was injected with human LECs, the circumference, lymphatic flow, and thickness of the skin of the rat tail became thinner than the groups injected with unpurified HDMECs or phosphate-buffered saline. Immunohistochemistry of the rat tails showed that the number of own lymphatic vessels was increased in the purified LEC transplantation group compared with the other groups. Furthermore, in the LEC transplantation group, some vessels were immunopositive for human-podoplanin or -LYVE-1 and the areas adjacent to the vessels were rat-podoplanin or -LYVE-1 immunopositive.

Conclusions

Our findings indicate that cell transplantation therapy using human LECs improved the secondary lymphedema in the nude rat tail. This therapeutic strategy may merit clinical investigation in patients with lymphedema.     

心脏淋巴管的分布、功能和病理意义

心脏淋巴引流对于维持心肌内微环境稳定起着重要作用。在心肌梗死和心力衰竭等疾病,淋巴管损伤或功能障碍可引起心脏淋巴水肿,从而导致心肌纤维化,加重炎症反应和严重影响心功能。近年来,人们对于心脏淋巴水肿与心脏疾病的相互关系以及心脏淋巴管新生的生理、病理意义的研究非常重视。靶向刺激心脏淋巴管新生可作为缓解心脏淋巴水肿的可靠疗法。然而,刺激心脏淋巴管新生的优化策略有待深入研究。本文主要综述心脏淋巴管的分布和功能特点,讨论心脏淋巴水肿的病理作用,心脏淋巴管新生的机制以及促进心脏淋巴管新生的措施和临床意义。     

血管生成素-2与大肠癌淋巴管密度的相关性研究

目的：通过检测大肠癌标本的血管生成素-2（ANG-2）mRNA的表达,并进行微淋巴管计数,探讨ANG-2 mRNA的表达与微淋巴管密度（MLVD）的关系,推断其在大肠癌微淋巴管生成中的作用。方法应用RT-PCR检测癌组织、良性病变及手术切缘正常组织的ANG-2 mRNA的表达;应用免疫组化技术,以LYVE-1标记微淋巴管并计数。结果ANG-2 mRNA在癌组织内阳性表达64.0%,良性病变内阳性表达8.3%,手术切缘正常组织内阳性表达7.5%。ANG-2 mRNA表达阳性的癌旁MLVD比阴性高（P<0.01）,且ANG-2 mRNA的表达水平与癌旁MLVD存在相关性（P<0.05,r=0.493）。癌旁MLVD及手术切缘正常组织MLVD比癌组织高（P<0.01）,且癌旁MLVD比手术切缘正常组织MLVD高（P<0.01）。Duke's A、B期的癌旁MLVD较C、D期低;有淋巴结转移的癌旁MLVD比无淋巴结转移的癌旁MLVD高（P<0.05）。结论ANG-2 mRNA在大肠腺癌组织内有高表达并与癌旁MLVD的表达水平密切相关。