亚甲蓝近红外荧光在大鼠口腔前哨淋巴结示踪中的应用

doi:10.19723/j.issn.1671-167X.2023.04.019

摘要/Abstract

摘要：

目的: 探索亚甲蓝用于近红外荧光成像的浓度范围及穿透深度，明确亚甲蓝近红外荧光在大鼠口腔淋巴引流及前哨淋巴结定位中的作用，为口腔癌前哨淋巴结的潜在临床研究和应用奠定基础。方法: 0.9%(质量分数)生理盐水稀释10%(质量分数)亚甲蓝注射液，配置29种不同浓度亚甲蓝溶液，使用近红外荧光成像仪测定亚甲蓝近红外荧光成像的浓度范围；制备不同厚度(1、2、3、4、5 mm)猪皮覆盖于亚甲蓝溶液，测定亚甲蓝近红外荧光的最大穿透深度；将0.2 mL亚甲蓝溶液注射到大鼠一侧舌侧缘黏膜下0.5 cm，用近红外荧光成像仪连续监测3 h，经皮观察到的第一个近红外荧光热点被认为是前哨淋巴结，标记后将大鼠处死；解剖头颈部，再次行近红外荧光成像，观察荧光组织是否与体外标记荧光热点一致；切除荧光组织行病理学检查以确认淋巴组织存在。结果: 除空白对照组未监测到荧光信号外，亚甲蓝近红外荧光强度随着其溶液浓度的减小呈现先增强后减弱的变化趋势，当亚甲蓝溶液浓度稀释到皮摩尔级时，依然可以探测到荧光信号。亚甲蓝近红外荧光的最大穿透深度为4 mm。亚甲蓝近红外荧光可以在大鼠口腔淋巴引流及前哨淋巴结中定位，荧光可持续3 h以上。筛选出在大鼠中用近红外荧光定位前哨淋巴结的亚甲蓝溶液浓度为3.34 mmol/L、6.68 mmol/L、13.37 mmol/L、26.74 mmol/L。结论: 亚甲蓝近红外荧光具有一定的穿透能力，能够经皮示踪大鼠口腔前哨淋巴结，有望进一步应用于口腔癌前哨淋巴结的研究中。

关键词: 口腔肿瘤, 前哨淋巴结, 亚甲蓝, 谱学，近红外线, 荧光

Abstract:

Objective: To explore the concentration range and penetration depth of methylene blue near-infrared fluorescence imaging, and to clarify the role of methylene blue in oral lymphatic drainage and sentinel lymph node localization, so as to lay a foundation for the potential research and application of sentinel lymph node in oral cancer. Methods: 10% (mass fraction) methylene blue injection was diluted into 29 different concentrations with 0.9% (mass fraction) normal saline, and the concentration range of methylene blue near-infrared fluorescence imaging was determined by near-infrared fluorescence imager. The maximum penetration depth of methylene blue near-infrared fluorescence was determined by covering pigskin with different thicknesses (1, 2, 3, 4 and 5 mm) in methylene blue solution. 0.2 mL methylene blue solution was injected into the submucosal 0.5 cm at the lateral margin of tongue on one side of the rats. The near-infrared fluorescence imager was used for continuously monitoring for 3 hours. The first near-infrared fluorescence hotspot was identified as sentinel lymph node and labeled by percutaneous observation. The rats were then sacrificed and dissected in the head and neck. Near-infrared fluorescence imaging was performed again to observe whether the fluorescent tissue was consistent with the labeled fluorescent hotspot in vitro, and the presence of lymphoid tissue was confirmed by pathological examination after resection. Results: Except that no fluorescence signals were detected in the blank control groups, the fluorescence intensity of methylene blue increased first and then decreased with its solution concentration decreased. When the concentration of methylene blue was diluted to the picomole level, the fluorescence signal could still be detected. The maximum penetration depth of methylene blue fluorescence was 4 mm. Methylene blue near-infrared fluorescence could be localized in oral lymphatic drainage and sentinel lymph node. The fluorescence was sustained for more than 3 hours after methylene blue injection. Methylene blue solution concentrations of 3.34 mmol/L, 6.68 mmol/L, 13.37 mmol/L and 26.74 mmol/L were selected in the rats to map sentinel lymph node by near-infrared fluorescence. Conclusion: Methylene blue near-infrared fluorescence has a certain penetrating ability and can transcuta-neously map the sentinel lymph node and their associated lymphatic vessels in rats, which is expected to be further applied in the study of sentinel lymph node in oral cancer.

Key words: Mouth neoplasms, Sentinel lymph node, Methylene blue, Spectroscopy, near-infrared, Fluorescence

中图分类号:

R739.8

吴雨筱,康一帆,毛茜潆,李梓萌,单小峰,蔡志刚. 亚甲蓝近红外荧光在大鼠口腔前哨淋巴结示踪中的应用[J]. 北京大学学报（医学版）, 2023, 55(4): 684-688.

Yu-xiao WU,Yi-fan KANG,Qian-ying MAO,Zi-meng LI,Xiao-feng SHAN,Zhi-gang CAI. Application of methylene blue near-infrared fluorescence imaging for oral sentinel lymph node mapping in rats[J]. Journal of Peking University (Health Sciences), 2023, 55(4): 684-688.

图/表 2

图1

图2

参考文献 28

1	D'CruzAK,VaishR,KapreY,et al.Elective versus therapeutic neck dissection in node-negative oral cancer[J].N Engl J Med,2015,373(6):521-529. doi: 10.1056/NEJMoa1506007
2	MelkaneAE,MamelleG,WyciskG,et al.Sentinel node biopsy in early oral squamous cell carcinomas: A 10-year experience[J].Laryngoscope,2012,122(8):1782-1788. doi: 10.1002/lary.23383
3	ThompsonCF,StJohnMA,LawsonG,et al.Diagnostic value of sentinel lymph node biopsy in head and neck cancer: A meta-analysis[J].Eur Arch Otorhinolaryngol,2013,270(7):2115-2122. doi: 10.1007/s00405-012-2320-0
4	HonsoulK,SangKL,YooMK,et al.Fluorescent iodized emulsion for Pre- and intraoperative sentinel lymph node imaging: Validation in a preclinical model[J].Radiology,2015,275(1):196-204. doi: 10.1148/radiol.14141159
5	CabanasRM.An approach for the treatment of penile carcinoma[J].Cancer,1977,39(2):456-466. doi: 10.1002/1097-0142(197702)39:2<456::AID-CNCR2820390214>3.0.CO;2-I
6	PfisterDG,SpencerS,AdelsteinD,et al.Head and neck can-cers, version 2.2020, NCCN clinical practice guidelines in onco-logy[J].Natl Compr Canc Netw,2020,18(7):873-898. doi: 10.6004/jnccn.2020.0031
7	KhallafE,AbdoonM,LotfyA,et al.Methylene blue 1% as a sensitive and safe alternative for sentinel lymph node biopsy in early stage breast cancer: Results of a large pilot study[J].Breast J,2019,5(25):1017-1019.
8	GyorkiDE,BarbourA,HanikeriM,et al.When is a sentinel node biopsy indicated for patients with primary melanoma? An update of the 'Australian guidelines for the management of cutaneous melanoma'[J].Australas J Dermatol,2017,58(4):274-277. doi: 10.1111/ajd.12662
9	Di StefanoAB,AcquavivaG,GarozzoG,et al.Lymph node mapping and sentinel node detection in patients with cervical carcinoma: A 2-year experience[J].Gynecol Oncol,2005,99(3):671-679. doi: 10.1016/j.ygyno.2005.07.115
10	HuangL,DuPY,ChenL,et al.Development of a near-infrared fluorescence imaging system based on fluorescence properties of methylene blue[J].J South Med Univ,2018,38(4):414-420.
11	MatsuiA,TanakaE,ChoiHS,et al.Real-time, near-infrared, fluorescence-guided identification of the ureters using methylene blue[J].Surgery,2010,148(1):78-86. doi: 10.1016/j.surg.2009.12.003
12	Al-TaherM,van den BosJ,ScholsRM,et al.Fluorescence ureteral visualization in human laparoscopic colorectal surgery using methylene blue[J].J Laparoendosc Adv Surg Tech A,2016,26(11):870-875. doi: 10.1089/lap.2016.0264
13	HoogstinsC,BurggraafJJ,KollerM,et al.Setting standards for reporting and quantification in fluorescence-guided surgery[J].Mol Imaging Biol,2019,21(1):11-18. doi: 10.1007/s11307-018-1220-0
14	D'AngeloDD,DicksonWD,PetrelliNJ,et al.Sentinel lymph node biopsy in breast cancer: A history and current clinical recommendations[J].Surg Oncol,2012,21(3):196-200. doi: 10.1016/j.suronc.2011.12.005
15	StoeckliSJ,PfaltzM,RossGL,et al.The second international conference on sentinel node biopsy in mucosal head and neck cancer[J].Ann Surg Oncol,2005,12(11):919-924. doi: 10.1245/ASO.2005.11.024
16	AlkureishiLW,RossGL,ShoaibT,et al.Sentinel node biopsy in head and neck squamous cell cancer: 5-year follow-up of a European multicenter trial[J].Ann Surg Oncol,2010,17(9):2459-2464. doi: 10.1245/s10434-010-1111-3
17	MehmetE,MetinK,LeventK,et al.A prospective comparative study to assess the contribution of radioisotope tracer method to dye-only method in the detection of sentinel lymph node in breast cancer[J].BMC Surgery,2013,13(13):1471-1477.
18	LuY,WeiJY,YaoDS,et al.Application of carbon nanoparticles in laparoscopic sentinel lymph node detection in patients with early-stage cervical cancer[J].PLoS One,2017,12(9):e0183834. doi: 10.1371/journal.pone.0183834
19	van der VorstJR,SchaafsmaBE,VerbeekFP,et al.Near-infrared fluorescence sentinel lymph node mapping of the oral cavity in head and neck cancer patients[J].Oral Oncol,2013,49(1):15-19. doi: 10.1016/j.oraloncology.2012.07.017
20	ToshiyukiK,TakuyaI,MitsuharuM,et al.Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer[J].Breast Cancer,2005,12(3):211-215. doi: 10.2325/jbcs.12.211
21	PengH,WangSJ,NiuX,et al.Sentinel node biopsy using indocyanine green in oral/oropharyngeal cancer[J].World J Surg Oncol,2015,13(1):278-284. doi: 10.1186/s12957-015-0691-6
22	BredellMG.Sentinel lymph node mapping by indocyanin green fluorescence imaging in oropharyngeal cancer: Preliminary expe-rience[J].Head Neck Oncol,2010,2(2):31-38.
23	TakeuchiM,SugieT,AbdelazeemK,et al.Lymphatic mapping with fluorescence navigation using indocyanine green and axillary surgery in patients with primary breast cancer[J].Breast J,2012,18(6):535-541. doi: 10.1111/tbj.12004
24	RamamurthyR,KottayasamySR,ShanmugamS,et al.A prospective study on sentinel lymph node biopsy in early oral cancers using methylene blue dye alone[J].Indian J Surg Oncol,2014,3(5):178-183.
25	MartinGR,PerezHM,SanchezCA,et al.Methylene blue staining and probing for fistula resection: Application in a case of bilateral congenital preauricular fistulas[J].Int J Oral Maxillofac Surg,2002,31(4):439-441. doi: 10.1054/ijom.2001.0062
26	MertesPM,MalinovskyJM,MoutonFC,et al.Anaphylaxis to dyes during the perioperative period: Reports of 14 clinical cases[J].J Allergy Clin Immunol,2008,122(2):348-352. doi: 10.1016/j.jaci.2008.04.040
27	SylvainG,HakSC,JohnVF,et al.Image-guided surgery using invisible near-infrared light: Fundamentals of clinical translation[J].Mol Imaging,2010,9(5):237-255.
28	ChuM,WanY.Sentinel lymph node mapping using near-infrared fluorescent methylene blue[J].J Biosci Bioeng,2009,107(4):455-459. doi: 10.1016/j.jbiosc.2008.11.011

相关文章 15

[1]	王磊,金香淑,董慧君,欧国敏,赖鑫源,庄辉,李彤,向宽辉. 基于COL1A1启动子和增强型绿色荧光蛋白基因建立人肝星状细胞活化的细胞模型[J]. 北京大学学报（医学版）, 2023, 55(5): 876-885.
[2]	谢尚,蔡志刚,单小峰. 全外显子测序及相关指标在口腔鳞状细胞癌精准治疗中的应用价值[J]. 北京大学学报（医学版）, 2023, 55(4): 697-701.
[3]	王微,李鑫,柳萍,董颖. 荧光原位杂交检测MDM2和DDIT3基因信号改变在诊断脂肪肉瘤中的价值[J]. 北京大学学报（医学版）, 2023, 55(2): 228-233.
[4]	杜强,洪锴,潘伯臣. 两种检测男性生殖道沙眼衣原体和解脲支原体方法的对比[J]. 北京大学学报（医学版）, 2021, 53(4): 785-788.
[5]	池彦廷,张延平,张秋露,刘翠苓,李斌斌. 唾液腺干燥综合征继发黏膜相关淋巴组织淋巴瘤的临床病理分析[J]. 北京大学学报（医学版）, 2021, 53(1): 40-45.
[6]	张树栋,洪鹏,王滨帅,邓绍晖,张帆,陶立元,曹财广,胡振华,马潞林. 吲哚菁绿标记的荧光实时显影技术在腹腔镜肾部分切除术中的应用[J]. 北京大学学报（医学版）, 2020, 52(4): 657-662.
[7]	周行红,黄莹,袁超,郑树国,张杰,张建国. 1 483名北京市居民对口腔癌及相关知识的认知情况调查[J]. 北京大学学报（医学版）, 2020, 52(2): 323-331.
[8]	徐韬,王佃灿,单小峰,蔡志刚. 不同部位口腔鳞癌的延迟诊断分析[J]. 北京大学学报（医学版）, 2019, 51(4): 748-752.
[9]	王嘉禹,赵美萍. 荧光分析法测定人体血液样品中脱嘌呤/脱嘧啶核酸内切酶1(APE1)的活性[J]. 北京大学学报（医学版）, 2019, 51(3): 487-492.
[10]	李聪,刘树铭,郑磊,黄明伟,石妍,吕晓鸣,张建国,张杰. 手术联合 ¹²⁵I粒子治疗口腔颌面部腺样囊性癌的疗效与相关预后因素分析[J]. 北京大学学报（医学版）, 2019, 51(1): 49-52.
[11]	刘婧寅，陈飞，葛严军，魏菱，潘韶霞，冯海兰. 选择性激光熔化种植体对早期骨矿化沉积率的影响[J]. 北京大学学报(医学版), 2018, 50(1): 117-122.
[12]	冯永良,范静慧,林宪娟,杨吉春,崔庆华,汤新景,徐国恒,耿彬. 荧光探针包被法测定血浆硫化氢浓度[J]. 北京大学学报(医学版), 2017, 49(6): 1060-1065.
[13]	王芳, 张琰琴, 丁洁, 俞礼霞. 多重竞争性荧光PCR检测X连锁Alport综合征大片段缺失突变[J]. 北京大学学报(医学版), 2017, 49(5): 760-767.
[14]	柳家园，彭翔，宁向辉，李腾，彭双鹤，王江宜，刘圣杰，丁义，蔡林，龚侃. 尿脱落细胞荧光原位杂交检查阳性在尿路上皮癌中的临床价值[J]. 北京大学学报(医学版), 2017, 49(4): 585-589.
[15]	陈志强，王莹，米贤军，陈昂，黄华勇，钟守军，邓文同，刘超凡，徐秀梅，代新珍. 聚羟基丙烯酸和Van-clear替代传统试剂在FISH法检测宫颈hTERC基因中的应用比较[J]. 北京大学学报(医学版), 2016, 48(2): 356-360.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed