中华泌尿外科杂志
中華泌尿外科雜誌
중화비뇨외과잡지
Chinese Journal of Urology
2015年
11期
860-864
,共5页
庞建智%闫三华%杨晓峰%苏西西%罗芳%李宏州%罗俊茜%张帆%张小雷
龐建智%閆三華%楊曉峰%囌西西%囉芳%李宏州%囉俊茜%張帆%張小雷
방건지%염삼화%양효봉%소서서%라방%리굉주%라준천%장범%장소뢰
膀胱癌%肿瘤导向肽%光学分子影像%靶向性
膀胱癌%腫瘤導嚮肽%光學分子影像%靶嚮性
방광암%종류도향태%광학분자영상%파향성
Bladder cancer%Tumor homing peptide%Optical molecular imaging%Targeting property
目的 验证导向肽NYZL1与膀胱癌BIU-87细胞株及其移植瘤结合的靶向特异性.方法 2014年4-12月固相合成导向肽NYZL1及阴性对照肽svNYZL1,采用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记,制备实验组荧光分子探针FITC-氨基己酸-CSSPIGRHC(FITC-NYZL1)和对照组荧光分子探针FITC-氨基己酸-CRIGSPHSC(FITC-svNYZL1).向膀胱癌BIU-87细胞株悬液中分别加入两种探针孵育,用流式细胞仪检测细胞平均荧光强度值,明确探针的亲和作用.将膀胱癌BIU-87细胞株爬片,分别加入两种探针孵育,用激光共聚焦显微镜(laser scanning confocal microscope,LSCM)观察探针与细胞结合的荧光图像,明确其亲和作用的特异性.选取16只SPF级BALB/c-nu雄性裸鼠.3～4周龄.体质量15 ～ 18 g.成功构建膀胱癌BIU-87细胞荷瘤裸鼠模型.采用完全随机分组法分为2组,每组8只,经尾静脉分别注入两种探针.4h后,实验组和对照组各取5只,剖取肿瘤组织、心、肝、脾、肺、肾、膀胱和胆囊,光学分子成像系统观察肿瘤及各组织的荧光图像;24 h后,实验组和对照组各取3只荷瘤裸鼠,剖取肿瘤组织、心、肝、脾、肺、肾和膀胱,制作冷冻切片,LSCM下观察肿瘤及各组织的荧光图像.结果 流式细胞仪检查实验组和对照组的平均荧光强度值分别为50.34±3.63和17.72±1.57,差异有统计学意义(P＜0.05).LSCM观察实验组细胞可见明显的绿色荧光,对照组细胞未见明显的绿色荧光.体内研究发现,光学分子成像系统观察肿瘤组织、胆囊及膀胱可见明显荧光,其余器官如心、肝、脾、肺和肾等未见明显荧光.LSCM观察下实验组的肿瘤组织中可见明显绿色荧光,而实验组其他器官(心、肝、脾、肺、肾和膀胱)和对照组肿瘤组织及器官中未见绿色荧光.结论 膀胱肿瘤导向肽NYZL1能够与膀胱癌BIU-87细胞株及其移植瘤靶向性结合,并可携带荧光素标记肿瘤组织.
目的 驗證導嚮肽NYZL1與膀胱癌BIU-87細胞株及其移植瘤結閤的靶嚮特異性.方法 2014年4-12月固相閤成導嚮肽NYZL1及陰性對照肽svNYZL1,採用異硫氰痠熒光素(fluorescein isothiocyanate,FITC)標記,製備實驗組熒光分子探針FITC-氨基己痠-CSSPIGRHC(FITC-NYZL1)和對照組熒光分子探針FITC-氨基己痠-CRIGSPHSC(FITC-svNYZL1).嚮膀胱癌BIU-87細胞株懸液中分彆加入兩種探針孵育,用流式細胞儀檢測細胞平均熒光彊度值,明確探針的親和作用.將膀胱癌BIU-87細胞株爬片,分彆加入兩種探針孵育,用激光共聚焦顯微鏡(laser scanning confocal microscope,LSCM)觀察探針與細胞結閤的熒光圖像,明確其親和作用的特異性.選取16隻SPF級BALB/c-nu雄性裸鼠.3～4週齡.體質量15 ～ 18 g.成功構建膀胱癌BIU-87細胞荷瘤裸鼠模型.採用完全隨機分組法分為2組,每組8隻,經尾靜脈分彆註入兩種探針.4h後,實驗組和對照組各取5隻,剖取腫瘤組織、心、肝、脾、肺、腎、膀胱和膽囊,光學分子成像繫統觀察腫瘤及各組織的熒光圖像;24 h後,實驗組和對照組各取3隻荷瘤裸鼠,剖取腫瘤組織、心、肝、脾、肺、腎和膀胱,製作冷凍切片,LSCM下觀察腫瘤及各組織的熒光圖像.結果 流式細胞儀檢查實驗組和對照組的平均熒光彊度值分彆為50.34±3.63和17.72±1.57,差異有統計學意義(P＜0.05).LSCM觀察實驗組細胞可見明顯的綠色熒光,對照組細胞未見明顯的綠色熒光.體內研究髮現,光學分子成像繫統觀察腫瘤組織、膽囊及膀胱可見明顯熒光,其餘器官如心、肝、脾、肺和腎等未見明顯熒光.LSCM觀察下實驗組的腫瘤組織中可見明顯綠色熒光,而實驗組其他器官(心、肝、脾、肺、腎和膀胱)和對照組腫瘤組織及器官中未見綠色熒光.結論 膀胱腫瘤導嚮肽NYZL1能夠與膀胱癌BIU-87細胞株及其移植瘤靶嚮性結閤,併可攜帶熒光素標記腫瘤組織.
목적 험증도향태NYZL1여방광암BIU-87세포주급기이식류결합적파향특이성.방법 2014년4-12월고상합성도향태NYZL1급음성대조태svNYZL1,채용이류청산형광소(fluorescein isothiocyanate,FITC)표기,제비실험조형광분자탐침FITC-안기기산-CSSPIGRHC(FITC-NYZL1)화대조조형광분자탐침FITC-안기기산-CRIGSPHSC(FITC-svNYZL1).향방광암BIU-87세포주현액중분별가입량충탐침부육,용류식세포의검측세포평균형광강도치,명학탐침적친화작용.장방광암BIU-87세포주파편,분별가입량충탐침부육,용격광공취초현미경(laser scanning confocal microscope,LSCM)관찰탐침여세포결합적형광도상,명학기친화작용적특이성.선취16지SPF급BALB/c-nu웅성라서.3～4주령.체질량15 ～ 18 g.성공구건방광암BIU-87세포하류라서모형.채용완전수궤분조법분위2조,매조8지,경미정맥분별주입량충탐침.4h후,실험조화대조조각취5지,부취종류조직、심、간、비、폐、신、방광화담낭,광학분자성상계통관찰종류급각조직적형광도상;24 h후,실험조화대조조각취3지하류라서,부취종류조직、심、간、비、폐、신화방광,제작냉동절편,LSCM하관찰종류급각조직적형광도상.결과 류식세포의검사실험조화대조조적평균형광강도치분별위50.34±3.63화17.72±1.57,차이유통계학의의(P＜0.05).LSCM관찰실험조세포가견명현적록색형광,대조조세포미견명현적록색형광.체내연구발현,광학분자성상계통관찰종류조직、담낭급방광가견명현형광,기여기관여심、간、비、폐화신등미견명현형광.LSCM관찰하실험조적종류조직중가견명현록색형광,이실험조기타기관(심、간、비、폐、신화방광)화대조조종류조직급기관중미견록색형광.결론 방광종류도향태NYZL1능구여방광암BIU-87세포주급기이식류파향성결합,병가휴대형광소표기종류조직.
Objective To determine the targeting property of tumor homing peptide NYZL1 and bladder cancer cell line BIU-87 both in vivo and in vitro.Methods From April 2014 to December 2014,peptide NYZL1 and its control peptide svNYZL1 were synthesized by solid-phase peptide synthesis from Fmoc chemistry.FITC-aminocaproic acid-CSSPIGRHC (FITC-NYZL1) and its control probe FITC-aminocaproic acid-CRIGSPHSC (FITC-svNYZL1) were also synthesized by labeled fluorescein isothiocyanate.Probes were added into BIU-87 bladder cancer cell suspensions and incubated for appropriate time.We detected fluorescence intensity value using Flow cytometry (FCM) to determine the affinity of probes.BIU-87 cells were incubated with experimental and control probes.The image of cells was observed by laser scanning confocal microscopy (LSCM) which could illustrate the specificity of affinity by detecting the fluorescent images of probes and cells.Sixteen BIU-87 bladder cancer xenograft models were randomly divided into 2 groups (n =8 each) and then injected experimental and control probes intravenously.After 4 hours of circulating, we excised tumors and control organs, such as heart, liver, spleen, lung, kidney, bladder and cholecyst from 5 models of each group to analyze targeting ability of probes by using optical molecular imaging system.After 24 hours, 3 rude mice bearing tumor were executed from experimental and control group each.Their frozen sections of tumors and control organs were prepared and examined for fluorescence by LSCM, in order to explore the targeting ability and specificity of probes by detecting the fluorescent images of tumors and control organs.Results Flow cytometry (FCM) study showed that the average fluorescence intensity values in experimental and control group were 50.34 ± 3.63 and 17.72 ± 1.57,respectively.According to LSCM images, green fluorescence can only been observed in experimental group.The in vivo study showed that tumor tissues, bladder and cholecyst presented green fluorescence through the optical molecular imaging system, while control organs, such as heart, liver, spleen, lung and kidney presented no fluorescence.LSCM study showed that green fluorescence can only been observed in tumor tissues in experimental group, while other organs from experimental and control group were negative.Conclusion The bladder cancer tumor homing peptide NYZL1 can specifically bound to BIU-67 bladder cancer cells.It can also mark xenografts and tumor tissues with fluorescein.
