小鼠原位膀胱肿瘤模型和肿瘤检测系统
Summary
这个协议描述在雌性C57BL / 6J小鼠和肿瘤生长的监测小鼠原位膀胱肿瘤的生成。
Abstract
这个协议描述膀胱肿瘤的生成中使用鼠膀胱癌细胞系MB49，其已被修饰以分泌人类前列腺特异性抗原（PSA）的雌性C57BL / 6J小鼠，对于肿瘤植入的确认的步骤。简言之，老鼠是使用注射药物麻醉，由背侧位置埋下了伏笔。尿从膀胱腾空和聚-L-赖氨酸（PLL）的50微升缓慢以10微升/ 20秒的速率用24克IV导管灌输。它是由塞住导管留在膀胱20分钟。导管被移除和PLL是通过在膀胱温和的压力腾空。这之后是鼠膀胱癌细胞系以10微升/ 20秒的速度（1×10 5个细胞/50μl）滴注。导管被塞住，以防止过早疏散。 1小时后，将小鼠恢复与逆转药物，和膀胱腾空。缓慢滴注速度是很重要的，因为它减少膀胱输尿管回流，这可能会导致肿瘤在上尿路和在肾脏发生。细胞系应该很好再悬浮，以减少细胞的聚集，因为这可能在植入后导致不均匀肿瘤大小。
这种技术诱导效率高的肿瘤。肿瘤生长是由泌尿系PSA分泌监控。 PSA标志的监测比超声波或荧光成像用于检测肿瘤的膀胱中存在的更可靠。如果不及时治疗4周 – 小鼠肿瘤约3一般达到最大大小负面影响健康。通过监测肿瘤的生长，有可能区分那些未成功地与肿瘤植入被治愈的小鼠。只有终点分析，后者可被误认为已通过疗法治愈。
Introduction
该方法的目标是产生鼠正位膀胱肿瘤，并尽可能准确地监测植入的肿瘤，所以没有肿瘤植入的小鼠都没有想到在终点分析已经固化。总体上，所示的方法将减少大量小鼠实验分析的需要，并确保在确定治疗结果更高的精度。
对于癌症的原位模型的发展是重要的，因为植入肿瘤细胞皮下不概括的临床疾病的环境或启用的治疗策略的开发。膀胱的结构允许膀胱癌治疗的滴注直接进入以最小的全身效应膀胱。因此，概括这种环境的动物模型，例如原位模型，是很重要的，以评估新疗法。从任何实验装置得出的结论是dependen在该模型的局限性吨。
几种技术已经开发了用于在小鼠中生产原位膀胱肿瘤。这些依赖于损伤膀胱的糖胺聚糖层，使肿瘤细胞被植入。所使用的技术包括电烙，这导致在膀胱壁损伤的单点，在膀胱1,2-导致肿瘤发展在一个地点。然而，使用电灼肿瘤植入的成功率是取决于运营商从10变 – 90％，并且它包括危险，即膀胱壁将被删截，从而导致肿瘤在腹腔显影。化学烧灼是用硝酸银，这损害了膀胱壁3进行。同样，酸已被用来损伤膀胱壁4。胰蛋白酶也被用来损伤膀胱以及5。这些方法可能会导致在一个以上的肿瘤在膀胱的发展。此外，存在严重损坏的膀胱的危险，如果化学品留在与膀胱壁接触时间过长。通过Ninalga 等人开发的方法。使用带正电荷的聚-L-赖氨酸（PLL）6分子涂覆膀胱壁;这使得带负电荷的肿瘤细胞粘到膀胱的糖胺聚糖层。这种方法通常导致在一个以上的肿瘤在膀胱显影，但肿瘤植入是在80 – 100％的4,7。从技术上讲，它也是以执行的最简单的过程。以保证该开发肿瘤即使在大小相当，它是重要的肿瘤细胞中不植入前大团块分组。
为了评估治疗功效，最好是具有相当类似大小的肿瘤对小鼠进行这项研究。因此，可以在植入后不久量化肿瘤大小良好的检测系统是重要的。几种策略有蜜蜂用于n评估肿瘤。这些包括磁共振成像（MRI）8-10，荧光11，生物发光12,13，超声14，和酶联免疫吸附试验（ELISA）15,16。而MRI和超声不需要的肿瘤细胞中的修改，有必要对敏感设备和造影剂用于MRI。的荧光 – ，luminescence-，和基于ELISA测定所需要的肿瘤细胞的修饰来表达，可以通过这些方法来检测标志物蛋白。为发光，需要用于检测荧光素酶活性的基板;因此，有一个附加的步骤，增加成本。这两个发光和荧光需要专门的设备。为了产生荧光，绿荧光蛋白（GFP）的环化，它是由分子氧催化的，是必需的。因此，GFP表达可能会因访问氧肿瘤块内的变量，使之成为一个相当可靠的指标<sup> 17。鼠膀胱癌细胞系MB49的修饰分泌人类前列腺特异性抗原（PSA）的15,16作为替代标志物是另一种策略。这些标志物还提供确认肿瘤存在在实验结束时，使它们的替代免疫组织化学的替代手段。这项研究报告原位肿瘤植入的PLL方法，并提出肿瘤检测系统，即ELISA，荧光和超声成像的比较。
Protocol
Representative Results
Discussion
在该协议中最关键的步骤是：1）成功地保持了细胞系的致瘤性; 2）确保小鼠肿瘤细胞植入前测PSA的分泌; 3）产生用于植入的单细胞悬浮液，从而减少肿瘤大小的变化;和4）灌输细胞以缓慢的速度，以防止膀胱输尿管回流，导致在肾肿瘤细胞植入。
体外延长传代后，MB49 / MB49 – PSA细胞可失去其致瘤性，这表现在它们不能在小鼠中产生肿瘤。这可能是一个混杂因素，以是?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.
Materials
| MB49-PSA cells | N/A | N/A | ref (Wu QH, 2004) |
| RPMI 1640 media | HyClone | SH30027.01 | |
| Dulbecco's Modified Eagle's Medium (DMEM) media | Biowest | L0102 | |
| Fetal Bovine Serum (FBS) South American | Biowest | S1810 | |
| Fetal Bovine Serum (FBS) South American, Premium | Biowest | S181B | |
| Fetal Bovine Serum (FBS) | HyClone | SH30088.03 | |
| L-glutamine | Biowest | X0550 | |
| Penicillin-Streptomycin | Biowest | L0022 | |
| Hygromycin B | Invitrogen | 10687-010 | |
| free PSA (Human) ELISA kit | Abnova | KA0209 | |
| TRIzol Reagent for RNA extraction | Ambion | 15596026 | |
| High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor | Applied Biosystems | 4374967 | |
| TaqMan Universal PCR Master Mix | Applied Biosystems | 4304437 | |
| TaqMan Gene Expression Assay – Mouse Actb | Applied Biosystems | 4331182 | Mm00607939_s1 |
| TaqMan Gene Expression Assay – Human KLK3 | Applied Biosystems | 4331182 | Hs00426859_g1 |
| C57BL/6J female mice | In Vivos | 4-6 wk old | |
| Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine) | Local pharmacy | ||
| Reversal drug (1mg/kg Atipamezole) | Local pharmacy | ||
| Ear punch | Electron Microscopy Sciences | 72893-01 | |
| Hartmann's solution or Compound sodium lactate | B Braun | ||
| Ophthalmic ointment – Duratears sterile ocular lubricant ointment | Alcon | ||
| Heat pack – HotHands handwarmers | Heatmax Inc | ||
| Introcan Certo IV catheter | B Braun | 4251300 | 24G x 3/4″ |
| Aquagel Lubricating jelly | Local pharmacy | ||
| Poly-L-lysine solution, 0.01%, | Sigma | P4707 | |
| cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | 4693159001 | |
| Quantichrom Creatinine Assay Kit | BioAssay Systems | DICT-50 | |
| Fluorescent dye – VivoTrack 680 | Perkin Elmer | NEV12000 | |
| RNAlater-ICE Frozen Tissue Transition Solution | Ambion | 4427575 | |
| Name | Company | Catalog number | Comments |
| Equipment and Software | |||
| 7500 Realtime PCR System | Applied Biosystems | ||
| 7500 Software v2.3 | Applied Biosystems | ||
| Metabolic Cage | Tecniplast | Vertical type rack for 12 cages | |
| BD FACSCanto I system | BD Biosciences | ||
| BD FACSDiva software v7 | BD Biosciences | ||
| IVIS SpectrumCT in vivo imaging system | Caliper Life Sciences | ||
| Living Image Software v3.1 | Caliper Life Sciences | ||
| Vevo 2100 imaging system | VisualSonics |
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