이 작품은 Associazione Italiana의 당 라 Ricerca 술 Cancro, 대학의 이탈리아어 연구부, 인쇄용 프로젝트와 PROGETTI 디 Ricerca 디 아테네 오, 부여 N ° CPDA137871, Fondazione Cariplo에 의해 지원되었다, N은 MDB에 2011-0485을 ° 및 Finanziamento 지오반니에 의해 부여 가이아 Codolo에 Studiosi, 파도바 대학.

동물 처리를위한 모든 절차는 건강의 이탈리아 교육부 (DM 2,011분의 204-B)에 의해 승인되었습니다. 1. 동물 <ol><li> microisolation 필터를 개별적으로 환기 케이지 연령 8 주에 C57BL6 / J 여성 쥐를 성장. 참고 : 자신의 외부 URO-생식기 장치의 해부학 적 형태가 남성에 비해 카테터 쉽게 렌더링하기 때문에 여성이 바람직하다. </li></ol> 2. 세포 배양 <ol><li> 가습 된 5 % CO 2에서 37 ° C에서 10 %의 열 - 불 활성화 된 소 태아 혈청 및 50 μg의 / ㎖ 젠타 마이신으로 보충 된 RPMI 1640 배지에서 마우스 urothelial 암종 세포주 MB49을 유지한다. </li><li> 80 %의 합류에 T-75 플라스크에 MB49 세포를 성장. 를 Trypsinize 0.5 × 105에서 재현 탁 - 마우스에 이식하기 전에 PBS 150 μL 당 0.5 × 10 6 세포. </li></ol> 3. 방광 내 종양 임플란트 <ol><li> (각각 33 ㎎ / ㎏ 및 20 ㎎ / ㎏)의 마취제 zoletil xylor과의 혼합을 사​​용하여 마우스를 마취, 복강 내 주사. </li><li> 앙와위에서 동물을 놓고 스카치 테이프로 패드에 뒷다리를 해결. 앞발을 충분히 잘 표시하고 카테터의 삽입을 위해 용이하게 액세스 가능한 요도를 렌더링하기 위해 분리되어야한다. </li><li> 참고 : 카테터를 들어, 24 G 소아 정맥 카테터는 8-10주 된 쥐에 적합합니다. 카테터의 선택은 요도 벽과 카테터 사이의 액 누설을 방지하기 위해 매우 중요하다. 큰 구멍 카테터는 큰 이상 마우스를 사용해야합니다. </li><li> 작은 집게를 사용하여, 요도의 외부 부분 중 하나 가장자리를 끼와 마우스의 &quot;머리 끝&quot;으로 아주 조심스럽게 비틀. 이 작업은 요도를 노출합니다. </li><li> 45 °의 각도로 요도에서 후자를 카테터의 내부 바늘을 제거하고 삽입 약간 지향마우스의 &quot;꼬리 끝&quot;으로. 카테터의 입구를 강요하지 마십시오 그 내부에 미끄러까지 요도 저항의 경우에는, 단지 매우 부드럽게 카테터 트위스트. 카테터의 약 0.5-0.8 cm가 요도에있을 때, 신중 마우스의 꼬리에 카테터를 병렬 배치하고 완전히 삽입. </li><li> 카테터 삽입되어야하는 바늘의 1 mL의 주사기를 이용하여, 멸균 PBS의 200-300 μl를 주입하여 잔류 뇨로부터 방광을 씻어 방광에서 모든 액체를 흡인. </li><li> 참고 : PBS를 주입하면 방광이 채워하고 붓기가 쥐의 뒷다리 사이에 볼 수 있습니다; 이 카테터가 올바른지 확인한다. 대부분 카테터 주입량을 계산할 때 고려되어야 데드 볼륨을 갖는다. </li><li> 1 mL의 멸균 주사기를 사용하여, 0.1 N의 HCl 200 μl를 주입. 전체 벽을 레코딩하기 위해 방광을 입력합니다. 10 초 후, 모든 산을 제거하고 즉시 neutrali멸균 주사기와 0.1 N NaOH를 200 μl를 주입하여 ZE. 셀의 가장 높은 숫자 (0.5 × 106)의 주입시, 산성화 단계는 필요하지 않다. </li><li> 기음 모든 잔류 액 즉시 멸균 PBS 300 μL와 공동을 세척. </li><li> 방광 - (0.5 × 106의 범위는 0.5 × 10 5) 흡인 방광을 비우고, MB49 및 세포를 함유하는 PBS 150 μl를 주입. 세포의 누설을 방지하기 위해 카테터에 조립 주사기를 남겨. 이 스카치 테이프 패드를 고정하여 주사기를 고정하는 것이 좋다. </li><li> 1 시간 후, 부드럽게로부터 요도 카테터를 추출하고 깨어까지 백열등, 케이지에서 마우스를 배치 이것은 보통 치료 종료 후의 1 ~ 2 시간이 발생한다. </li></ol> HP-NAP 4. 방광 내 배달 <ol><li> 주 : 셀 점안 후 최소 3 일, 그것은 시작하는 것이 가능하다치료. </li><li> 단계 3.1-3.4에 설명 된대로 카테 테르를 꽂다. 이 단계에서, 그것은 HCl로 방광 벽을 구울 필요가 없습니다. </li><li> 1 ML의 주사기를 사용하여 멸균 PBS의 200 ~ 300 μl를 주입하여 잔류 소변에서 방광 세척과 방광의 모든 액체를 대기음. </li><li> 방광에 150 μL PBS 당 HP-NAP의 50 μg의를 주입한다. 단백질 용액의 누출을 방지하는 카테터에 부착 된 주사기를 남겨. 스카치 테이프로 패드에 고정하여 주사기를 고정합니다. </li><li> 그들이 깨어 때까지, 백열 램프에서 1 시간 후, 부드럽게 요도에서 카테터를 추출하고 새장에 마우스를 놓습니다. </li></ol> 5. 종양의 Explants 및 조직 학적 분석 <ol><li> 처리의 끝에서, 동물을 희생 누운 위치에 배치하고 스카치 테이프 패드를 고정 뒷다리. </li><li> 외과 용 메스를 사용하여, 피부를 통해 약 1.52 cm 긴 수직 절개를복막 바로 마우스의 &quot;헤드 엔드&quot;외부 생식기 위에서 시작. 방광 손상의 위험을 최소화하기 위해, 절삭 깊이에 특히주의. </li><li> 방광은 절단의 중앙 왼쪽에 배치되고 종양 어두운 핑크 / 바이올렛 질량으로 표시됩니다. 그 절제시, 조직 학적 새장에 전체 방광을 배치하고 파라핀에 삽입하기 전에 10 % 포르말린에 고정합니다. </li><li> 조직 학적 및 면역 조직 화학적 분석을 위해, 표준 마이크로톰을 이용하여 방광 (46 μm의 두께)의 시리얼 섹션을 준비합니다. </li><li> 헤 마톡 실린 / 에오신으로 섹션 얼룩과 종양 크기 (D XD는 2), 컴퓨터 보조 화상 분석기를 사용 단면적 당 괴사의 비율을 결정하는 계산을 진행. </li><li> 용기 카운트, immunoperoxidase 표준 기술을 이용하여, 내피 세포 마커 CD31 / PECAM에 대한 부분을 염색하여 진행. 섹션을 스캔가장 높은 혈관 밀도의 영역을 식별하기위한 저전력 (× 4)를 t. 이 지역 내에서 높은 전력 (20 ×)에서 다섯 별도의 임의의 필드에서 개별 미세 혈관을 계산합니다. </li></ol>

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

암 치료에서의 진보의 대부분은 임상 시험을 시작하기 전에 동물 모델에서 시험을 요구한다. 생체 내 종양 생물학을 연구 동물 모델을 활용 가능성이 다른 치료 적 접근 방식을 평가할 수있는 암 발병 조사 연구를위한 중요한 도구를 나타낸다. 동 소성 모델은 금본위 제 14 ~ 15을 유지, 모두 때문에 종양 모델을 설정하고 할 수 세포주의 거대한 양의가 자연적으로 발생하는 종양 ...

방광암 USA 1 거의 75,000 매년 새로운 사례로, 비뇨 생식 관의 가장 흔한 암이다. 재발의 높은 비율은 방광암 치료하는 비싼 암 중 하나를 만드는 평생 추적 관찰을 필요로한다. 고급 NMIBC의 황금 표준 치료는 BCG와 방광 면역 다음, 트랜스 요도 절제술이다. BCG의 항 종양 활성의 정확한 메카니즘은 완전히 밝혀지지 그대로지만, 그것은 T 헬퍼 (토륨) (1) 및 세포 독성 T (TC) 한 세포가 풍부한 세포 매개 면역 반응의 활성화에 중요한 것으로 받아 들여 이 요법의 성공. BCG는 NMIBC위한 선택의 처리는 남아 있다는 사실에도 불구하고, 치료에 반응하지 않는 환자의 높은 비율; 또한, 부작용의 수를 일으킬 수있다 : 처리 된 종양의 약 70 %의 근육 침습적 형태 ~ 15 %의 진행을 일정 시간 후 재발 및질병. BCG 치료와 관련된 다른 부작용은 배뇨 장애, 방광염, 신장 감염 3-6을 포함한다. 신규 한 치료 전략의 개발은 초기 시험 관내 평가 다음, 고려 전임상 모델을 사용한다; 이것은 누구의 미세 크게 치료에 자신의 개발 및 응답에 영향을 미칠 수있는 종양에 특히 관련이있다. 지난 10 년간, 우리는 HP-NAP, 헬리코박터 파일로리 균에 의해 생성 된 단백질의 면역 변조 활동의 여러 측면을 언급 한 초기 다형 핵 세포 (백혈구) (7)의 내피 부착을 촉진 할 수있는 것으로 확인. 구조적으로, HP-NAP는 공복 상태 (DPS) 가족 (8)에서 DNA를 보호하는 단백질에 속하고 dodecameric 쉘에 배치 (12)와 동일한 서브 유닛으로 구성되어 있습니다. 우리는 HP-NAP가 있음을 증명하고있다T의 분화를 구동 할 책임 강력한 면역 조절 활성을 갖는 수신자 같은 수용체 (TLR) 2 효능 제는 시험 관내 및 생체 내 9-10 모두에서, 한 Th1 표현형으로 림프구. 이 활동의 미덕에서, HP-NAP는 알레르기 성 천식 (11)의 쥐 모델에서 더 유리한받은 Th1 반응에 TH2 면역 반응을 리디렉션 할 수 있습니다. HP-NAP의받은 Th1 종속 항 종양 가능성을 평가하기 위해 BCG 투여의 영향을 평가하기위한 O&#39;Donnel 동료 (12)에 의해 수년 전에 개발 방광암의 마우스 모델을 이용했다. 이 프로토콜로, 우리는 HP-NAP는 방광암에 대한 강력한 항 종양 가능성을 가지고 및 HP-NAP 관리의 효능 (인터페론을 생산 모두받은 Th1과에 T 림프구의 종양 및 지역 림프절 내에서 상당한 축적을 평행을 입증 IFN)은 13 -γ </&gt; SUP. HP-NAP-처리 된 마우스로부터 분리 된 종양을 더 괴사 미처리 대조 미만 혈관 신생을 나타내었다. 본 보고서는 동물의 준비, 그들의 요도 카테터, 방광 점막 세포의 부착을 위해 필요한 화학적 연소 및 종양 세포의 주입을 자세하게, stepbystep으로 프로토콜을 제공한다. 또한 방광암의 치료를 위해 개발 된 임의의 치료제의 원형으로 간주 할 수있는 HP-NAP의 국소 투여를 기술한다. 제어 및 HP-NAP 처리 된 동물로부터 분리 된 종양을 비교하여 얻은 증거는 단지 HP-NAP 방광 암 면역 요법을위한 좋은 후보뿐만 아니라 셋업 실험의 전반적인 효율성이 될 수 있다는 사실을 강조한다.

<table><tbody><tr><td colspan="4">Materials</td></tr><tr><td>C57BL/6J female mice</td><td>Harlan Italy (Udine, Italy)</td><td></td><td></td></tr><tr><td>MB49 Cells</td><td></td><td></td><td>Obtained from Prof. O&#039;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td></tr><tr><td>RPMI</td><td>Sigma-Aldrich (St. Louis, Missouri, USA)</td><td>R8758</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D1408</td><td>10X, to be diluted in apyrogenic water</td></tr><tr><td>Flask</td><td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td><td>353135</td><td></td></tr><tr><td>Syringe 1ml</td><td>Becton Dickinson</td><td>301358</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies (Waltham, Massachusetts, USA)</td><td></td><td></td></tr><tr><td>Gentamycin</td><td>Life Technologies</td><td>15710-049</td><td></td></tr><tr><td>Xilor</td><td>Bio 98 s.r.l. (Milano, Italy)</td><td></td><td>2% Xylazin</td></tr><tr><td>Zoletil</td><td>Virbac (Carros, France)</td><td>359713301992</td><td>5% Zolazepam + 5% Tiletamine</td></tr><tr><td>24G Catheter</td><td>Terumo (Rome, Italy)</td><td>SR+DM2419PX</td><td></td></tr><tr><td>HCl</td><td>Carlo Erba Reagents (Milano, Italy)</td><td>403871</td><td>Liquid</td></tr><tr><td>NaOH</td><td>JT Baker (Center Valley, Pennsylvania, USA)</td><td>10095011</td><td>Powder</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>Surgical Scalpel</td><td>Albion Surgical Limited (Sheffield, England)</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica Microsystem ( Wetzlar, Germany)</td><td>RM2235</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR International (Radnor, Pennsylvania, USA)</td><td>631-0108</td><td></td></tr><tr><td>Image Analyzer</td><td>Zeiss (Jena, Germany)</td><td>Cyres System</td><td></td></tr></tbody></table>

evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

도 1은 카테터 기법을 나타낸 것이다; 마우스는 카테터 0.5 × 10 6 MB49 세포를 주입한다. HP-NAP와 치료는 방광 벽에 ​​부착하고 증식 할 수 있도록하기 위해 3 일 종양 세포의 주입 후 시작된다. 대조군에 속한 모든 동물들은 종양의 개발; 그들 중 일부는 의한 요도의 폐색에 13 일의 기간이 종료하기 전에 다이있다. HP-NAP와 첫번째 주입 후에, 동물은 네 주사...

Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

효능 평가<em&gt; H. 파이로리</em동소 이식 쥐 모델에서 방광암의 치료를위한 치료 도구로&gt; 단백질 HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

7.2K 조회수.  University of Padua. Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

비디오: Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

쥐 동소 이식 방광 종양 모델 및 종양 탐지 시스템

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

연구

암 연구학

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

마우스 블레더 월 주입

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

의학

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Murine 방광 암의 Orthotopic 모델

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Orthotopic 방광 종양 모델 및 Intravesical saRNA 트리 트먼트의 평가

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

유전자 전달 연구 동소 방광암 모델

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

면역 본래대로 사람 MUC1 유전자 변형 마우스의 침략 과도 셀 방광 암의 유도 : 면역 요법 개발을위한 모델

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

쥐과 동 소성 방광 이종 이식의 최소 침습 설립

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

한 단계의 부정적인 크로마토 그래피 정제<em&gt; 헬리코박터 파이로리</em&gt; 호중구 - 활성화 단백질 과발현<em&gt; 대장균</em&gt; 배치 모드에서

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

면역학 및 감염병학

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration Technique

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. After anesthetizing female C57BL/6J mice in a supine position, a 24 G intravenous needle is inserted into the bladder to evacuate its contents. A 34 G dispensing needle is then introduced through the catheter, rotated five times to create a focal injury to the bladder dome mucosa, and subsequently removed. The MB49 cell suspension is aspirated and connected to a 30 G dispensing needle, which is inserted into the bladder via the catheter. Tumor cells are injected submucosally into the bladder under pressure. This technique results in minimal trauma to the mice, a high tumor take rate, and a fixed tumor location. It is characterized by simplicity and excellent reproducibility. This model provides an ideal experimental platform for developing intravesical therapies for bladder cancer, facilitating the advancement and optimization of treatment strategies for this malignancy.

This protocol describes a unique method for constructing an orthotopic model of superficial bladder cancer.

Establishing a Murine Superficial Bladder Cancer Model via an Intravesical Cell Administration 기법

This study presents an innovative method for establishing an orthotopic murine bladder tumor model with high efficiency and precise tumor localization. ...

Evaluating the Anti-Tumor Efficacy of a Bacterial Immunomodulatory Protein in a Murine Model

Source: Codolo, G. et al. <a href="https://app.jove.com/v/52743">Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates a technique to assess the anti-tumor activity of Helicobacter pylori neutrophil-activating protein, or HP-NAP, in a mouse bladder cancer model. HP-NAP interacts with tumor-cell neutrophils to produce pro-inflammatory cytokines, inducing the differentiation of na&#239;ve T cells into type 1 T helper (Th1) and type 1 cytotoxic (Tc1) T cell phenotypes. The Th1 and Tc1 cells secrete interferon-gamma, inhibiting tumor angiogenesis and promoting cytotoxic T cell-mediated tumor destruction, reducing tumor volume.

Evaluating the anti-tumor efficacy of a bacterial immunomodulatory protein in a murine model(쥐 모델에서 박테리아 면역조절 단백질의 항종양 효능 평가)

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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