この作業は、研究Scholarのアメリカの癌協会からの補助金（116403-RSG-09から082-01-MGO）とGSのウェイクフォレスト大学健康科学の学内資金によって部分的にサポートされていました。 DBSは、NCI訓練助成金5T32CA079448によってサポートされていました。

<ol>
	<li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+vector-based+RNAi+technology+to+suppress+gene+expression+in+mammalian+cells.">A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550-553 (2002).</li><li>Zhang, Q., Stovall, D. B., Inoue, K., Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncogenic+role+of+Yin+Yang+1.">The oncogenic role of Yin Yang 1.</a> Crit. Rev. Oncog. 16, 163-197 (2011).</li><li>Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+plays+an+essential+role+in+breast+cancer+and+negatively+regulates+p27.">Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.</a> Am. J. Pathol. , (2012).</li><li>Sui, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+a+DNA+vector-based+RNAi+technology.">Gene silencing by a DNA vector-based RNAi technology.</a> Methods in Molecular Biology. 309, 205-218 (2005).</li><li>Trower, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+PCR-based+colony+screening+protocol+for+cloned+inserts.">A rapid PCR-based colony screening protocol for cloned inserts.</a> Methods Mol. Biol. 58, 329-333 (1996).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+controlled+conditions+in+lentiviral+vector+titration+and+in+the+use+of+multiplicity+of+infection+(MOI)+for+predicting+gene+transfer+events.">The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.</a> Genet Vaccines Ther. 2, 6 (2004).</li><li>Geran, R. I., Greenberg, N. H., MacDonald, M. M., Schumacher, A. M., Abbott, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+screening+chemical+agents+and+natural+products+against+animal+tumors+and+other+biological+systems.">Protocols for screening chemical agents and natural products against animal tumors and other biological systems.</a> Cancer Chemother. Rep. 3, 1-103 (1972).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96, 23-28 (1990).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li><li>Rubinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33, 401-406 (2003).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+negative+regulator+of+p53.">Yin Yang 1 is a negative regulator of p53.</a> Cell. 117, 859-872 (2004).</li></ol>

利害の衝突が宣言されません。

このプロトコルは、shRNAを用いて遺伝子をノックダウンし、in vivoでの腫瘍細胞増殖の生物発光イメージングを使用して、その生物学的効果を可視化する方法について説明します。shRNAのターゲットサイトが使用される3つの制限部位のいずれか（をBamHI、HindIII及びEcoRI）を含めるべきではありませんサブクローニングのために。以下のいずれのサイトが存在するときにまれなシナリオでは、追加の制限酵素は、コンストラクトを生成する際にそれを置き換えるために使用することができます。 このプロトコルのいくつかの重要なステップは、shRNAレンチウイルスベクターの構築をスピードアップします。最初に、U6またはH1プロモーターを含むプラスミドのPCRテンプレートがレンチウイルスベクター（典型的にはアンピシリン）とは異なる抗生物質耐性遺伝子（カナマイシンなど）があります。これはプラスミドテンプレートによって引き起こされる転換の背景を減らすことができます。第二に、 大腸菌コンピを使用する必要がある変換の高効率を持つ大腸菌細胞。カリウムを使用するプロトコルNDマンガンイオンは大腸菌コンピを生成するために使用することができます非常に高い能力12と大腸菌細胞。第三に、選択マーカーは、3断片のサブクローニングを容易にすることができます。例えば、レンチウイルスベクターは、PCR断片を挿入することによって置き換えられ、P3/P4のオリゴヌクレオチドを​​アニールされるβ-ガラクトシダーゼの発現カセット（LacZ遺伝子）を含むように設計することができます。 X-galを（5 - ブロモ-4 - クロロ-3 - インドリル-β-D-ガラクト - ピラノシド）にさらされたときに挿入せずに、これらは、青になりながら、このケースでは、挿入した組換えプラスミドは、白色コロニーを形成します。 レンチウイルスベクターと3パッケ​​ージングプラスミドの品質は、効率的なレンチウイルスの生産に不可欠である。リン酸カルシウム沈殿を使用して非常に経済的かつ効率的なトランスフェクション法が提供されています。ポリエチレンイミン13またはほとんどの市販のトランスフェクション試薬は、レンチウイルスの生産に使用することもできます。適切なMOI FOを使用するにはRの感染は、それが生産レンチウイルスの力価を決定することが重要です。 すべての実験群のマウスは、異種移植片腫瘍イメージングの間に生物発光の変化を引き起こす可能性があり、その概日リズムの違いを排除するために同じ部屋に収容する必要があります。マウスの安楽死のアプローチは、イソフルランによるCO 2窒息と過剰摂取を含め、制度のACUCによって承認されるべきである。

RNAとタンパク質の解析や細胞培養に使用される一般的な実験装置に加えて、以下の材料および試薬は、このプロトコルが必要になります。 <table border="1"><tbody><tr><td> 試薬の名前 </td></tr><tr><td>バイオセーフティレベル2の封じ込めに適したバイオセーフティキャビネット</td></tr><tr><td> PCRサーマルサイクラー</td></tr><tr><td>超遠心</td></tr><tr><td>イソフルランスカベンジャーによる麻酔誘導室</td></tr><tr><td>デジタルノギス</td></tr><tr><td> IVISイメージングシステム</td></tr><tr><td>有能DH5a大腸菌</td></tr><tr><td>をEcoRI、BamHI部位、及びHindIII制限酵素</td></tr><tr><td> T4 DNAリガーゼ</td></tr><tr><td>第三世代レンチウイルスパッケージングプラスミド、VSV-G、PRSV-RevおよびpMDLg / pRRE </td></tr></tbody></table> 材料リスト

dna vector-based rna interference to study gene function in cancer

RNA干渉（RNAi）が特異的に分解する標的mRNAによる遺伝子発現を阻害する。遺伝子サイレンシング1の二本鎖の小さな干渉RNA（siRNA）の発見以来、RNAiは遺伝子機能の研究において強力な研究ツールとなっています。遺伝子欠失に比べて、RNAiによる遺伝子サイレンシングは、そのようなことが行われると使いやすさと、ほとんどの細胞株への適合性など多くの利点を持っています。複数の研究は癌研究でのRNAi技術の応用を実証した。特に、U6またはH1プロモーターにより駆動される小さなヘアピンRNA（shRNA）を生成するDNAベクターベースの技術の開発が可能2,3サイレン長期および誘導性遺伝子を行いました。このようなレンチウイルスなどの遺伝子組み換えウイルスベクターと組み合わせて、その使用は、安定したshRNA発現のためにゲノムDNAにshRNAの配信、および/または統合の高効率​​を容易にします。 我々はdetaileを記述するdの手順では、shRNAは、レンチウイルスの生産と細胞感染、マウス異種移植モデルを用いた機能研究を表現するレンチウイルスベクターの構築を含む、遺伝子機能を決定するDNAベクターベースのRNAi技術を使用します。 様々な戦略がshRNAコンストラクトを生成する際に報告されている。プロトコルは、PCR増幅を採用し、3断片ライゲーションが直接かつ効率的にshRNAをコードする配列に余分な塩基の隣接を離れることなく、レンチウイルスコンストラクトをshRNAを含む生成するために使用することができますここで説明する。この戦略によって作成されたshRNAの発現カセットを制限酵素によって切断することができるので、それらは容易に異なる蛍光または抗生物質マーカーを有する他のベクターに移動​​することができます。ほとんどの市販のトランスフェクション試薬は、レンチウイルス生産に使用することができます。しかし、このレポートでは、我々は90％のトランスフェクション効率を上に実現することができますリン酸カルシウム沈殿を用いた経済的な方法を提供する293T細胞。構成のshRNA発現ベクターに比べ、誘導性のshRNAシステムは、細胞増殖に必須の遺伝子をノックダウンに特に適しています。我々は、陰陽1（YY1）、TETオン誘導shRNAのシステムおよび腫瘍形成に及ぼす影響によって乳癌4,5、の潜在的な癌遺伝子の遺伝子サイレンシングを示しています。レンチウイルスを用いた研究は、研究者の所属機関のバイオセーフティ委員会によるバイオプロトコルのレビューと承認を必要とします。動物モデルを用いた研究では、アニマルケア、動物プロトコルのレビューと承認を必要とし、研究者の機関の委員会（ACUC）を使用します。

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA干渉（RNAi）は遺伝子ノックアウトよりも多くの利点を有し、広範に遺伝子機能研究のツールと​​して使用されています。 DNAベクターベースのRNAi技術の発明は、長期的および誘導性遺伝子のノックダウンを可能にした、また、遺伝子サイレンシングの可能性を増加している<emin vivoでの&gt;</em&gt;。

癌における遺伝子機能を研究するDNAベクターベースのRNA干渉

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

dna-vector-based-rna-interference-to-study-gene-function-in-cancer

Universidad San Sebastian

Research

JoVE Journal

Medicine

20.4K ビュー.  Wake Forest University School of Medicine. RNA干渉（RNAi）は遺伝子ノックアウトよりも多くの利点を有し、広範に遺伝子機能研究のツールと​​して使用されています。 DNAベクターベースのRNAi技術の発明は、長期的および誘導性遺伝子のノックダウンを可能にした、また、遺伝子サイレンシングの可能性を増加している。

ビデオ: DNA Vector-based RNA Interference to Study Gene Function in Cancer

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

ビボ犬筋肉の機能アッセイに

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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.

遺伝子送達研究のための同所膀胱癌モデル

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

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

マウス肺の病理生理学を研究するための非侵襲的気管内挿管

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三次元文化：乳細胞の悪性形質転換正常対腺房アーキテクチャを研究するためのツール

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

正常および病的脳機能を研究するために脳深部刺激でSaccadometryを使用します

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...