Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

sRANKL MC DNA의 1. 유체 역학 배달 <ol><li> 마우스의 꼬리 정맥을 통해 유체 역학 배달 <ol><li> 꼬리 정맥 주입하기 전에 마우스의 무게를. 마우스의 체중의 10 % ~의 총 부피에 sRANKL 또는 링거액에 녹색 형광 단백질 (GFP) MC (미리 따뜻한 37 ° C에서)를 희석. </li><li> 혈관을 팽창하고 눈에 보이는 측면 혈관 (정맥 주사)를 확인하기 위해 주사 전에 10 분 동안 새장에 마우스를 따뜻하게. 탈수와 고열을 피하기 위해 신중하게 마우스를 모니터링합니다. </li><li> 즉시 정맥 주사가 넓혀진 눈에 보이는대로, 제한 수단에 마우스를 전송하고 이동을 억제하는 통에 충분히 플러그를 삽입합니다. 같은 호흡으로 정상 활동에 마우스를 모니터링합니다. 필요한 경우 제한 기의 플러그를 조정합니다. </li><li> 와이프 주류로 꼬리의 끝으로부터 주입 영역 3 / 4을 소독. 마우스 꼬리 정맥 주입을 위해 27 ~ ​​30 G 바늘을 사용합니다. 한 손으로 단단히 꼬리를 잡고있는 바늘을 삽입꼬리 정맥에. 주사기에 압력을 적용하고 5-7 초 내에 주입을 완료합니다. </li><li> 꼬리 정맥에서 바늘을 제거하고 출혈을 멈추게하는 주사 부위에 면봉으로 압력을 적용합니다. 호흡 속도가 정상 속도로 감소되면 다음 동식물 사육장에 다시 마우스를 전송, 15 ~ 30 분 동안 마우스를 모니터링합니다. </li></ol></li><li> 마우스 혈청 TRACP5b 포스트 유전자 전송의 전신 마우스 sRANKL 표현과 정량화의 확인 <ol><li> 혈관을 팽창하고 정맥 주사를 볼 수 있도록하기 위해 5 ~ 10 분간의 장에 마우스를 따뜻하게. 탈수와 고열을 피하기 위해 신중하게 마우스를 모니터링합니다. </li><li> 꼬리의 끝에서 앞으로 사이트 한 곳에서 마우스의 꼬리에 잎을 가진 작은 절개를합니다. </li><li> 혈청 분리 튜브에 혈액의 약 100 μl를 수집합니다. </li><li> 실온에서 30 분 동안 혈청 분리 튜브를 배양한다. </li><li> 원심 분리기 5 분 10,000 XG에 샘플 및 혈청을 수집합니다. STOR추가 분석을 위해 -80 ° C에서 E 세럼. </li><li> 상용 가능한 키트를 사용하여 혈청 샘플에 sRANKL ELISA와 마우스 혈청 TRACP5b 분석을 수행합니다. </li></ol></li></ol> 마우스 BMMs 2. 체외 파골 세포 생성 <ol><li> 분리 및 BMMs의 배양 <ol><li> 경골과 대퇴골 뼈의 분리 : CO 2 기증자 마우스를 안락사, 70 % 에탄올로 다리를 소독. 그대로 대퇴골과 경골 뼈를 제거하기 위해, 종골 뼈에 치골에서 해부. </li><li> 뼈를 손상시키지 않고 조심스럽게 피부와 근육을 제거합니다. 인산염 완충 생리 식염수 (PBS)를 포함하는 배양 접시에서 처리 대퇴골과 경골 뼈를 놓습니다. </li><li> 골수 제거 : 대퇴골과 경골 뼈의 한쪽 끝을 잘라 및 α-MEM은 1 % 페니실린 / 스트렙토 마이신을 포함하는로드 25 G 바늘을 1 ㎖ 주사기를 사용하여 50 ㎖ 튜브에 골수를 플러시 (파골 세포 배양액 / OCM). </li><lI&gt; 프로세스 골수 : 혼합 및 70 μm의 나일론 셀 스트레이너를 통해 전달하여 새로운 50 ㎖ 튜브에 골수 현탁액을 전송합니다. 원심 분리기 300 XG와의 세포를 1 % 페니실린 / 스트렙토 마이신과 10 % FBS (파골 세포 배양액 / OCM (+))를 포함하는 α-MEM에 다시 일시 중지합니다. </li></ol></li><li> 대 식세포의 문화 <ol><li> 혈구 또는 자동 세포 계수기를 사용하여 세포를 계산합니다. </li><li> 96 - 웰 플레이트에 넣고 37 ℃에서 부화 유리 커버 슬립 (5 ㎜) 또는 상아질 조각에 6 잘 판 3 × 10 5 세포를 잘 당 플레이트 1 X 10 6 세포 </li><li> 기음 비 부착 세포를 포함하는 48 시간 동안 25 NG / ML 마우스 M-CSF를 포함하는 신선한 OCM (+)와 37 ° C에서 세포를 품어 모든 미디어. </li><li> 25 NG / ML 마우스 M-CSF 및 파골 세포를 시작하는 30 NG / ML 마우스 sRANKL를 포함 기음 모든 미디어와 신선한 OCM으로 37 ° C에서 세포를 배양 (+) 미디어. 필요에 따라 매 3 일마다 미디어를 보충. </li><li> 세포 성장약 6~8일 뼈를 resorbing 할 수있는 거대한 멀티 핵 세포를 형성하는 데 필요한 권한입니다. 즉시 다핵 세포가 나타날 때, 상용 가능한 키트를 사용하여 염색 TRAP (주석 산염 저항하는 산성 인산 가수 분해 효소)를 수행하고, 완전히 성숙 할 때, 기능 분석을 수행합니다. phalloidin도 스테인 및 이미지 (19) 이전에 설명한 바와 같이 주사 전자 현미경 (SEM)에 의해 슬라이스 상아질 부착 세포를 이용하여 커버 슬립에 F-액틴 링 시각화. </li></ol></li></ol> 인간 PBMC를 3. 체외 파골 세포 생성 <ol><li> 인간 PBMC를의 분리 <ol><li> 50 ㎖ 튜브에 미리 예열 Histopaque-1077의 10 ML을 추가합니다. </li><li> 빈 백혈구 필터는 50 ML 튜브 (PBS와 혈액의 1:1 비율)로 멸균 PBS와 혈액 은행에서 얻을. 혈액이 histopaque와 혼합하지 않도록하고, 아주 천천히 histopaque 용액에 희석 된 혈액을 레이어. </li><li> 18 ° C에서 20 분 1000 XG에 원심 분리기 샘플 따라원심 분리를하여 보내고, 정중 백혈구 함유 백색 버피 코트 층을 분리하고 50 ㎖의 새로운 튜브로 옮긴다. </li><li> 세포를 수집하는 5 분 650 XG에 다시 PBS와 원심 분리기로 세포를 희석. </li><li> 미디어 및 셀 카운터를 사용하여 세포를 계산 (-) OCM에 뜨는에 resuspend 세포를 제거합니다. </li></ol></li><li> PBMC를 도금 및 배양 <ol><li> OCM에있는 세포를 재현 탁 (+) 미디어 판 (8) × 10 / ㎖ 당 잘 6 잘 플레이트 또는 1 X 10 6 세포 / ㎖ 당 잘 96 - 웰 플레이트에있는 6 개의 세포. 로 면역 형광 이미징뿐만 아니라 골 흡수 분석에 적합한 뼈 조각에 대한 필요에 따라 유리 커버 슬립 (5 ㎜)에 플레이트 세포는 이전에 (20)을 설명했다. </li><li> 변경 배양액 인간 M-CSF 2-3 일마다 또는 필요에 따라 세포를 보충. 그런 osteoclastogenes을 시작 OCM (+) 미디어 25 NG / ML 인간의 M-CSF와 함께 인간의 sRANKL 30 겨 / ㎖를 추가하여 5 일에 분화 단계로 진행이다. </li><li> 거대한 멀티 핵 세포를 형성하는 데 약 14-21일에 세포를 성장. 마자 다핵 세포가 나타나는 바와 같이, 이들 세포는 TRAP 표지 할 수 있고, 완전히 성숙 할 때, 기능적 분석법이 수행 될 수있다. F-액틴 링은 phalloidin도 얼룩을 사용하여 커버 슬립에 시각화 할 수 있습니다. 세포 형태는 SEM에 의해 조각을 상아질에 부착 된 세포에서 검사 할 수 있습니다. </li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+5b:+a+novel+serum+marker+of+bone+resorption.">Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 15, 1337-1345 (2000).</li><li>Adamopoulos, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-23+is+critical+for+induction+of+arthritis,+osteoclast+formation,+and+maintenance+of+bone+mass.">IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass.</a> J Immunol. 187, 951-959 (2011).</li><li>Suda, T., Takahashi, N., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine reviews. 13, 66-80 (1992).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123, 2600-2602 (1988).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods Mol Biol. 455, 19-35 (2008).</li><li>Arai, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+and+differentiation+of+osteoclast+precursor+cells+by+the+sequential+expression+of+c-Fms+and+receptor+activator+of+nuclear+factor+kappaB+(RANK)+receptors.">Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.</a> The Journal of experimental medicine. 190, 1741-1754 (1999).</li><li>Fuller, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+colony-stimulating+factor+stimulates+survival+and+chemotactic+behavior+in+isolated+osteoclasts.">Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.</a> The Journal of experimental medicin. 178, 1733-1744 (1993).</li><li>Edwards, J. 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The authors have nothing to disclose.

근골격계 조건은 병적 상태와 장애의 원인을 선도하고 150 개 이상의 질병 및 증후군 구성되어 있습니다; 현재 약 90 만 명의 미국인에 영향을 미치는. 관절 염증 및 골 파괴는 관절염과 골다공증 등의 근골격 조건의 주된 기능이다. 골다공증은 주로 뼈의 골절로 이어지는 뼈의 무결성을 약화 조건이다. 관절염은 부어, 부드럽고 뻣뻣한 제한 정상적인 운동이되어 장애로 이어질 수있는 관절의 염증?...

근골격계 질환은 국가 및 지역 보건 시스템 1에 대한 미국과 현재의 심각한 결과에있는 사람들의 수백만에 영향을 미칩니다. 이 질환은 뼈의 손실과 광범위한 치료 및 회복의 오랜 기간을 필요로 관절 기능을 특징으로한다. 일반적으로, 파골 세포의 수 및 / 또는 활성의 상대적 증가는 세포가 골다공증, 뼈 재 흡수하도록 특수화 및 관절염이 관찰된다. 생리적 조건에서 파골 세포의 수와 활성을 골아 세포에 의해 생산되는 핵 인자 κ B-리간드 (RANKL)의 수용체 활성에 의해 조절된다. 게린 (OPG), RANKL에 대한 미끼 수용체는 조골 세포 (3)에 의해 생성되는 조직 sRANKL의 과발현 또는 OPG의 삭제를 포함하는 생체 내 동물 모델 골다공증 연구에 매우 유용합니다.; 그러나,이 방법은 형질 전환 마우스 4,5의 생성을 필요로합니다. 여기서, 새로운 대안근골격계 관련 질환의 연구 sRANKL를 과발현하는 방법이 설명된다. 특히, minicircle (MC) DNA 기술 및 유체 전달 방법은 생체 내에서 sRANKL의 유전자 전달을 달성하고 체계적으로 6 마우스 sRANKL를 과발현하는 데 사용되었습니다. 이 방법은 또한 낮은 칼슘 다이어트 (8)에 의해 난소 7식이 개입을 다음 파골 세포의 호르몬 변조 골다공증의 다른 생체 모델을 보완. 이 모델들은 수술 절차가 필요하고 상당한 비용 9에서 몇 달까지 걸릴 수 있습니다 그러나 근골격계 관련 질환의 다양한 측면을 연구하는 것은 매우 유용하다. 난소 절제 (OVX) 설치류 모델은 난소를 제거함으로써 인간의 폐경 후 골다공증 (10)을 흉내 낸 에스트로겐 결핍에 이르게 실험 동물 모델입니다. 인간의 폐경 후 골다공증, 에스트로겐 defici 조건ency은 골절의 위험이 증가로 연결 골다공증은 미국에서만 약 8 백만명의 여자에 영향을 미칩니다. OVX 모델은 폐경 후 골다공증에 유용하지만 그것은 일반적으로 골다공증을 연구하는 제한된 이점을 제공합니다. 에스트로겐 따라서 그것의 부재에서 증가 된 파골 세포 활성은 10-12을 관찰하고, 파골 세포 및 조골 세포 유도 세포 사멸을 억제함으로써, 골 손실을 억제한다. 골 흡수 호의 RANKL-OPG 비율 불균형도 13를 관찰된다. 성장 인자 β (TGF β의), 증가 된 인터루킨 12 (IL-12) 및 TNF, IL-1 및 IL-6 (14, 15)를 변형의 수준을 감소함으로써 그러나, 생체 내에서 에스트로겐 결핍도 수반한다. 이러한 사이토 카인은 RANKL 경로의 뼈 리모델링 조절 성 기능을 독립적 알고있다, 그것은 전적으로 RANKL-RANK 축에있는 파골 세포의 활성화를 속성 수 없다. 이 문서에 설명 된 모델은 비브에서 공부하는 연구자 수오 전 염증성 사이토 카인없이 파골 세포와 뼈 손실에 RANKL-RANK 축이 OVX 쥐 모델에 비해. 또한, 체외 파골 세포 기술은 근골격계 질환의 잠재적 인 치료 트리트먼트의 파골 세포의 활성을 연구하는 중요한 도구입니다. 이전 연구는 또한 마우스 대 식세포 집락 자극 인자 (M-CSF)와 마우스 sRANKL와 마우스 골수 유래 대 식세포 (BMMs)를 배양하는 파골 세포의 분화 3,16,17로 이어질 수있는 것으로 나타났습니다. 여기서, 마우스 골수로부터뿐만 아니라 체외 (18)에서 인간의 말초 혈 단핵 세포 (PBMC를)까지 다핵 파골 세포 - 유사 세포를 생성하는 프로토콜이 설명된다. 성숙한 말기 차별화 된 완벽한 기능 파골 세포를 정의하는 데 필요한 세포 기반 분석도 간략하게 설명되어 있습니다. 체외 기술은 생체 방식의 소설을 보완하고 함께 P 역할owerful 조사 도구는 파골 세포의 분화 및 활성화를 공부한다. 이러한 시스템을 사용하여, 과학자들은 생체 내 및 시험관 내에서 파골 세포 생성 및 증식 및 활성화에 필요한 자극 및 신호를 정의 할뿐만 아니라 약리학 적 및 생물학적 억제제의 효능을 시험 할 수있다.

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">alpha-MEM</td>
			<td style="width:71px;">Life Technologies&#160;</td>
			<td style="width:119px;">12561-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Human M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-096-492</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse M-CSF</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-643</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Human RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td>130-094-631</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Mouse RANK-Ligand - soluble</td>
			<td style="width:71px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-094-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tailveiner Restrainer for mice</td>
			<td style="width:71px;">Braintree</td>
			<td style="width:119px;">TV-150 STD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&#160;</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">MTR00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">387A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">MouseTRAP assay&#160;</td>
			<td style="width:71px;">immunodiagnostic systems</td>
			<td style="width:119px;">SB-TR103</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

여기서, 파골 세포에 대한 사이토 카인의 효과를 연구하기위한 방법으로 체외로 파골 전구 세포 분화에 대한 생체 내 및 세포 배양 프로토콜에서 파골 세포의 분화를위한 신규 한 유전자 전달 기술이 설명된다. 그림 1에서, 마우스에있는 GFP 마우스 sRANKL MC의 성공적인 유전자 전달의 대표적인 결과가 표시됩니다. 그림 2에서, 마우스의 골수 또는 시야 ?...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K 조회수.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

비디오: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

같은 자폐증과 정신 분열증과 같은 일반적인 장애에 드 노보 변이를 식별하는 전략

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

연구

의학

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.

면역 결함 마우스의 확립과 인간 Retinoblastoma 종양의 전파

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

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

소설을 사용하여 척수 상해 후에 마우스 뒷다리 뼈 손실의 세로 평가, 생체내에 방법론

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

암의 생산 및 반응 산소 종의 감지 (ROS)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

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.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

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

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

태아 마우스 모델에서 Bioactive 에이전트의 Intratracheal 관리를위한 소설 외과 접근

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

오디오 기반의 가상 게임 환경의 개발은 맹인의 탐색 기술을 지원하는

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

근골격계 초음파 이미징의 소설 응용 프로그램

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

세포 유전 분광법을 사용하여 특발성 척추 측만증의 예후 예측을위한 세포 기반 분석 프로토콜

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...