Name: repression of multiple myeloma cell growth in vivo by single-wall carbon nanotube (swcnt)-delivered malat1 antisense oligos
Uploaded: 2018-12-13T13:00:00
Description: Watch this Scientific Journal Video about Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos at JoVE.com

作者感谢勒纳研究所的蛋白质组学、基因组和成像核心的帮助和支持。资金: 这项工作得到了 NIH/NCI 赠款 r00 ca17292 (给 j. z.) 和启动资金 (jj. z.) 和凯斯西储备大学核心利用试点赠款 (j. z.) 临床和转化科学合作 (ctsc) 的财政支持。这项工作使用了 leica sp8 共聚焦显微镜, 该显微镜是在国家卫生研究院 sig 赠款1s10od01992-01 下购买的。

所有涉及动物的实验都是由克利夫兰诊所 iacuc (机构动物护理和使用委员会) 预先批准的。
1. 功能化 swcnt 的合成
<ol><li>将1毫克的 swcnt、5毫克的 dspe-peg2000 胺和5毫升的灭菌无核水混合在玻璃闪烁小瓶中 (20 毫升)。摇匀, 完全溶解所有试剂。</li>
	<li>将小瓶放入水浴声纳中, 在室温下功率为 40 w, 1小时 (rt, 20分钟 x 3, 每20分钟更换一次水, 以避免过热)。然后, 以 24, 000 x g离心...

<ol>
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有证据表明, ncrna 参与调节癌症的多种生理和病理生理程序, 包括 mm7,8,9;它们有可能成为癌症治疗的目标, 而反义寡核苷酸20、21、22 可以实现。美国食品药品监督管理局 (fda) 已批准了几种反义寡核苷酸药物, 包括用于巨细胞病毒视网膜炎23的 fomivirsen、用于纯...

swcnt 是一种新型的纳米材料, 可提供各种类型的药物, 如蛋白质、小分子和核酸, 在体外 1和体内2具有理想的耐受性和最小毒性。功能化的 swcnt 具有很强的生物相容性和水溶性, 可作为小分子的穿梭点, 并可携带小分子穿透细胞膜 3,4,5.
lnrna 是一组 rna (gt;200 nt), 从基因组转录到 mrna, 但不能转化为蛋白质。越来越多的证据表明, ncrna 参与了基因表达的调节 6, 并参与了大多数类型癌症的发生和发展, 包括 mm7,8,9。mallat1 是一种富核非编码记录 2 (neat2) 和高度保守的 lncRNA 10。mat1 最初被认为是转移性非小细胞肺癌 (nsclc)11, 但已在许多肿瘤5,12,13过度表达;它是 mm8,14中表达最严重的信息之一, 与预后差有关。与仅确诊为 mm 15 的患者相比, 致命病程外髓质 mm 患者的 malat1 表达水平明显高于.
在先前的一项研究中, 我们已经证实, 抗 mat1 寡核苷酸在 mm 16 中使用 gapmer dna 反义寡核苷酸靶向 mm1 (抗 amalat1) 强致 mm 16 中的 dna 损伤和凋亡。gapmer dna 由反义 dna 组成, 并由 2 '-ome-rna 连接, 这可能会促使 mirat1 通过 rnase h 活性一旦结合 17.反义寡核苷酸的体内传递效率仍然限制了其临床应用。
为了测试 swcnt 对抗 amalat1 变指寡核苷酸的传递效果, 将抗 malat1 报的 dna 与 dspe-peg2000-胺功能化的 cnt 结合。然后将 swcnt 抗 matrat1 静脉注射到 mm 扩散小鼠模型中;经过四种治疗后观察到明显的抑制。

<table border="0" cellpadding="0" cellspacing="0" style="width:1168px;" width="1167">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">SWCNTs</td>
			<td style="width:199px;">Millipore-Sigma</td>
			<td style="width:209px;">704113</td>
			<td style="width:436px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DSPE-PEG2000-Amine</td>
			<td>Avanti Polar Lipids</td>
			<td>880128</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bath sonicator</td>
			<td>VWR</td>
			<td>97043-992</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4 mL centrifugal filter</td>
			<td>Millipore-Sigma</td>
			<td>Z740208-8EA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UV/VIS spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>accuSkan GO UV/Vis Microplate Spectrophotometer</td>
			<td>extinction coefficient of 0.0465 L/mg/cm at 808 nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sulfo-LC-SPDP</td>
			<td>ProteoChem</td>
			<td>c1118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DTT solution</td>
			<td>Millipore-Sigma</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NAP-5 column</td>
			<td>GE Healthcare</td>
			<td>17-0853-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">in vivo imaging system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NOD.CB17-Prkdcscid/J mice</td>
			<td>Charles River lab</td>
			<td>250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton Dickinso</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>Lipofectamine2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RMPI-1640 medium</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MALAT1-QF:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- GTTCTGATCCCGCTGCTATT - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MALAT1-QR:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- TCCTCAACACTCAGCCTTTATC - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GAPDH-QF:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- CAAGAGCACAAGAGGAAGAGAG - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GAPDH-QR:</td>
			<td>synthesized by IDT Company</td>
			<td style="width:209px;"></td>
			<td>5&#8217;- CTACATGGCAACTGTGAGGAG - 3&#8217;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Quantitative PCR using SYBR Green PCR master mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25780</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RevertAid first-stand cDNA synthesis kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-MALAT1</td>
			<td>synthesized by IDT Company</td>
			<td></td>
			<td>5&#8217;-mC*mG*mA*mA*mA*C*A*T*T 
			*G*G*C*A*C*A*mC*mA*mG*mC*mA-3&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Viability Assay Kit</td>
			<td>Promega Corporation</td>
			<td>G7570</td>
			<td>CellTiter-GloLuminescent Cell Viability Assay Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">accuSkan GO UV/Vis Microplate Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifugal filter</td>
			<td>Millipore-Sigma</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPSS software</td>
			<td>IBM</td>
			<td>version 24.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin</td>
			<td>Millipore-Sigma</td>
			<td>L9504</td>
			<td></td>
		</tr>
	</tbody>
</table>

repression of multiple myeloma cell growth in vivo by single-wall carbon nanotube (swcnt)-delivered malat1 antisense oligos

单壁碳纳米管 (swcnt) 是一种新型的纳米粒子, 已被用于向细胞输送多种药物, 如蛋白质、寡核苷酸和合成小分子药物。swcnt 具有可定制的尺寸, 一个大的表面积, 并可以灵活地结合药物通过不同的修改, 在其表面;因此, 它是一个理想的系统, 运输药物到细胞。长非编码 rna (lnrna) 是一组时间超过200nt 的非编码 rna 群, 不能转化为蛋白质, 但在生物和病理生理过程中起着重要作用。转移性与肺腺癌相关的转录 1 (malat1) 是一种高度保守的 lncRNA。结果表明, 较高的 malat1 水平与各种癌症的预后不良有关, 包括多发性骨髓瘤 (mm)。我们发现, matr1 调节 dna 修复和细胞死亡在 mm;因此, matr1 可被视为 mm 的治疗靶点。然而, 反义寡核苷酸在体内有效传递给抑制-击倒 malat1 仍然是一个问题。在这项研究中, 我们修改了 swcnt 与 peg-2000, 并结合抗 mat1 寡核苷酸到它, 测试这种化合物的体外交付, 注入静脉注射 mm 小鼠模型, 并观察到一个显著的抑制 mm 进展, 这表明swcnt 是抗马拉特1口角 dna 的理想穿梭机。

为了证明抗 amat1 gapmer dna 在 mm 中的抑制作用, 我们击倒了菌与高级101的表达, 并将其应用于 h929 和 mm. 1s 细胞中。48小时后, 收集细胞, 分析细胞的击倒效率和细胞凋亡状态转染抗 mallat1 口吃或控制 dna。qrt-pcr 结果表明, 抗 makat1 间隙酶 dna 有效地降低了 h929 和 mm. 1s 细胞中的 halat1 表达 (图 2a)。流式细胞仪测定细胞凋亡的状态, 显示下调节的 ...

Watch this Scientific Journal Video about Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos at JoVE.com

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos

这篇手稿描述了单壁碳纳米管 (swcnt)-共轭 malat1 反义 gapgmer dna 寡核苷酸 (swcnt 反材料-malat1) 的合成, 这表明了 swcnt 的可靠交付和强大的治疗效果。抗马拉特1在体外和体内。介绍了用于 swcnt 抗 malat1 的合成、改性、共轭和注射的方法。

单壁碳纳米管 (sscnt) (mallat1 反义寡 3号) 抑制多骨髓瘤细胞在体内的生长

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as ...

我们在这里使用的方法可以帮助将寡核苷酸与单壁碳纳米管进行包装，这是一种用于药物输送的纳米粒子。这种技术的主要优点是更有效地将寡核苷酸药物输送到体内的细胞中。这项技术的影响延伸到研究多发性骨髓瘤，因为它引入了纳米粒子，以改善寡核苷酸药物的输送。通过这种方法可以提供多发性骨髓瘤的治疗洞察。它也可以应用于其他疾病，并与其他货物结合，如蛋白质和小分子。首先，将一毫克单壁碳纳米管、5毫克DSPE-PEG 2000胺和5毫升灭菌无核糖水混合在20毫升玻璃闪烁小瓶中。在室温下，在90瓦的功率水平上将小瓶在水浴声波器中声波化一小时。声波化后，将小瓶以24，000倍g离心6小时。然后收集上一点溶液。将单壁碳纳米管溶液的一毫升添加到 100 千吨分子量截止离心过滤装置中，然后加入 4 毫升灭菌无核素水。在室温下以4000倍g的离心10分钟，以去除多余的胺。在 450 微升胺功能化单壁碳纳米管中加入 0.5 毫克 Sulfo-LC-SPDP。然后加入50微升10xPBS，并在室温下孵育两小时。接下来，将反应混合物加入到100千托氏分子量截止离心过滤装置中，然后加入四毫升无核酸酶水。在室温下以4000倍g离心样品10分钟，以去除多余的S sulfo-LC-SPDP。收集过滤器中留下的 Sulfo-LC-SPDP 处理胺单壁碳纳米管溶液，然后用 500 微升以前准备的抗 MALAT1-Cy3 溶液稀释。然后在四摄氏度下孵育样品过夜。为了获得最佳的比较结果，随机将14只小鼠安排成试验组或对照组，每组7只小鼠，每组男女比例相同。接下来，清洁操作台，用 70%乙醇消毒。使用加热灯加热其中一只小鼠，帮助尾部静脉出现。使用尾部喷射抑制器正确约束鼠标。通过尾静脉在50微升PBS中注入MM.1S-luc/mCherry细胞。用酒精拭子按注射部位 30 秒。然后标记注射的鼠标，并返回到干净的笼子。第七天，MM1S细胞注射后，将50微升含有单壁碳纳米管抗MALET1的PBS注射到小鼠的尾静脉中。然后用酒精拭子按注射部位 30 秒。观察尾巴上的局部出血和小鼠的一般行为一分钟，然后返回到干净的笼子。在将笼子返回到机架之前，再次观察所有注射小鼠，以确保注射的耐受性良好。解剖前称量每只鼠标。从心脏收集外周血，由血细胞计数器机进行完整的血液计数测定。接下来，收集骨髓、脾脏、淋巴结、肾脏和具有可见转移的组织的组织。从骨髓样本中提取RNA和蛋白质。将所有剩余的组织修复在形式素中。固定24小时后，将骨骼样本浸入0.5摩尔EDTA溶液中5天，进行脱钙。然后将所有样品浸入75%乙醇中，进行长期储存。QRT PCR结果表明，抗 MALAT1 gapmeR DNA 在 H929 和 MM.1S 细胞中显著地击倒了 MALAT1 表达并诱导凋亡。单壁碳纳米管抗MA拉特1-Cy3被传递到MM细胞核，显著抑制了H929和MM.1S细胞的内源性MA拉特1水平。BMEC-1细胞在治疗单壁碳纳米管抗MA拉特1-Cy3或不同剂量和时间点控制后，其生存能力无显著差异。这表明单壁碳纳米管抗MA拉特1-Cy3的毒性在高剂量下对正常细胞具有有限且可接受的毒性。建立了人类MM传播小鼠模型，以评估单壁碳纳米管抗MART1在体内的治疗效果。单壁碳纳米管抗MART1治疗组中小鼠的荧光素信号与21天治疗后对照值明显降低。从这些结果中得出结论，通过静脉注射进行单壁碳纳米管抗MAOL1治疗，可以有效地将抗MAOLT1寡糖送到肿瘤细胞。未观察到治疗的显著副作用，表明单壁碳纳米管抗MART1注射是小鼠的安全治疗。在尝试此程序时，重要的是要记住，细胞分子组成将影响寡核苷酸药物的传递、效率和治疗效果。按照这个程序，其他方法，如蛋白质或小分子的结合，可以执行，以回答额外的问题。比如使用单壁碳纳米管来提供其他药物。

Research

JoVE Journal

Cancer Research

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...