作者想感谢曼迪 Loebert 和蒂娜 Czajka 的优秀技术支持。我们感谢弗洛拉罗 (哈佛医学院, 波士顿, 马萨诸塞州, 美国) 的语言编辑帮助。

1. 制备突变 pcDNA3.1/pcDNA3.1/GAA 结构
注意: 玻璃和GAA编码序列 (cd) 的克隆策略已在较早的15、18中报告。
<ol>
	<li>用定点诱变的定点诱变
	<ol>
		<li>分别使用参照序列 NM_000169.2 和 NM_000152.4 作为玻璃化和 GAA基因突变的模板。有一套高纯度无盐底漆 (25-37-市面) 由一个商业供应商合成, 与意义和反义引物运载其中一个各自序列修改中央对他们的长度单独地介绍变异。使用免费入门设计工具支持入门设计19。</li>
		<li>对于反应混合物, 使用制造商提供的反应溶液和 PCR 条件的标准条件。<ol>
			<li>混合5µL 10x 反应缓冲器, 10 ng 双模板质粒 DNA (pcDNA3.1/pcDNA3.1/GAA), 每个底漆 125 ng, 1 µL 提供 dNTP 混合物, 3 µL 的亚甲基亚砜试剂在适当量的去离子水 (最终反应量:50 µL)。最后, 添加 2.5 U 的 DNA 聚合酶和混合由移上下。作为一个负控制, 随身携带一个 no-primer 样本。</li>
		</ol>
		</li>
		<li>使用以下程序启动 PCR: 步骤 1:95 ° c 为1分钟, 步 2:95 ° c 为五十年代, 步 3:60 ° c 为五十年代, 步 4:68 ° c 为 8 min, 重复步骤 2-4 18 次, 并且步骤 5:68 ° c 为 10 min。<ol>
			<li>以下 PCR, 添加1µL 的DpnI 限制性酶 (10 U/µL) 和进一步孵化反应瓶在37° c 为 1 h。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>所需克隆的转换和筛选
	<ol>
		<li>根据制造商的建议, 转换分的 ultracompetent 细胞。使用 SOC 介质 (胰 2% (w/v), 酵母提取物 0.5% (w/v), 氯化钠10毫米, 氯化钾2.5 毫米, 在121° c 消毒, 然后添加无菌过滤解决方案的氯化镁2和葡萄糖高达最终浓度10和20毫米, 分别) 而不是制造商的中.程序后, 250 µL 的样本突变的 LB 板含有100µg/毫升氨苄西林和孵化在37° c 18 小时。</li>
		<li>确保转化的数量和 #62; 10 和反应产生至少三倍的殖民地作为 no-primer 控制反应,例如, 使用发光板, 以促进菌落计数。然后选择3殖民地和准备3毫升的隔夜文化在 LB 肉汤培养基。</li>
		<li>第二天, 用标准的试剂盒进行质粒制备, 并使用 T7 (5ʹ-TAA TAC GG-3ʹ) 和 BGHr (5ʹ-标记 AAG-TCG AGG-3ʹ) 通过标准桑格测序对整个序列进行分析。</li>
		<li>使用合适的分子生物学工具分析序列。当检测到所需的突变, 并没有进一步的序列异常相比, NM_000169.2 (α-乳糖 A) 或 NM_000152.4 (酸α-酶), 选择克隆的转染级质粒纯化。</li>
		<li>通过测量分光光度计的吸收度来确定 DNA 的纯度。 注: 只允许生产质粒纯度与 #62 的260/280 吸光度比的制剂; 1.8 用于细胞培养实验。</li>
	</ol>
	</li>
</ol>
2. HEK293H 细胞的培养
<ol>
	<li>维持 HEK293H 细胞在高葡萄糖 (4.5 克/升) Dulbecco´s 改良鹰培养基 (DMEM) 补充10% 胎牛血清 (FBS) 和1% 青霉素/链霉素。将细胞保持在37° c 以下 5% CO2大气压下的套培养箱中。</li>
	<li>培养细胞到密度为 80-90%。</li>
	<li>在没有 Ca2 +和 Mg2 +的情况下, 使用磷酸盐缓冲盐水 (PBS) 吸入培养基并进行洗涤。</li>
	<li>通过加入0.05% 胰蛋白酶-EDTA 和孵育5分钟在37° c 和 5% CO2。</li>
	<li>将新培养基和种子中的1:15 细胞分裂成一种新型的 T75 烧瓶, 以维持永久的文化。不要使用超过25通道的细胞。</li>
</ol>
3. pcDNA3.1/pcDNA3.1/GAA 质粒转染治疗 HEK293H
<ol>
	<li>在转染前24小时, 用 PBS2 +、Mg2 +将 T75 细胞培养瓶中的 HEK293H 细胞洗涤一次。收获的细胞与0.05% 胰蛋白酶-EDTA 如上所述, 种子 1.5 x 105细胞在24井培养板的洞使用500µL DMEM 培养基补充 10% FBS 没有抗生素。</li>
	<li>根据制造商手册执行转染协议。通常, 使用1µg 质粒 DNA 和2.5 µL 转染试剂的混合物在100µL 无血清 DMEM。在室温下孵育20分钟, 随后以 drop-wise 的方式添加到细胞中。</li>
	<li>在37° c/5% CO2的4小时后, 取出含有转染试剂的培养基, 并加入 500 FBS/10% µL 的新鲜 DMEM。 注: 在此步骤中, 可将 1-Deoxygalactonojirimycin 盐酸盐 (DGJ) 或 1-素盐酸盐 (DNJ) 添加到培养基中 (使用10毫米的水溶液, 以获得20µM DGJ 和 DNJ 的最终浓度。).新的 DGJ 或 DNJ 在质粒转染后增加42小时。</li>
</ol>
4. 细胞收获和α-乳糖 A 或酸α-酶活动测量
<ol>
	<li>细胞收获
	<ol>
		<li>在收割的当天, 从孵化箱中取出细胞, 并吸入培养基。使用带有 Ca2 +和 Mg2 +的 PBS 仔细清洗单元格2次。 注意: 这一步是至关重要的, 因为 DGJ 和 DNJ 分别是α-乳糖和α-酶的强力抑制剂, 任何残留物都将使测试无效。</li>
		<li>在细胞顶部直接添加200µL 的去离子水。从盘子里冲洗细胞, 然后将它们转移到1.5 毫升的反应管。</li>
	</ol>
	</li>
	<li>冻融均匀化
	<ol>
		<li>将样品放入适当的泡沫机架和涡流中5秒, 使裂解更加有效。在十年代和室温水浴中, 将样品交替放入液氮中, 直到解冻完成 (5 分钟)。</li>
		<li>重复此步骤5次, 然后旋转样本5分钟, 在 1万 x g。</li></ol></li></ol>在新的反应管中保留上清和吸管。
	
	
	<li>用二辛可宁酸测定蛋白质浓度
	<ol>
		<li>为每个样品准备一个新的管, 其中包含40µL 的去离子 H2O, 并添加10µL 的样品。混合溶液由涡流简要地和转移10µL 入一个96井板的洞 (每个样品三份)。在去离子 H2O 中稀释2毫克/毫升牛血清白蛋白 (bsa) 的储存液, 如下所示获取标准曲线:50 µL H2O/50 µL BSA;60µL H2O/40 µL BSA;70µL H2O/30 µL BSA;80µL H2O/20 µL BSA;90µL H2O/10 µL BSA;100µL H2O。</li>
		<li>开始反应, 加入200µL 的综合分析试剂 (试剂 a 和试剂 B 混合在一个50:1 的比率) 和孵化1小时在黑暗中, 在37° c 下轻微搅拌在一个轨道振动筛 (300 rpm)。在平板阅读器中测量 560 nm 的吸光度。 注: 样品通常包含每µL 1 至1.5 µg 蛋白。</li>
	</ol>
	</li>
	<li>人工 4-甲基基质 (4 杯) 的酶活性测定
	<ol>
		<li>稀释每个样品的计算量和吸管到新鲜的1.5 毫升反应管获得 0.05 (α-乳糖 A) 或 0.5 (对于酸性α-酶) µg 蛋白/µL 溶液。漩涡样品为 5 s 再和吸管10µL 这稀释入96井板 (每个样品在一重复)。</li>
		<li>开始反应, 加入20µL 各自的基板解决方案: α-乳糖甲: 2 毫米 4-甲基-α-d-乳糖 (4-亩-加仑) 在0.06 米磷酸盐柠檬酸盐缓冲, pH 4.7。 酸性α-酶: 2 毫米 4-甲基α-d-喃 (4-亩-谷氨酸) 在0.025 米醋酸钠, pH 4.0。</li>
		<li>在37° c 下, 在轨道振动筛 (300 rpm) 的轻微搅动下, 在黑暗中孵育 1 h 的酶反应。终止反应由增加200µL 1.0 M, pH 10.5 被调整的甘氨酸-氢氧化钠缓冲。</li>
		<li>从0.01 毫克/毫升的库存中制备 4-伞形 (4-亩) 的标准曲线, 如下所示: 100µL H2O/4-亩;80µL H2O/20 µL 4-亩;60µL H2O/40 µL 4-亩;40µL H2O/60 µL 4-亩;20µL H2O/80 µL 4-亩;没有 H2O/100 µL 4-亩, 吸管10µL 每稀释入96井板 (在重复) 和增加200米甘氨酸氢氧化钠缓冲液每井为了调整容量和 pH 值。</li>
		<li>在装有适当过滤器设置的荧光阅读器中测量酶活性, 并使用合适的软件对荧光读写器进行数据分析。 注: 在接触α-乳糖 A 或酸性α-酶的过程中, 两个4杯基片的衬底都减少到4μ。释放4亩是一个荧光, 可以分别测量为360和 465 nm 的激发和发射波长, 使用微荧光阅读器。</li>
	</ol>
	</li>

<ol>
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A., 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=Oral+pharmacological+chaperone+migalastat+compared+with+enzyme+replacement+therapy+in+Fabry+disease:+18-month+results+from+the+randomised+phase+III+ATTRACT+study.">Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.</a> J Med Genet. 54, 288-296 (2017).</li><li>Parenti, G., Andria, G., Valenzano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Chaperone+Therapy:+Preclinical+Development,+Clinical+Translation,+and+Prospects+for+the+Treatment+of+Lysosomal+Storage+Disorders.">Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.</a> Mol Ther. 23, 1138-1148 (2015).</li><li>Parenti, G., 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=A+Chaperone+Enhances+Blood+&#945;-Glucosidase+Activity+in+Pompe+Disease+Patients+Treated+With+Enzyme+Replacement+Therapy.">A Chaperone Enhances Blood &#945;-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy.</a> Mol Ther. 22, 2004-2012 (2014).</li></ol>

作者声明他们没有竞争的金融利益。

本文所述的协议为遗传性溶酶体代谢疾病的酵素损伤评估提供了健壮的结果。本手稿是对先前发布的15协议的修正。最关键的修改涉及严格 (即, 在突变体的载体构造准备过程中), 细胞培养协议 (即, HEK293H 细胞维护和转染条件) 的标准化, 以及高实验重复次数 (至少 5), 对结果的重现性有很高的贡献。总之, 两个目标基因都很容易获得诱变和多样性的突变可以并行组装。...

有十几个溶酶体存储紊乱 (LSDs) 与糖苷功能障碍相关的主要基因突变的结果。在法布里 (OMIM #301500) 和庞贝氏病 (OMIM #232300) 疾病, 超过500和200义突变1,2,3分别报告, 这相当于大约60% 的总变异计数。许多新的基因变种仍在被识别, 其中很多是未知的意义。广泛的生物化学研究表明, 某些基因型不会导致一个完整的功能的玻璃基因 (OMIM * 300644) 在法布里病, 但导致相应的酶未能达到一个热力青睐的折叠状态 4.这导致 ER 保留和过早退化, 否则功能酶。类似的结论也在其他 LSDs 包括庞贝氏病5中得出。此外, 分子特性的酶变体可以促进临床解释的突变在诊断时的6, 这表明 LSD 进展是一个个体的过程中, 基于突变的性质。因此, 为了简化临床咨询和治疗决策, 应重新评估传统的2到3种不同临床类型的分类。
两种疾病都有酶替代疗法。然而, 在受影响的组织/器官, 如大脑和骨骼肌肉的功效有限。此外, 专家审评可以引起免疫的反应, 危及其治疗效益。药理陪护 (PCs) 是一种有吸引力的治疗方案, so-called 反应性突变患者。pc 作为正确的蛋白质折叠和稳定的分子支架, 从而防止内质网 (er) 保留和 er 相关的酶降解。此外, 个人电脑可以口服和潜在的能够跨越血脑屏障。因此, PCT 可能是一个更可行的选择, 以治疗患者的某些基因型。有关 LSDs 中 PC 应用程序的广泛审查, 请参阅 Parenti7的优秀评审。
发现数以百计的疾病, 导致突变等位基因挑战前临床药物测试, 并要求一个简单, 快速, 和高度标准化的评估, 以适合病人的个性化药物的方法。为了评估 LSD 基因突变的不利影响, 并测试候选突变, 以预测适合的患者 PCT, 一个高度标准化的表达系统的 HEK293H 细胞, 允许快速和可靠的酶活性测量开发.类似的表达系统曾被描述为法布里和庞贝氏病, 使用 COS-78,9,10,11, HeLa 细胞12, 或 HEK29313 ,14,15,16糖苷基因的单元格。
一个非常类似的方法甚至被授予专利作为 "预测反应的方法, 药理陪护治疗疾病"17表明的相关性, 细胞培养系统能够融入临床实践。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL Site-Directed Mutagenesis Kit&#160;</td>
			<td>Stratagene, La Jolla, CA, USA</td>
			<td>#200522</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-frw: 5&#180;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-rev: 5&#180;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-rev: 5&#180;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A143T]-rev: 5&#180;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-frw: 5&#180;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-rev: 5&#180;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-frw: 5&#180;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[L552P]-frw: 5&#180;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer T7: 5&#180;-TAA TAC GAC TCA CTA TAG GG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer BGHrev: 5&#180;-TAG AAG GCA CAG TCG AGG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>103,753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,049,360,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,058,330,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,083,422,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,980,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>3908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X863.3</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,065,860,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,062,640,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>ZymoResearch, Freiburg, Germany</td>
			<td>D4020</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAfilter Plasmid Midi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12245</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium&#160;</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum&#160;</td>
			<td>GE Healthcare, South Logan, Utah, USA</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td>
			<td>VWR International GmbH, Hannover, Germany</td>
			<td>82050-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (24 well)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>831,836</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 96 well plate</td>
			<td>Greiner bio one, Frickenhausen, Germany</td>
			<td>655185</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1,5mL&#160;</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9641</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Toronto Research Chemicals, Toronto, Canada</td>
			<td>D236500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxynojirimycin&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9305</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Calcium, w/o Magnesium</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-galactopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M7633</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-glucopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M9766</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incubator type T6</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminous Plate Gr. 2 E</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems, Darmstadt, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GFL-3032 bacterial shaker</td>
			<td>GFL, Burgwedel, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-25 centrifuge</td>
			<td>Beckman/Coulter, Krefeld, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraspec 3100 pro Spectrophotometer</td>
			<td>Amersham Biosciences, Buckinghamshire, United Kingdom</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>water-jacket incubator&#160;</td>
			<td>Binder, Tuttlingen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 1 touch mixer</td>
			<td>Scientific Industries, Bohemia, NY, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Z 233 MK-2 refrigerated microtube centrifuge</td>
			<td>Hermle, Tuttlingen, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboStar ultrapure water device</td>
			<td>Siemens, Berlin, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker PST-60HL-4 orbital shaker</td>
			<td>Biosan, Riga, Latvia</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>HI 223 Microprocessor pH Meter</td>
			<td>HANNA Instruments, Arvore, Portugal</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY 
			Cell Counter + Analyser System 
			Model TT</td>
			<td>Innovatis AG, Cham/Zug, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.04</td>
			<td>GraphPad Software, Inc., La Jolla, CA; USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>BioEdit Alignment Editor, V7.0.1</td>
			<td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbreviations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>frw = forward; rev = reverse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n.a. = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

使用个性化药物治疗罕见的基因疾病, 如溶酶体存储紊乱 (LSDs) 是挑战的复杂的临床试验设计, 高成本, 和低的病人人数。在大多数的 LSDs 中, 有数百个变种等位基因被牵连。根据病情轻重, 这些疾病通常分为2到3种不同的临床类型。此外, 基因型的分子特性可以帮助预测临床结果和告知病人护理。因此, 我们开发了一个简单的细胞培养方法的基础上 HEK293H 细胞 heterologously 发酵的突变发现的法布里和庞贝氏病。最近还引入了类似的检测方法作为临床前检查, 以确定在法布里病中药物陪护治疗 (PCT) 的易变异。这份手稿描述了一个修订的细胞培养法, 使位变异的快速表型评估, 以确定符合标准的患者, 并可能有助于发展新的 pharmacochaperones。

诱变过程 为了评估玻璃化基因突变的效率, 突变被归类为以下类别之一. 这种产生突变的方法显示, 在第一次尝试中, 大约有66.5% 的玻璃化突变得到了. 另有25% 可在稍加修饰的第二 PCR 后获得。
1类: 突变 PCR 在第一次尝试中是有效的。
类别 2 : 第一诱变 PCR...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

有一个要求, 使临床前测试的一个新的类 "孤儿" 药物称为药理伴侣可再生, 快速, 高效。我们开发了一个简单, 高度标准化, 多功能细胞文化检测, 以筛选合格的病人以及新的药理陪护药物。

体外酶法检测药物陪护与庞贝氏症的反应

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

in-vitro-enzyme-measurement-to-test-pharmacological-chaperone

Research

JoVE Journal

Medicine

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

8.0K 次观看.  University Rostock Medical Center. 有一个要求, 使临床前测试的一个新的类 "孤儿" 药物称为药理伴侣可再生, 快速, 高效。我们开发了一个简单, 高度标准化, 多功能细胞文化检测, 以筛选合格的病人以及新的药理陪护药物。

视频: In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

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.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

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 Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...