一本小说<em&gt;防爆大毛</em&gt;带技术来改变心内血流动力学在鸡胚胎系统

摘要

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

摘要

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

引言

Abnormally formed outflow valves are the most common type of congenital heart defects ¹. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies ². Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighted.

The primitive heart tube, which is the first functioning organ in an embryo, exhibits two distinct layers - an endothelial endocardium surrounded by myocardium - separated by extracellular matrix (cardiac jelly) which is mostly produced and secreted by the myocardium ^3-5. As development continues, valve primordia (endocardial cushions) are formed, after rightward looping of the embryonic heart, by local expansion of the cardiac jelly at the atrioventricular (AV) canal and the outflow tract (OFT) ^4,6. This expansion is mediated by the highly regulated process of epithelial-mesenchymal transition (EMT), during which the cardiac jelly becomes populated by endocardially-derived mesenchymal cells ⁶. In addition to the mesenchymal population derived through EMT, neural crest cells are also involved in valvulogenesis of the OFT ³.

Hemodynamic stimuli, such as shear stress, are important epigenetic factors that regulate heart development in the embryo ^7,8. Using a 3D in vitro system, we have previously shown shear stress to be a factor influencing the expression and deposition of fibrous extracellular matrix (ECM) proteins in AV and OFT cushions ^9,10. Moreover, studies carried out by several researchers have demonstrated that altered blood flow leads to improper valves and septa formation ^11-16. Recently, using the novel banding procedure presented here, we have shown that changing hemodynamics in the embryonic chick heart affects the early processes involved in OFT valve formation ¹⁷.

The technique described here provides a novel model for altering hemodynamics in the developing chick heart by partially constricting the OFT ex ovo. This reproducible procedure is relatively quick and allows researchers to obtain a sufficient number of embryos/whole hearts/OFT tissue, etc. for downstream analyses including gene expression studies. Moreover, this new model can be used to study 'chronic' effects of altered hemodynamics on OFT valve development.

研究方案

禽流感胚胎不在IUCAC法规考虑脊椎动物。

1.获取外科手术胚胎

1. 孵育80 - 在一个加湿（60％）的摇杆培养箱90受精Bovan鸡卵（钝端上）在40℃下大约72小时在哈密尔顿和汉堡（11H）阶段获得的胚胎17确定基于鸡蛋的确切数目功耗分析和胚胎¹⁷的成活率。使用塑料托盘鸡蛋孵化，以便有足够的空气流通。
2. 一旦这个期望发育阶段被获得，倒入约20ml温水（37℃）的Tyrode缓冲液（补充有碳酸氢钠（1克/升））的成100毫米×26毫米的培养皿。
3. 消毒用70％乙醇蛋壳。
4. 破解轻轻用手术刀手柄的外壳和精心释放其内容为包含台氏缓冲的菜。丢弃如果胚胎（i）它们在视觉上出现异常（ⅱ）它们未在合适的发育阶段（ⅲ）它们不正确定向在蛋黄和/或（iv）任何出血发生。保留未进行手术的任何胚胎在40℃放置前卵 ，以便不损害发展之后。
   注:分期是根据汉密尔顿和汉堡¹⁸进行。鸡胚异常通过肉眼而根据解剖范围来确定。确保胚胎有适当的折叠和弯曲和血管。

2. OFT捆扎

1. 从单一的11/0尼龙手术缝合Tweeze有个别长1厘米的线程，形成一个松散的结。紫外线消毒所有预制节。
2. 在解剖镜下，在视觉上保证心脏以正常速度（〜120次/分）击败。如果不是，丢弃的胚胎。
3. 刚好手术前，应用5 - 第6毫升温（37℃）台氏缓冲液中，使用移液管胚胎表面。
4. 通过预形成的结中的一个自由端OFT下，在OFT /心室结（OVJ）（或期望的位置）缝线定位，和自由端通入结从而缩窄OFT包括围绕心脏的膜。
5. 手术后，湿蛋黄的表面5 - 第6毫升的温用移液管（37℃）蒂罗德缓冲液中。
6. 保持控制胚胎前大毛作为带状胚胎;然而，不使其受到了外科手术。
7. 在加湿（60％）培养箱孵育胚胎，对时间的期望的量，在40℃。
8. 一旦达到所希望的时间点，切除从使用直剪刀蛋黄胚胎。用细镊子解剖的心脏，并使用了几个下游的研究，如心脏形态，由于血流动力学改变¹⁷的变化。

3.确认捆扎干预导致血流动力学中的变更

注意:所引起的条纹干涉的部分收缩导致在OVJ增加血流速度。此血液动力学参数是使用二维超声成像，这是在实验的所需时间点上执行方便评估。

1. 由于只有单个的胚胎可以一次成像，保持在40℃的培养箱中指定用于超声成像的所有其它胚胎。
2. 放置在设定在40℃的加热垫待成像含有胚胎的培养皿。
3. 填写的菜，到边缘，用温水（37℃）台氏缓冲区。慢慢倒入缓冲溶液放入盘中，以便保持蛋黄完好无损。然而，如果蛋黄的完整性在该步骤中受到损害，丢弃胚胎。
4. 东方胚胎，使得胚胎的中心轴是垂直于从一个可调节的支架悬浮超声波探头。
5. 随着超声机歌剧亭在B模式，获得跳动的心脏在屏幕上的2D图像和移动台（加热板），使得OFT，心室和OVJ都清晰可见。
6. 切换到脉冲波（PW）模式，在所需的脉冲重复频率（ 例如 ，20千赫），并在OVJ准确获得速度数据。在屏幕上得到心脏跳动的B模式图像。
   1. 移动舞台上看到的OFT，心室和OVJ。使用超声波机软件，获得准确的OVJ速度测量。

请注意:如果一个胚胎的心脏速率成像期间降低，这样获得的速度数据不应该被用于分析。用于速度测量所有胚胎应优选地不被用于超声成像后的任何其它实验。

结果

如图1中所示，建议需要OFT绑扎工具。含有胚胎的前培养皿（ 图1A） 卵内应该足够深，以便当与盖覆盖，以不破坏胚胎。深培养皿（ 图1C）应也可用于超声成像以允许台氏缓冲液的适当体积要浇卵黄顶上。

单1厘米螺纹（ 图2A）是从11/0尼龙线（ 图1A）tweezed<...

讨论

这种技术是相对快速和容易执行，然而某些关键点需要牢记以便获得准确的下游结果。胚胎应保持前卵在包含蒂罗德缓冲以提供足够的补液的培养皿。同样重要的是水合蛋黄手术后与台氏缓冲液，以确保孵育室充分水合。手术不应该在胚胎如有出血可见或蛋黄甚至略有破损进行。这将影响胚胎存活率，特别是对于长期的实验。杀伤力的胚胎另一个潜在原因，是各地OFT乐队是否太紧时首次应?...

材料

Name	Company	Catalog Number	Comments
Fertilized Bovan chicken eggs	Clemson University, Clemson, SC
11 / 0 Nylon suture	Ashaway	S30001	UV sterilize knots before surgery
100 x 26 mm petri dish	VWR	25387-030
Transfer pipettes	Thermo Scientific	232-20S
Scalpel handle #3	Fine Science Tools	91003-12
Straight scissor	Roboz	RS-6702
Dumont #5 fine forceps	Fine Science Tools	11254-20
Tyrodes buffer	Sigma-Aldrich	2145-10L	Filter sterlize before use
Sodium bicarbonate	Fisher Scientific	S233-500
Vevo 770 Ultrasound Imaging system	VisualSonics, Inc.	VS-11392
708 Ultrasound transducer	VisualSonics, Inc.	VS-11171