我们对藏医学院“十四五”规划内涵建设项目（2022ZYYGH12）、藏医药大学教育部藏医与基础教育重点实验室2022年开放课题（ZYYJC-22-04）、宁夏市重点研发计划（2023BEG02012）的资金支持表示衷心感谢。 成都中医药大学杏林学者研究推广项目（XKTD2022013）。

将化合物溶液、蛋白质溶液和染料引入PBS溶液中。随后，使用数字加热振荡干浴对样品进行逐步加热，并通过测量复杂系统内荧光强度的变化来评估蛋白质的热稳定性。下面概述了详细的步骤， 图 1 说明了该协议的概述。
1. 溶液制备
<ol><li>通过将0.1mg伞形酮加入616.75μLDMSO溶液中来制备1mM伞形酮溶液（参见 材料表）。</li>
	<li>通过将 0.1 mg CD40 加入 500 μL ddH2O 溶液中来制备 200 μg/mL CD40 蛋白溶液（参见 材料表）。</li>
	<li>通过将 1 μL 5000x 橙色染料加入 9 μL 磷酸盐缓冲盐水 （PBS） 中来制备 500x 橙色染料溶液（参见 材料表）。 注意：在冰上执行上述操作，由于移液器的最小量程为 0.1 μL，因此最初将橙色蛋白染料稀释 10 倍以方便后续使用。</li>
</ol>2.染料性能检测
<ol><li>将 500x 橙色染料加入 PBS 溶液中并稀释以获得染料：PBS 比例为 1：500、1：1000、1：2000 和 1：4000。 注意：确保 DMSO 的最终浓度不超过 2% （v/v）。</li>
	<li>打开荧光酶标仪并预热10分钟。</li>
	<li>依次打开计算机和数据采集软件（见 材料表）。</li>
	<li>点击 仪器，选择对应的荧光微孔板型号，在数据采集软件中选择 确定 。 注意： 等待荧光微孔板完成预热并在面板上显示温度，然后再打开数据采集软件，以确保仪器与软件成功连接。</li>
	<li>单击 “采集设置 ”，然后在数据采集软件中选择“ 荧光 ”。</li>
	<li>在波长接口中编辑程序：Lm1 = 470 nm、570 nm，然后选择确定。 注：橙色染料可以用 300 nm 的紫外线或 470 nm 的可见光激发，发射可以在 570 nm 处测量。</li>
	<li>将不同浓度的橙色染料（1：500、1：1000、1：2000 和 1：4000）和 PBS 以 100 μL/孔的浓度加入 96 孔板中。 注意：在将溶液添加到PBS之前，将溶液溶解在DMSO中，并在制备过程中以2000× g 彻底涡旋，以确保由于染料的沉淀倾向而完全溶解。</li>
	<li>将96孔板放入仪器的检测台上，点击数据采集软件中的 读取 按钮，在室温下测量每种染料浓度的荧光13次，每次间隔2分钟。</li>
	<li>每次测定后单击“导出”按钮，选择“导出到 XML XLS TXT”，选择“所有板”，然后选择“输出格式的板”，最后单击“确定”以“XLS”格式将数据保存在文件夹中以供进一步分析。 注：这些步骤有助于确定连续测量对室温下橙色染料自发荧光的影响，并确定最佳染色浓度。</li>
	<li>打开数字加热摇动干浴，设置加热温度为35°C，加热时间为2分钟。</li>
	<li>在 1.5 mL 微量离心管中制备染料的最佳稀释比例，并将其置于数字加热振荡干浴中 2 分钟。</li>
	<li>将PBS溶液和在35°C加热的染色溶液以每孔100μL的体积加入96孔板中。</li>
	<li>将 96 孔板放入仪器的检测台，然后单击数据采集软件中的 读取 按钮。</li>
	<li>将加热温度设置为 40 °C，加热时间为 2 分钟。</li>
	<li>将 96 孔板中的染料溶液移回 1.5 mL 微量离心管中，并在仪器中加热 2 分钟。</li>
	<li>重复步骤2.12-2.15和步骤2.9的操作，分别测试染料溶液在35°C、40°C、45°C、50°C、55°C、60°C、65°C、70°C、75°C、80°C、85°C、90°C和95°C下的吸光度。 注：这些步骤有助于确定橙色染料在连续加热下自发荧光的稳定性，梯度为35°C至95°C。 至关重要的是，必须迅速工作，以避免温度快速下降或添加错误的液体，这可能导致实验错误。</li>
</ol>3. 检测蛋白质的温度熔融（Tm）
<ol><li>将 CD40 蛋白和橙色染料与 PBS 溶液混合，最终浓度分别为 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL 和 1：500。</li>
	<li>重复步骤 2.2-2.6 和步骤 2.10-2.16 中概述的操作，分别评估 CD40 蛋白溶液在 35 °C、40 °C、45 °C、50 °C、55 °C、60 °C、65 °C、70 °C、75 °C、80 °C、85 °C、90 °C 和 95 °C 的温度下的吸光度。</li>
	<li>重复步骤 2.9 保存和分析数据，然后使用数据分析软件计算 Tm 值（参见 材料表）。</li>
	<li>向 PBS 溶液中加入 CD40 蛋白、伞形酮和橙色染料，分别获得 5 μg/mL、10 μg/mL、15 μg/mL、20 μg/mL、10 μM 和 1：500 的终浓度。 注意：在溶液制备过程中剧烈涡旋对于确保染料、CD40 蛋白和伞形酮在 PBS 中的均匀分布至关重要。</li>
	<li>重复步骤3.2-3.3的操作，分别在35°C，40°C，45°C，50°C，55°C，60°C，65°C，70°C，75°C，80°C，85°C，90°C和95°C的温度下测量CD40-伞形酮复合物溶液的吸光度。 注：执行这些步骤是为了确定 CD40 蛋白和 CD40-伞形酮复合物的 Tm 值，旨在确定与伞形酮的最佳 CD40 蛋白结合浓度。</li>
</ol>

使用荧光高灵敏度检测化合物-蛋白质结合

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作者没有什么可透露的。

DSF，也称为热位移测定或热荧光测定，是一种用于检测样品中蛋白质的热变性过程的技术，通过监测测试样品或染料的荧光信号在缓慢的程序化温度升高期间的变化。DSF 最初由 Pantoliano14 建立，是一种高通量方法。主要步骤包括在计算机控制的加热板上升高温度，使用长波长紫外线灯发射激发光，并通过电荷耦合器件相形成相机捕获荧光染料与蛋白质结合产生的荧光信号强度变?...

Vicatia thibetica H. Boissieu是伞形植物科的一种植物，在藏药中常用，是五种基本成分（Polygonatum sibiricum Delar. ex Redoute， Asparagus cochinchinensis （Lour.）Merr、 Vicatia thibetica H. Boissieu、 Oxybaphus himalaicus Edgew. 和 Gymnadenia conopsea （L.） R. Br.）1.主要分布在我国西南地区，如云南西北部、川西、西藏等地区，其干根作为当归1的局部替代品。这种根以其香味而闻名，经常被用作炖菜调味料，而被称为西藏芹菜的叶子则有助于制作美味的菜肴。因此， 紫 薇不仅作为一种独特的药用植物具有重要意义，而且作为西藏的食物来源也具有重要意义。
国内外研究表明，硫苜蓿具有补血和气（力量或能量）的特性，有利于调节月经。它用于解决由心悸和血虚引起的月经不规则痛经症状，具有抗氧化调节身体免疫力等药理作用2。Vicatia thibetica 的酒精提取物已证明能够恢复环磷酰胺诱导的免疫功能低下小鼠的体重和器官指数。此外，它还增加了血清中超氧化物歧化酶的活性，降低了丙二醛的含量，表明抗氧化能力有所提高，对脂质过氧化有平衡作用2,3。同时，它增强了血细胞和血红蛋白的数量，这不仅客观地反映了人体的造血功能，而且在免疫系统中起着至关重要的作用2,3。
Umbelliferone的特点是针状结晶和苦味，分子量小，易挥发，可以用蒸汽蒸馏。易升华，在水中溶解度低，在有机物中的溶解度高。作为 Vicatia thibetica 的主要化学成分之一，伞形酮在氢化可的松诱导的免疫抑制小鼠模型中对细胞免疫、体液免疫和非特异性免疫具有免疫调节作用 4,5。
细胞表面分子 CD40 是肿瘤坏死因子受体超家族的成员，在免疫细胞中广泛表达6.其同源配体 CD154，也称为 CD40L，是一种由活化的 T 淋巴细胞表达的 II 型跨膜蛋白。CD40 激活具有上调树突状细胞 （DC） 和单核细胞表面共刺激分子表达的能力。该过程促进主要组织相容性复合物 （MHC） 分子的抗原呈递功能，并进一步激活 CD8+ T 细胞7。
巨噬细胞在肿瘤微环境的形成和调控中起着至关重要的作用，CD40的激活可以增强巨噬细胞对肿瘤微环境的重塑8。CD40信号激活显著影响B细胞的增殖和活化。当 B 细胞被 CD40 激活时，可以作为有效的抗原呈递细胞发挥作用。它们呈递抗原，产生效应 T 细胞活性，从而有助于抗肿瘤作用9。此外，肿瘤细胞中的CD40激活可以诱导细胞凋亡并抑制肿瘤生长10。CD40 主要通过控制非受体酪氨酸蛋白激酶的活性来转导信号，包括 Lyn、Fyn、Syk 等。此外，它还具有刺激 Bcl-xL、Cdk4 和 Cdk6 蛋白、激活 Rel/NF-kB 转录因子以及磷酸化 CG-2 和 PI3K10 的能力。
差示扫描荧光 （DSF） 方法被广泛用于评估各种环境条件（如缓冲液组成、温度和小分子配体）对蛋白质结构热稳定性的影响。DSF 常用的染料是一种橙色、环境敏感的疏水染料。在正常情况下，蛋白质结构被折叠，在内部隐藏了其疏水部分。随着温度的升高，蛋白质的疏水区域变得更加暴露，导致天然蛋白质结构逐渐分解。染料选择性地与这种暴露的蛋白质部分结合，放大其荧光。然后通过监测荧光信号检测11的变化来计算Tm值。在一定程度上，Tm 值的变化可以衡量由于突变、缓冲液变化或配体结合导致的蛋白质稳定性变化。此外，它可以指示蛋白质折叠过程中的结构改变12。这种方法提供精确的数据、宽广的温度范围、高灵敏度和最小的蛋白质样品损失13。
在这项研究中，使用荧光酶标仪而不是荧光定量聚合酶链反应 （PCR） 仪器进行荧光测定。这种改进使得在没有荧光定量PCR仪器的实验室中进行DSF检测，使方法不那么复杂，减少了仪器设置所需的步骤，从而简化了实验过程。但是，这种方法存在某些缺点。虽然复杂性降低了，但过程变得更加繁琐。在不同温度下进行手动荧光检测是必要的，并且无法实现从系统中自动连续收集荧光。因此，本研究利用DSF技术探索CD40蛋白与伞形酮之间的相互作用，为藏医分子机制提供了新的见解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

研究不同分子之间的相互作用是理解疾病发病机制和筛选药物靶点的关键方面。Umbelliferone 是藏药 Vicatia thibetica 中的一种活性成分，具有未知机制的免疫调节作用。CD40蛋白是免疫反应的关键靶标。因此，本研究采用差示扫描荧光技术原理，利用荧光酶标记物分析CD40蛋白与伞形酮之间的相互作用。最初，通过实验验证了蛋白质荧光橙染料的稳定性，并确定了最佳稀释比例为1：500。随后观察到CD40蛋白的温度熔解（Tm）值随着浓度的增加而趋于降低。有趣的是，发现CD40蛋白与伞形酮之间的相互作用增强了CD40蛋白的热稳定性。这项研究代表了使用荧光微孔板和荧光染料检测小分子化合物和蛋白质结合潜力的首次尝试。该技术具有灵敏度和准确度高的特点，在蛋白质稳定性、蛋白质结构和蛋白质-配体相互作用方面取得了可观的进展，从而促进了进一步的研究和探索。

Vicatia thibetica H. Boissieu, a plant in the umbelliferous family, is commonly used in Tibetan medicine and represents one of the essential components of the five basic ingredients (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew., and Gymnadenia conopsea (L.) R. Br.)1. Mainly distributed in southwest China, such as northwest Yunnan, western Sichuan, Tibet, and other areas, its dried root serves as a local substitute for Angelica1. The root, known for its fragrance, is often utilized as a stew seasoning, and the leaves, referred to as Tibetan celery, contribute to delectable dishes. Thus, Vicatia thibetica holds significance not only as a unique medicinal plant but also as a food source in Tibet.
Both domestic and international research indicates that Vicatia thibetica possesses blood-replenishing and qi (power or energy)-invigorating properties, beneficial for regulating menstruation. It is employed to address symptoms of irregular menstrual dysmenorrhea caused by palpitation and blood deficiency, exhibiting pharmacological effects such as antioxidant regulation of body immunity2. The alcohol extract of Vicatia thibetica has demonstrated the ability to restore body mass and organ index in immunocompromised mice induced by cyclophosphamide. Additionally, it increases the activity of superoxide dismutase in serum and reduces the content of malondialdehyde, suggesting an improvement in antioxidant capacity and a balancing effect on lipid peroxidation2,3. Simultaneously, it enhances the number of blood cells and hemoglobin, which not only objectively reflects the body&#39;s hematopoietic function but also plays a crucial role in the immune system2,3.
Umbelliferone, characterized by acicular crystals and a bitter taste, possesses a small molecular weight, is volatile, and can be distilled with steam. It sublimates easily, has low solubility in water, and high solubility in organic matter. As one of the main chemical components of Vicatia thibetica, umbelliferone exhibits immunomodulatory effects on cellular immunity, humoral immunity, and non-specific immunity in hydrocortisone-induced immunosuppression mouse models4,5.
The cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed in immune cells6. Its homologous ligand, CD154, also known as CD40L, is a type II transmembrane protein expressed by activated T lymphocytes. CD40 activation has the capacity to up-regulate the expression of co-stimulatory molecules on the surface of dendritic cells (DC) and monocytes. This process promotes the antigen presentation function of major histocompatibility complex (MHC) molecules and further activates CD8+ T cells7.
Macrophages play a crucial role in the formation and regulation of the tumor microenvironment, and CD40 activation can enhance the remodeling of the tumor microenvironment by macrophages8. CD40 signal activation significantly influences the proliferation and activation of B cells. B cells, when activated by CD40, can function as effective antigen-presenting cells. They present antigens, generate effector T cell activity, and thereby contribute to anti-tumor effects9. Moreover, CD40 activation in tumor cells can induce apoptosis and inhibit tumor growth10. CD40 primarily transduces signals by controlling the activity of non-receptor tyrosine protein kinases, including Lyn, Fyn, Syk, and others. Additionally, it has the ability to stimulate Bcl-xL, Cdk4, and Cdk6 proteins, activate Rel/NF-kB transcription factors, and phosphorylate CG-2 and PI3K10.
The Differential Scanning Fluorescence (DSF) method is widely employed to assess the impact of various environmental conditions, such as buffer composition, temperature, and small molecule ligands, on the thermal stability of protein structures. The commonly utilized dye for DSF is an orange, environmentally sensitive, hydrophobic dye. Under normal conditions, the protein structure is folded, concealing its hydrophobic segment internally. As the temperature increases, the protein&#39;s hydrophobic region becomes more exposed, resulting in a gradual breakdown of the natural protein structure. The dye selectively binds to this exposed protein portion, amplifying its fluorescence. Tm values are then calculated by monitoring changes in the fluorescence signal detection11. To a certain extent, variations in Tm values can gauge shifts in protein stability due to mutations, changes in buffers, or ligand binding. Furthermore, it can indicate structural alterations during the protein folding process12. This approach offers precise data, a broad temperature range, high sensitivity, and minimal protein sample loss13.
In this study, fluorescence determination was conducted using a fluorescent microplate reader instead of a fluorescence quantitative polymerase chain reaction (PCR) apparatus. This modification enables DSF detection in laboratories lacking a fluorescent quantitative PCR instrument, making the method less complex and reducing the steps required for instrument setup, thereby simplifying the experimental process. However, there are certain drawbacks to this approach. While the complexity is reduced, the procedure becomes more cumbersome. Manual fluorescence detection at different temperatures is necessary, and automatic and continuous collection of fluorescence from the system cannot be achieved. Thus, this study utilized the DSF technique to explore the interaction between CD40 protein and umbelliferone, providing novel insights into the molecular mechanisms of Tibetan medicine.

作者聚焦：简化 PCR 方法以提高可及性和效率

通过差示扫描荧光检测CD40蛋白-umbellefone相互作用

Our protocol provides a significant advantage by allowing use without specialized equipment. Unlike traditional fluorescence quantitative PCR methods, it simplifies the experimental process, requiring fewer steps and reducing overall complexity, making it safer and easier to conduct. This experiment requires the operator to be very familiar with the experimental procedure and the fluorescent microplate reader, and not to waste too much time during the solution heating and the fluorescence detection.We will focus on the molecular mechanism by which umbelliferone binds to CD14 protein to produce immune effects.

橙色染料在室温和高温下均在 Ex = 470 nm 和 Em = 570 nm 处始终表现出稳定的荧光激发。确定了1：500的最佳稀释比例（图2A，B）。当CD40蛋白浓度低于15μg/ mL时，Tm值的检测被证明是具有挑战性的（图3A，B）。然而，在浓度为15μg/ mL时，可检测到51.82°C的稳定Tm值，随着CD40蛋白浓度的增加，该值降至45.79°C（图3C，D</st...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

该协议描述了一种用于检测化合物和蛋白质分子之间结合的独特方法，具有最小的蛋白质样品损失和高数据准确性的优点。

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。

detection-cd40-protein-umbelliferone-interaction-via-differential

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

356 次观看.  University of Tibetan Medicine. 我们的协议提供了一个显着的优势，允许在没有专门设备的情况下使用。与传统的荧光定量PCR方法不同，它简化了实验过程，需要更少的步骤并降低了整体复杂性，使其更安全、更易于进行。这个实验要求操作者对实验程序和荧光酶标仪非常熟悉，不要在溶液加热和荧光检测过程中浪费太多时间。我们将重点介绍伞形酮与 CD14 蛋白结合产生免疫效应的分子机制。