Name: exploring the two herb combination strategy to treat injured pc12 cells
Uploaded: 2022-11-18T14:40:00
Description: Watch this Scientific Journal Video about Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells at JoVE.com

本研究由四川省科技厅重点研发项目（2020YFS0325）资助。

1. 试剂的制备
<ol><li>通过在 125 mL 无菌培养瓶中加入 90 mL DMEM 高糖培养基、10 mL 胎牛血清 （FBS） 和 1 mL 青霉素-链霉素溶液来制备完整培养基。混合完整的培养基，并在封闭条件下储存在4°C。</li>
	<li>通过在 10 mL DMEM 无糖培养基（完全溶解）中加入 0.02 g CoCl 2 粉末（准确称量）来制备 CoCl2 溶液，以获得 8.4 mM 储备溶液。用0.22μm细菌过滤器过滤溶液，密封，并将其储?...

<ol>
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现代临床实践中治疗脑缺血的理想药物尚缺乏2.在补气、活血化血法的指导下，Ast、Eri等制剂在中医临床实践中联合使用，取得了良好的综合优势13、14、15。大量研究表明，Ast可以改善血脑屏障的通透性，增加脑血流量，提高神经细胞耐受缺氧和抵抗氧自由基损伤的能力，并能显著改善神经功能障碍<sup class="x...

慢性脑缺血，由脑灌注不足引起，是中老年人常见病1。作为一种长期隐匿性缺血性疾病，可导致进行性或持续性神经功能障碍。主要病理机制包括细胞凋亡和氧化损伤，导致进行性或持续性神经功能障碍;这是阿尔茨海默病、血管痴呆等疾病的病理基础，严重影响患者的生活质量。然而，现代医学中仍然缺乏治疗慢性脑缺血的理想药物2。同时， 蒙古黄芪 （Ast）和 短黄芪 （Eri）的组合已广泛应用于中医（TCM）的临床实践中4。联合策略在促进脑缺血后神经功能恢复方面有明显改善，优于单一药物;但是，两种药物组合的剂量比例存在较大差异4。其有效成分和作用机制尚不明确，是制约其临床应用的关键问题。
已有研究证明了Ast注射液和Eri注射液制剂组合治疗大鼠脑缺血的协同作用。它可以上调p-Akt蛋白的表达，下调Bcl-2相关死亡启动子（BAD）蛋白5，6的表达，Bcl-2是B淋巴母细胞瘤2（Bcl-2）家族蛋白之一，具有促进细胞凋亡的作用。但其协同效应的物质基础和机制尚不清楚。此外，利用CoCl2组合无糖培养基建立了PC12细胞的损伤模型，筛选并定义了Ast和Eri的最佳成分组合7。
在这项研究中，通过使用受伤的PC12细胞模型筛选Ast和Eri的有效剂量。利用该模型结合均质设计方法筛选两种药物的最佳组合。细胞模型用于进一步评估制备组合和组分组合在受伤PC12细胞中的作用和机制的差异。该策略旨在通过PI3K/Akt/Nrf2信号通路探索两类组合对损伤PC12细胞的保护作用和调控机制，以确定最佳组合模式。本研究为优化中药最佳应用模式提供参考。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1300 series class II biosafety cabinet</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>1374</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>1334GR001</td>
			<td>MTT</td>
		</tr>
		<tr>
			<td>ACEA NovoExpress&#160;1.4.0</td>
			<td>ACEA Biosciences, Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>10176-2-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical flow cytometry</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>62-2-1810-1027-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V-FITC apoptosis detection kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>C1062L</td>
			<td></td>
		</tr>
		<tr>
			<td>Astragalus injection</td>
			<td>Heilongjiang Zhenbao Island Pharmaceutical Company</td>
			<td>A03190612144</td>
			<td>Ast injection</td>
		</tr>
		<tr>
			<td>Astragaloside A</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>84687-43-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bax</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>60267-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Bcl-2</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>26593-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubators</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>0816-2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-3</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>19677-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorogenic acid</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>327-97-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride hexahydrate</td>
			<td>Merck Biotechnology, Inc.</td>
			<td>7791-13-1</td>
			<td>CoCl2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>2020112701</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate dodecahydrate</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2020090101</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high sugar medium</td>
			<td>Thermo scientific Hyclone</td>
			<td>2110050</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM sugar free medium</td>
			<td>Beijing Solarbio life sciences Company</td>
			<td>2029548</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL luminous fluid</td>
			<td>Lianshuo Biological Company</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Haozhuang Hengping Scientific Instrument Co., Ltd., Shanghai, China</td>
			<td>FA1204</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis instrument</td>
			<td>Bio-Rad Laboratories (Shanghai) Co., Ltd</td>
			<td>1658026</td>
			<td></td>
		</tr>
		<tr>
			<td>Erigeron breviscapus capsule</td>
			<td>Yunnan Biogu Pharmaceutical Company</td>
			<td>Z53021671</td>
			<td>Eri capsule</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Four Seasons Institute of Biological Engineering</td>
			<td>20210402</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympms Corporation</td>
			<td>IX71-F32PH</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imager</td>
			<td>Bio-Rad Laboratories (Shanghai) Co., Ltd</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>SA00001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary antibody</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>SA00001- 2</td>
			<td></td>
		</tr>
		<tr>
			<td>IBM SPSS Statistics version 26.0</td>
			<td>International Business Machines Corporation, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.8.0</td>
			<td>National Institutes of Health, USA</td>
			<td>-</td>
			<td>imaging software</td>
		</tr>
		<tr>
			<td>Immunol&#160;fluorescence&#160;staining kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0196</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2021070901</td>
			<td></td>
		</tr>
		<tr>
			<td>Marker</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Perkin Elmer Corporate Management (Shanghai) Co.</td>
			<td>HH35L2018296</td>
			<td></td>
		</tr>
		<tr>
			<td>Motic Inverted microscope</td>
			<td>Nanda Scientific Instruments Co.</td>
			<td>AE2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2014081301</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat milk</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0216</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrf2</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>16396-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>P-Akt</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>66444-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>PC12 cells</td>
			<td>Chinese Academy of Sciences</td>
			<td>CBP60430</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Thermo scientific Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase protease inhibitor mixture</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P1045</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2015082901</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive oxygen species assay kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>RI-PA lysis solution</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scutellarin</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>27740-01-8</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE sample loading buffer, 5x</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0015L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter tips (0.22 &#181;m)</td>
			<td>Merck Biotechnology, Inc.</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>1115GR500&#160;</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo scientific Hyclone</td>
			<td>J190013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2021051301</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex oscillator</td>
			<td>OHAUS International Co., Ltd., Shanghai, China</td>
			<td>VXMNAL</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>66009-1-Ig</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring the two herb combination strategy to treat injured pc12 cells

针对中药联合治疗脑缺血的优势，我们研究了制剂组合和成分组合在疗效和机制上的差异，以探讨两种中药联合治疗损伤PC12细胞的策略。氯化钴（CoCl2）与无葡萄糖培养基结合诱导PC12细胞的氧化损伤。然后，选择 蒙古黄 芪（Ast）和 短黄芪 （Eri）注射液的最佳组合，在筛选其在PC12细胞上安全有效浓度后，按照统一的设计方法进行组合。此外，筛选的组分组合包括两种草药中的10μM黄芪甲苷A、40μM黄芩苷和75μM绿原酸。然后，采用MTT、膜联蛋白V-FITC/PI、免疫荧光和蛋白质印迹分析，评价制剂组合和组分组合对损伤PC12细胞的疗效和机制。结果表明，细胞促存活的最佳制备组合是Ast注射液和Eri胶囊，浓度为6：1.8（μM）。组分组合（10 μM黄芪甲苷A、40 μM黄芩苷和75 μM绿原酸）比制剂组合更有效。两种组合均显著降低细胞凋亡速率、半胱天冬酶-3的荧光强度和细胞内活性氧（ROS）水平;同时，他们上调了p-Akt/Akt、Bcl-2/Bax和Nrf2的表达水平。这些影响在组件组合中更为明显。总之，两种组合都可以抑制CoCl2 与无糖培养基联合对PC12细胞诱导的损伤，从而促进细胞存活。然而，组分组合相对于制备组合的效率可能是由于其对与氧化应激和细胞凋亡相关的PI3K/Akt/Nrf2信号通路的更强调节。

Ast注射液和Eri胶囊的最佳组合的筛选如图1所示。Ast注射液和Eri胶囊在正常PC12细胞上的细胞存活率如图1A所示。浓度大于12μM的Ast注射液（图1A）和浓度大于5μM的Eri胶囊（图1A）的细胞活力低于95%，表明最大无毒浓度分别为12μM和5μM。它们的细胞毒性大于单独使用的黄芪甲苷A和黄芩苷。Ast注射液和Eri胶囊对CoCl...

Watch this Scientific Journal Video about Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells at JoVE.com

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

该协议提供了两种草药的组合策略来治疗受伤的PC12细胞。该方案为优化中药最佳应用模式提供了参考。

探索两种草药组合策略治疗受损PC12细胞

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

本研究的方案提供了两种草药治疗缺氧损伤PC12细胞的组合策略，并为优化草药的最佳组合模式提供了参考。该技术可以提供一种简单、可靠、高效的体外实验方法，用于从中草药中筛选有效成分组合，对抗缺氧细胞。该策略也可用于筛选来自其他草药制剂组合的缺氧细胞的组分组合，并阐明其保护机制。首先，在补充有5%二氧化碳的培养箱中，每两到三天在37摄氏度下传代培养PC12细胞，以获得4至8的传代，用于所有后续实验。用一毫升0.25%胰蛋白酶处理70至80%汇合的PC12细胞，并在显微镜下观察细胞以进行细胞分离。然后通过加入三毫升完整培养基来停止胰蛋白酶消化。在15毫升离心管中以840×g离心转移细胞旋转五分钟。弃去上清液后，用完整的培养基重悬细胞沉淀，并将细胞悬液转移到1.5毫升微量离心管中。要计数细胞，请启动流式细胞术软件并在计数设置中选择细胞溶液的相应稀释倍数。加载细胞样本后单击密度图。将细胞群绕出远离 x 轴和 y 轴的圈子。右键单击图下方的数据表，然后选择 X、Y、计数和 Abs。计数以获得Abs.Count数据表下的细胞计数结果。用完全培养基将细胞浓度调节至每毫升约1乘以10至第5个细胞后，将每孔100微升细胞悬液加入96孔板中，并在37摄氏度和5%二氧化碳下孵育24小时。第二天，向上清液丢弃的平板中加入120微升每毫升MTT溶液120微升，并在37摄氏度下孵育4小时。孵育后，弃去上清液后，向每个孔中加入150微升DMSO。将板在涡旋振荡器中以每分钟 240 次的速度振荡 10 分钟。然后在酶标仪中测量490纳米处的吸光度。为了通过均质设计方法筛选药物的最佳组合，如前所述培养细胞，并用六种不同比例浓度的黄芪注射液和短囊治疗受伤的PC12细胞。为了评估两种最佳组合的保护作用，用两种类型的药物组合治疗受伤的PC12细胞。如前所述，孵育细胞24小时并计算细胞活力。将PC12细胞接种在12孔板中，并在37摄氏度下孵育24小时。药物治疗后，每孔用250微升胰蛋白酶处理细胞，并在室温下以840×g离心细胞5分钟。然后用500微升结合缓冲液重悬细胞沉淀。每组加入5微升膜联蛋白V-FITC和10微升碘化丙啶，一式三份，并在室温下在黑暗中孵育细胞15分钟，以使用流式细胞术检查细胞凋亡。在流式细胞仪软件中，单击“开始”菜单中的“自动补偿”，在“选择通道”中选择“FITC、PE”、“PerCP”和“APC”，然后选择“高空补偿”。然后在统计项目：中值中单击确定。单击密度图并使用前面演示的细胞计数方法圈出有效细胞群。使用 FITC 荧光作为 x 轴参数，将其他荧光作为 y 轴参数，创建密度图。单击“开始”菜单中的“补偿矩阵”和“溢出矩阵中的系数”。将PC12细胞接种到盖玻片上，一式三份放置在24孔板孔上，并在37摄氏度下孵育24小时。药物治疗后，用PBS冲洗盖玻片两次，每次5分钟，用4%多聚甲醛固定15分钟，并在室温下用0.5%Triton X-100透化20分钟。冲洗细胞后，在室温下用10%山羊血清封闭一小时。向每个盖玻片中加入200微升稀释的一抗半胱天冬酶-3，并在4摄氏度下孵育过夜。第二天，用TBST缓冲液洗涤三次，每次三分钟，并在室温下在黑暗中与200微升二抗孵育一小时。孵育后，用300微升每毫升0.5微克DAPI孵育盖玻片10分钟，并用TBST洗涤细胞三次，每次5分钟。在荧光显微镜下捕获盖玻片图像，并使用成像软件统计分析荧光强度。用PBS洗涤药物治疗的PC12细胞盖玻片三次，每次三分钟，并与400微升10微摩尔二氯二氢荧光素二乙酸酯在37摄氏度下孵育20分钟。用无血清DMEM再次洗涤细胞两次，每次三分钟。加入荧光淬灭剂，立即在荧光显微镜下捕获盖玻片图像，用成像软件计算相对荧光强度。用PBS在6孔板中洗涤药物治疗的PC12细胞三次，每次5分钟，并在每孔100微升的制备裂解缓冲液中在冰上孵育30分钟。裂解后，将细胞在4摄氏度下以16， 000×g离心20分钟以收集上清液。通过Bradford测定法检测蛋白质浓度后，在每个泳道中用25微克蛋白质运行12%SDS-PAGE电泳，并将凝胶转移到PVDF膜上。在室温下用5%脱脂牛奶封闭膜1.5小时，并在4摄氏度下与5毫升相应的一抗孵育24小时。用TBST孵育和洗涤膜三次后，每次10分钟，在室温下与5毫升二级HRP偶联抗体孵育2小时。最后，在TBST洗涤的膜上，按照制造商的说明加入化学发光HRP底物进行蛋白质条带检测。然后使用化学发光成像系统捕获图像，以β-肌动蛋白作为内部对照来量化蛋白质的灰度值。使用黄芪注射液时，正常PC12细胞的细胞活力低于95%，浓度大于12微摩尔，短囊大于5微摩尔。黄芪注射液可提高6-12微摩尔浓度范围内损伤PC12细胞的存活率，2-5微摩尔浓度范围内的短囊可提高存活率。而比例为6至1.8微摩尔的药物组合表现出最高的细胞活力。两种组合对损伤PC12细胞的细胞活力得到显著提升，且组分组合优于制备组合。细胞凋亡率显示，模型组早凋亡细胞、晚期细胞和总凋亡细胞的百分比显著高于正常组。与模型组相比，治疗组各分期凋亡细胞百分比显著降低。与模型组相比，各处理组半胱天冬酶-3的荧光强度显著降低，组分组合组的荧光强度相对较低。基因Akt、Bcl-2和Bax的相对表达表明，组分组合在促进细胞存活方面优于制剂组合，这与更强的抗细胞凋亡作用有关。与制剂组合相比，组分组合表现出更好的抗氧化损伤效果，Nrf2蛋白的表达显著提高。该方法可用于筛选和评估成分组合与草药组合中的其他潜在药理活性，例如抗炎，抗菌等。

Research

JoVE Journal

Medicine

A Strategy to Identify de Novo Mutations in 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, ...

科研

医学

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

结合消融房颤左心耳封堵

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

The Measurement and Treatment of Suppression in Amblyopia

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 ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization

双相位锥束CT看看，到达，并在药物洗脱珠Transarterial化疗栓塞治疗原发性肝癌

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of Recombinant Proteins to Treat Chronic Pain

重组蛋白治疗慢性疼痛的研究进展

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

三维胶原基质支架植入作为肝脏再生策略

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

血管保留性显微外科纵向套叠血管表皮造口术治疗附睾梗阻性无精子症

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...