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Chinese Academy of Medical Sciences and Peking Union Medical College

18 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Jing Xu 1, Mansoor Amiji 1
1Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

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Biology

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries
Yi Yu 1,2, Yuyan Xiong 1,2, Jean-Pierre Montani 2,3, Zhihong Yang 1,2, Xiu-Fen Ming 1,2
1Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg, 2Kidney Control of Homeostasis, Swiss National Centre of Competence in Research, 3System Physiology, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.−) respectively, in freshly isolated intact aortas of an obesity mouse model.

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Developmental Biology

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors
Wei Wen 1, Jian-Ping Zhang 1, Wanqiu Chen 2, Cameron Arakaki 2, Xiaolan Li 1,2, David Baylink 2, Gary D. Botimer 3, Jing Xu 1, Weiping Yuan 1, Tao Cheng 1,4,5,6,7, Xiao-Bing Zhang 1,2
1State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Division of Regenerative Medicine, Department of Medicine, Loma Linda University, 3Department of Orthopaedic Surgery, Loma Linda University, 4Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, 5Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, 6Collaborative Innovation Center for Cancer Medicine, 7Tianjin Key Laboratory of Blood Cell Therapy and Technology

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

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Medicine

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Guihao Chen 1, Yuejin Yang 1, Chuansheng Xu 2, Shuo Gao 2
1State Key Laboratory of Cardiovascular Disease, Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

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Education

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition
Zhenyi Jia 1, Jun Yang 1, Huanlong Qin 1,2
1Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 2Department of General Surgery, Shanghai Tenth People's Hospital Affiliated to Tongji University

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

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Environment

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle
Yan-Ru Zhang 1,2, Hai-Li Qiao 3, Li-Li Ren 1, Rong Wang 4, Peng-Fei Lu 1
1The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University, 2School of Forestry, Inner Mongolia Agricultural University, 3Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 4School of Forestry, Fujian Agriculture and Forestry University

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

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Medicine

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice
Fuliang Luo 1, Xin Wang 1, Xiaokang Luo 1, Bo Li 1, Desheng Zhu 2, Hansong Sun 3, Yue Tang 1
1Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2Laboratory Animal Center, Peking University, 3Center of Cardiac Surgery for Adults, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Centre for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

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Behavior

Assessment of Sexual Behavior of Male Mice
Zi-Wei Liu 1, Ning Jiang 2, Xue Tao 2, Xiao-Ping Wang 1, Xin-Min Liu 2, Shui-Yuan Xiao 3
1Department of Psychiatry, The Second Xiangya Hospital, Central South University, National Clinical Research Center for Mental Disorders and National Technology Institute on Mental Disorders, Hunan Key Laboratory of Psychiatry and Mental Health, 2Research Center for Pharmacology & Toxicology, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, 3Department of Social Medicine and Health Management, Xiangya School of Public Health, Central South University

This article describes how to perform sexual behavior tests in male mice.

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Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development
Yong Wang *1, Ding Zhang *2, Fang Zhou 2, Meijun Zhou 2, Qiujie Li 1, Jingyu Chen 3, Jun Yang 1,2
1Department of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Wuxi Lung Transplant Center, Wuxi People's Hospital affiliated to Nanjing Medical University

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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Medicine

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma
Chunlong Fu *1,2, Yaqun Zhang *1, Xin Wang 1, Shengcai Zhu 1, Huimin Hou 1, Shengjie Liu 1, Cheng Pang 1, Xinda Song 1, Jia Chen 1, Jianye Wang 1, Ming Liu 1,2
1(Urology) Beijing Hospital, National Center of Gerontology, The Joint Unit of the Chinese Academy of Medical Sciences, 2Graduate School of Peking Union Medical College, Beijing Hospital

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

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Neuroscience

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder
Rui Wang 1, Zi-tong Huang 3, Wen-kang Ren 1, Jiao Zhang 1, Yao Zhang 1, Bo Tan 3, Ping Huang 1,2, Hong-ying Cao 1,2
1School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, 2Dongguan & Guangzhou University of Chinese Medicine Cooperative Academy of Mathematical Engineering for Chinese Medicine, Guangzhou University of Chinese Medicine, 3School of Basic Medical Sciences, Guangzhou University of Chinese Medicine

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

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Immunology and Infection

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies
Sean Graham 1, Lei Gao 1, Rui Wang 1
1AbbVie, Cambridge Research Center

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

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Bioengineering

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy
Peyman Obeidy 1, Haoqing Wang 1,2,3, Mingqin Du 1, Huiqian Hu 1,4, Fang Zhou 1, Haoruo Zhou 5, Hao Huang 5, Yunduo Charles Zhao 1,2, Lining Arnold Ju 1,2,3
1School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, 2Charles Perkins Centre, The University of Sydney, 3Heart Research Institute, 4Department of Chemistry, The Hong Kong University of Science and Technology, 5School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering, The University of Sydney

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

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Medicine

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion
Ruijie Tang *1, Kaihao Wang *1, Yuyan Xiong 1, Jian Meng 2, Yuejin Yang 1
1State Key Laboratory of Cardiovascular Disease, Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 2State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

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Medicine

Laparoscopic Radical Antegrade Modular Pancreatosplenectomy via Dorsal-Caudal Artery Approach for Pancreatic Neck-Body Cancer
Zhantao Shen 1,2, Guihao Chen 1,2, Chunbao Zhu 2, Xiang Wu 1,2, Yifeng Liu 1,2, Zhangyuanzhu Liu 1,2, Zhijian Tan 1,2, Xiaosheng Zhong 1,2
1The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2The Second Clinical College of Guangzhou University of Chinese Medicine

With advancements in laparoscopic techniques, laparoscopic radical antegrade modular pancreatosplenectomy (L-RAMPS) has been widely recognized. However, owing to several technical difficulties in this procedure, the artery-first approach in L-RAMPS still remains uncommon. Here, we developed the dorsal-caudal artery approach for L-RAMPS, which might be safe and beneficial for pancreatic neck tumors.

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Neuroscience

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells
Sue Han *1, Xin Wang *1, Nicholas Cordero 1, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

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Neuroscience

A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice
Xiao-Wei Pang 1, Man Chen 1, Yun-Hui Chu 1, Yue Tang 1, Chuan Qin 1, Dai-Shi Tian 1
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

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Cancer Research

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression
Lulu Zhang *1, Siqi Zhang *2, Wenyu Wu 1, Xingkai Wang 2, Jieting Shen 2, Dongyuan Wang 4, Kuan Hu *2,3, Ming-Rong Zhang *3, Feng Wang *1, Rui Wang *2
1Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, 2State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Department of Advanced Nuclear Medicine Sciences, Institute for Quantum Medical Science, National Institutes for Quantum Science and Technology, 4Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

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