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TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts

Neural Regeneration Research 2019年第10期14卷 1765-1771页

作者： Mei-Ge Zheng Wen-Yuan Sui Zhen-Dan He Yan Liu Yu-Lin Huang Shu-Hua Mu Xin-Zhong Xu Ji-Sen Zhang Jun-Le Qu Jian Zhang Dong Wang Department of Orthopedics The Seventh Hospital of Sun Yat-sen UniversityShenzhenGuangdong ProvinceChina Department of Orthopedics The Second Hospital of Anhui Medical UniversityHefeiAnhui ProvinceChina School of Medicine Shenzhen UniversityShenzhenGuangdong ProvinceChina Department of Scientific Research The Seventh Hospital of Sun Yat-sen UniversityShenzhenGuangdong ProvinceChina Psychology & Social College of Shenzhen University ShenzhenGuangdong ProvinceChina Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Optoelectronic EngineeringShenzhen UniversityShenzhenGuangdong ProvinceChina

We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the regeneration and functional recovery of the peripheral nerve.In the present study,we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts.Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA,TrkA-shRNA or the respective control.The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect.Then,8 weeks after surgery,hematoxylin and eosin staining showed that compared with the control groups,the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged,whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group.Western blot assay showed that compared with the control groups,the TrkA overexpressing group had higher expression of the myelin marker,myelin basic protein and the axonal marker neurofilament 200.The TrkA overexpressing group also had higher levels of various signaling molecules,including TrkA,pTrkA(Tyr490),extracellular signal-regulated kinases 1/2(Erkl/2),pErk1/2(Thr202/Tyr204),and the anti-apoptotic proteins Bcl-2 and Bcl-xL.In contrast,these proteins were downregulated,while the pro-apoptotic factors Bax and Bad were upregulated,in the TrkA-shRNA group.The levels of the TrkA effectors Akt and pAkt(Ser473)were not different among the groups.These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway.All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University,China in December 2014(approval No.AEWC-2014-001219).

关键词： nerve regeneration bone marrow stromal stem cells tropomyosin receptor kinase A receptor lentiviral vector shRNA extracellular signal-regulated protein kinases 1/2 Bcl-2 nerve grafts peripheral nerve regeneration survival neural regeneration

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Adipose-derived stem cells modified by BDNF gene rescue erectile dysfunction after cavernous nerve injury

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Neural Regeneration Research 2020年第1期15卷 120-127页

作者： Mei Yang Jiang-Yang Sun Cheng-Cheng Ying Yong Wang Yong-Lian Guo Department of Endocrinology Tongren Hospital of Wuhan University(Wuhan Third Hospital)WuhanHubei ProvinceChina Department of Hepatobiliary Surgery The Central Hospital of WuhanTongji Medical CollegeHuazhong University of Science and TechnologyWuhanHubei ProvinceChina Department of Urology The Central Hospital of WuhanTongji Medical CollegeHuazhong University of Science and TechnologyWuhanHubei ProvinceChina

Cavernous nerve injury is the main cause of erectile dysfunction following radical prostatectomy.The recovery of erectile function following radical prostatectomy remains challenging.Our previous studies found that injecting adipose-derived stem cells(ADSCs)into the cavernosa could repair the damaged cavernous nerves,but the erectile function of the treated rats could not be restored to a normal level.We evaluated the efficacy of ADSCs infected with a lentiviral vector encoding rat brain-derived neurotrophic factor(lenti-rBDNF)in a rat model of cavernous nerve injury.The rats were equally and randomly divided into four groups.In the control group,bilateral cavernous nerves were isolated but not injured.In the bilateral cavernous nerve injury group,bilateral cavernous nerves were isolated and injured with a hemostat clamp for 2 minutes.In the ADSCGFP and ADSCrBDNF groups,after injury with a hemostat clamp for 2 minutes,rats were injected with ADSCs infected with lenti-GFP(1×106 in 20μL)and lenti-rBDNF(1×106 in 20μL),respectively.Erectile function was assessed 4 weeks after injury by measuring intracavernosal pressures.Then,penile tissues were collected for histological detection and western blot assay.Results demonstrated that compared with the bilateral cavernous nerve injury group,erectile function was significantly recovered in the ADSCGFP and ADSCrBDNF groups,and to a greater degree in the ADSCrBDNF group.Neuronal nitric oxide synthase content in the dorsal nerves and the ratio of smooth muscle/collagen were significantly higher in the ADSCrBDNF and ADSCGFP groups than in the bilateral cavernous nerve injury group.Neuronal nitric oxide synthase expression was obviously higher in the ADSCrBDNF group than in the ADSCGFP group.These findings confirm that intracavernous injection with ADSCs infected with lenti-rBDNF can effectively improve erectile dysfunction caused by cavernous nerve injury.This study was approved by the Medical Animal Care and Welfare Committee of Wuhan University,China(approval No.2017-1638)on

关键词： adipose-derived stem cells brain-derived neurotrophic factor cavernous nerve injury erectile dysfunction infection intracavernous injection lentiviral vector neuronal nitric oxide synthase radical prostatectomy

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On the development of optical peripheral nerve interfaces

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Neural Regeneration Research 2019年第3期14卷 425-436页

作者： Hans E.Anderson Richard F.ff.Weir Department of Bioengineering University of Colorado Denver|Anschutz Medical Campus

Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in the world. These techniques are both dependent on a robust interface with the peripheral nerve. Current methods for interfacing with the peripheral nerve tend to suffer from low specificity, high latency and insufficient robustness for a chronic implant. An optical peripheral nerve interface may solve some of these problems by decreasing invasiveness and providing single axon specificity. In order to implement such an interface three elements are required:(1) a transducer capable of translating light into a neural stimulus or translating neural activity into changes in fluorescence,(2) a means for delivering said transducer and(3) a microscope for providing the stimulus light and detecting the fluorescence change. There are continued improvements in both genetically encoded calcium and voltage indicators as well as new optogenetic actuators for stimulation. Similarly, improvements in specificity of viral vectors continue to improve expression in the axons of the peripheral nerve. Our work has recently shown that it is possible to virally transduce axons of the peripheral nerve for recording from small fibers. The improvements of these components make an optical peripheral nerve interface a rapidly approaching alternative to current methods.

关键词： peripheral nerve interfaces optogenetics optical neural interface optical peripheral nerve interface GCaMP ArcLight adenoassociated viral vector lentiviral vectors viral vectors implantable microscopy

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E2F-1 overexpression inhibits human gastric cancer MGC-803 cell growth in vivo

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World Journal of Gastroenterology 2015年第2期21卷 491-501页

作者： Wei-Yuan Wei Lin-Hai Yan Xiao-Tong Wang Lei Li Wen-Long Cao Xiao-Shi Zhang Ze-Xu Zhan Han Yu Yu-Bo Xie Qiang Xiao Department of Surgery The First Affiliated Hospital of Guangxi Medical University Department of Surgery People’s Hospital of Guangxi Zhuang Autonomous Region Department of Anaesthesiology The First Affiliated Hospital of Guangxi Medical University

AIM: To evaluate the influence of E2F-1 on the growth of human gastric cancer(GC) cells in vivo and the mechanism involved. METHODS: E2F-1 recombinant lentiviral vectors were injected into xenograft tumors of MGC-803 cells in nude mice, and then tumor growth was investigated. Overexpression of transcription factor E2F-1 was assessed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting analysis. Apoptosis rates were determined using a terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) assay. Expression levels of certain cell cycle regulators and apoptosis-related proteins, such as Bax, survivin, Bcl-2, cyclin D1, S-phase kinaseassociated protein 2, and c-Myc were examined by Western blotting and RT-PCR. RESULTS: Xenograft tumors of MGC-803 cells in nude mice injected with E2F-1 recombinant lentiviral vectors stably overexpressed the E2F-1 gene as measured by semi-quantitative RT-PCR(relative m RNA expression: 0.10 ± 0.02 vs 0.05 ± 0.02 for control vector and 0.06 ± 0.03 for no infection; both P lentiviral vectors significantly inhibited the growth of human GC xenografts(2.81 ± 1.02 vs 6.18 ± 1.15 in control vector infected and 5.87 ± 1.23 with no infection; both P lentiviral vector-mediated E2F-1 overexpression increased theexpression of Bax and suppressed survivin, Bcl-2, cyclin D1, Skp2, and c-Myc expression in tumor tissue.CONCLUSION: E2F-1 inhibits growth of GC cells via regulating multiple signaling pathways, and may play an important role in targeted therapy for GC.

关键词： E2F-1 Gastric cancer lentiviral vector Mouse model

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STUB1基因RNA干扰慢病毒载体的构建与鉴定

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浙江大学学报（医学版） 2010年第6期39卷 623-627页

作者：赵虹张惊宇徐万海杨子超赵庆杰哈尔滨医科大学附属第四医院神经内科黑龙江哈尔滨150001 哈尔滨医科大学附属第一医院神经内科黑龙江哈尔滨150001 哈尔滨医科大学附属第四医院泌尿外科黑龙江哈尔滨150001

目的:构建人STUB1基因RNA干扰(RNA interference,RNAi)慢病毒表达载体并进行鉴定。方法:针对筛选确定的人STUB1基因RNAi有效靶点序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeI和EcoRI酶切后的pMagic 4.0载体连接,产生短发卡RNA... 详细信息

目的:构建人STUB1基因RNA干扰(RNA interference,RNAi)慢病毒表达载体并进行鉴定。方法:针对筛选确定的人STUB1基因RNAi有效靶点序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeI和EcoRI酶切后的pMagic 4.0载体连接,产生短发卡RNA慢病毒载体。PCR筛选阳性克隆,测序鉴定,并包装成慢病毒颗粒。结果:PCR鉴定与DNA测序证实,合成的含STUB1 shRNA慢病毒载体寡核苷酸链插入正确。STUB1 shRNA慢病毒载体在293T细胞中成功包装成慢病毒颗粒。结论:成功构建人STUB1基因RNAi慢病毒载体以及包装成功慢病毒颗粒,为研究STUB1在胶质瘤发生发展过程中相关信号通路的作用,提供了稳定感染细胞的载体。

关键词：基因 RNA干扰慢病毒载体构建与鉴定 RNA interference lentiviral vector 慢病毒颗粒慢病毒表达载体构建人包装 RNAi 胶质瘤发生寡核苷酸链 shRNA DNA测序载体连接阳性克隆信号通路筛选确定合成

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Gene editing for corneal disease management

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World Journal of Translational Medicine 2016年第1期5卷 1-13页

作者： Sudhanshu P Raikwar Apoorva S Raikwar Shyam S Chaurasia Rajiv R Mohan Department of Veterinary Medicine and Surgery College of Veterinary MedicineUniversity of Missouri Harry S.Truman Memorial Veterans’ Hospital Department of Microbiology Roy J.and Lucille A.Carver College of MedicineUniversity of Iowa Mason Eye Institute School of MedicineUniversity of Missouri Ophthalmology and Molecular Medicine One-health One-Medicine Ophthalmology and Vision ResearchUniversity of Missouri

Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.

关键词： Adeno-associated virus Clustered Regularly-Interspaced Short Palindromic Repeats associated protein 9 Cornea Clustered regularly interspaced short palindromic repeat Double strand breaks Gene editing sgRNA Gene targeting Homology directed repair Homologous recombination Indels lentiviral vector Protospacer-adjacent motif Transcription activator like effector nucleases Zinc finger nucleases

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Inhibition of SW620 human colon cancer cells by upregulating mi RNA-145

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World Journal of Gastroenterology 2016年第9期22卷 2771-2778页

作者： Chen Li Na Xu Yu-Qiang Li Yu Wang Zhi-Tu Zhu Biobank The First Affiliated Hospital of Liaoning Medical University Department of Radiology The First Affiliated Hospital of Liaoning Medical University Department of Oncology The First Affiliated Hospital of Liaoning Medical University

AIM: To investigate the targeted inhibition of proliferation and migration of SW620 human colon cancer cells by upregulating mi RNA-145(mi R-145).METHODS: Forty-five samples of colon cancer tissues and 45 normal control samples were obtained from the biological database of the First Affiliated Hospital of Liaoning Medical University. We performed quantitative analysis of mi R-145 and N-ras expression in tissues; reverse transcriptase polymerase chain reaction analysis of mi R-145 expression in SW620 colon cancer cells and normal colonic epithelial cells; construction of mi R-145 lentiviral vector and determination of mi R-145 expression in SW620 cells transduced with mi R-145 vector; analysis of the effect of mi R-145 overexpression on SW620 cell proliferation; analysis of the effect of mi R-145 overexpression on SW620 cell migration using a wound healing assay; and analysis of the effect ofmi R-145 on N-ras expression using Western blotting. RESULTS: mi R-145 expression was significantly downregulated in colon cancer tissues, with its expression in normal colonic tissues being 4-5-fold higher(two sample t test, P < 0.05), whereas N-ras expression showed the opposite trend. mi R-145 expression in SW620 cells was downregulated, which was significantly lower compared to that in colonic epithelial cells(two sample t test, P < 0.05). mi R-145 vector and control were successfully packaged; expression of mi R-145 in SW620 cells transduced with mi R-145 was 8.2-fold of that in control cells(two sample t test, P < 0.05). The proliferation of mi R-145-transduced SW620 cells was significantly decreased compared to control cells(two sample t test, P < 0.05). At 48 h in the wound healing experiment, the migration indexes and controls were(97.27% ± 9.25%) and(70.22% ± 6.53%), respectively(two sample t test, P < 0.05). N-ras expression in mi R-145-tranduced SW620 cells was significantly lower than others(one-way analysis of variance, P < 0.05). CONCLUSION: mi R-145 is important in inhibiting colon cancer cell proliferation and

关键词： mi RNA-145 Colorectal cancer Targeted therapy N-ras lentiviral vector

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Leukemogenic Fusion Gene (p190 BCR-ABL) Transduction into Hematopoietic Stem/Progenitor Cells in the Common Marmoset

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Open Journal of Blood Diseases 2012年第1期2卷 1-10页

作者： Yan Dong Seiichiro Kobayashi Yamin Tian Manabu Ozawa Takafumi Hiramoto Kiyoko Izawa Yuansong Bai Yasushi Soda Erika Sasaki Toshio Itoh Yoshiro Maru Satoshi Takahashi Kaoru Uchimaru Naoki Oyaizu Arinobu Tojo Chieko Kai Kenzaburo Tani Central Institute for Experimental Animals Kawasaki Japan Department of Laboratory Medicine Research Hospital Institute of Medical Science the University of Tokyo Tokyo Japan Department of Molecular Genetics Medical Institute of Bioregulation Kyushu University Fukuoka Japan Department of Pharmacology Tokyo Woman’s Medical University Japan Division of Molecular Therapy Institute of Medical Science the University of Tokyo Tokyo Japan Laboratory Animal Research Center Institute of Medical Science the University of Tokyo Tokyo Japan.

Patients with Philadelphia chromosome (p190 BCR-ABL fusion gene)-positive acute lymphoblastic leukemia have a poor prognosis despite intensive therapeutic intervention.In this study, we attempted to develop a leukemia nonhuman primate model that mimics various human systems. Hematopoietic stem/progenitor cells in the common marmoset were transduced with a lentiviral vector containing the p190 BCR-ABL fusion gene by ex vivo transduction or in vivo direct bone marrow injection. In the latter model, BCR-ABL gene expression was maintained for more than one and a half years. One marmoset unexpectedly developed myelofibrosis-like disease. However, none of the marmosets have developed leukemia to date. In conclusion, we successfully achieved sustained p190 BCR-ABL gene expression in vivo. However, a genetic mutation in addition to p190 BCR-ABL may be required for the malignant transformation of hematopoietic stem/progenitor cells in the common marmoset during the short observation period. This novel in vivo approach will help develop a marmoset leukemia model in the future.

关键词： Leukemia lentiviral Vector Myelofibrosis Common Marmoset

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Identification of host miRNAs that may limit human rhinovirus replication

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World Journal of Biological Chemistry 2014年第4期5卷 437-456页

作者： Victor Paky Bondanese Ana Francisco-Garcia Nicole Bedke Donna E Davies Tilman Sanchez-Elsner LGL Lab Ex LIO ENS Lyon Site Monod 69364 Lyon France Academic Unit of Clinical and Experimental Sciences University of Southampton Faculty of Medicine University Hospital Southampton Southampton SO16 6YD United Kingdom Immunocore Limited Milton Park Abingdon Oxon OX14 4RY United Kingdom

AIM: To test whether the replication of human rhinovirus(HRV) is regulated by micro RNAs in human bronchial epithelial cells.METHODS: For the present study, the human cell line BEAS-2B(derived from normal human bronchial epithelial cells) was adopted. DICER knock-down, by si RNA transfection in BEAS-2B cells, was performed in order to inhibit micro RNA maturation globally. Alternatively, antisense oligonucleotides(anti-mi Rs) were transfectedto inhibit the activity of specific micro RNAs. Cells were infected with HRV-1B. Viral replication was assessed by measuring the genomic viral RNA by reverse transcription quantitative polymerase chain reaction(RT-q PCR). Association between micro RNA-induced-silencing-complex and viral RNA was detected by Ago2 co-immunoprecipitation followed by RT-q PCR. Targetscan v.6 was used to predict micro RNA target sites on several HRV strains.RESULTS: Here, we show that micro RNAs affect replication of HRV-1B. DICER knock-down significantly reduced the expression of mature micro RNAs in a bronchial epithelial cell line(BEAS-2B) and in turn, increased the synthesis of HRV-1B RNA. Additionally, HRV-1B RNA co-immunoprecipitated with argonaute 2 protein, an important effector for micro RNA activity suggesting that micro RNAs bind to viral RNA during infection. In order to identify specific micro RNAs involved in this interaction, we employed bioinformatics analysis, and selected a group of micro RNAs that have been reported to be under-expressed in asthmatic bronchial epithelial cells and were predicted to target different strains of rhinoviruses(HRV-1B,-16,-14,-27). Our results suggest that, out of this group of micro RNAs, mi R-128 and mi R-155 contribute to the innate defense against HRV-1B: transfection of specific anti-mi Rs increased viral replication, as anticipated in-silico.CONCLUSION: Taken together, our results suggest that pathological changes in micro RNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovir

关键词： Human Rhinovirus Micro RNAs Mi R-155 Si RNA lentiviral transduction Antiviral innate immunity

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