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Semaphorin 3A-hypoxia inducible factor 1 subunit alpha co-overexpression enhances the osteogenic differentiation of induced pluripotent stem cells-derived mesenchymal stem cells in vitro

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Chinese Medical Journal 2020年第3期133卷 301-309页

作者： Jing-Yi Li Ting-Ting Wang Chong Li Zhi-Fang Wang Shan Li Li Ma Li-Li Zheng Department of Plastic Surgery Capital Medical University Affiliated Beijing Tiantan HospitalBeijing 100050China Department of Endocrinology and Metabolism The First Affiliated Hospital of Zhengzhou UniversityZhengzhouHenan 450000China Department of Plastic Surgery China-Japan Friendship HospitalBeijing 100029China.

Background:Mesenchymal stem or stromal cells(MSCs)derived from the induced pluripotent stem cells(iPSCs)have uniform biological activity,which makes the clinical application of MSCs in bone repair possible.Culturing the iPSC-MSCs onto osteoconductive materials is a promising tissue engineering-based strategy in bone regeneration.The aim of this work was to evaluate the effects of semaphorin 3A(Sema3A)and hypoxia inducible factor 1 subunit alpha(HIF1α)co-overexpression on the survival and osteogenic differentiation of iPSC-MSCs.Methods:Sema3A and HIF1αwere linked together with the three(GGGGS;G,glycine;S,serine)peptide fragment,and their co-expression in iPSC-MSCs was mediated by a lentiviral vector.The fusion protein retained the immune reactivity for both Sema3A and HIF1αas determined with Western blotting.iPSC-MSCs were infected with overexpression lentivirus(oeLenti)as negative control,oeLenti-Sema3A,oeLenti-HIF1αor oeLenti-Sema3A-HIF1αlentiviruses.Results:Sema3A overexpression alone promoted the osteogenic differentiation of iPSC-MSCs(the activity and/or expression of osteoblast markers,such as alkaline phosphatase,osteopontin,and osteocalcin,were upregulated),and suppressed cell survival.The Sema3A-HIF1αfusion protein showed a comparable osteoconductive effect to that of Sema3A without reducing cell survival.We further seeded iPSC-MSCs modified by SemaA-HIF1αoverexpression onto hydroxyapatite(HA)scaffolds,and evaluated their growth and differentiation on this three-dimensional material.Additional data indicated that,as compared to iPSC-MSCs cultured in ordinary two-dimensional dishes,cells cultured in HA scaffolds grew(blank vs.HA scaffolds:0.83 vs.1.39 for survival)and differentiated better(blank vs.HA scaffolds:11.29 vs.16.62 for alkaline phosphatase activity).Conclusion:Modifying iPSC-MSCs with pro-osteogenic(Sema3A)and pro-survival(HIF1α)factors may represent a promising strategy to optimize tissue engineering-based strategy in bone repair.

关键词： Semaphorin 3A Hypoxia inducible factor 1 subunit alpha Induced pluripotent stem cells Mesenchymal stems Cell survival Osteogenic differentiation Hydroxyapatite scaffolds

Rapid construction of a whole-genome mutant library by combining haploid stem cells and inducible self-inactivating PiggyBac transposon

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Protein & Cell 2020年第6期11卷 452-457页

作者： Junjie Mao Kai Xu Jiabao Han Guihai Feng Ying Zhang Wei Li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for Stem Cell and Regeneration Chinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Dear Editor,Genome-scale screening is a powerful method used to explore phenotypes that are of interest,and numerous screening systems have been built for functional sites identification.Classical genetic screening in eukaryotes has been performed using chemical mutagens(Chen et al.,2000),transposon mediated gene trapping(Dupuy et al.,2005),and CRISPR-Cas9 system(Wang et al.,2014).

关键词： PiggyBac inducible inactivating

Prognostic value of hypoxia-inducible factor-1 alpha and prolyl 4-hydroxylase beta polypeptide overexpression in gastric cancer

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World Journal of Gastroenterology 2018年第22期24卷 2381-2391页

作者： Jun Zhang Yue Wu Yu-Hang Lin Shuai Guo Pei-Fang Ning Zhi-chao Zheng Yue Wang Yan Zhao Department of Gastric Cancer Liaoning Cancer Hospital and Institute (Cancer Hospital of China Medical University) Department of Emergency Sheng Jing Hospital of China Medical University Department of Pancreatic and Thyroid Surgery Sheng Jing Hospital of China Medical University Department of Pathology Liaoning Cancer Hospital and Institute (Cancer Hospital of China Medical University)

AIM To investigate the relationship between hypoxia-inducible factor-1α(HIF-1α), prolyl 4-hydroxylase beta(P4 HB) expression, and clinicopathologic parameters, as well as the prognostic value of these genes for patients with gastric cancer(Gc).METHODS Hypoxia is a critical factor that shapes the Gc microenvironment. In previous reports, we have demonstrated that P4 HB is a potential target of HIF-1α. In the present study, gene expression profiling interactive analysis(GEPIA) was used to analyze the relationship between P4 HB and hypoxia-associated genes. To this end, 428 Gc tissue samples were used to analyze the expression of HIF-1α and P4 HB via immunohistochemical staining. Patient samples were classified as having weak-expression or over-expression both in terms of HIF-1α and P4 HB. Correlations between biomarkers and clinicopathological factors were analyzed to predict survival. RESULTS P4 HB demonstrated a positive correlation with hypoxiaassociated genes(P 0.05).CONCLUSION Overexpression of HIF-1α and P4 HB is associated with poor prognosis in patients with Gc. Thus, these genes may be potential prognostic biomarker candidates in GC.

关键词： Gastric cancer Hypoxia inducible factor-1α Prolyl 4-hydroxylase beta polypeptide Overall survival Clinicopathological predictors Disease free survival

Investigating the function of Akt by tet-off inducible expression system

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Chinese Science Bulletin 2001年第3期46卷 222-225页

作者： Qiwei Zhai Hongbin Ji Zhongcheng Zheng Lanying Sun Xinyuan Liu Chinese Acad Sci Shanghai Inst Biol Sci Inst Biochem & Cell Biol Shanghai 200031 Peoples R China

A tet-off inducible cell line named BBT derived from BA/F3β cell line was constructed and the effect of this inducible expression system was significant when detected by tet-off responded luciferase reporter gene assay. Then tet-off responded Akt expression plasmid was transfected into BBT cells, and the stable cell lines were screened. The result of Northern blot showed that the expression of akt was significantly inducible. The clone with the best inducible effect was selected and named BBA for investigating the function of Akt. We found that Akt could significantly inhibit zinc-induced decrease of cell viability when assayed by MTT method. And the flow cytometric analysis showed that Akt could markedly repress zinc-induced apoptosis.

关键词： inducible expression system Akt apoptosis.

A report on infection dynamics of inducible clindamycin resistance of Staphylococcus aureus isolated from a teaching hospital in India

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Asian Pacific Journal of Tropical Biomedicine 2013年第2期3卷 148-153页

作者： Debasmita Dubey Shakti Rath Mahesh C.Sahu Subhrajita Rout Nagen K.Debata Rabindra N.Padhy Microbiology Department IMS & Sum HospitalSiksha 'O' Anusandhan University Central Research Laboratory IMS & Sum HospitalSiksha 'O' Anusandhan University

Objective:To investigate the infection of hospital-and community-acquired"erythromycininduced clindamycin resistant"strains or D-test positives of clinical isolates of Staphylococcus aureus(S.aureus)(with and without methicillin resistance)in a hospital.Methods:Strains of S.aureus isolated from clinical specimens were subjected to D-test and antibiotic profiling.Results:Of the total 278 isolates,140(50.35%)were D-test positives and the rest were D-test negatives.Further,of 140(100%)pesitives,87(62.14%)and 53(37.85%)strains were from males and females,respectively.Of 140(100%)pesitives,117(83.57%)were methicillin resistant S.aureus and23(16.42%)were methicillin sensitive S.aureus;of 140 strains,103(73.57%)strains front persons with and 37(26.42%)were without related infections;of 140 strains,91(65%)and 49(35%)were from hospital-and community-acquired samples,respectively.In 140 strains,118(84.28%)with comorbidities and 22(15.71%)without comorbidities cases were recorded;similarly,persons with prior antibiotic uses contributed 108(77.14%)and without 32(22.85%)positive strains.These binary data of surveillance were analyzed by a univariate analysis.It was evident that the prior antibiotic uses and comorbidities due to other ailments were the determinative factors in D-test positivity,corroborated by low P values,P=0.001 1 and 0.0024,respectively.All isolates(278)were resistant to17 antibiotics of nine groups,in varying degrees;the minimum of 28%resistance for vancomycin and the maximum of 97%resistance for gentamicin were recorded.Further,of 278 strains,only42(15.1%)strains were resistant constitutively to both antibiotics,erythromycin resistant and clindamycin resistant,while 45(16.2%)strains were constitutively sensitive to both antibiotics(erythromycin sensitive,and clindamycin sensitive).Further,of the rest 191(68.7%)strains were with erythromycin resistant and clindamycin resistant,of which only 140(50.35%)strains were D-test positives,while the rest 51(18.34%)strains were D-test negatives.Conclusions:In view o

关键词： Antibiotics Community-acquired D-test Erythromycin resistance Hospital-acquired inducible clindamycin resistance MRSA MSSA Staphylococcus aureus

Abnormal expression of inducible nitric oxide synthase in Duchenne muscular dystrophy muscles

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Chinese Science Bulletin 1998年第10期43卷 860-863页

作者： Minsheng Zhu Yuhua Fan Xiangyu Xu Gang Wang Yue Shen Ying Pan Shubiao Ding Nanjing Mil Inst Med Sci Nanjing 210002 Peoples R China Nanjing Children Hosp Dept Neurol Nanjing 210005 Peoples R China

Duchenne muscular dystrophy (DMD) is a fatal genetic disease for the youth and children. 8 biopsies of DMD patients were determined and demonstrated that the membrane_binding nitric oxide synthase was enriched in normal skeletal muscles and was little in DMD muscles. The results from Western blot and immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was overexpressed in DMD muscle fibers, while a small amount of highly localized iNOS can be found in normal fibers. Based on these findings, it is proposed that the mechanism of progressive injury in DMD muscle might be associated with the abnormal expression of iNOS.

关键词： Duchenne muscular dystrophy inducible nitric oxide synthase human.

Requirement for endogenous heat shock factor 1 in inducible nitric oxide synthase induction in murine microglia

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中国药理学通报 2015年第B11期31卷 76-77页

作者： LUXu TONGLi-juan WANGJi-li ZHANGWei HUANGChao DeptofPharmacology SchoolofPharmacynatongUniversityNantongJiangsu226001

Aim inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of KB- α （IKB-α） -nuclear factor-KB （NF-KB） cascade, whereas interferon-γ （IFN-γ） acts through Janus kinase （ JAK）- signal transducer and activator of transcription 1 （ STAT1 ） signals. Heat shock factor 1 （ HSF1 ）, a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6）. But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide （NO） content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation （CHIP） was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.

关键词： heat shock factor 1 lipopolysaccharide interferon--y inducible nitric oxide synthase nuclear factor-KB signal transducer and activator of transcription 1

Quercetin modulates iron homeostasis and iNOS expression of splenic macrophages in a rat model of iron deficiency anemia

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Chinese Journal of Natural Medicines 2018年第8期16卷 580-589页

作者： Maryam Mazhar Nurul Kabir Shabana U Simjee Dr. Panjwani Center for Molecular Medicine and Drug Research International Centerjbr Chemical and Biological SciencesUniversity of Karachi Karachi 75270 Pakistan H.E.J. Research Institute. for Chemistry International CenterJor Chemical and Biological Sciences University of Karachi Karachi 75270 Pakistan

Iron deficiency anemia is one of the most common micronutrient deficient conditions around the globe with various consequences,including the weakened immune system.Quercetin is widely distributed bioflavonoid;it has been debated for its dual roles in iron regulation.Quercetin-iron interaction in the body is a complex mechanism which has not been completely understood.The present study aimed to investigate the effect of quercetin on iron supplementation in iron deficiency anemia and on i NOS expression in splenic macrophages.The rat model of iron deficiency anemia was induced by feeding low iron diet to weanling rats for 20 days.The animals were then administered with ferrous sulfate,quercetin,and their combination for 30 days.Blood parameters,histopathological analysis,iron storage,CD68,i NOS and SLC40 expression in rat spleen were investigated.Our results showed that quercetin regulated iron absorption,despite SLC40 down-expression,indicating possible alternate route of iron transport,and that quercetin modulated i NOS production in splenic macrophages..

关键词： Iron deficiency anemia inducible nitric oxide synthase Macrophages Spleen Quercetin

Overexpression of p27^(KIP1)induced cell cycle arrest in G_1 phase and subsequent apoptosis in HCC-9204 cell line

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World Journal of Gastroenterology 2000年第4期6卷 513-521页

作者： Jiang Li Xin Ke Yang Xin Xin Yu Meng Liang Ge Wen Liang Wang Jie Zhang Yun De Hou Department of Pathology,Fourth Military Medical University,Xi’an 710033,Shaanxi Province,China State Key Laboratory for Molecular Virology and Genetic Engineering,Beijing 100052,China Department of Dermatology,Beijing Hospital,Beijing 100016,China Institute of Radiation Medicine,Beijing 100085,China 第四军医大学陕西西安中国预防医学科学院病毒所北京中国预防医学科学院病毒所分子病毒基因工程国家重点实验室北京北京医院北京解放军军事医学科学院放射医学所北京

AIM We have previously reported that inducibleover-expression of Bak may prolong cell cycle inG1 phase and lead to apoptosis in HCC-9204 cells.This study is to investigate whether p27KIP1playsan important role in this process.METHODS In order to elucidate the exactfunction of p27KIP1in this process,a zinc induciblep27KIP1stable transfectant and transient p27KIP1-GFP fusion transfectant were constructed.Theeffects of inducible,p27KIP1on cell growth,cellcycle arrest and apoptosis were examined in themock,control pMD vector,and pMD-KIP1transfected HCC-9204 cells.RESULTS This p27KIP1-GFP transfectant maytransiently express the fusion gene.The cellgrowth was reduced by 35% at 48 h of p27KIP1induction with zinc treatment as determined bytrypan blue exclusion assay.These differencesremained the same after 72 h of p27KIP1expression,p27KIP1caused cell cycle arrest after24 h of induction,with 40% increase in G1population.Prolonged p27KIP1expression in thiscell line induced apoptotic cell death reflected byTUNEL assay.Fourty-eight h and 72 h of p27KIP1expression showed a characteristic DNA ladder onagarose gel electrophoresis. CONCLUSION Bak may induce cell cycle arrest inG1 phase through upregulating expression ofp27KIP1and subsequently lead to apoptosis inHCC-9204 cells.The p27KIP1-GFP fusion proteincan be transiently expressed in HCC-9204 cells.The inducible p27KIP1-expressing cell line providesa model to assess p27KIP1function.

关键词： p27KIP1 apoptosis cell cycle inducible expression system carcinoma hepatocellular liver neoplasms