MM： To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes, METHODS： Hepatic progenitor cells and then mature hepatocytes from mou...MM： To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes, METHODS： Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem （ES） cells were obtained in a sequential manner, induced by valproic acid （VPA） and cytokines （hepatocyte growth factor, epidermal growth factor and insulin）, Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy, Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepa- tocytes, Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells, Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells.RESULTS： Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The dif- ferentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers,in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells to-wards hepatic lineages. CONCLUSION： Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.展开更多
Progression,relapse,and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell(CSC)population.This enigmatic subpopulation of cancer cells has...Progression,relapse,and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell(CSC)population.This enigmatic subpopulation of cancer cells has therefore emerged as promising therapeutic candidate.We earlier reported enrichment of CSC-like side population(SP)with increasing resistance towards Cisplatin and Paclitaxel either alone or in combination in epithelial ovarian cancer(EOC)cells.This SP population is a small proportion of the total population of cancer cells characterised with high expression of drug transporters,a unique feature of stem cells and thereby can be isolated through their efflux properties of DNA binding dyes.While the bulk non-SP(NSP)population of the cancer cells lack overexpression of the drug transporters and thus can be identified as the dye containing population.In this study,we show that increased expression of Runt related transcription factor 1(RUNX1)maintains undifferentiated state of CSC-like SP cells through upregulation of inhibitors of DNA binding/differentiation genes(ID1 and ID3)in late cisplatinpaclitaxel resistant cells.Higher RUNX1 expression was found to correlate with decreased median overall survival and disease-free survival in The Cancer Genome Atlas(TCGA)data set of high grade serous ovarian cancer(HGSOC)patients.The protein-protein interaction network analysis of 397 upregulated genes in RUNX1-high samples of TCGA data show significant enrichment of pathways known to negatively regulate CSC differentiation.Intriguingly RUNX1 inhibition not only induces CSC differentiation but also downregulates anti-apoptotic protein BCL2 in both SP and NSP cells and potentiates cytotoxic effects of Cisplatin-Paclitaxel in chemoresistant EOC cells.Inhibition of BCL2 through Venetoclax treatment,a small molecule BH3 mimic,sensitized these cells to platinum taxol treatment.Altogether,our data reveal new regulatory roles by RUNX1 to modulate CSC differentiation via ID1 and ID3 and to promote chemoresistance through BCL2 upregulation.展开更多
AIM:To investigate the expression and clinical relevance of inhibitor of differentiation(ID)proteins in biliary tract cancer.METHODS:ID protein expression was analyzed in129 samples from patients with advanced biliary...AIM:To investigate the expression and clinical relevance of inhibitor of differentiation(ID)proteins in biliary tract cancer.METHODS:ID protein expression was analyzed in129 samples from patients with advanced biliary tract cancer(BTC)(45 extrahepatic,50 intrahepatic,and 34 gallbladder cancers),compared to normal controls and correlated with clinical an pathological parameters.RESULTS:ID1-3 proteins are frequently overexpressed in all BTC subtypes analyzed.No correlation between increased ID protein expression and tumor grading,tumor subtype or treatment response was detected.Survival was influenced primary tumor localization(extrahepatic vs intrahepatic and gall bladder cancer,OS 1.5 years vs 0.9 years vs 0.7 years,P=0.002),by stage at diagnosis(OS 2.7 years in stageⅠv s 0.6 years in stageⅣ,P<0.001),resection status and response to systemic chemotherapy.In a multivariate model,ID protein expression did not correlate with clinical prognosis.Nevertheless,there was a trend of shorter OS in patients with loss of cytoplasmic ID4 protein expression(P=0.076).CONCLUSION:ID protein expression is frequently deregulated in BTC but does not influence clinical prognosis.Their usefulness as prognostic biomarkers in BTC is very limited.展开更多
The formation of osteolytic bone lesions is a key process for osteolytic cancer to metastasize to the bone and is under the control of a set of transcription factors. Recently, the inhibitor of differentiation 1 （Id1...The formation of osteolytic bone lesions is a key process for osteolytic cancer to metastasize to the bone and is under the control of a set of transcription factors. Recently, the inhibitor of differentiation 1 （Id1） has been linked with angiogenesis, tumorigenesis, metastasis and bone formation. However, the function of Id1 during the process of bone destruction caused by cancer in vivo has not yet been elucidated. We, therefore, examined whether and how Id1 affects the ability of cancer to form osteolytic lesion in vivo. The study used a lentiviral vector overexpressing short hairpin RNA （shRNA） targeting Id1 gene. PC3 cells, a prostate cancer cell line, were transduced with Id1 shRNA or negative control （NC） shRNA before implantation in BALB/c mice. Cells were implanted in a tibial injection model. Tumor formation in bone was monitored by X-ray. The relationship between parathyroid hormone-related protein （PTHrP）, an osteolytic factor, and Id1 was analyzed by using immunohistochemistry in tissue sections from osteolytic lesion of the BALB/c mice. Our results showed that Id1 shRNA delivery to PC3 cells by lentivirus caused efficient and stable Id1 gene silencing. In the intratibial model, PC3 cells produced primarily osteolytic lesions in the bone. Eleven of 14 mice in Id1 shRNA group but only 4 of 14 mice in the NC shRNA group developed osteolytic lesions with cortical destruction at 4th week. Mice treated with Id1 shRNA had larger tumor volume in the bone and larger cortical destruction. The expression of PTHrP protein in PC3 cells was not affected by Id1 knockdown in vivo. These results indicate that Id1 may down-regulate the ability of PC3 cells to form osteolytic lesions in vivo and the signal pathway needs to be further investigated.展开更多
AIM:To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer(NSCLC) cells and whether a hypoxia-inducible factor(HIF) inhibitor is able to suppress the process.METHODS:Human lung adenocar...AIM:To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer(NSCLC) cells and whether a hypoxia-inducible factor(HIF) inhibitor is able to suppress the process.METHODS:Human lung adenocarcinoma A549 cells and squamous carcinoma QG56 cells were cultured under normoxic(21%O_2) or hypoxic(4%or 1%O_2) conditions.The expression of the following genes were examined by reverse transcription-polymerase chain reaction,Western blotting and/or immunofluorescence:HIF-1α and HIF-2αsubunits;differentiation marker genes,namely surfactant protein C(SP-C)(type Ⅱ alveolar cell marker),CC10(type I alveolar cell marker) and aquaporin 5(AQP5)(Clara cell marker);and stem cell-associated genes,namely CD133,0CT4,and Musashi-1(MSI1).The tumor sphere-forming ability of the cells was evaluated by culturing them in serum-free growth factor-rich medium containing epidermal growth factor(EGF) and fibroblast growth factor(FGF).CD133 expression in hypoxic regions in A549 tumors was examined by double-immunostaining of tissue cryosections with an anti-2-nitroimidazole EF5 antibody and an anti-CD133 antibody.The metastatic ability of A549 cells was examined macroscopically and histologically after injecting them into the tail vein of immunocompromised mice.RESULTS:A549 cells primarily expressed SP-C,and QG56 cells expressed CC10 and AQP5.Exposure of A549 cells to hypoxia resulted in a marked downregulation of SP-C and upregulation of CD133,OCT4,and MSI1 in a time-dependent manner.Moreover,hypoxia mimetics,namely desferrioxamine and cobalt chloride,elicited similar effects.Ectopic expression of the constitutively active HIF-la subunit also caused the downregulation of SP-C and upregulation of CD133 and MSI1 but not OCT4,which is a direct target of HIF-2.Hypoxia enhanced the sphere-forming activity of A549 cells in serum-free medium containing EGF and FGF.Similarly,hypoxia downregulated the expression of CC10 and AQP5 genes and upregulated CD133,OCT4,and MSI1 genes in QG56 cells.TX-402(3-amino-2-quinoxalinecarbonitrile 1,4-dioxide),which is a small molecule inhibitor of the expression of HIF-1α and HIF-2αsubunits under hypoxic conditions,inhibited the upregulation of SP-C'and hypoxia-induced down-regulation of CD133,OCT4,and MSI1.Notably,TX-402 significantly suppressed the hypoxia-enhanced lung-colonizing ability of A549 cells.CONCLUSION:Hypoxia induces the de-differentiation of NSCLC cells into cancer stem cell-like cells,and HIF inhibitors are promising agents to prevent this process.展开更多
With the wide application of immunotherapy drug PD-1/PD-L1 inhibitor in China and the continuous clinical research,a major problem that we have to face is the immune-related pneumonia caused by PD-1/PD-L1.At present,w...With the wide application of immunotherapy drug PD-1/PD-L1 inhibitor in China and the continuous clinical research,a major problem that we have to face is the immune-related pneumonia caused by PD-1/PD-L1.At present,western medicine mainly treated it by hormone therapy,which may cause side effects,such as obesity,osteoporosis and osteonecrosis for hormone therapy by long-term,and increase the patients'pain.Under the guidance of the theory of syndrome differentiation,traditional Chinese medicine(TCM)advocates the methods of expelling wind and clearing away cold,resolving phlegm and relieving asthma,relieving heat from lung,and invigorating the spleen and tonifying the kidney,etc.Exact differentiation of symptoms and rational usage of drugs can play an important role in the early prevention and treatment of immune-related pneumonia,reflecting the important role of TCM in the prevention and treatment of side effects of new drugs.In order to provide an effective clinical reference for clinicians in the practice of using PD-1/PD-L1,this paper systematically reviewed the use of TCM in the treatment of immune-related pneumonia induced by PD-1/PD-L1 inhibitors.展开更多
BACKGROUND Cardiovascular diseases particularly myocardial infarction(MI)are the leading cause of mortality and morbidity around the globe.As cardiac tissue possesses very limited regeneration potential,therefore use ...BACKGROUND Cardiovascular diseases particularly myocardial infarction(MI)are the leading cause of mortality and morbidity around the globe.As cardiac tissue possesses very limited regeneration potential,therefore use of a potent small molecule,inhibitor Wnt production-4(IWP-4)for stem cell differentiation into cardiomyocytes could be a promising approach for cardiac regeneration.Wnt pathway inhibitors may help stem cells in their fate determination towards cardiomyogenic lineage and provide better homing and survival of cells in vivo.Mesenchymal stem cells(MSCs)derived from the human umbilical cord have the potential to regenerate cardiac tissue,as they are easy to isolate and possess multilineage differentiation capability.IWP-4 may promote the differentiation of MSCs into the cardiac lineage.AIM To evaluate the cardiac differentiation ability of IWP-4 and its subsequent in vivo effects.METHODS Umbilical cord tissue of human origin was utilized to isolate the MSCs which were characterized by their morphology,immunophenotyping of surface markers specific to MSCs,as well as by tri-lineage differentiation capability.Cytotoxicity analysis was performed to identify the optimal concentration of IWP-4.MSCs were treated with 5μM IWP-4 at two different time intervals.Differentiation of MSCs into cardiomyocytes was evaluated at DNA and protein levels.The MI ratmodel was developed.IWP-4 treated as well as untreated MSCs were implanted in the MI model,then the cardiac function was analyzed via echocardiography.MSCs were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiI)dye for tracking,while the regeneration of infarcted myocardium was examined by histology and immunohistochemistry.RESULTS MSCs were isolated and characterized.Cytotoxicity analysis showed that IWP-4 was non-cytotoxic at 5μM concentration.Cardiac specific gene and protein expression analyses exhibited more remarkable results in fourteen days treated group that was eventually selected for in vivo transplantation.Cardiac function was restored in the IWP-4 treated group in comparison to the MI group.Immunohistochemical analysis confirmed the homing of pre-differentiated MSCs that were labeled with DiI cell labeling dye.Histological analysis confirmed the significant reduction in fibrotic area,and improved left ventricular wall thickness in IWP-4 treated MSC group.CONCLUSION Treatment of MSCs with IWP-4 inhibits Wnt pathway and promotes cardiac differentiation.These pre-conditioned MSCs transplanted in vivo improved cardiac function by cell homing,survival,and differentiation at the infarcted region,increased left ventricular wall thickness,and reduced infarct size.展开更多
目的:探索分化抑制因子(inhibitor of differentiation,ID)家族在慢性髓系白血病(chronic myeloid leukemia,CML)中的表达和启动子甲基化水平,并分析其临床意义。方法:应用定量PCR及定量甲基化特异性PCR的方法检测2010年1月至2017年12...目的:探索分化抑制因子(inhibitor of differentiation,ID)家族在慢性髓系白血病(chronic myeloid leukemia,CML)中的表达和启动子甲基化水平,并分析其临床意义。方法:应用定量PCR及定量甲基化特异性PCR的方法检测2010年1月至2017年12月期间江苏大学附属人民医院就诊的非恶性血液病患者(对照组)和CML患者骨髓单个核细胞中ID2/ID3/ID4表达及ID4启动子甲基化水平,通过分组分析ID家族异常的临床意义。结果:ID2及ID3表达在CML患者中均呈现显著上调(P<0.001,P<0.05),而ID4表达在CML患者中呈现显著下调(P<0.01)。其中,接受者操作特征曲线分析揭示ID2表达可作为CML鉴别的潜在分子标志物(AUC=0.895,P<0.001)。CML患者中ID4启动子高甲基化概率显著高于对照组患者(P=0.001),且ID4启动子甲基化与ID4表达呈现负相关(r=-0.424,P=0.002)。通过分组分析发现ID2高表达较易发生于男性患者中(P=0.040);ID4低表达/高甲基化较易发生于加速/急变期患者(P=0.003,P<0.001)。此外,CML加速/急变期患者ID4表达水平低于慢性期患者(P<0.001),而ID4甲基化水平高于慢性期患者(P<0.001)。通过单因素及多因素Logistic回归分析发现ID4高甲基化是CML患者疾病进展的独立危险因素(P=0.007)。结论:ID家族在CML患者中表达态势不同,其中ID2/ID3表达上调;而ID4表达下调,与ID4启动子高甲基化相关。ID4表达/甲基化与CML疾病进展相关,其中ID4甲基化可能是CML疾病进展的独立危险因素。展开更多
目的探讨分化抑制因子1(inhibitor of differentiation factor 1,Id1)对人骨肉瘤细胞MG63恶性逆转向成骨分化的影响。方法用重组腺病毒调控MG63细胞中Id1过表达后,RT-PCR及Western blot检测验证Id1的表达情况,CCK-8检测细胞增殖能力,划...目的探讨分化抑制因子1(inhibitor of differentiation factor 1,Id1)对人骨肉瘤细胞MG63恶性逆转向成骨分化的影响。方法用重组腺病毒调控MG63细胞中Id1过表达后,RT-PCR及Western blot检测验证Id1的表达情况,CCK-8检测细胞增殖能力,划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力,流式细胞术检测细胞周期,ALP读数法检测成骨分化早期指标ALP的活力。结果 Ad Id1重组腺病毒可以使MG63细胞中的Id1表达量增高,Adsi Id1重组腺病毒可以使MG63细胞中的Id1表达量降低(P<0.05);Id1过表达后,MG63细胞的增殖能力、迁移能力、侵袭能力等恶性生物学行为均增强,G1期细胞比例降低,Id1表达量下调后则逆转MG63细胞的恶性生物学行为,G1期细胞比例增高(P<0.05);Id1表达量下调后,正常成骨分化的早期指标碱性磷酸酶(ALP)表达量增高(P<0.05)。结论抑制Id1基因表达可以促使骨肉瘤的恶性生物学行为逆转并诱导骨肉瘤细胞发生成骨分化。展开更多
基金Supported by A grant from Medicine and Health Key Project of Zhejiang Province, Science and Technology Foundation of Ministry of Health of the People’s Republic of China, No. WKJ2007-2-037Shaoxing Key Project for Science and Technology, No. 2007A23008, 2005141
文摘MM： To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes, METHODS： Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem （ES） cells were obtained in a sequential manner, induced by valproic acid （VPA） and cytokines （hepatocyte growth factor, epidermal growth factor and insulin）, Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy, Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepa- tocytes, Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells, Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells.RESULTS： Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The dif- ferentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers,in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells to-wards hepatic lineages. CONCLUSION： Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.
文摘Progression,relapse,and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell(CSC)population.This enigmatic subpopulation of cancer cells has therefore emerged as promising therapeutic candidate.We earlier reported enrichment of CSC-like side population(SP)with increasing resistance towards Cisplatin and Paclitaxel either alone or in combination in epithelial ovarian cancer(EOC)cells.This SP population is a small proportion of the total population of cancer cells characterised with high expression of drug transporters,a unique feature of stem cells and thereby can be isolated through their efflux properties of DNA binding dyes.While the bulk non-SP(NSP)population of the cancer cells lack overexpression of the drug transporters and thus can be identified as the dye containing population.In this study,we show that increased expression of Runt related transcription factor 1(RUNX1)maintains undifferentiated state of CSC-like SP cells through upregulation of inhibitors of DNA binding/differentiation genes(ID1 and ID3)in late cisplatinpaclitaxel resistant cells.Higher RUNX1 expression was found to correlate with decreased median overall survival and disease-free survival in The Cancer Genome Atlas(TCGA)data set of high grade serous ovarian cancer(HGSOC)patients.The protein-protein interaction network analysis of 397 upregulated genes in RUNX1-high samples of TCGA data show significant enrichment of pathways known to negatively regulate CSC differentiation.Intriguingly RUNX1 inhibition not only induces CSC differentiation but also downregulates anti-apoptotic protein BCL2 in both SP and NSP cells and potentiates cytotoxic effects of Cisplatin-Paclitaxel in chemoresistant EOC cells.Inhibition of BCL2 through Venetoclax treatment,a small molecule BH3 mimic,sensitized these cells to platinum taxol treatment.Altogether,our data reveal new regulatory roles by RUNX1 to modulate CSC differentiation via ID1 and ID3 and to promote chemoresistance through BCL2 upregulation.
文摘AIM:To investigate the expression and clinical relevance of inhibitor of differentiation(ID)proteins in biliary tract cancer.METHODS:ID protein expression was analyzed in129 samples from patients with advanced biliary tract cancer(BTC)(45 extrahepatic,50 intrahepatic,and 34 gallbladder cancers),compared to normal controls and correlated with clinical an pathological parameters.RESULTS:ID1-3 proteins are frequently overexpressed in all BTC subtypes analyzed.No correlation between increased ID protein expression and tumor grading,tumor subtype or treatment response was detected.Survival was influenced primary tumor localization(extrahepatic vs intrahepatic and gall bladder cancer,OS 1.5 years vs 0.9 years vs 0.7 years,P=0.002),by stage at diagnosis(OS 2.7 years in stageⅠv s 0.6 years in stageⅣ,P<0.001),resection status and response to systemic chemotherapy.In a multivariate model,ID protein expression did not correlate with clinical prognosis.Nevertheless,there was a trend of shorter OS in patients with loss of cytoplasmic ID4 protein expression(P=0.076).CONCLUSION:ID protein expression is frequently deregulated in BTC but does not influence clinical prognosis.Their usefulness as prognostic biomarkers in BTC is very limited.
基金supported by a grant from the National Natural Science Foundation of China(No.30772206)
文摘The formation of osteolytic bone lesions is a key process for osteolytic cancer to metastasize to the bone and is under the control of a set of transcription factors. Recently, the inhibitor of differentiation 1 （Id1） has been linked with angiogenesis, tumorigenesis, metastasis and bone formation. However, the function of Id1 during the process of bone destruction caused by cancer in vivo has not yet been elucidated. We, therefore, examined whether and how Id1 affects the ability of cancer to form osteolytic lesion in vivo. The study used a lentiviral vector overexpressing short hairpin RNA （shRNA） targeting Id1 gene. PC3 cells, a prostate cancer cell line, were transduced with Id1 shRNA or negative control （NC） shRNA before implantation in BALB/c mice. Cells were implanted in a tibial injection model. Tumor formation in bone was monitored by X-ray. The relationship between parathyroid hormone-related protein （PTHrP）, an osteolytic factor, and Id1 was analyzed by using immunohistochemistry in tissue sections from osteolytic lesion of the BALB/c mice. Our results showed that Id1 shRNA delivery to PC3 cells by lentivirus caused efficient and stable Id1 gene silencing. In the intratibial model, PC3 cells produced primarily osteolytic lesions in the bone. Eleven of 14 mice in Id1 shRNA group but only 4 of 14 mice in the NC shRNA group developed osteolytic lesions with cortical destruction at 4th week. Mice treated with Id1 shRNA had larger tumor volume in the bone and larger cortical destruction. The expression of PTHrP protein in PC3 cells was not affected by Id1 knockdown in vivo. These results indicate that Id1 may down-regulate the ability of PC3 cells to form osteolytic lesions in vivo and the signal pathway needs to be further investigated.
基金Supported by Grant-in-Aid from the Ministry of Education,Culture,Sports,Science and Technology,(No.18590280)the Foundation for the Promotion of Cancer Research in Japan+1 种基金Grant-in-Aid for Japan Arteriosclerosis Research FoundationShimane University"S-TAKUMI Medical Nanotechnology"Project
文摘AIM:To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer(NSCLC) cells and whether a hypoxia-inducible factor(HIF) inhibitor is able to suppress the process.METHODS:Human lung adenocarcinoma A549 cells and squamous carcinoma QG56 cells were cultured under normoxic(21%O_2) or hypoxic(4%or 1%O_2) conditions.The expression of the following genes were examined by reverse transcription-polymerase chain reaction,Western blotting and/or immunofluorescence:HIF-1α and HIF-2αsubunits;differentiation marker genes,namely surfactant protein C(SP-C)(type Ⅱ alveolar cell marker),CC10(type I alveolar cell marker) and aquaporin 5(AQP5)(Clara cell marker);and stem cell-associated genes,namely CD133,0CT4,and Musashi-1(MSI1).The tumor sphere-forming ability of the cells was evaluated by culturing them in serum-free growth factor-rich medium containing epidermal growth factor(EGF) and fibroblast growth factor(FGF).CD133 expression in hypoxic regions in A549 tumors was examined by double-immunostaining of tissue cryosections with an anti-2-nitroimidazole EF5 antibody and an anti-CD133 antibody.The metastatic ability of A549 cells was examined macroscopically and histologically after injecting them into the tail vein of immunocompromised mice.RESULTS:A549 cells primarily expressed SP-C,and QG56 cells expressed CC10 and AQP5.Exposure of A549 cells to hypoxia resulted in a marked downregulation of SP-C and upregulation of CD133,OCT4,and MSI1 in a time-dependent manner.Moreover,hypoxia mimetics,namely desferrioxamine and cobalt chloride,elicited similar effects.Ectopic expression of the constitutively active HIF-la subunit also caused the downregulation of SP-C and upregulation of CD133 and MSI1 but not OCT4,which is a direct target of HIF-2.Hypoxia enhanced the sphere-forming activity of A549 cells in serum-free medium containing EGF and FGF.Similarly,hypoxia downregulated the expression of CC10 and AQP5 genes and upregulated CD133,OCT4,and MSI1 genes in QG56 cells.TX-402(3-amino-2-quinoxalinecarbonitrile 1,4-dioxide),which is a small molecule inhibitor of the expression of HIF-1α and HIF-2αsubunits under hypoxic conditions,inhibited the upregulation of SP-C'and hypoxia-induced down-regulation of CD133,OCT4,and MSI1.Notably,TX-402 significantly suppressed the hypoxia-enhanced lung-colonizing ability of A549 cells.CONCLUSION:Hypoxia induces the de-differentiation of NSCLC cells into cancer stem cell-like cells,and HIF inhibitors are promising agents to prevent this process.
文摘With the wide application of immunotherapy drug PD-1/PD-L1 inhibitor in China and the continuous clinical research,a major problem that we have to face is the immune-related pneumonia caused by PD-1/PD-L1.At present,western medicine mainly treated it by hormone therapy,which may cause side effects,such as obesity,osteoporosis and osteonecrosis for hormone therapy by long-term,and increase the patients'pain.Under the guidance of the theory of syndrome differentiation,traditional Chinese medicine(TCM)advocates the methods of expelling wind and clearing away cold,resolving phlegm and relieving asthma,relieving heat from lung,and invigorating the spleen and tonifying the kidney,etc.Exact differentiation of symptoms and rational usage of drugs can play an important role in the early prevention and treatment of immune-related pneumonia,reflecting the important role of TCM in the prevention and treatment of side effects of new drugs.In order to provide an effective clinical reference for clinicians in the practice of using PD-1/PD-L1,this paper systematically reviewed the use of TCM in the treatment of immune-related pneumonia induced by PD-1/PD-L1 inhibitors.
文摘BACKGROUND Cardiovascular diseases particularly myocardial infarction(MI)are the leading cause of mortality and morbidity around the globe.As cardiac tissue possesses very limited regeneration potential,therefore use of a potent small molecule,inhibitor Wnt production-4(IWP-4)for stem cell differentiation into cardiomyocytes could be a promising approach for cardiac regeneration.Wnt pathway inhibitors may help stem cells in their fate determination towards cardiomyogenic lineage and provide better homing and survival of cells in vivo.Mesenchymal stem cells(MSCs)derived from the human umbilical cord have the potential to regenerate cardiac tissue,as they are easy to isolate and possess multilineage differentiation capability.IWP-4 may promote the differentiation of MSCs into the cardiac lineage.AIM To evaluate the cardiac differentiation ability of IWP-4 and its subsequent in vivo effects.METHODS Umbilical cord tissue of human origin was utilized to isolate the MSCs which were characterized by their morphology,immunophenotyping of surface markers specific to MSCs,as well as by tri-lineage differentiation capability.Cytotoxicity analysis was performed to identify the optimal concentration of IWP-4.MSCs were treated with 5μM IWP-4 at two different time intervals.Differentiation of MSCs into cardiomyocytes was evaluated at DNA and protein levels.The MI ratmodel was developed.IWP-4 treated as well as untreated MSCs were implanted in the MI model,then the cardiac function was analyzed via echocardiography.MSCs were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiI)dye for tracking,while the regeneration of infarcted myocardium was examined by histology and immunohistochemistry.RESULTS MSCs were isolated and characterized.Cytotoxicity analysis showed that IWP-4 was non-cytotoxic at 5μM concentration.Cardiac specific gene and protein expression analyses exhibited more remarkable results in fourteen days treated group that was eventually selected for in vivo transplantation.Cardiac function was restored in the IWP-4 treated group in comparison to the MI group.Immunohistochemical analysis confirmed the homing of pre-differentiated MSCs that were labeled with DiI cell labeling dye.Histological analysis confirmed the significant reduction in fibrotic area,and improved left ventricular wall thickness in IWP-4 treated MSC group.CONCLUSION Treatment of MSCs with IWP-4 inhibits Wnt pathway and promotes cardiac differentiation.These pre-conditioned MSCs transplanted in vivo improved cardiac function by cell homing,survival,and differentiation at the infarcted region,increased left ventricular wall thickness,and reduced infarct size.
文摘目的:探索分化抑制因子(inhibitor of differentiation,ID)家族在慢性髓系白血病(chronic myeloid leukemia,CML)中的表达和启动子甲基化水平,并分析其临床意义。方法:应用定量PCR及定量甲基化特异性PCR的方法检测2010年1月至2017年12月期间江苏大学附属人民医院就诊的非恶性血液病患者(对照组)和CML患者骨髓单个核细胞中ID2/ID3/ID4表达及ID4启动子甲基化水平,通过分组分析ID家族异常的临床意义。结果:ID2及ID3表达在CML患者中均呈现显著上调(P<0.001,P<0.05),而ID4表达在CML患者中呈现显著下调(P<0.01)。其中,接受者操作特征曲线分析揭示ID2表达可作为CML鉴别的潜在分子标志物(AUC=0.895,P<0.001)。CML患者中ID4启动子高甲基化概率显著高于对照组患者(P=0.001),且ID4启动子甲基化与ID4表达呈现负相关(r=-0.424,P=0.002)。通过分组分析发现ID2高表达较易发生于男性患者中(P=0.040);ID4低表达/高甲基化较易发生于加速/急变期患者(P=0.003,P<0.001)。此外,CML加速/急变期患者ID4表达水平低于慢性期患者(P<0.001),而ID4甲基化水平高于慢性期患者(P<0.001)。通过单因素及多因素Logistic回归分析发现ID4高甲基化是CML患者疾病进展的独立危险因素(P=0.007)。结论:ID家族在CML患者中表达态势不同,其中ID2/ID3表达上调;而ID4表达下调,与ID4启动子高甲基化相关。ID4表达/甲基化与CML疾病进展相关,其中ID4甲基化可能是CML疾病进展的独立危险因素。
文摘目的探讨分化抑制因子1(inhibitor of differentiation factor 1,Id1)对人骨肉瘤细胞MG63恶性逆转向成骨分化的影响。方法用重组腺病毒调控MG63细胞中Id1过表达后,RT-PCR及Western blot检测验证Id1的表达情况,CCK-8检测细胞增殖能力,划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力,流式细胞术检测细胞周期,ALP读数法检测成骨分化早期指标ALP的活力。结果 Ad Id1重组腺病毒可以使MG63细胞中的Id1表达量增高,Adsi Id1重组腺病毒可以使MG63细胞中的Id1表达量降低(P<0.05);Id1过表达后,MG63细胞的增殖能力、迁移能力、侵袭能力等恶性生物学行为均增强,G1期细胞比例降低,Id1表达量下调后则逆转MG63细胞的恶性生物学行为,G1期细胞比例增高(P<0.05);Id1表达量下调后,正常成骨分化的早期指标碱性磷酸酶(ALP)表达量增高(P<0.05)。结论抑制Id1基因表达可以促使骨肉瘤的恶性生物学行为逆转并诱导骨肉瘤细胞发生成骨分化。
