AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin(GFX) to stromal fibroblasts(SFs) in vitro.METHODS: SFs were treated with GFX at different concentrations(0.009375%-0.3%), and their viability was ...AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin(GFX) to stromal fibroblasts(SFs) in vitro.METHODS: SFs were treated with GFX at different concentrations(0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine(PS) externalization, and mitochondrial transmembrane potential(MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins.RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2(Bcl-2) and B-cell leukemiaXL(Bcl-XL), and upregulate Bcl-2 assaciated X protein(Bax), Bcl-2-associated death promoter(Bad), Bcl-2 interacting domain(Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs.CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.展开更多
The organization of the compartment of mesenchymal stem cells is still obscure. Two types of human stromal precursor cells are known. Both of them are analyzed in in vitro system: mesenchymal multipotent stromal cells...The organization of the compartment of mesenchymal stem cells is still obscure. Two types of human stromal precursor cells are known. Both of them are analyzed in in vitro system: mesenchymal multipotent stromal cells (MMSC) and fibroblast colony forming units (CFU-F). The aim of this study was to compare the main characteristics of MMSC and CFU-F derived from the bone marrow of 24 healthy donors. Growth and differentiation parameters, as well as relative expression levels of different genes were analyzed in MMSC and CFU-F. MMSC were cultivated for 5 passages. CFU-F concentration was determined for each bone marrow sample. The data obtained demonstrated the heterogeneity and hierarchical organization of both studied populations of stromal precursor cells-MMSC and CFU-F. These two types of stromal precursor cells turned to be different in most parameters studied. Altogether MMSC seemed to be more immature cells than CFU-F and took up the higher position in hierarchical tree of mesenchymal stem cells. The rate of differentiation and proliferative potential decreased with the donor’s age in both populations MMSC and CFU-F.展开更多
The tumor microenvironment(TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous popula...The tumor microenvironment(TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous population of tumor cells and TME cells. This review will focus on two stromal cell types, the cancerassociated adipocyte(CAA) and the cancer-associated fibroblast(CAF). In the clinic, the presence of CAAs and CAFs in the TME translates to poor prognosis in multiple tumor types. CAAs and CAFs have an activated phenotype and produce growth factors, inflammatory factors, cytokines, chemokines, extracellular matrix components, and proteases in an accelerated and aberrant fashion. Through this activated state, CAAs and CAFs remodel the TME, thereby driving all aspects of tumor progression, including tumor growth and survival, chemoresistance, tumor vascularization, tumor invasion, and tumor cell metastasis. Similarities in the tumorpromoting functions of CAAs and CAFs suggest that a multipronged therapeutic approach may be necessary to achieve maximal impact on disease. While CAAs and CAFs are thought to arise from tissues adjacent to the tumor, multiple alternative origins for CAAs and CAFs have recently been identified. Recent studies from our lab and others suggest that the hematopoietic stem cell, through the myeloid lineage, may serve as a progenitor for CAAs and CAFs. We hypothesize that the multiple origins of CAAs and CAFs may contribute to the heterogeneity seen in the TME. Thus, a better understanding of the origin of CAAs and CAFs, how this origin impacts their functions in the TME, and thetemporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.展开更多
Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral m...Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing （RNA-Seq） was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 （LOXL4）, the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts （P〈0.05）. In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells （HUVECs）. There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues （r=0.882）. These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.展开更多
目的探讨衰老的卵巢间质成纤维细胞对卵巢癌细胞系SKOV3的增殖、侵袭能力的影响。方法原代培养人卵巢正常成纤维细胞(NOF),在体外持续传代诱导细胞衰老,用衰老相关的β半乳糖苷酶染色(SA-b-gal)试验检测细胞是否发生衰老。收集正常成纤...目的探讨衰老的卵巢间质成纤维细胞对卵巢癌细胞系SKOV3的增殖、侵袭能力的影响。方法原代培养人卵巢正常成纤维细胞(NOF),在体外持续传代诱导细胞衰老,用衰老相关的β半乳糖苷酶染色(SA-b-gal)试验检测细胞是否发生衰老。收集正常成纤维细胞的条件培养基(CMn)及衰老成纤维细胞的条件培养基(CMs)。用CMn和CMs分别处理SKOV3细胞,利用CCK8试剂盒、Tra ns we ll小室及三维培养试验检测细胞的增殖、侵袭能力的变化。结果卵巢正常成纤维细胞在体外持续传代会导致细胞复制性衰老,在SA-b-gal试验中有明显的深蓝色沉淀产物。用CMs处理SKOV3细胞,CCK8试验显示细胞培养第5天的OD(2.067±0.058)高于CMn组(1.073±0.056)(t=21.19,P<0.01),表明细胞的增殖能力增强。Tra ns we ll试验显示CMs组穿透Ma trig e l胶的细胞数(235.00±29.68)个,较CMn组的(129.30±15.52)个明显增多(t=5.47,P<0.01);三维培养中形成的克隆球CMs组较CMn组明显增大、数量明显增多(27.00±2.16 vs 11.67±2.50;t=8.04,P<0.01),向外侵袭能力增强。结论衰老的卵巢间质成纤维细胞在体外培养中能促进卵巢癌细胞的增殖和侵袭能力。展开更多
基金Supported by the National High Technology R&D Program of China(No.2006AA02A132)
文摘AIM: To reveal the cytotoxicity and related mechanisms of gatifloxacin(GFX) to stromal fibroblasts(SFs) in vitro.METHODS: SFs were treated with GFX at different concentrations(0.009375%-0.3%), and their viability was detected by MTT method. The cell morphology was observed using light/transmission electron microscope. The plasma membrane permeability was measured by AO/EB double-staining. Then cell cycle, phosphatidylserine(PS) externalization, and mitochondrial transmembrane potential(MTP) were analyzed by flow cytometry. DNA damage was analyzed by electrophoresis and immunostaining. ELISA was used to evaluate the caspase-3/-8/-9 activation. Finally, Western blotting was applied for detecting the expressions of apoptosis-related proteins.RESULTS: Morphological changes and reduced viability of GFX-treated SFs demonstrated that GFX above 0.009375% had cytotoxicity to SFs with dependence of concentration and time. GFX-treating cells also showed G1 phase arrest, increased membrane permeability, PS externalization and DNA damage, which indicated that GFX induced apoptosis of SFs. Additionally, GFX could activate the caspase-8, caspase-9, and caspase-3, induce MTP disruption, downregulate B-cell leukemia-2(Bcl-2) and B-cell leukemiaXL(Bcl-XL), and upregulate Bcl-2 assaciated X protein(Bax), Bcl-2-associated death promoter(Bad), Bcl-2 interacting domain(Bid) and cytoplasmic cytochrome C in SFs, suggesting that caspase-dependent extrinsic and intrinsic pathways were related to GFX-contributed apoptosis of SFs.CONCLUSION: The cytotoxicity of GFX induces apoptosis of SFs through triggering the caspase-dependent extrinsic and intrinsic pathways.
文摘The organization of the compartment of mesenchymal stem cells is still obscure. Two types of human stromal precursor cells are known. Both of them are analyzed in in vitro system: mesenchymal multipotent stromal cells (MMSC) and fibroblast colony forming units (CFU-F). The aim of this study was to compare the main characteristics of MMSC and CFU-F derived from the bone marrow of 24 healthy donors. Growth and differentiation parameters, as well as relative expression levels of different genes were analyzed in MMSC and CFU-F. MMSC were cultivated for 5 passages. CFU-F concentration was determined for each bone marrow sample. The data obtained demonstrated the heterogeneity and hierarchical organization of both studied populations of stromal precursor cells-MMSC and CFU-F. These two types of stromal precursor cells turned to be different in most parameters studied. Altogether MMSC seemed to be more immature cells than CFU-F and took up the higher position in hierarchical tree of mesenchymal stem cells. The rate of differentiation and proliferative potential decreased with the donor’s age in both populations MMSC and CFU-F.
基金Supported by In part by the NIH/NCI(R01 CA148772,ACL)the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs(Merit Awards,ACL)the Hollings Cancer Center(Translational Research Pilot Project,P30 CA138313,ACL)
文摘The tumor microenvironment(TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous population of tumor cells and TME cells. This review will focus on two stromal cell types, the cancerassociated adipocyte(CAA) and the cancer-associated fibroblast(CAF). In the clinic, the presence of CAAs and CAFs in the TME translates to poor prognosis in multiple tumor types. CAAs and CAFs have an activated phenotype and produce growth factors, inflammatory factors, cytokines, chemokines, extracellular matrix components, and proteases in an accelerated and aberrant fashion. Through this activated state, CAAs and CAFs remodel the TME, thereby driving all aspects of tumor progression, including tumor growth and survival, chemoresistance, tumor vascularization, tumor invasion, and tumor cell metastasis. Similarities in the tumorpromoting functions of CAAs and CAFs suggest that a multipronged therapeutic approach may be necessary to achieve maximal impact on disease. While CAAs and CAFs are thought to arise from tissues adjacent to the tumor, multiple alternative origins for CAAs and CAFs have recently been identified. Recent studies from our lab and others suggest that the hematopoietic stem cell, through the myeloid lineage, may serve as a progenitor for CAAs and CAFs. We hypothesize that the multiple origins of CAAs and CAFs may contribute to the heterogeneity seen in the TME. Thus, a better understanding of the origin of CAAs and CAFs, how this origin impacts their functions in the TME, and thetemporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.
基金supported by the National Natural Science Foundation of China (grant nos. 81030018, 30872900 and 30901680)the Doctoral Fund of Ministry of Education of China (grant no. 20120001110043)
文摘Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing （RNA-Seq） was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 （LOXL4）, the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts （P〈0.05）. In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells （HUVECs）. There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues （r=0.882）. These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.
文摘目的探讨衰老的卵巢间质成纤维细胞对卵巢癌细胞系SKOV3的增殖、侵袭能力的影响。方法原代培养人卵巢正常成纤维细胞(NOF),在体外持续传代诱导细胞衰老,用衰老相关的β半乳糖苷酶染色(SA-b-gal)试验检测细胞是否发生衰老。收集正常成纤维细胞的条件培养基(CMn)及衰老成纤维细胞的条件培养基(CMs)。用CMn和CMs分别处理SKOV3细胞,利用CCK8试剂盒、Tra ns we ll小室及三维培养试验检测细胞的增殖、侵袭能力的变化。结果卵巢正常成纤维细胞在体外持续传代会导致细胞复制性衰老,在SA-b-gal试验中有明显的深蓝色沉淀产物。用CMs处理SKOV3细胞,CCK8试验显示细胞培养第5天的OD(2.067±0.058)高于CMn组(1.073±0.056)(t=21.19,P<0.01),表明细胞的增殖能力增强。Tra ns we ll试验显示CMs组穿透Ma trig e l胶的细胞数(235.00±29.68)个,较CMn组的(129.30±15.52)个明显增多(t=5.47,P<0.01);三维培养中形成的克隆球CMs组较CMn组明显增大、数量明显增多(27.00±2.16 vs 11.67±2.50;t=8.04,P<0.01),向外侵袭能力增强。结论衰老的卵巢间质成纤维细胞在体外培养中能促进卵巢癌细胞的增殖和侵袭能力。
