Our previous study has proven that tea polyphenol has a role in lung neoplasms. The present communication was to investage the anti-proliferation effect of tea polyphenol on the PG cells, which was a high metastatic h...Our previous study has proven that tea polyphenol has a role in lung neoplasms. The present communication was to investage the anti-proliferation effect of tea polyphenol on the PG cells, which was a high metastatic human lung carcinoma cell line, by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) cell viability assay, and to study the change of intracellular calcium concentration, connexin43 (Cx43) expression, gap junctional intercellular communication (GJIC) and cell cycle distribution after the tea polyphenol treatment by laser scanning confocal microscopy and flow cytometry. The results showed that 1) tea polyphenol could kill the PG cells in a dose-depent manner via inhibiting the PG cell proliferation and blocking the PG cell cycle progression staying in G0/G1 phase and not transfering in S and G2/M phases to reduce the PG cell proliferation index;2) the increases of intracellular calcium concentration, GJIC and Cx43 expression were related with the tea polyphenol doses. The data suggested that tea polyphenol could inhibit the growth of PG cells, which mechanism was associated with the up-regulation of GJIC.展开更多
Recent results have shown that the level of gap junction coupling could modulate the induction of apoptotic reactions. We previously observed that 1H-[1,2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a block- er of gua...Recent results have shown that the level of gap junction coupling could modulate the induction of apoptotic reactions. We previously observed that 1H-[1,2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a block- er of guanylyl cyclase, inhibited gap junction coupling and thereby promoted activation of characteristic apoptotic reactions such as chromatin condensation, DNA strand breaking, and formation of blebs in GFSHR-17 granulosa cells, the in vitro model for granulosa cells of the maturing ovular follicle. In the present report, we focus on the effects of ODQ with respect to the cell cycle in GFSHR-17 granulosa cells. In synchronised GFSHR-17 granulosa cells, the double whole-cell patch-clamp technique revealed that gap junction conductance in mitotic cells was reduced in comparison to cells in interphase. This reduction of gap junction conductance correlated with a reduction of non-phosphorylated Cx43 in mitotic cells. We compared the stimulation of apoptotic reactions by ODQ between cells in mitosis and in interphase. We observed that the induction of both chromatin condensation and DNA strand breaking by ODQ was increased in mitotic cells, as compared to cells in interphase. The effects of ODQ were not observed in He-La cells that do not express connexins. The results in- dicate that reduction of gap junction coupling in mitotic GFSHR-17 granulosa cells depends on phosphor- rylation of Cx43 and raises the sensitivity to stimulation of apoptosis. We propose that gap junction coupling is involved in regulation of apoptosis of granulosa cells in maturing ovular follicle.展开更多
AIM:To investigate mediating and regulatory effects of osteoblastic gap juncti onal intercellular communication(GJIC) on low-dose parathyroid hormones(PTH)-s timulated bone formation activities in vitro.METHODS:Rat ca...AIM:To investigate mediating and regulatory effects of osteoblastic gap juncti onal intercellular communication(GJIC) on low-dose parathyroid hormones(PTH)-s timulated bone formation activities in vitro.METHODS:Rat calvarial osteoblasts ( ROBs) in cultures were divided into three groups according to the different mode of exposure.Group A: vehicle (sodium acetate, SA)-treated group; Group B:1×10-8 mol/L hPTH(1-34) intermittent exposure group;Group C:1×10-8 mol/L hPTH(1-34) +1×10-7mol/L TPA exposure group.48 h incubation cycles in three groups were repeated for eight times.GJIC and mineralized bone nodules formation in thr ee groups were detected using Lucifer Yellow (LY) scrape loading dye transfer (S LDT) and mineralized nodule staining together with nodule index,respectively. RE SULTS:At various measuring time points of SA×6 h in group A,PTH×6 h in group B ,PTH×6 h+1 h in group B and PTH×6 h+TPA×1 h in group C,LY(+) cell numbers were 6.8±2.5,19.5±6.5,14.0±3.6 and 5.7±2.4,respectively.Diffusion and transf er of LY fluorescent probe was much more noticeably discerned in group B than in group A and C(P< 0.01).Mineralized bone nodule indices were 45.2±12.5, 88.0±1 5.3 and 38.5±17.9 in group A,B and C respectively.Bone formation activity was m uch better revealed in group B than in group A and C (P< 0.01),whereas no statis tically significant difference of bone formation activities were found in group A compared with group C(P=0.465).CONCLUSION:Mediations and regulations of the co ordinating signals in osteoblastic network via GJIC essentially contribute to PT H-stimulated bone anabolism.However,disruption of GJIC not only hinders osteobl astic intercellular coordination but also frustrates PTH-induced bone formation activities in vitro.Therefore,GJIC may evidently play important roles in regula tions on low-dose PTH-induced bone formation.展开更多
Objective: To explore the pathophysiological mechanism of the changes in gap junctionalintercellular communication (GJIC) in the myocardial cells after burns. Methods: After the myocardial cellswere cultured and injur...Objective: To explore the pathophysiological mechanism of the changes in gap junctionalintercellular communication (GJIC) in the myocardial cells after burns. Methods: After the myocardial cellswere cultured and injured with hypoxia and burn serum, the GJIC in the cells was detected with scrapeloading and dye transfer. Meanwhile, the viability, cytosolic free Ca2+ concentration and Ca2+ influx of themyocardial cells were determined. Results: The cytosolic free Ca2+ concentration and the cellulartransmembrane Ca2+ influx were significantly increased but the viability of the cells markedly decreased afterthe injury. The LY fluorescence reached 4 rows of cells from the scrape line in the normal myocardial cells.The GJIC was blocked at the first hour after hypoxia or hypoxia and burn serum injury. The LY fluorescencewas limited to the primary loads cells at the sixth hour after hypoxia and the third hour after hypoxia andburn serum injury. Conclusion: The function of GJIC in the myocardial cells is to maintain high ordersynchronous contraction of the myocardium. After burns, the runaway calcium homeostasis and impairmentof GJIC function would be accused to be the pathological basis for myocardial heterogeneous behavior.展开更多
Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs ...Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.展开更多
Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endo...Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.展开更多
Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by sin...Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.展开更多
Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescen...Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably transfected clones have been obtained. A stable Cx43-transfectant clone RDL6/C4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is demonstrated. Forced expression of Cx43 not only inhibits cell growth but also correlates with the improved myogenic differentiation of RD cells.展开更多
文摘Our previous study has proven that tea polyphenol has a role in lung neoplasms. The present communication was to investage the anti-proliferation effect of tea polyphenol on the PG cells, which was a high metastatic human lung carcinoma cell line, by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) cell viability assay, and to study the change of intracellular calcium concentration, connexin43 (Cx43) expression, gap junctional intercellular communication (GJIC) and cell cycle distribution after the tea polyphenol treatment by laser scanning confocal microscopy and flow cytometry. The results showed that 1) tea polyphenol could kill the PG cells in a dose-depent manner via inhibiting the PG cell proliferation and blocking the PG cell cycle progression staying in G0/G1 phase and not transfering in S and G2/M phases to reduce the PG cell proliferation index;2) the increases of intracellular calcium concentration, GJIC and Cx43 expression were related with the tea polyphenol doses. The data suggested that tea polyphenol could inhibit the growth of PG cells, which mechanism was associated with the up-regulation of GJIC.
文摘Recent results have shown that the level of gap junction coupling could modulate the induction of apoptotic reactions. We previously observed that 1H-[1,2, 4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a block- er of guanylyl cyclase, inhibited gap junction coupling and thereby promoted activation of characteristic apoptotic reactions such as chromatin condensation, DNA strand breaking, and formation of blebs in GFSHR-17 granulosa cells, the in vitro model for granulosa cells of the maturing ovular follicle. In the present report, we focus on the effects of ODQ with respect to the cell cycle in GFSHR-17 granulosa cells. In synchronised GFSHR-17 granulosa cells, the double whole-cell patch-clamp technique revealed that gap junction conductance in mitotic cells was reduced in comparison to cells in interphase. This reduction of gap junction conductance correlated with a reduction of non-phosphorylated Cx43 in mitotic cells. We compared the stimulation of apoptotic reactions by ODQ between cells in mitosis and in interphase. We observed that the induction of both chromatin condensation and DNA strand breaking by ODQ was increased in mitotic cells, as compared to cells in interphase. The effects of ODQ were not observed in He-La cells that do not express connexins. The results in- dicate that reduction of gap junction coupling in mitotic GFSHR-17 granulosa cells depends on phosphor- rylation of Cx43 and raises the sensitivity to stimulation of apoptosis. We propose that gap junction coupling is involved in regulation of apoptosis of granulosa cells in maturing ovular follicle.
文摘AIM:To investigate mediating and regulatory effects of osteoblastic gap juncti onal intercellular communication(GJIC) on low-dose parathyroid hormones(PTH)-s timulated bone formation activities in vitro.METHODS:Rat calvarial osteoblasts ( ROBs) in cultures were divided into three groups according to the different mode of exposure.Group A: vehicle (sodium acetate, SA)-treated group; Group B:1×10-8 mol/L hPTH(1-34) intermittent exposure group;Group C:1×10-8 mol/L hPTH(1-34) +1×10-7mol/L TPA exposure group.48 h incubation cycles in three groups were repeated for eight times.GJIC and mineralized bone nodules formation in thr ee groups were detected using Lucifer Yellow (LY) scrape loading dye transfer (S LDT) and mineralized nodule staining together with nodule index,respectively. RE SULTS:At various measuring time points of SA×6 h in group A,PTH×6 h in group B ,PTH×6 h+1 h in group B and PTH×6 h+TPA×1 h in group C,LY(+) cell numbers were 6.8±2.5,19.5±6.5,14.0±3.6 and 5.7±2.4,respectively.Diffusion and transf er of LY fluorescent probe was much more noticeably discerned in group B than in group A and C(P< 0.01).Mineralized bone nodule indices were 45.2±12.5, 88.0±1 5.3 and 38.5±17.9 in group A,B and C respectively.Bone formation activity was m uch better revealed in group B than in group A and C (P< 0.01),whereas no statis tically significant difference of bone formation activities were found in group A compared with group C(P=0.465).CONCLUSION:Mediations and regulations of the co ordinating signals in osteoblastic network via GJIC essentially contribute to PT H-stimulated bone anabolism.However,disruption of GJIC not only hinders osteobl astic intercellular coordination but also frustrates PTH-induced bone formation activities in vitro.Therefore,GJIC may evidently play important roles in regula tions on low-dose PTH-induced bone formation.
文摘Objective: To explore the pathophysiological mechanism of the changes in gap junctionalintercellular communication (GJIC) in the myocardial cells after burns. Methods: After the myocardial cellswere cultured and injured with hypoxia and burn serum, the GJIC in the cells was detected with scrapeloading and dye transfer. Meanwhile, the viability, cytosolic free Ca2+ concentration and Ca2+ influx of themyocardial cells were determined. Results: The cytosolic free Ca2+ concentration and the cellulartransmembrane Ca2+ influx were significantly increased but the viability of the cells markedly decreased afterthe injury. The LY fluorescence reached 4 rows of cells from the scrape line in the normal myocardial cells.The GJIC was blocked at the first hour after hypoxia or hypoxia and burn serum injury. The LY fluorescencewas limited to the primary loads cells at the sixth hour after hypoxia and the third hour after hypoxia andburn serum injury. Conclusion: The function of GJIC in the myocardial cells is to maintain high ordersynchronous contraction of the myocardium. After burns, the runaway calcium homeostasis and impairmentof GJIC function would be accused to be the pathological basis for myocardial heterogeneous behavior.
基金supported by the National Natural Science Foundation of China(30471753)
文摘Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.
基金supported by grants from the National Natural Science Foundation of ChinaNo.8117111281371272 to MCL
文摘Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.
基金This work was supported by a grant from the National Natural Science Foundation of China (grant no. 39840017).
文摘Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.
基金Project supported by the National Natural Science Foundation of China and the exchange program between China and Canada.
文摘Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably transfected clones have been obtained. A stable Cx43-transfectant clone RDL6/C4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is demonstrated. Forced expression of Cx43 not only inhibits cell growth but also correlates with the improved myogenic differentiation of RD cells.
