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
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
Objective: To study gap junction intercellular communication (GJIC), H ras oncogene expression and ras oncogene product (P 21 ras protein) expression in four human solid tumor cell lines, W1-38,CACO 2,A549 and...Objective: To study gap junction intercellular communication (GJIC), H ras oncogene expression and ras oncogene product (P 21 ras protein) expression in four human solid tumor cell lines, W1-38,CACO 2,A549 and PaCa, and the effects of four compounds, Salvia miltiorrhiza derivative (SMD), d Limonene, Turmeric derivative I (TD I) and Turmeric derivative II (TD II), on them. Methods: The abilities of the four solid tumor cell lines to transfer dye to adjacent cells were examined by the scrape loading/dye transfer technique, and the H ras oncogene expression by Northern blotting and P 21 ras protein expression by Western blotting. Results: The results showed the loss of intercellular coupling in PaCa cells, slight GJIC in A549 and CACO 2 cells, and a good GJIC in W1-38 cells. The four compounds could improve the GJIC of PaCa to different extents. The amount of total and membrane associated P 21 ras in PaCa cells were decreased after treatment with SMD, d Limonene and TD I (2.5 μg/ml) for 48 h. Concomitantly, the growth of PaCa cells decreased in soft agar and had enhanced GJIC. The relative potency was found to be:d Limonene>SMD >TD I=TD II. There was no significant effect of the four compounds on H ras oncogene expression. Conclusion: It was suggested that there was an excellent correlation between loss of Lucifer Yellow dye transfer and ras gene mutation rate in the four solid tumor cell lines (ras gene mutation rate inversely correlated with average cell number coupled, r=0.98) i.e., the high ras gene mutation was closely correlated with loss of GJIC in these malignant human tumor cells; The antitumor effect of the monoterpene d Limonene and the phenol compound, SMD, might be related to inhibition of P 21 ras membrane association and enhancement of GJIC, whilst that of the others may be by a different mechanism; The inhibition of P 21 ras membrane association was directly related to the enhancement of gap junction intercellular com munication.展开更多
Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quan...Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions. High incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement. With the subsidence of cell movements, both dye coupling and gap junctions were reduced to lower levels. It was, therefore, suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap junctions of altered configuration occurred in notochord cells in late tailbud stage. The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.展开更多
Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work conce...Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By. using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was otherved that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels.The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal celis, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.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
【目的】探讨丹参酮ⅡA(TanshinoneⅡA)对人胎脐静脉上皮细胞(HUVEC)缝隙连接细胞通讯(GJIC)的影响及其机制。【方法】采用四甲基偶氮唑盐(MTT)法检测丹参酮ⅡA对HUVEC细胞生长的影响,荧光显微镜观察及流式细胞仪荧光示踪法分析GJIC功...【目的】探讨丹参酮ⅡA(TanshinoneⅡA)对人胎脐静脉上皮细胞(HUVEC)缝隙连接细胞通讯(GJIC)的影响及其机制。【方法】采用四甲基偶氮唑盐(MTT)法检测丹参酮ⅡA对HUVEC细胞生长的影响,荧光显微镜观察及流式细胞仪荧光示踪法分析GJIC功能变化,并与阳性对照药全反式维甲酸(ATRA)比较,采用荧光实时定量PCR(q RT-PCR)法分析Cx37、Cx40 m RNA的表达。【结果】丹参酮ⅡA作用HUVEC 24、48 h的半数致死量(IC50)分别为15、8.5μmol/L,32、64μmol/L丹参酮ⅡA作用HUVEC 48 h有明显细胞毒性,抑制率大于70%;选用0、4、8、16、32μmol/L丹参酮ⅡA作用HUVEC48 h,流式细胞仪检测结果显示:各组接受绿色荧光Calcein-AM的细胞数量与总细胞数比值明显升高,其中8、16、32μmol/L组可显著提高GJIC功能(P<0.01)。q RT-PCR分析结果显示:与空白对照组比较,8、16、32μmol/L丹参酮ⅡA组可显著提高Cx37、Cx40 m RNA表达(P<0.05或P<0.01),并有一定的浓度依赖关系。【结论】丹参酮ⅡA能够提高Cx37、Cx40 m RNA表达,这可能是丹参酮ⅡA增强上皮细胞GJIC功能的机制之一。展开更多
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.
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Objective: To study gap junction intercellular communication (GJIC), H ras oncogene expression and ras oncogene product (P 21 ras protein) expression in four human solid tumor cell lines, W1-38,CACO 2,A549 and PaCa, and the effects of four compounds, Salvia miltiorrhiza derivative (SMD), d Limonene, Turmeric derivative I (TD I) and Turmeric derivative II (TD II), on them. Methods: The abilities of the four solid tumor cell lines to transfer dye to adjacent cells were examined by the scrape loading/dye transfer technique, and the H ras oncogene expression by Northern blotting and P 21 ras protein expression by Western blotting. Results: The results showed the loss of intercellular coupling in PaCa cells, slight GJIC in A549 and CACO 2 cells, and a good GJIC in W1-38 cells. The four compounds could improve the GJIC of PaCa to different extents. The amount of total and membrane associated P 21 ras in PaCa cells were decreased after treatment with SMD, d Limonene and TD I (2.5 μg/ml) for 48 h. Concomitantly, the growth of PaCa cells decreased in soft agar and had enhanced GJIC. The relative potency was found to be:d Limonene>SMD >TD I=TD II. There was no significant effect of the four compounds on H ras oncogene expression. Conclusion: It was suggested that there was an excellent correlation between loss of Lucifer Yellow dye transfer and ras gene mutation rate in the four solid tumor cell lines (ras gene mutation rate inversely correlated with average cell number coupled, r=0.98) i.e., the high ras gene mutation was closely correlated with loss of GJIC in these malignant human tumor cells; The antitumor effect of the monoterpene d Limonene and the phenol compound, SMD, might be related to inhibition of P 21 ras membrane association and enhancement of GJIC, whilst that of the others may be by a different mechanism; The inhibition of P 21 ras membrane association was directly related to the enhancement of gap junction intercellular com munication.
文摘Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye, Lucifer Yellow. Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions. High incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement. With the subsidence of cell movements, both dye coupling and gap junctions were reduced to lower levels. It was, therefore, suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap junctions of altered configuration occurred in notochord cells in late tailbud stage. The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.
文摘Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By. using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was otherved that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels.The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal celis, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.
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.
文摘【目的】探讨丹参酮ⅡA(TanshinoneⅡA)对人胎脐静脉上皮细胞(HUVEC)缝隙连接细胞通讯(GJIC)的影响及其机制。【方法】采用四甲基偶氮唑盐(MTT)法检测丹参酮ⅡA对HUVEC细胞生长的影响,荧光显微镜观察及流式细胞仪荧光示踪法分析GJIC功能变化,并与阳性对照药全反式维甲酸(ATRA)比较,采用荧光实时定量PCR(q RT-PCR)法分析Cx37、Cx40 m RNA的表达。【结果】丹参酮ⅡA作用HUVEC 24、48 h的半数致死量(IC50)分别为15、8.5μmol/L,32、64μmol/L丹参酮ⅡA作用HUVEC 48 h有明显细胞毒性,抑制率大于70%;选用0、4、8、16、32μmol/L丹参酮ⅡA作用HUVEC48 h,流式细胞仪检测结果显示:各组接受绿色荧光Calcein-AM的细胞数量与总细胞数比值明显升高,其中8、16、32μmol/L组可显著提高GJIC功能(P<0.01)。q RT-PCR分析结果显示:与空白对照组比较,8、16、32μmol/L丹参酮ⅡA组可显著提高Cx37、Cx40 m RNA表达(P<0.05或P<0.01),并有一定的浓度依赖关系。【结论】丹参酮ⅡA能够提高Cx37、Cx40 m RNA表达,这可能是丹参酮ⅡA增强上皮细胞GJIC功能的机制之一。
