Objective To explore the effect of TGF-a1 on the mRNA and prot ei n expression of connective tissue growth factor (CTGF) in human peritoneal mesot helial cells (HPMCs) and the probable mechanism. Methods Greater oment...Objective To explore the effect of TGF-a1 on the mRNA and prot ei n expression of connective tissue growth factor (CTGF) in human peritoneal mesot helial cells (HPMCs) and the probable mechanism. Methods Greater omenta from hea lthy adults were used for primary culture of HPMCs. The third generation cells w ere stimulated by 5 ng/ml TGF-a1. Immunohistochemistry, Western blot, ELISA an d RT-PCR were employed to examine the follows:the mRNA and protein expression o f CTGF, the mRNA and protein expression of fibronectin (FN) and collagen type Ⅰ (ColⅠ), the protein expression of p-Smad2/3 and its migration in HPMCs. Result s (1)The mRNA expression of CTGF in the treatment groups increased obviously a s compared to the control group, and peaked at 48 h. (2)The protein expression of intracellular CTGF in the control group was very little, whereas it increase d markedly 24 h after TGF-a1 stimulation, and peaked at 48 h. The mRNA express ion of intracellular FN, and the protein expression of supernatent FN, as well a s the mRNA and protein expression of intracellular ColⅠin the treatment group u p-regulated significantly in a time dependent manner as compared to those in th e control group. (3)p-Smad2/3 in HPMCs hardly expressed in the control group( 3%p-Smad2/3-positive cells), and remarkably expressed 15 min after TGF-a1 s timulation(29%), peaked at 1 h (84%) and then decreased after 2 h(37%). Meanw hile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus at 1 h, and distributed in cytoplasm again at 2 h. Co nclusions TGF-a1 can induce the mRNA and protein expression of CTGF in the pro gression of human peritoneal fibrosis. The probably mechanism is that signaling through Smad pathway in HPMCs can be activated by TGF-a1 stimulation.展开更多
AIM： To determine the inhibitory effect of Astragalus memebranaceushas on gastric cancer cell supernatant- induced apoptosis of human peritoneal mesothelial cells. METHODS： Human peritoneal mesothelial cell （HPMC）...AIM： To determine the inhibitory effect of Astragalus memebranaceushas on gastric cancer cell supernatant- induced apoptosis of human peritoneal mesothelial cells. METHODS： Human peritoneal mesothelial cell （HPMC） line HMrSV5 was co-incubated with gastric cancer cell supernatant （MKN45） and/or Astragalus memebranaceushas. Morphological changes in gastric cancer cells were observed under phase-contrast microscope. Quantitative cell damage was determined by MTT assay. Apoptosis was determined under transmission electron microscope and quantified by detectinr, acridine orange/ethidium bromide-stained （AO/EB） condensed nuclei under fluorescent microscope or by flow cytometry. Expressions of Bcl-2 and Bax were evaluated with immunostaining.展开更多
This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cance...This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cancer cells. Morphological changes of HMrSV5 cells were observed. The cell damage was quantitatively determined by MTT assay. The apoptosis of HMrSV5 cells was observed under transmission electron microscope. Acridine orange/ethidium bromide-stained condensed nuclei was detected by fluorescent microscopy and flow cytometry. The expressions of Bcl-2 and Bax was immunochemically evaluated. The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. The supematants could induce apoptosis of HMrSV5 cells in a time-dependent manner. The supernatants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells. These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis, The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis. Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.展开更多
文摘Objective To explore the effect of TGF-a1 on the mRNA and prot ei n expression of connective tissue growth factor (CTGF) in human peritoneal mesot helial cells (HPMCs) and the probable mechanism. Methods Greater omenta from hea lthy adults were used for primary culture of HPMCs. The third generation cells w ere stimulated by 5 ng/ml TGF-a1. Immunohistochemistry, Western blot, ELISA an d RT-PCR were employed to examine the follows:the mRNA and protein expression o f CTGF, the mRNA and protein expression of fibronectin (FN) and collagen type Ⅰ (ColⅠ), the protein expression of p-Smad2/3 and its migration in HPMCs. Result s (1)The mRNA expression of CTGF in the treatment groups increased obviously a s compared to the control group, and peaked at 48 h. (2)The protein expression of intracellular CTGF in the control group was very little, whereas it increase d markedly 24 h after TGF-a1 stimulation, and peaked at 48 h. The mRNA express ion of intracellular FN, and the protein expression of supernatent FN, as well a s the mRNA and protein expression of intracellular ColⅠin the treatment group u p-regulated significantly in a time dependent manner as compared to those in th e control group. (3)p-Smad2/3 in HPMCs hardly expressed in the control group( 3%p-Smad2/3-positive cells), and remarkably expressed 15 min after TGF-a1 s timulation(29%), peaked at 1 h (84%) and then decreased after 2 h(37%). Meanw hile, p-Smad2/3 mainly distributed in cytoplasm at 15 min, concentrated in cell nucleus and peri-nucleus at 1 h, and distributed in cytoplasm again at 2 h. Co nclusions TGF-a1 can induce the mRNA and protein expression of CTGF in the pro gression of human peritoneal fibrosis. The probably mechanism is that signaling through Smad pathway in HPMCs can be activated by TGF-a1 stimulation.
基金Supported by The National Natural Science Foundation of China, NSFC, 30672050
文摘AIM： To determine the inhibitory effect of Astragalus memebranaceushas on gastric cancer cell supernatant- induced apoptosis of human peritoneal mesothelial cells. METHODS： Human peritoneal mesothelial cell （HPMC） line HMrSV5 was co-incubated with gastric cancer cell supernatant （MKN45） and/or Astragalus memebranaceushas. Morphological changes in gastric cancer cells were observed under phase-contrast microscope. Quantitative cell damage was determined by MTT assay. Apoptosis was determined under transmission electron microscope and quantified by detectinr, acridine orange/ethidium bromide-stained （AO/EB） condensed nuclei under fluorescent microscope or by flow cytometry. Expressions of Bcl-2 and Bax were evaluated with immunostaining.
基金supported by a grant from the National Natural Sciences Foundation of China (No.30672050)
文摘This study examined the mechanism by which the gastric cancer cells lead to early peritoneal metastasis. HMrSV5 cells, a human peritoneal mesothelial cell line, were co-incubated with the supernatants of gastric cancer cells. Morphological changes of HMrSV5 cells were observed. The cell damage was quantitatively determined by MTT assay. The apoptosis of HMrSV5 cells was observed under transmission electron microscope. Acridine orange/ethidium bromide-stained condensed nuclei was detected by fluorescent microscopy and flow cytometry. The expressions of Bcl-2 and Bax was immunochemically evaluated. The results showed that conspicuous morphological changes of apoptosis were observed in HMrSV5 cells 24 h after treatment with the supernatants of gastric cancer cells. The supematants could induce apoptosis of HMrSV5 cells in a time-dependent manner. The supernatants could up-regulate the expression of Bax and suppress that of Bcl-2 in HMrSV5 cells. These findings demonstrated that gastric cancer cells can induce the apoptosis of HPMCs through supernatants in the early peritoneal metastasis, The abnormal expressions of Bcl-2 and Bax may contribute to the apoptosis. Anti-apoptosis drugs promise to be adjuvant chemotherapeutic agents in the treatment of peritoneal metastasis of gastric cancer.
