Impact of caffeine on ß cell proliferation and apoptosis under the influence of palmitic acid.

We examined the influence of caffeine on the proliferation and apoptosis of ß cells cultured in vitro in the presence of the free fatty acid palmitic acid (PA). Different concentrations of caffeine (1-100 mM) and free fatty PA were added to cultured ß cells. The MTT assay was used to analyze cell proliferative activity; flow cytometry was used to measure apoptosis and calculate the apoptosis rate. Compared with the blank control group, cells cultured with 500 mM PA for 24, 48, 72, and 96 h showed inhibition of pancreatic ß cell proliferative activity. In the 10 and 25 mM caffeine groups cultured for 48, 72, and 96 h, ß cell proliferative activity was much higher than that in the 500 mM PA group. The apoptosis rate in the 500 mM PA group was 40.55 ± 20.33%, which was higher than that in the blank control group. The apoptosis rates in the 10 and 25 mM caffeine group and the PA group were 19.12 ± 10.56 and 20.97 ± 9.75%, respectively, which was lower than that in the 500 mM PA group. At some concentrations, caffeine can improve free fatty PA levels and guide pancreatic ß cell proliferation inhibition and cell apoptosis.

Expression characteristics of MMP-2 and MMP-9 in guinea pig ovaries during the estrous cycle.

We investigated the expression characteristics of matrix metalloproteinases (MMP)-2 and MMP-9 in the ovaries of normal adult guinea pigs during the estrous cycle. The estrous cycle was classified according to the appearance of the vaginal orifice and by vaginal exfoliated cell smears. Guinea pigs that had experienced at least two normal estrous cycles were selected. Ovaries were collected from guinea pigs at proestrus or metestrus (N = 6). The expression characteristics of steroidogenic acute regulatory protein (StAR), MMP-2, and MMP-9 were investigated by hematoxylin and eosin staining and immunohistochemical staining. The results showed that MMP-2, MMP-9, and StAR were significantly expressed in the granulosa and thecal cells of the ovarian atretic follicles during proestrus, and were strongly expressed in the corpus luteum during metestrus.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3.

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60 percent of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.