Study on characteristic spectrum of ingredients from different species Cordyceps and its anti-fibrotic activity on human embryonic fibroblasts / 中国中药杂志

In this study, 10 samples of parasites, cursive, and the whole from six different species of Cordyceps were determined and compared by HPLC and LC-MS methods. Uridine, adenosine, and cordycepin were selected as the main evaluation index. The anti-fibrotic activity of different species Cordyceps extracts was observed using in vitro TGF-β1-induced ECM accumulation in human embryonic fibroblasts CCC-ESF-1. The results demonstrated that the number of atoms and hyphae ingredients of different species showed little difference, however, the content distribution of each component has obvious significance. The in vitro anti-fibrotic activities of different species were as follow： Cordyceps flower > Cicada Cordyceps (Cicada flower)> Silkworm Cordyceps> Tussah Cordyceps>natural Cordyceps. Our preliminary data could serve as reference for the discovery of artificial alternatives of natural Cordyceps.

Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages

Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 micrometer) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 micrometer of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.

Isolation,cultivation and identification of human embryonic fibroblasts and expressions of cytokines essential for growth of human embryonic germ cells in vitro / 吉林大学学报(医学版)

Objective To isolate,cultivate and identify the human embryonic fibroblasts(hEFs) derived from the gonadal ridges and dorsal mesenteries of human embryos,and to detect the expression by hEFs of cytokines crucial for the growth of human embryonic germ cells(hEG) in vitro.Methods The hEFs were isolated by enzyme digestion from gonadal ridges and dorsal mesenteries of 5-to 9-week old human embryos.The cells were then cultivated.The biologic characteristics(morphology,growth characteristics and cell cycle) of these cells were also studied.The reverse transcription-polymerase chain reaction(RT-PCR) was used to seek the expressions of a specific fibroblast marker,prolyl 4-hydroxylase ?,and a specific marker of epithelial cells,cytokeratin-4,in the cells.The expressions of cytokines essential for the growth of hEG,namely basic fibroblast growth factor(bFGF) and leukemia inhibitory factor(LIF),were also examined by RT-PCR.Results The hEFs were successfully isolated and cultivated from gonadal ridges and dorsal mesenteries of human embryos.They could be passaged beyond the 25th generation.The biologic characteristics of the cells did not change,even in high-passage cells or frozen-thawed cells.The cells expressed prolyl 4-hydroxylase ?, but not cytokeratin-4,which was similar to the fibroblasts.The cultured cells expressed bFGF and LIF.Conclusion The hEFs derived from gonadal ridges and dorsal mesenteries of human embryos are successfully isolated and cultivated,and the cells express the cytokines essential for the growth of hEG in vitro.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.