ABSTRACT
BACKGROUND:Currently, the enzymatic digestion combined with magnetic activated cel sorting for isolating microvascular endothelial cel s are cumbersome and do harm to cel s. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cel s to obtain highly purified endothelial cel s in vitro becomes a hotspot. OBJECTIVE:To explore a simple and effective cultivation method of microvascular endothelial cel s from diabetic patient skins in vitro, and to detect the cel growth. METHODS:Diabetic patients with chronic foot wounds after amputation were enrol ed to col ect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cel s were obtained using adherent method and trypsin method, fol oewd by purified utilizing trypsin digestion and repeated attachment method when passage culture. RESULTS AND CONCLUSION:Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10th day, and the cell concentration reached 80%at the 12th-13th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a“cobblestone”arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100%positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the invertedSshape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin method.
ABSTRACT
Objective To observe the influence of Huiyang Shengji Ointment and its modified formulae on interleukin-1 (IL-1α) and thromboxane B2 (TXB2) in diabetic rats with chronic skin ulcers, and explore the mechanism for promoting the healing of ulcer.Methods Six out of 160 rats were randomly selected as a blank group, without any further processing. The remaining rats were made diabetic model and randomly divided into five groups after 2 weeks:1 d, 3 d, 5 d, 7 d and 14 d groups. Then, these groups were further divided into normal group (Vaseline ointments), model group (Vaseline ointments), Huiyang Shengji Ointment group (whole formula Ointment), Wenyang Yiqi group (Yiqi group, modified Wenyang Yiqi formula ointments) and Huoxue Shengji group (Huoxue group, modified Huoxue Shengji formula ointments). Normal group and model group were given Vaseline ointments;whole formula group, Yiqi group and Huoxue group were given corresponding ointment. Normal group used the method of skin excision, while other groups used STZ injection-hydrocortisone interference-skin excision-foreign body embedded preparation of composite factors for chronic skin ulcer model. After the appropriate treatment period, the rats were executed and tested for the contents of IL-1α and TXB2 in serum by enzyme-linked immunosorbent assay of five time points.Results In treatment 3 d, the contents of IL-1α in Yiqi group were significantly higher than the blank group, model group, whole formula group and Huoxue group (P<0.05). In treatment 5 d, the contents of IL-1α in whole formula group were significantly higher than the blank group and model group (P<0.05). In treatment 7 d, the contents of IL-1α in each treatment group were significantly higher than blank group and model group (P<0.05), and the whole formula group was higher than the Yiqi group and Huoxue group. In treatment 14 d, the contents of IL-1α in model group and Huoxue group were lower than the blank group (P<0.05). In treatment 3 d, the contents of TXB2 in normal group and the whole formula group were higher than the blank group (P<0.05). In treatment 5 d, the contents of TXB2 in whole formula group were higher than the blank group and the normal group (P<0.05). In treatment 7 d, the contents of TXB2 in Yiqi group were higher than the blank, the model, the whole formula and Huoxue groups (P<0.05). In treatment 14 d, the contents of TXB2 in Huoxue group were higher than the blank and model group (P<0.05), and the contents of TXB2 in the blank group and normal group was lower than those treatment groups (P<0.05).Conclusion Huiyang Shengji Ointment and its modified formulae could promote inflammation, stimulate secretion of inflammatory cytokines, while Yiqi Wenyang ointments played a more active role in promoting inflammation of the early phase of wound surface.