Expression Of Topo I In Human Pathological Scar Tissue And The Effect Of Hydroxycamptothecin On Fibroblasts Of Human Pathological Scar

Goal: There is a substantial interest in preventing and treating pathological scar. Our study is aiming at understanding the pharmacological effect of hydroxycamptothecin (HCPT) on fibroblasts by determining the difference in TopoI expression between pathological scar and normal skin tissue and identifying the effect of HCPT on the proliferation, apoptosis and cell cycle regulation of fibroblasts from pathological scar.Methods: (1) 21 pathological scar samples were surgically taken from patients (15 male, 6 female; age range 18～40) after 6～9 months recovery from burn injury without any scar treatment. Normal skin tissue taken from the same patient was served as control. Immunohistochemistry staining was used to measure the expression of TopoⅠ. (2) In vitro culture of fibroblasts from pathological scar 1)MTT assay was used to determine the inhibitory effect of hydroxycamptothecin on the proliferation of fibroblasts under 10 different doses(2～1000ng/ml) and 3 different times(24hour,48hour,72hour). 2)Flow cytometry was used to measure the cell cycle and apoptosis index of fibroblasts after treated with hydroxycamptothecin for 24 hours under 5 different concentration(31, 63, 125, 250, 500ng/ml). 3)DNA Fragment Detection was used to study the apoptosis induction by 500ng/ml hydroxycamptothecin on fibroblasts from pathological scar.Results:(1) Topo I is expressed in karytinocytes, fibroblasts of human pathological scar and normal skin tissue. In normal skin, 3 positive were found in both karytinocytes and fibroblasts; in pathological scar, 7 positive were found in karytinocytes and 12 positive were found in fibroblasts. The percentage of Topo I positive karytinocytes was 14.3% in normal skin and 33.3% in pathological scar, which has no significant difference (p=0.1473). The percentage of Topo I positive fibroblasts was 14.3% in normal skin and 57.1% in pathological scar which has significant difference (p=0.0038). (2) 1)Hydroxycamptothecin has been shown to inhibit the proliferation of fibroblasts in a dose-dependent manor (Correlation coefficient is 0.8675, P=0.0024). IC50 was 233ng/ml for 24 hour, 176ng/ml for 48 hour and 103ng/ml for 72 hour. 2)Hydroxycamptothecin can increase the percentage of cells in the S phase,.induce fibroblasts from pathological scar to undergo apoptosis. There was an apoptotic peak after 250ng/ml hydroxycamptothecin treatment for 24 hour. The number of apoptotic fibroblasts increase with the rise of hydroxycamptothecin concentration. 3)Characteristic DNA ladder of apoptosis shown by 1% agarose electrophoresis appeared after 24 hour treatment of 500ng/ml hydroxycamptothecin on fibroblasts.Conclusion: The expression of Topo I in fibroblasts from pathological scar is higher than fibroblasts from normal skin. In vitro hydroxycamptothecin can inhibit the proliferation of fibroblasts from pathological scar, increase percentage of cells in the S phase and induce apoptosis when the concentration achieves 250ng/ml. The number of apoptotic fibroblasts increase with the rise of hydroxycamptothecin concentration.