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AIM: To detect the effect of connective tissue growth factor (CTGF) on the apoptosis in the diabetic retina with small interfering RNAs (siRNA) targeting CTGF. METHODS: A total of 60 rats were divided into 6 groups including control group, diabetic 4, 8, 12, 16 weeks groups, and interference group. Diabetic rats were induced by intraperitoneal streptozotocin (STZ). Retinas were obtained from control, diabetic rats and diabetic rats of interference group treated by intravitreal injection of CTGFsiRNA to suppress the expression of CTGF mRNA. Retinal cells apoptosis was detected by Tunnel staining and mRNA expression of CTGF was analyzed by RT-PCR. RESULTS: The levels of CTGF and the apoptosis in the retinas of diabetic rats were significantly higher than those in the controls. Apoptosis occurred at 4 weeks after a diabetic model being set up, became serious with the diabetes developing, while CTGF elevated at 8 weeks. The apoptosis cell counts increased to 25.8cells/mm(2) at 24weeks of diabetes. SiRNA-mediated inhibition of CTGF mRNA resulted in a significant decrease in apoptosis. Significant correlations were found between CTGF and apoptosis in the retina. CONCLUSION: It was suggested that CTGF might be involved in retinal cells apoptosis which is a characteristic of early diabetic retina. SiRNA targeting CTGF seems to have the advantage of ameliorating retinal cells apoptosis.
RESUMO
OBJECTIVE: To develop an HCT-8/5-FU multidrug-resistant colorectal cancer cell line and to elucidate the effect of Andrographolid (AG), an extract from Andrographis paniculate, a medicinal herb on the HCT-8/5-FU multidrug-resistant colorectal cancer cell line. METHODS: An HCT-8 colorectal cancer cell line was used and a high concentration of 5-Fluorouracid (5-FU) was introduced at the beginning to induce drug resistance, then the concentration of 5-FU was increased in gradients. Approximately 7 months later, the cells grew stably in 2.0 microg/mL of 5-FU, and the cell line was named HCT-8/5-FU multidrug-resistant colorectal cancer cell line. The resistant index of HCT-8/5-FU cells to 5-FU, adriamycin (ADM), cisplatin (DDP) was checked by MTT test, and a growth curve was drawn. The morphological changes were observed by both light and electron microscope. The function of P-170 was detected by rhodamine staining. After the application of AG and co-administration of 5-FU, ADM and DDP, the growth curves and inhibition rate as well as apoptosis rate of HCT-8/5-FU at different concentrations of AG were evaluated by MTT and flow cytometry. Rhodamine staining was used to investigate the possible mechanism involved by AG. RESULTS: The resistance index of HCT-8/5-FU to 5-FU was 16.6, and a cross-resistance to ADM and DDP was noticed. Compared with parental cells, HCT-8/5-FU cell's growth rate did not change significantly but the cell's morphology was remarkably changed as compared with parental cells. Overexpression of P-170 by HCT-8/5-FU cell was indicated through rhodamine staining. AG at a low concentration showed weak inhibitory effect on HCT-8/5-FU. However, a remarkable inhibitory and apoptosis rate was shown when AG was co-administered with 5-FU, ADM and DDP, respectively. Interestingly AG alone could not induce apoptosis and change the cell cycles. AG might affect the expression of P-170, which was indicated by rhodamine staining. CONCLUSIONS: The HCT-8/5-FU multidrug-resistant colorectal cancer cell line has been successfully developed and because it has cross-resistance to 5-FU, ADM and DDP, it might serve as an ideal multidrug resistance (MDR) model for colorectal cancer research. The mechanism of HCT-8/5-FU resistance to chemotherapeutic agents might be related to the overexpression of P-170. Low concentrations of AG alone have no significant inhibition on HCT-8/5-FU and fail to induce apoptosis and to change cell cycles. AG might act as a chemosensitizer when co-administered with 5-FU, ADM and DDP, and the mechanism of reversal modulation of multidrug resistance by AG in the HCT-8/5-FU resistant cell line might be related to its downregulation of overexpression of P-170.
