| Hepatocellular carcinoma is one of the most common neoplasms in China. It ranks third in incidence and second in mortality among malignant tumors. Hepatocellular carcinoma has a high recurrence if surgically ablated, and has a low sensitivity to radiotherapy and chemotherapy. Immunotherapy and intervention therapy cannot always achieve ideal result. With the development of molecular biology, genetherapy in combination with drugs will be an ideal way for treatment of carcinoma.1,25-(OH)2D3, the active form of vitamin D3, can regulate phosphorus and calcium balance and maintain homeostasis in the body. It also has a role of regulation in cell growth and differentiation. But at the same time it can cause hypocalcaemia. This has limited its clinical application. EB1089 is an analog of vitamin D3. It has a strong activity against cellular proliferation of carcinomas such as breast, prostate and colon carcinomas, and can induce apoptosis of many kinds of carcinoma cells. Though hypocalcaemia cannot be totally avoided, the probability drops about 50% compared with 1,25-(OH)2D3. Therefore, EB1089 as a vitamin D analog is under extensive research. Cell cycle is mainly regulated by cyclin, cyclin dependent kinase(CDK) and cyclin dependentkinase inhibitor (CDKI). P57Kip2, a CDK inhibitory protein of Cip/Kip family is discovered recently. The function of P57Kip2 suggests its inhibition to tumor and a potential role of tumor suppressor gene. P57Kip2, a member of CDKI, can inhibit cellular proliferation, promote apoptosis, induce differentiation and be taken as a prognostic marker of malignancy. The research on its carcinoma-inhibiting mechanism can provide some basis for differential diagnosis, prognosis evaluation and genetherapy, and it has become an important topic in the biomedical field. There has been no report on the relationship of EB1089 with the HCC growth and proliferation. By using techniques .such as cell culture, tumor implant experiment of nude mice, immunohistochemistry, RT-PCR, TUNEL assay, flow cytometry and Western-blot, the effect of EB1089 on hepatocellular carcinoma and its underline mechanisms and relationship to p57Kip2 gene are studied in current thesis. The major experimental results are as follows:1. To investigate the expression and clinical significance of P57Kip2 in HCC, S-ABC immunohistochemical staining method was used to evaluate its expression in 40 cases of HCC (including adjacent tissues) The positive rates of P57Kip2 expression were 57.5% (23/40), 80% (32/40) in HCC and adjacent tissues, respectively (p<0.05). The P57Kip2 protein expression rate was significantly (p<0.05) higher in well-differentiated HCC (75%) than that in moderate (58%) and poorly-differentiated HCC samples (37.5%).2. In 15 hepatocellular carcinoma and adjacent tissues, RT-PCR results showed no significant difference in the p57Kip2 mRNA level by pair-matching t-test. There was no relationship between p57Kip2 mRNA level and hepatocellular carcinoma histology grade.3. When exposed to EB1089 at 0, 1, 10, 100nmol/L for 2, 4, 6, 8 days, HepG2 cells were significantly inhibited in terms of cell growth and proliferation detected by colorimetric analysis and Trypan blue exclusiontest. The inhibitory rate to cells growth was between 16.9%-74.3%. EB1089 at 10nmol/L also showed in vivo inhibitory effect on tumor growth in nude mice with an inhibitory rate of 53.60%.4. When cultured with 10 nmol/L EB1089 for 4 days, the HepG2 cells showed its G1 phase was 71.0% while the control group was 63.2% in flow cytometry. Cells in S/G2 phase were 28.9% and 36.8% in EB1089 and control groups, respectively. TUNEL assay showed that the number of TUNEL-positive cells increased after 4 days' treatment of 10 nmol/L EB1089. The apoptosis peak was also observed by FCM in the EB1089 group with apoptotic rate of 21.4%.5. EB1089 treatment for 4 days caused p57Kip2 protein up-regulated without parallel changes in p57Kip2 mRNA level.The above results showed that there was a positive correlation between p57Kip2 protein level and can...
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