| Breast cancer is the most common cancer and it accounts for 31.5% of total cancer incidence and 18.4% of total cancer mortality in U.S. women. In spite of the lower incidence, now the incidence of breast cancer is increasing steadily in Chinese women. It becomes the second common cancer and accounts for 7%~8% of total cancer in women. In recent years, breast cancer is the most common cancer in some big cities ( Beijing, Shanghai ,etc). In the clinical therapy, the role of endocrine therapy and chemotherapy is equal to the operation and radiotherapy in the breast cancer, especially in the palliative treatment of elderly and late patients.For many years, we aided the new tumor markers (c-erbB-2/neu,pS2, etc) to the routine detection (ER, PR), for instructing the use of hormone therapy and prognostic prediction. It makes the hormone therapy more effective and selective. But, there are also some new problems in this area, the mutation or variation of the steroid receptor, drug resistance to chemo-endocrine therapy, and the optimization of co-detecting(mixed-detecting) tumor markers for prognostic prediction. We must investigate the biological features of breast cancer cell in gene levels.Not only ER and PR, but also pS2, Hsp70 and ST13 were detected by immunohistochemistry (IHC) in this study. Coupled with the follow-up data, we also analysis the correlation among the molecular markers, and the interaction with the single or co-detect and survival of the breast cancer patients. The main goal is investigating the role of Hsp70 and ST13 on biological features of breast cancer using antisense nucleic acid technique.Part I : The expression and correlation of ER, PR, pS2, HspTO and ST13, and their interaction with survival on breast cancer patients1. Preparation of the ST13 monoclonal antibody(McAb):After culturing the ST13 hybridoma cell(lF2) with routine condition, the cells were injected into the abdominal cavity to induce the ascites. The ST13 McAb were purificated from ascites by saturation ammonium sulfate(ASA).2. ER, PR, pS2, HspTO and ST13 were detected on primary site in 45 cases of breast cancer with follow-up data during 1993 ~ 1994. Results: i The percentage of positive expression is similar in all markers. ER was 77.8%(the highest), HSP70 and ST13 were 62.2% and 64.4%/espectively.3. The study showed a positive correlation among ER, PR, pS2 and ST13, especially between ER and ST13 (P = 0.000) or ER and PR (P = 0.000). But the negative correlation was be found between LN(+) ^4 and postmenopausal state (P <0.05). But non association between Hsp70 and other biomarkers was be found.4. In Kaplan-Meier survival analysis and Log Rank test, only the patients with expression of ST13 and Hsp70 have the good 5-year survival rate (P <0.05). The patients with LN(+) < 4 and postmenopausal state also have the good 5-year survival rate (P <0.01 and P <0.05, respectively).5. In COX survival analysis model, also it was be found that the patients with expression of ST13 and Hsp70 have the good 5-year survival rate (P <0.05).Part II: Construction of the eukaryotic expression recombinant plasmid pcDNA-AsHsp (antisense hsp70-pcDNA3.1(+))1. Construction of the prokaryotic expression recombinant vector pGEM-Hsp (Hsp70-pGEM-T Easy): The fragment of Hsp70 375bp cDNA from RT-PCR wasgotten by agarose gel electrophoresis and was purified with column. The HspVO cDNA fragments were ligated into pGEM-T Easy vector. pGEM-Hsp recombinant vector was confirmed by DNA sequence.2. Construction of pcDNA-AsHsp: 375bp Hsp70 fragments were gotten from pGEM-Hsp recombinant vector by EcoR I digestion. After recover with 1.2% agarose gel electrophoresis and purification with column, it also was ligated into pcDNA3.1(+). The pcDNA-AsHsp vector was screened out and confirmed by DNA sequence.Part III: The effect of antisene Hsp70 and antisene ST13 on biological feature of breast cancer cell1. The breast cancer Bcap-37 cells were transfected with recombinant pcDNA-AsHsp vector, pcDNA-...
|
PDF Full Text Request