Experimental Studies On The Expression Of Survivin In Hepatocarcinoma And Survivin-mut Targeting Genetherapy

Liver cancer, one of the most common malignancies, which has poor prognosis, threatens the health of mankind seriously. The technology of apoptotic intervention aiming at selectively inducing apoptosis of tumor cells has become the basic strategy of its genetherapy. Survivin is a new member of the inhibitor apoptosis protein families(IAPs) and researched hotly at present because of its unique molecular structure, particular distribution in different tissues and notable anti-apoptotic effects. By now, the mechanisms of survivin in the original occurrence, progression and development of hepatocarcinoma have not clear throughly and the researches about the relationship with survivin and hepatocarcinoma are few.Our study is to make clear the expression of survivin in heptocarcinoma cell lines and tumors, to investigate the killing effects of survivin dominant-negative mutant (survivin-mut) gene expressed in vivo, whose plasmid constructed by pLEGFP-Cl retroviral vector and transfected into common human hepatocarcinoma cell lines, in order to give a green light to the next researches based by the expression of survivin-mut and offer a new method of the survivin targeting genetherapy.Part 1 Expression of Survivin and Its Relationship to Apoptosis in Human Hepatocarcinoma objective: To explore and master the using method of real time quantitative PCR(RQ-PCR), and detect the expression of survivin and apoptosis index(AI) in human hepatocarcinoma cell lines and tumors, then analyze their interaction. Method: DNA combined SYB Green I dyeing method has been used to quantitatively detect the survivin expression in HepG2, SMMC7721 and 36 cases of surgically resected specimens, tumorous tissues and their neighboringnontumorous tissues contained. Flow cytometry has been used to detect the AI in hepatocarcinoma cell lines, and the AI of tumorous tissues and their neighboring nontumorous tissues were assessed by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL). The results obtained were analyzed and compared to explore the relationship between the expression of survivin and apoptosis in cell lines and tumors. Results: The natural AI of HepG2 and SMMC7721 are 5.46% ±0.97% and 5.88% ±0.77%, with no significant differentiation(P>0.05), and its' Survivin/p-actin are respectively 11.05 + 2.36 and 12.21+3.11, which also have no notable differentiation (P>0.05). The survivin/p-actin of tumorous tissues is notablely higher than their neighboring nontumorous tissues(P<0.001). The expression of survivin in tumorous tissues and neighboring tissues have no significant differentiation in different groups divided by gender, age, size, with or without embolus in portal vein and metastatic conditions(P>0.05), the same as the AI in different groups divided by gender, age, with or without embolus in portal vein(P>0.05). But the AI of tumorous tissues whose size under 5 centimeter are significantly lower than their neighboring nontumorous tissues(PO.Ol), which show apoptotic inhibition, on the other hand, the higher AI go with the higher expression of survivin in tumorous tissues whose tumor size larger than 5 centimeter. Cone I ut ions: RQ-PCR is a fitting method of measuring the expression of survivin quantitatively in hepatocarcinoma cell lines and tumors. Survivin expressed highly in tumorous tissues and lowly in their neighboring nontumorous tissues. The expressional conditions of survivin in tumorous and nontumorous tissues are not relate to gender, age, size, portal vein invasion and metastatic condition of the patients. The AI of tumorous tissues is not relate to the patients' gender, age and portal vein invasion, but has relationship with tumor size and metastatic condition. The highly expressed survivin contributes to the apoptotic inhibition in tumorous tissues, but does not induce the AI of all tumors decreasing. Part 2 Experimental Study of Survivin-mut Transfected into Human Hepatocarcinoma Cell Lines Modulate the Apoptotisisobject i ve: To investigate the apoptotic modulating action of survivin-mut-Thr34Ala after transfected into human hepatocarcinoma cell lines, and explore the method and mechanism of survivin-mut targeting genetherapy. Method: Transfect survivin-mut-Thr34Ala, constructed by pLEGFP-Cl retroviral vector, into common human hepatocarcinoma cell lines HepG2 and SMMC7721 by lipofecTAMINE 2000, then observe the transfectional outcomes and expression of survivin by means of RT-PCR and western blotting, detect the apoptotic changes of these transfected cell lines, and analyze the regulating action of survivin-mut to apoptosis. Using the method of colorimetric MTT assay to observe cells growth after chemotherapeutic drugs added and analyze cell growth curve to make out the killing effects affected by survivin-mut. Resu I ts: ?Enhanced Green Fluorecent Protein(EGFP) was observed in two cell lines after transfection; the outcome of RT-PCR was 528bp in size, in accord with what was expected; the sequencing result was about 99% same as the nucleic acid sequence of survivin described in Genebank; 16.5kDa protein was detected by western blotting, indicating the survivin-mut-Thr34Ala transfected availably and expressed stably in hepatocarcinoma cell lines. ?Abnomal mitosis and mitotic catastrophe were detected in two availably transfected hepatocarcinoma cell lines, and were not detected in the control groups. ?The Al of HepG2 24h and 48h after transfection were 28.0%±3.5% and 38.7%±8.4%, while it were 20.7%±3.4% and 33.6%±4.6% in SMMC7721, both notably increased highly compared with the control groups(P<0.01). ?The quantity of survival cells sorted descendingly, after dealt with chemotherapeutic drugs and detected by colorimetric MTT, was empty vector groups(lumol/L) without drugs, empty vector groups(lumol/L), three survivin-mut recombinant groups(0.1umol/L, lumol/L, lOumol/L), furthermore, this trend was increasing with time going; on the other hand, the quantity of alive cells increased slowly in groups of higher survivin-mut concentration, but the final quantity of alive cells was not relate to their orginal survivin-mut concentration. Cone I ut ions: The expression of survivin-mut-Thr34Ala can induce apoptosis of human hepatocarcinoma cell lines and result in abnomal mitosis and mitotic catastrophe; The expression of survivin-mut-Thr34Ala can upgrade the sensitivity to common chemotherapeutic drugs.