| Objective To acquire a ribozyme aginst the E6 gene of Human Papillomaviruses type 16 , study the ma1ignant phenotype, the immune characterization, apoptosis and gene expression of the cultured cervical cancer cell line transfected with anti-HPV I 6E6-ribozyme, and investigate the possibility and practicality of ribozyme in tr tment of cervical cancer. Methods (1) According to Symon's hanmierhead structure, anti-HPV16E6 ribozyme was designed by using computer programs. (2) The DNA sequence encoding the ribozynie was synthesized, and incorporated into a prokaryotic expressing vector. Ribozyme and HPV 1 6E6 mR.NA were acquired by transcription in vitro, and the ribozyme's activity was identified by cleavage experiment in vitro. (3) The ribozyme was aloned into an eukaryotic vector. With the method of lipofectin transfection, the anti-HPV16E6-ribozyme and empty eukaryotic expressing plasmids were transfected into CaSK1 cell, which named as CaSKi-R, CaSKi-P respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by northern blot. (4) The morphology and the soft agar forming ability, were studied in CaSK1 cell and the transfected cells. The ability of each cell to form tumor was detected by injecting cells under the nude mice's skin. The expression of E6, PCNA and C-erbB-2 proteins were studied by using Flow Cytometry (FCM). (5) DNA and apoptosis rates of the three cervical cancer cell lines were detected by FCM. The expression of apoptosis related proteins, including c-myc, bcl-2 p53 and Fas, was also detected. (6) Antigens of tumor cells, HLA-1, HLA-2, B7-1, and B7-2 were detected by FCM. NK, LAX, CD3AK cells, and LAK cell stimulated by tumor cells were induced. Their cytotoxicity was detected on CaSK1-R, CaSKi-P, and CaSKi cells. Results (1) The 170 site of HPVI6E6 gene was chosen as cleavage site. The hammerhead ribozyme has two arms of 9 nt. (2) In vitro cleavage reaction demonstrated that anti-i 6HRz could site-specifically cleave the target HPV16E6 mRNA. (3) Anti-HPV16E6-ribozyme eukaryotic plasmid could be expressed stably in transfected CaSKi cells detected by RNA dc;t blot. Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSK1. (4) There was no distinct difference of the morphology and growth rate between CaSKi and CaSKi-P, but the growth rate of CaSKi-R decreased. The soft agar-forming rate of CaSKi-P was similar with that of CaSKi cells, while that of CaSKi-R was found decreased. The result of flow cytometric analysis showed that anti-HPV 1 6E6-ribozyme can reduce the expression of E6, PCNA and C-erbB-2 proteins on CaSKi-R cells, while this phenomenon was not found on the CaSKi-P cells. The ability of CaSKi-R to form tumor in nude mice was decreased compared with CaSKi and CaSKi-P cells. (5) The result of FCM analysis showed that the apoptosis rate of CaSKi-R cell was much higher than CaSKi and CaSKi-P. Anti-HPV16E6-ribozyme can reduce the expression of E6, c-myc and bcl-2 proteins on CaSKi-R cells, and increase the expression of p53. While this phenomenon was not found on the CaSKi-P cells. The expression of Fas was similar in the three kinds of cells. (6) The result of FCM analysis showed that anti-HPV 1 6E6-ribozyme could increase the expression of HLA-2 B7-l.. B7-2 antigens on CaSKi-R cells. The expressing rates of HLA-1 were very high in the three kinds of cells. The cytotoxicity of NK, LAK and CD3AK cells to CaSKi-R was much higher...
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