| Objective To investigate the p27kip1 protein expression in carcinoma of the esophagus and its relation to clinicopathological features and construct the recombinant adenoviral vector carrying human p27kip1 cDNA, study inhibitory effect of p27kipl gene transfere on esophageal carcinoma in vivo and vitro and study antitumor mechanisms of p27kipl.Methods (1)Twenty-seven cases of carcinoma of esophagus were studied by immunohistochemical technique. (2)The shuttle plasmid encoding p27kip1cDNA was constructed by cloning p27kip1cDNA fragment in the obverse direction into the pACCMVPLPA. Then the plasmid pJM17 and the shuttle plasmid were contransferred into 293 cells with lipofectamine for homologous recombination to acquire recombinant adenovirus-Ad-p27kip1. Ad-p27kip1 and Ad-LacZ were transfected to 293 cells to amplify the recombinant adeno virus. A significant number of Ad-p27kip1 and Ad-LacZ were acquired by CsCl density gradient separation and purification. (3)Before and after infected by Ad-p27kipl, effect of proliferation on cells was evaluated with MTT assay, 3H-TdR and 3H-Leucine incorporation, cell cycle and apoptosis were detected by flow cytometry(FCM), DNA fragment analysis was used to confirm the presence of apoptosis, activity of telomerase was examined by TRAP PCR-ELISA, expression of p27kipl and Survivin were detected by immunocytochemical technique, Western blotting was used to detected expression of Survivin. (4)Eca9706 cells were injected into subcutaneous tissue of nude mice to establish tumors. The transplanted rumors were reproduced among the nude mice contionually after original grafts grew well. The model of esophageal carcinoma in nude mice was established by transplanting the tumor tissue mass into the subcutaneous tissue of nude mice. Ad-p27kip1 and Ad-LacZ were directly injected into the esophageal tumors in nude mice respectively. Compared with control group, the growth curves of tumor were drawed and the growth inhibition rates of tumor were calculated. Expression of p27kipland Survivin in tumors were detected by immunohistochemical technique. Western blotting was used to detected expression of Survivin.Results (1)The positive rates of p27kipl expression in carcinoma of the esophagus were 40.7%.But there was over expression in noncancerous specimens of esophagus. (2)A significant number of Ad-p27kipl and Ad-LacZ with high purity were obtained. (3)After infected by Ad-p27kipl, survival rate of Eca9706 cells was decreased, the incorporation of 3H-TdR and 3H-Leucine was lower than that in control group(P<0.001), the ratio of G0/G1 phase cells increased and G2/M phase and S phase cells decreased (P<0.01) and a obviously apoptotic sub-G1 peak was found, agarose electrophoresis showed marked ladder, the activity of telomerase was obviously inhibited(P<0.01), expression of p27kipl was low and expression of Survivin was high in Eca9706 cells, but expression of p27kipl was increased and expression of Survivin was decreased after Eca9706 cells were transfected with Ad-p27kip1. (4)The model of esophageal carcinoma in nude mice was successfully established. The growth of tumors in gene therapy group with p27kipl was obviously suppressed , it had significant difference compared with control group (P<0.001 ) . The growth inhibition rate of tumor reached 64.1%. The tumors in nude mice were low differentiation esophageal squamous carcinoma proved by pathological detection. In tumors of control group, expression of p27kipl was low and expression of Survivin was high. In tumors of p27kipl gene therapy group, expression of p27kipl was high and expression of Survivin was low.Conclusions These findings suggested that the downexpression of p27kipl may play a crucial role in the carcinogenesis and development of esophageal carcinoma.p27kip1 gene transfere mediated by adenovirus has significant inhibitory effect on esophageal carcinoma in vivo and vitro. The antitumor mechanisms are suppressing Survivin expression, causing G1 blocking, inhibiting activity of telomerase and inducing apoptosis. It in...
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