| Objective: To construct adenovirus vector of RNA interference targeting Her2/neu(HER2-si RNA) and package recombine adenovirus, then to observe the changes of human ovarian cancer xenografts infected by recombinant adenovirus combination with cisplatin. and to monitor the interference effect of HER2-si RNA in vivo with SPECT imaging as the basis for gene therapy of human ovarian cancer in the further.Methods: ① In accordance with the previous researches in this laboratory, we inserted the specific HER-2/neu-sh RNA sequence with the highest interference efficiency and a one scrabble sequence into adenovirus vector. The sequencing was used to identify. ②The above vectors were transfected into HEK293 cells using Lipofectamine 2000, and the recombinant adenovirus were packaged by Ad Max adenovirus package system. The adenovirus were purified by means of Adeno-X? Virus Purification Kit. The titer of adenovirus was tested by End-point dilution. ③ After the BALB/c nude mice xenograft models being established by subcutaneous injection of SKOV-3 cells, they were divided into three treatment groups randomly, One was Ad-HER2-RNAi group being injected intratumorly and subcutaneously around the tumor with specific HER2-si RNA adenovirus, another was NC group being injected negative adenovirus at the same method and the other was CON group being injected PBS. The size and weight of xenografts in the groups were measured to evaluate the therapy efficacy, and the protein HER2 expression in xenografts of each group were determined by immunohistochemistry(IHC). Mice in three groups were injected with131I-Herceptin for SPECT imaging and T/B(tumor/background) ratios were calculated by ROI technique in their planar images. The mice in CON group and Ad-HER2-RNAi group were scanned again with131I-Herceptin using SPECT 15 days later. The other mice in Ad-HER2-RNAi group were taken SPECT imaging with 131I-Herceptin at different of dose. ④ The nude mice with ovarian cancer xenograft are allocated into 5 groups to carry out combination thrapy experiments, among which three groups were similar to treatment groups mentioned above, namely Ad-HER2-RNAi group, NC group and CON group, and the new groups were Ad-HER2-RNAi+DDP group and DDP group respectively. The difference was that normal saline was added in each group except the new two groups The DDP and normal saline were administered intraperitoneally, while others are injected intratumorly and subcutaneously around the tumor. During the period of thrapy, several evaluation data such as the size and weight of xenografts, weight of mice and mental state of mice were recorded. The expression of HER2 protein in each group was detected by IHC. Finally, the tumor/background(T/B) ratios in all experimental groups were calculated by the SPECT images analysis.Results: ① T he shuttle plasmids of HER2-si RNA and t h e s c r a bb l e were e s t a b l i s h e d r e s p e c t i v e l y a n d t h e s e q u e n c e s we r e c o n fi r me d b y s e q u e n c i n g. ②The recombinant adenovirus were packaged successfully with high-titer 1×1011 PFU/m L. ③The average volume and weight of xenografts of Ad-HER2-RNAi group were obviously decreased(P<0.05) compared with those of the CON group and NC group, The results of IHC showed that the protein HER2 expression in Ad-HER2-RNAi group was obviously reduced. The xenograft images could be seen at the time of 1 hour after 131I-Herceptin were injected through tail vein and the radioactivity in xenograft accumulated gradually. There were no differences of T/B ratios between the first SPECT imaging and the second imaging 15 days later. It was no significant effect to images using different dose of radiopharmaceutical.. Therefore, the 131I-Herceptin with medium dosage 7.4MBq would be used as imaging dose in each group in the following experiments. Compared the T/B ratios at different time and the T/B ratios adjusted by tissue thickness of Ad-HER2-RNAi group with those of CON group and NC group, the differences were significantly(P<0.01). ④The combination therapy experiment suggested that the weight of mice with DDP decreased significantly(P<0.05),Compared three groups of Ad-HER2-RNAi group, DDP group and Ad-HER2-RNAi+DDP group with two groups of CON group and NC group, the average tumor volume and weight of xenografts of three groups were obviously decreased(P<0.01), especially those of the Ad-HER2-RNAi+DDP group were much lower than those of the two others. The IHC results showed that the protein HER2 expressions in the combination group were down-regulated obviously. The results of SPECT imaging showed that the T/B ratios and T/B adjusted ratios at each point of time in the three treatment groups were lower than those of CON group and NC group respectively(P<0.01). In addition, compared the T/B original ratios of Ad-HER2-RNAi+DDP group at each point of time with those of Ad-HER2-RNAi group and DDP group, the differences were significantly(P<0.05). After adjustment, T/B adjusted ratios in combination groups at each point of time were lower than those in single administrated groups except those at the point of 1h(P<0.05).Conclusions: The growth of xenograft could be inhibited effectively after specific HER2-si RNA recombinant adenovirus infected xenograft of human ovarian cancer with high-expression HER2. It should enhance inhibition effect of tumor when HER2-si RNA recombinant adenovirus combined with cisplatin. SPECT imaging could detect and evaluate the interference effect of HER2-si RNA in vivo. |
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