Comparison Of Effects Of SiRNA Transfection Mediated By Polyethyleneimine And Ultrasound Microbubble Targeting Technique On Accumulation Of Protoporphyrin IX In Human Endometrial Cancer Cells

ObjectiveWe established animal models of endometrial cancer, and used in vivo small animal imaging system to detect the fluorescence intensity caused by the accumulation of protoporphyrin IX(Pp IX) in the endometrial cancer cells after treatment with small-dose 5-aminolevulinic acid(ALA) combined with si RNA transfection mediated by polycation polyethyleneimine(PEI) and/or ultrasound microbubble targeting technique, so as to explore the feasibility of ultrasound microbubble targeting technique replacing transfection agents in the photodynamic diagnosis of endometrial cancer.Methods1 Human endometrial stromal cells(ESC) were isolated by collagenase digestion and cultured conventionally.2 MTT assay was employed to detect the proliferation toxicity of PEI on ESC.3 N-methyl-N’-nitro-N-nitrosoguanidine combined with estrogen was used to induce in situ tumor in the endometrium of ICR mice. The cultured and amplified HEC-1-A cell line was inoculated to build a model of human endometrial cancer xenograft in nude mice.4 In vivo small animal imaging system was employed to detect the effects of various concentrations of ALA on the animal models of endometrial cancer.5 Knockdown of ferrochelatase(FECH) in human endometrial cancer xenograft in nude mice was performed by transfection with FECH-si RNA mediated by polycation PEI and ultrasound microbubble targeting technique alone or in combination, and then local injection of small-dose ALA was conducted in xenograft model.6 In vivo small animal imaging system was employed to detect the fluorescence intensity changes of Pp IX in the nude mice with different treatments.7 Fluorescence microscopy was employed to detect the fluorescence intensity changes of Pp IX in frozen section of the tumor from the nude mice with different treatments.8 Fluorescence spectrophotometer was employed to semi-quantitatively detect the content changes of Pp IX in the tumor from the nude mice with different treatments.9 Pathological changes of non-targeted organs of the nude mice transfected with FECH-si RNA were observed.Results1 PEI showed an in vitro toxic effect on human normal ESC, and the IC50 was(9.461±0.081) μg/ml.2 Complex endometrial hyperplasia occurred in ICR mice 9 months after the experiment began. Canceration occurred in ICR mice 18 months after the experiment began. Tumorigenesis was observed in all the 31 nude mice after one-time cell inoculation, without death.3 The ICR mice model of in situ endometrial carcinoma didn’t show detectable fluorescence by in vivo small animal imaging modality, but the nude mice model of endometrial transplanted carcinoma did.4 There was apparent fluorescence in the nude mice given more than 6.251 mg/kg of ALA, but without significant difference between tumor and other tissues. When the dose of ALA was more than 50 mg/kg, there was significant difference between tumor and other tissues.5 After transfection with si RNA mediated by PEI and/or ultrasound microbubbles, combined with 1 mg/kg ALA, the tumor showed significantly higher Pp IX fluorescence intensity than that treated with 1 mg/kg ALA alone(P<0.05). The transfection efficiency ranged from high to low as follows: PEI combined with ultrasound microbubbles, PEI, and ultrasound microbubbles.6 After the treatment with PEI, ultrasound microbubbles or si RNA alone(no si RNA transfection), combined with 1 mg/kg ALA, the fluorescence intensity was not higher than that treated with 1 mg/kg ALA alone(P>0.05).7 The results of fluorescence microscopy were the same as those of in vivo small animal imaging modality. The fluorescence intensity showed significant differences between the three groups with si RNA transfection and the group with 1 mg/kg ALA treatment alone(P<0.05). There was no significant difference about fluorescence intensity between the three groups without si RNA transfection and the group with 1 mg/kg ALA treatment alone(P>0.05).8 The semi-quantitative detection of the Pp IX content in tumor extract by fluorospectrophotometer demonstrated that there were significant differences between the three groups with si RNA transfection and the group with 1 mg/kg ALA alone(P<0.05).9 After transfected with FECH-si RNA, the non-target organs of the nude mice in each group exhibited intact histological structure, without hemorrhage, inflammatory cell infiltration, cellular edema or degeneration.Conclusion1 PEI has an in vitro toxic effect on human normal ESC, and the IC50 is(9.461±0.081) μg/ml.2 The method of using N-methyl-N’-nitro-N-nitrosoguanidine combined with estrogen to induce endometrial in situ tumor in ICR mice is feasible, but it takes over 9 months of estrogen treatment so that it requires improvement.3 PEI as a carrier could knock down the expression of FECH by mediating FECH-si RNA transfection into human endometrial cancer xenograft in nude mice and enhance the red fluorescence intensity of Pp IX in combination with small dose of ALA.4 Ultrasound microbubble targeting technique could also knock down the expression of FECH by mediating FECH-si RNA transfection into human endometrial cancer xenograft in nude mice and enhance the red fluorescence intensity of Pp IX in combination with small dose of ALA.But transfection efficiency of ultrasound microbubble targeting technique alone is worse than that of PEI alone.5 No damage is found in non-targeted organs of the nude mice treated with PEI, ultrasound microbubbles or si RNA.6 Ultrasound microbubble targeting technique could replace PEI under certain conditions to induce Pp IX fluorescence imaging in endometrial cancer cells and reduce the toxic effect on normal cells.