| BackgroundColorectal cancer is one of the major diseases that threatenes the health of human,with the incidence increasing in China.The conventional cancer therapies are accompanied by their intrinsic limitations,therefore newly treatment methods are urgently needed.With the development of molecular technology and deep recognition of colorectal cancer,gene therapy represents a novel treatment model in cancer therapy.More and more data showed that the abnormality of cell apoptosis was very important in cancer development as well as malignant cell proliferation and differentiation hindering.In general condition,apoptosis can destroy DNA-damaged cells and cycle-disordered cells.The proliferation rate of tumor cells was not higher than that of normal cells and the accumulation of tumor cells result from cells escaping apoptosis.The disorder of regulation in apoptosis may well prolong the survival of cells aberrantly,being subject to accumulation of mutation which leads to transformation,and thereby involves in carcinogenesis.So,tumor is not only due to the disorder of cell proliferation and differentiation but also owing to the abnormality of cell apoptosis.The inducing apoptosis is a new therapy method for tumor and transferring apoptosis-activating gene or apoptosis-inactivating suppressor gene into tumor cells has become one of most intriguing therapeutic strategies.Survivin is the most effective inhibitor factor. Survivin is a newly discovered member of inhibitor of apoptosis proteins family and play an important role in the regulation of apoptosis.It expresses in human embryonic tissue and takes part in the histological differentiation,organ formation, but almost not in normal differentiated human tissues.Meanwhile,survivin low-expresses in non-proliferation vascular endothelial cells and over-expresses in neogenetic vascular endothelial cells.Therefore,survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.Antisense oligonucleotides(ASODN) can adjust the message transfer from gene to protein and inhibit the protein expression through specifically combination with target mRNA.ASODN adjust the genetic information transfer on the upper stream, it has good function with lower dosage.ASODN can specifically block the single gene expression without the side effect on normal gene.Survivin only expresses in human embryonic tissue and tumor tissue,it makes the anti-survivin gene therapy a specific and safe therapy.The important aim of gene therapy is the continually and steadily expression of obtained genetic material in transferred cell.ASODN is a kind of hydrophilic anion ionic polymer,it has poor permeability to biomembrane and be degraded easily by the mucleicacidase,it can't be absorbed and accumulated in the target cell.All this reason restrict the extensive application of ASODN and need a effective carrier.Gene transfect efficiency is high for traditional virus vector,but the virus posed immunogenic and cytotoxic.The transfection efficiency is low for non-virus vector. Nanometer particle is a kind of natural or synthetic high polymer particle,their diameter are the same with virus among 10-100nm,smaller than normal cell.They can finish the gene therapy through carrying DNA,RNA and oligonucleotide and absorbed by target cell.Polyamidoamine dendrimer(PAMAM) is a kind of new man-made namometer material.Oligonucleotide/PAMAM Complex will form by electrostatic interaction between surface charged positive electricity of the amidocyanogen group and the DNA main chain charged negative electricity of the phosphoric group,and then the complex can enter the cell by phagocytosis with the help of interaction between cation of the complex surface and the negative electricity of the glycoprotein, phospholipids on the cell surface.The absorb of complex can be promoted and the persistence can be delayed by PAMAM.In order to elevate the survivin ASOND transfer and strengthen the gene therapy effect,we use PAMAM as a surviving ASODN nanometer carrier,detect its feature and gene transfer rate and release characteristic,observe the killer function of survivin-asODN to colorectal cancer cell SW620 in vitro and vivo.ObjectivesThe feasibility of transfecting survivin-asODN mediated by PAMAM and the effects on the expression of survivin and apoptosis to colorectal cancer cells SW620 were investigated.Further,tumors derived from SW620 cells were established in nude mice and the anti-tumor effect of survivin-asODN in vivo was studied.Methods1.The preparation of PAMAM antisense gene complex and the investigation of its feature.PAMAM antisense gene complex was prepared by combining 300μg/L survivin-asODN and 4.06μg/L PAMAM.Similarly,cationic liposome antisense gene complex was prepared as control.The shape of the complex were observed by transmission electron microscope,the size was determined by laser particle size analyzer and the zeta potential was measured by zeta potential analyzer.The encapsulating efficiency,DNA loading level and releasing progress in vitro were determined by ultraviolet spectrophotometer in centrifuge method.2.The investigation of expression of survivin and apoptosis through transfecting survivin-asODN mediated by PAMAM to colorectal cancer cells SW620 in vitro.Both of the above gene transfect complexes were used to transfect colorectal cancer cells and transfect efficiencies of survivin-asODN in SW620 cells were measured.Survivin protein expression was checked by Western blot.For both group cells,the apoptosis was assessed by flow cytometry.3.The investigation of the anti-tumor effect of survivin-asODN mediated by PAMAM to tumors derived from SW620 cells in nude mice.To establish SW620 carcinoma transplanted subcutaneously in nude mice,4-to 6-week-old female Balb/c nude mice were used as hosts xenografts,SW620 cells were injected subcutaneously into the abdominal region.All tumors were 0.5cm mean diameter and mice were grouped randomly according to treatment regimen.The tumor and around tissue were injected by LipolifectmainTM2000 and PAMAM (group A:negative control),lipidosome--survivin-asODN(group B) and PAMAM-survivin-asODN(group C).The tumor diameter was measured before and after treatment.The weight of tumors was studied and the growth inhibition rate was calculated after treatment.The change of tunour cells were observed by Transmission Electron Microscope(TEM),the expression of surviving gene in tissue and pathological change in heart,liver,spleen,lung and kidney were observed at the same time.ResultsDNA releasing lasted 14 days for PAMAM,but only 5 days for liposome. Transfect efficiency of PAMAM-survivin-asODN on colorectal cancer cells was higher compared with that of liposome-survivin-asODN(P＜0.05).After the colorectal cancer cells transfected,survivin protein expression was lower(P＜0.05),and apoptosis rate was higher(P＜0.05) compared with control.1.Observation of the feature and diameter of transfected complex and measurement of zeta potential of transgected complex.The complex of liposome-survivin-asODN and PAMAM-survivin-asODN were round or round like particle under the TEM,the diameter of PAMAM-survivin-asODN complex was smaller(t=7.874,P=0.000,P＜0.001) compared with that of liposome-survivin-asODN complex,but zeta potential was higher(t=4.158,P=0.002,P＜0.01).2.Measurement of nuclear acid encapsulating efficiency,loading level and gene releasing in vitro of transfected complex,There were no significant differences between the two groups in terms of DNA loading level(t=0.178,P=0.863,P＞0.05) and encapsulating efficiency(t=0.582, P=0.573,P＞0.05).There are blasting releasing of gene in the complex of liposome -survivin-asODN in 24～48 hours and decreased quickly.The release of DNA can not be measured in 6 hours.There are blasting releasing of gene in the complex of PAMAM-survivin-asODN in 24～48 hours,and the releasing decrease slowly and last for more than 14 days.3.Cell transfection and transfection efficiency24 hours after transfection,green fluorescence could be seen under fluorescence microscope for both groups of cells.48 hours later,the highest transfection efficiency was reached.The fluorescence intensity(46.46±9.84) from liposome group was lower than that from PAMAM group(63.90±10.29),and the difference was statistically significant.(t=2.739,P=0.025,P＜0.05)4.The growth suppression effect of surviving-asODN to SW620 cells.There are significant difference between the treatment group of survivin-asODN and the bland control group,the suppression effect of survivin-asODN to SW620 cells was significant(P＜0.001).There growth suppression of cells treated by PAMAM-survivin-asODN and liposome-survivin-asODN,but growth suppression of the PAMAM-survivin-asODN complex was more strong(P＜0.05).5.The expression of survivin mRNA in SW620 cells.There were specific surviving strap in 191bp of survivin-asODN treated group and blank control group.The survivin mRNA of SW620 was 0.85±0.06(in blank control group),0.56±0.10(liposome-survivin-asODN group) and 0.36±0.10(PAMAM-survivin-asODN).Compared with the blank control group,there were significant difference in both treated group(P＜0.001).The expression of surviving in PAMAM-survivin-asODN treated group was obviously lower than that in liposome-survivin-asODN treated group(P＜0.01).6.Effect of Survivin-asODN on survivin protein expression of SW620 cells.Survivin protein expression from SW620 cells of blank control group was (72.78±6.79),while for the liposome group and PAMAM group were(35.02±6.28)and(25.36±7.15) separately,both of them were significantly lower than the blank control group(P＜0.01);Survivin protein from PAMAM group was significantly lower than that of liposome group(P＜0.05).7.Effect of Survivin-asODN on the apoptosis rate of SW620 cells.Compared with empty control group,the cell apoptosis rates from both liposome group and PAMAM group were significantly increased(P＜0.05).While the apoptosis rate of PAMAM group was higher than that of liposome,the difference is statistically significant(P＜0.05).8.The modeling of nude rate mouse with SW620 tumors.SW620 cells were injected subcutaneously into the abdominal region of nude mouse and formed tumor successfully.There were no redness and swelling in the injected region.The tumor form time was 15.7±1.14 days.9.The tumor suppression effect of Survivin-asODN to SW620 cells in nude mouse.There were no significant difference in gross tumor volume and weight before treatment among blank control group,liposome-survivin-asODN treated group and PAMAM-survivin-asODN treated group(F=0.229,P=0.798,P＞0.05).At the end of treatment,the gross tumor volume was decreased obviously in Lipo-survivin-asODN treated group(t=7.918,P=0.000,P＜0.001) and PAMAM-survivin-asODN treated group(t=11.854,P=0.000,P＜0.001).The gross tumor volume in treated group were significantly lower than that in the blank control group(P＜0.001).Compared with the liposome-survivin-asODN group,the gross tumor volume in PAMAM-survivin-asODN group was significantly lower(P＜0.05).At the end of treatment,the weight of tumor in both of the treated group was significantly decreased compared with the blank control group(P＜0.001).Compared with the liposome-survivin-asODN group,the weight of tumor in PAMAM-survivin-asODN group was significantly lower(P＜0.05).The tumor suppression effect of PAMAM-survivin-asODN was significantly higher than that of liposome-survivin-asODN(t=48.550,P=0.000,P＜0.01).10.The surviving protein expression in the transplanted tumor in nude mouse.There expression of surviving protein in the transplanted tumor in nude mouse was high.There were significant difference between the blank control group and treated group(P＜0.05).The expresson of surviving protein was significantly lower in PAMAM group than that in liposome group(P＜0.05)。11.The expression of survivin mRNA in transplanted tumor of nude mouse.There were specific surviving strap in 191bp in blank control group and survivin-asODN treated group through the RT-PCR measurement.The survivin mRNA was 0.85±0.06(blank control group),0.56±0.10 (liposome-survivin-asODN group) and 0.36±0.10(PAMAM-survivin-asODN group). Both them in the treated group were significantly lower than that in blank control group(P＜0.001).It was lower in the PAMAM-survivin-asODN treated group compared with liposome-survivin-asOD treated group(P＜0.01). 12.The observation of transplanted tumor cells in nude mouse under electron microscope.The tumor cells were crimpled and the microvillus were disappeared after the gene therapy under the transmission electron microscope.The membranes of cell nucleus were crimpled and vanished,some of the cell nucleus were broken,the chromatin were condensed or broken.Pyknotic apoptosis bodys were observed in the tumor cells.13.The observation of main organ in nude mouse after the survivin-asODN complex treatment.There were no obvious change in the nude mouse heart,lung,liver,spleen,and kidney observed through routine pathological check after the PAMAM-survivin-asODN and liposome-survivin-asODN treatment.Conclusion1.PAMAM-survivin-asODN transfect complex was synthesized in this experiments, the complex was round or round like nanometer particle with small diameter and high potential.2.There were no significant differences between the PAMAM-survivin-asODN transfect complex and the liposome-survivin-asODN transfect complex in terms of DNA loading level and encapsulating efficiency.But the liposome-survivin-asODN transfect complex can release the target gene slowly and last long time.3.The electric charge in PAMAM-survivin-asODN transfect complex were compounded stable and completely.The PAMAM can protect the survivin-asODN through resisting the degradation effect by nucleicacidase.4.PAMAM can transfect survivin-asODN to SW620 cells successfully,its transfect efficiency was high than liposome-survivin-asODN.5.There were high level expression of survivin in SW620 cells,survivin-asODN transfected by PAMAM decreased the surviving expression through mRNA and protein path.6.The growth of SW620 treated by PAMAM-survivin-asODN and liposome-survivin-asODN were inhibited through inducing apoptosis.7.Survivin-asODN obviously suppressed the growth of the SW620 tumor in nude mouse.There were stronger effects in PAMAM-survivin-asODN complex than that in liposome-survivin-asODN complex.8.There were high level expression of survivin in transplanted SW620 cells in nude mouse,survivin-asODN transfected by PAMAM decreased the surviving expression in transplanted SW620 cells in nude mouse through mRNA and protein path.9.The growth of transplanted SW620 cells in nude mouse treated by survivin-asODN were inhibited through inducing apoptosis.10.There were no obvious change in the nude mouse heart,lung,liver,spleen,and kidney observed through routine pathological check after the PAMAM-survivin-asODN treatment,which showed that the PAMAM-survivin-asODN treatment system were safe to the nude mouse. |
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