Research Of Interference With XIAP Gene And 5-FU On Growth Suppression Of Transplanted Cholangiocarcinoma In Nude Mice And Enhanced The Sensitivity To Chemotherapeutics

Objective:Through transfected antisense oligonucleotide of x linked inhibitor of apoptosis protein into transplanted human cholangiocarcinoma QBC939 cells in nude mice by cationic liposomes, to explore the effect of ASODN on human cholangiocarcinoma cells inhibition of XIAP gene in nude mice, proliferation and induced apoptosis of cancer cells;To observate the sensitivity of cholangiocarcinoma cells to chemotherapeutical drugs fluorouracil(5-FU) after the expression of antisense oligonucleotides inhibit XIAP gene.Methods:planted the cells of the proliferative logarithmic phase of QBC939 into the area of left forlimb armpit of nude mice,each 0.2m1(4 X 10⁷). after 10 days constructed tumor model(5mm X 5mm),the mice were divided into 5 groups randomly:control group,liposomes (lip) group,5-FU group,ASODN/lip group and ASODN/lip5-FU group. Each group were injected transfer dye liquid 600μl (serum without antibiotics),Lip 10μg/ml 100μl add transfer liquid to 600μl,transfer dye liquid 600μl,ASODN/Lip final concentration 1μM+Lipfectin 10μg/ml 100μl add transfer liquid to 600μl and ASODN/Lip final concentration 1μM+Lipfectin lOμg/ml 100μl add transfer liquid to 600μl into the tumor or around the tumor, respectively.4 times injections were given,one time per week. Each related group injected 5-FU 1Omg/Kg on the first,third,fifth day after transferred,the other group were injected normal saline. mice were killed after 4 weeks.The expression of XIAP mRNA was determined by RT-PCR; Apoptosis and cells cycle changes were determined by flow cytometry.Immunohistochemical SP staining method were used to detect the expression of XIAP and PCNA protein, TUNEL in situ apoptosis. Cell proliferation index and apoptosis index.were calculated.Results:The subcutaneous tumor model in nude mice was successfully established. Using cationic liposome transfected ASODN into nude mice successfully. Gross specimen shows the tumor volume of ASODN/Lip and ASODN/Lip-5FU decreased during treatment, the rate of tumor suppression reached 54.55% and 65.00%,which were significantly increased in the group of ASODN/Lip and ASODN/Lip-5FU compared with other 3 groups(p< 0.05);while the tumor volume of the other three groups increase.The weight of tumor were significantly different in each group(p< 0.05). The inhibition rates of tumor weight were 60.60%,39.02% in the group of ASODN/Lip-5FU and ASODN/Lip respectively, the different was significant(p< 0.05).There are no statistical difference among the groups in body weight changes of 5 groups mice. RT-PCR results show that the expression of XIAP mRNA decreased significangtly in ASODN/Lip and ASODN/Lip-5FU than other groups(P<0.05). Flow cytometry showed that ASODN/Lip group and ASODN/Lip-5FU group QBC939 cells were found to represent the sub-G₁ apoptotic peak (15.11±2.25)%, (60.36±2.17)%, and the Go/G₁ phase cells was higher than control group, the difference was significant (P<0.05). Immunohistochemical results showed after transfection ASODN, ASODN/Lip-5FU group and ASODN/Lip group XIAP protein expression was (27.83±4.97)%,(31.62±6.85)%, significantly lower than the control group (62.54±8.32)%.The difference was statistically significant (P <0.05). PCNA immunohistochemistry showed the proliferation index of the transfection ASODN and combined 5-FU group was significantly lower than the control group, the difference was statistically significant (P<0.05). TUNEL results showed that the apoptosis index of ASODN/Lip-5FU group, ASODN/Lip group and 5-FU group were (18.93±0.21)%,(10.19±0.24)%,(10.22±0.18)%, compared with the control group the differences were statistically significant (P <0.05),ASODN/Lip-5FU group and 5-FU group were statistically. significant(P <0.05).Conlusion:The liposomal transfection of ASODN can down-regulate the expression of XIAP gene,Which can inhibit cells growth and induce apoptosis of transplanted cholangiocarcinoma QBC939 cells in nude mice, and increase the chemotherapeutic sensitivity of transplanted cholangiocarcinoma cells of QBC939 after XIAP inhibition. So it may be the new target for gene therapy in cholangiocarcinoma.