| Objective: Based on the cooperation of growth-suppression of Adenovirus-mediated human ING4 gene (Ad-hING4) combined sustaining low dose-rateγray (125I seed) on pancreatic cancer in vitro, we established subcutaneous tumor models of pancreatic cancer (PANC-1) in nude mice, to futher study the cooperation of Ad-hING4 and 125I seeds in tumor-suppression on human pancreatic cancer xenograft in vivo, and to explore possible molecule mechanisms.Methods: (1) Ad-hING4 recombinant adenovirus vector and adenovirus vector Ad-GFP were transfected into QBI-293A cells to obtain high titre adenovirus, and then detected their titre and identified their expression;(2) Then twenty-five subcutaneous tumor models in nude mice were established with human pancreatic cancer cell line PANC-1.When the tumors grew up to 810mm in dimensions, the nude mice were randomly divided into five groups, PBS control group (PBS group), Ad carrier group (Ad group), 125I seeds brachytherapy group (125I group), Ad-hING4 gene treatment group (Ad-hING4 group),125I seeds combined Ad-hING4 group (combination group) ,and five animals in every group for treatment study of tumor- suppression. The mice of Ad-hING4 group were intratumorally injected with Ad-hING4 virus (1×107pfu/50μl) , and the mice of PBS group and Ad group were intratumorally injected with equivalent PBS and Ad-GFP, once every two days for six times. One 125I seed with 14.8MBq was implanted into each mouse in 125I group. In combination group, each mouse was intratumorally implanted with one 125I seed (14.8MBq) and injected with Ad-hING4 virus(1×107pfu/50μl, once every two days for six times).The mice were monitored daily.(3) The volumes (in cubic centimeters) were measured with a vernier caliper every 5 days after treatment,and calculated by the formula, tumor volume=ab2×0.52, where a was the larger and b was the smaller of the two dimensions. After 20 days, 25 mice were sacrificed and subcutaneous tumors were taken out. The volumes and the weights of tumor were measured, and the ratios of tumor-suppression and Jin-Shi q value were analyzed.(4) The morphological changes of tumor cells, the extent and range of tissue injure and apoptotic index (AI) were examined on routine pathological sections.(5) The expression of CD34(MVD), VEGF, PCNA, Bcl-2, Bax, Bcl-2/Bax, Fas, Survivin, Caspase-3 and hING4 gene were detected by Immunohistochemistry SP method.Results:(1) After infecting and increasing the recombinant virus many times, the virus titre of Ad-hING4 and Ad-GFP both reached to109pfu/ml by fluorescence counting method. RT-PCR identified the recombinant virus. Its result indicated that adenovirus-mediated hING4 gene could be transcripted successfully in PANC-1 cells.(2) The subcutaneous tumor models were successfully established with human pancreatic cancer cell line PANC-1. During treatment, no mice was dead and no toxic reaction appea- red.(3) The ratios of tumor-suppression of three treatment groups of 125I group, Ad-hING4 group and the combination group were respectively (34.19±10.80)%, (31.50±13.36)%, (67.15±5.15)%. Compared with PBS group and Ad group, they all had very prominent difference (P<0.01). The ratio of the combination group was higher than that of 125I group and Ad-hING4 group (q=1.22).(4) Routine pathological examination found degenerative necrosis at the site nearby the seeds but the alive tumor cell still presented faraway from the seeds. The degenerative necrosis of in other groups had no obvious associations with distance. The apoptosis index (AI) of three treatment groups were obviously higher than that of PBS group and Ad group (P<0.001). And AI of the combination group were the highest in three treatment groups (P<0.001).(5) Immunohistochemistry:①Effects of Ad-hING4 or/and 125I seed on the angiogenesis of human pancreatic cancer in nude miceMVD marked by CD34 and VEGF of three treatment groups were obviously lower than that of PBS group and Ad group (P<0.05). The MVD and VEGF of the combination group were the lowest in three treatment groups (P<0.01).②Effects of Ad-hING4 or/and 125I seed on the proliferation of human pancreatic cancer in nude miceThe number of positive cells of PCNA of three treatment groups were distinctly lower than that of PBS group and Ad group (P<0.05), and the combination group were the lowest in three treatment groups (P<0.05).③Effects of Ad-hING4 or/and 125I seed on the expression of protein associated with the apoptosis of human pancreatic cancer in nude miceThe number of positive cells of Bcl-2 of three treatment groups were distinctly lower than that of PBS group and Ad group (P<0.05). But the expressions of Bcl-2 of the combination group had no evident difference compared with other two treatment groups (P>0.05). The number of positive cells of Bax of three treatment groups was obviously higher than that of PBS group and Ad group (P<0.05), which of the combination group was the highest in three treatment groups (P<0.05). And Compared with PBS group and Ad group, the ratios of Bcl-2 and Bax of three treatment groups were prominently low (P<0.001), and the ratio of combination group was the lowest in three treatment groups (P<0.001). The number of positive cells of Survivin of three treatment groups were notably lower than that of PBS group and Ad group (P<0.001), and that of combination group also had obvious difference compared with other two treatment groups (P<0.001). The number of positive cells of Fas and Caspase-3 were prominently higher than that of PBS group and Ad group (P<0.01). The Fas and Caspase-3 of the combination group also had obvious difference compared with other two treatment groups in corresponding index (P<0.05).The all above-mentioned indexes had no prominent discrimination between 125I group and Ad-hING4 group, Ad group and PBS group (p>0.05).④The expression efficiency of hING4 gene intracorpreal transfanctionThe expression efficiency of hING4 gene intracorpreal transfanction of Ad-hING4 group and the combination group were respectively 34.38±8.49 and 32.16±7.51, higher than that of PBS group, Ad group and 125I group.Conclusion: (1)125I seed and Ad-hING4 inhibit the growth of PANC-1 pancreatic cancer in nude mice significantly. Combined treatment has cooperation in tumor-suppression (q=1.22) and Ad-hING4 is hope to a novel radiosensitizer. (2)The possible mechanisms of cooperation in tumor-suppression:①suppressing angiogenesis of human pancreatic cancer in nude mice, such as suppressing the expression of CD34 and VEGF;②suppressing proliferation of human pancreatic cancer in nude mice, such as down- regulating the expression of PCNA;③promoting apoptosis of human pancreatic cancer in nude mice, such as down-regulating the ratio of Bcl-2/Bax, suppressing the expression of Survivin; inducing the expression of Fas and Caspase-3. |
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