Experimental Study Of Dauricine Regulation Of The Hedgehog Signaling Pathway To Pancreatic Cancer-related Genes And Proteins Influence

Objective:Experimental research by dauricine(Dau)anti-pancreatic cancer effects In vivo and vitro,to observed Dau on human pancreatic cancer cells BxPC-3 and nude mouse transplantation tumor inhibition,ultrastructural pathology change and the influence of the cell cycle,apoptosis;Observe Dau regulating the Hedgehog signaling pathway in Shh,Ptch1,Smo,Gli-1 mRNA and protein expression effect on inhibition of pancreatic cancer,Delve into effect and mechanism of pancreatic cancer.By cell biology、pathology Supermicro、immunochemical、Western blot、real-time quantitative PCR,flow cytometry technology,from the biological characteristics、the protein and gene level protein and gene level elaborated Dau inhibition of pancreatic cancer and the Hedgehog signaling pathway correlation.Methods:BxPC-3 pancreatic cancer cells were cultured and passaged,divided into four groups:model group,Dau high and low dose group and 5-FU group,build xenografts in nude mice were randomly divided into five groups:model group,the control group,Dau high and low dose group and 5-FU group,drug intervention detection:(1)In vitro experiments by trypan blue staining、MTT assay detect Dau to human BxPC-3 pancreatic cancer cell growth of the curve and proliferation(2)In vivo detection of Dau BxPC-3 human pancreatic cancer xenografts affect inhibition rate and spleen index.(3)By By transmission electron microscope Dau impact on human pancreatic cancer BxPC-3 cells and xenografts ultrastructural changes.(4)FCM detection BxPC-3 cells and xenografts changes of cell cycle and apoptosis.(5)Using real-time quantitative PCR、immunohistochemistry,Western blot detection of Hedgehog signaling pathway in major molecular Shh、Ptch1、Smo、Gli1 mRNA and protein expression levels.Result:(1)Dau in vitro assay could inhibit the proliferation of BxPC-3 cells,Dau inhibition rate of high-dose groups、Dau low-dose group、5-FU group were 70.32%、42.68、56.07%,Compared with model group(P<0.01);with a time and dose-dependent manner(P<0.01).(2)In vivo experiments Dau for pancreatic cancer BxPC-3 transplanted tumor inhibition rate is high、low-dose group,the 5-FU group were 50.44%、31.74%and 33.64%.Compared with the model group were significantly different(P<0.01);Dau high and low dose group and the control group compared with no significant effect on the spleen index of tumor-bearing nude mice(P>0.05),and comparing 5-FU group and the control group(P<0.05).(3)Dau BxPC-3 pancreatic cancer cells and xenografts ultrastructural changes in organelle different degrees of damage by TEM,and accompanied by apoptosis occurs.(4)In vivo and in vitro assay FCM,each group BxPC-3 cells and xenografts drug intervention are different degrees of retardation,manifested as G1 phase growth,S shortening;Dau concentration and with the apoptosis rate increased in a dose-dependent manner;High doses group、5-FU group of the cell cycle compared with model group were significantly different(P<0.01),Dau low dose compared with model group(P<0.05);apoptosis in each group compared with the model group were significantly different(P<0.01).(5)Using real-time quantitative PCR,immunohistochemistry,Western blot detection of Hedgehog signaling pathway in major molecular Shh、Ptch1、Smo、Gli1 mRNA and protein expression,Dau high and low dose group 5-FU group expression are under withered,Dau high-dose group and model group significant difference was statistically significant(P<0.01).Conclusion:(1)Dau effects on BxPC-3 human pancreatic cancer cells was significantly inhibited,inhibition of cell proliferation,and has a time and dose-dependent manner.(2)Dau effectively inhibit BxPC-3 pancreatic tumor growth in nude mice,spleen without obvious injury.(3)After Dau intervention cells and transplantation tumor,can induce apoptosis of tumor and cell cycle arrest.(4)Dau regulation of tumor cells in vivo and vitro can be reduced in Shh、Ptch1、Smo、Gli1 mRNA and protein expression of Hedgehog signaling pathway,Therefore,we can infer that the mechanism of action of anti-pancreatic cancer Dau is One of through the inhibition of the Hedgehog signaling pathway achieved.The mechanism may be through down Hedgehog signaling pathway-related genes、protein involved in apoptosis induction of tumor cells and cell cycle arrest,thereby inhibiting tumor growth and proliferation.