Research On Efficacy And Mechanism Of CA4P Combined With Ginsenoside Rd And Construction Of A New DNA Fluorescence Detection Method

In chapter one,we explored the inhibitory effects and possible mechanisms of the combination of combretastatin A4 phosphate(CA4P)and ginsenoside Rd on HepG2 cells and HepG2 xenograft tumors.Combretastatin A4 phosphate(CA4P),a vascular disrupting agent,can cause rapid tumor vessel occlusion.Subsequently,extensive necrosis is discovered in tumor center,which inducing widespread hypoxia and the rise of the a subunit of hypoxia-inducible factor-1(HIF-1?).Ginsenoside Rd is classified into 20(S)-protopanaxadiol and has significant anti-proliferative effects on a variety of tumor cells.Recently,it has been reported that ginsenoside Rd could down-regulate the expression of HIF-la and inhibit PI3K/AKT/mTOR signaling pathway,which leads retarded growth of MDA-MB-231 cell in vitro and in vivo.Therefore,combination of CA4P and Rd may be a complementary strategy of anti-tumor.In this part,the effect of CA4P and ginsenoside Rd on the survival rate of HepG2 cells when using them alone or in combination was detected and the combined index of the two drugs was analyzed.TUNEL kit was used to analyze the effect of CA4P and ginsenoside Rd on HepG2 cell apoptosis when used alone or in combination.A HepG2 xenograft tumor model was established in nude mice to investigate the antitumor effect of CA4P and ginsenoside Rd combined in vivo.The expression of Ki67 and TUNEL in tumor tissues were detected with immunohistochemistry,MRI was used to image the tumor of nude mice in each group and the effect of CA4P and ginsenoside Rd on tumor necrosis was analyzed.HypoxyprobeTM Plus Kit was used to detect the expression level of exogenous hypoxia marker pimonidazole HCI in tumor,and western blot was used to detect the expression level of endogenous tumor marker HIF-la protein.The expression of PI3K/AKT/mTOR signaling pathway-related proteins were analyzed by western blot.The results showed that the combination of CA4P and ginsenoside Rd enhanced the inhibition of HepG2 cell proliferation,and the two drugs were synergistic.Compared to monotherapy,CA4P combined with ginsenoside Rd can enhance HepG2 cell apoptosis.Animal experiments showed that CA4P combined with ginsenoside Rd synergistically inhibited tumor growth in nude mice bearing tumors.The combination of CA4P and ginsenoside Rd significantly reduced the expression level of Ki67 in tumors,significantly increased the expression level of TUNEL,and increased the necrosis rate,indicating that the combination of CA4P and ginsenoside Rd promoted apoptosis of tumor cells in vivo and inhibited proliferation,increased tumor necrosis.The combination of CA4P and ginsenoside Rd reduced the hypoxic area inside the tumor and the expression level of HIF-1?,indicating that this strategy improved the hypoxic microenvironment in the tumor.The results of western blot showed that CA4P combined with ginsenoside Rd inhibited the expression of p-PI3K,p-AKT,p-mTOR and HIF-1? protein.After adding mTOR activator MHY1485,the combined effect of the two drugs on the expression level of HIF-1? protein decreased,but compared with the model group,it still significantly inhibited the expression of HIF-1? protein.These results indicated that the combination of CA4P and ginsenoside Rd can effectively inhibit HepG2 cell proliferation and induce apoptosis in vitro and in vivo.The mechanism may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.In chapter two,a DNA fluorescence capture probe based on activators generated by electron transfer for atom transfer radical polymerization(AGET ATRP)signal amplification strategy was established,and the ability of this new strategy to detect target DNA was investigated.Sensitive detection of DNA is conducive to enhance the accuracy of diseases diagnosis and risk prediction.In this work,we report the use of AGET ATRP as a novel on-chip amplification strategy for the fluorescence detection of DNA.More specifically,the target DNA was captured by the immobilized hairpin DNA probes on-chip.Upon hybridization,exposed 3'-N3 of the hairpin was used to attach AGET ATRP initiators onto the silicon surface by click chemistry.Then,the formation of long chain polymers of fluorescein o-acrylate led to numerous fluorescents labeling with the end of the probes,which in turn amplified the fluorescence signal for DNA detection.Under optimal conditions,it showed a good linear range from 100 fM to 1 ?M in DNA detection,with the detection limit as low as 4.3 fM.Moreover,this strategy showed good detection performance in complexes real serum samples,the fluorescence intensity of 0.1 nM tDNA in 1%fetal bovine serum samples was 97.6%of that in TE buffer.Quantitative detection of HER2 gene in nude mouse serum showed that tumor-bearing nude mice have higher serum HER2 levels than the normal group,which indicated the detection method we established has good practicality in real samples.Based on its high sensitivity,reduced cost and simplicity,the proposed signal amplification strategy displays translational potential in clinical application.