Construction Of Tumor And Gut On Chips And Evaluation Of Bioactivity Of Chitooligosaccharides

The new drug development cycle under the existing technology system requires 5-10 years,and the R&D expenditure needs more than billions of dollars.The successful market rate is only about 1 in 10,000.Because of the differences in species between animals and humans,the success rate of drug clinical testing is only 8%.Establishment of an in vitro model that can simulate the human body to perform a rapid round of active screening of drug candidates and eliminate most of the ineffective drug candidates has become a hot spot for scientific researchers to chase.Organ chips can be used to simulate human organs from "composition","structure" and "environment".It has become a new platform for pharmacodynamics,toxicology and pharmacokinetic research.Chitooligosaccharides(COS),as a low-grade polymeric sugar,are generally made up of 2-10 monosaccharides connected by glycosidic bonds.COS has attracted much attention due to the diversity and specificity of its physiological effects,it has significant effects on inhibiting tumor growth,promoting the growth of beneficial bacteria in the intestine and improving gut microenvironment.In order to further explore the effect of COS on human tumor cells metastasis and human intestinal inflammatory diseases,this dissertation constructs a 3D tumor-vessel models and a peristaltic intestine models in vitro,and evaluates the functions of the models firstly.Later,using the tumor metastasis model,we explored the inhibitory effect of COS with a degree of acetylation of 46%(paCOS)on liver tumor cells metastasis.Using the human gut inflammation-on-a-chip,we explores the effect of low-polymerization COS on the intestinal mucus barrier,intestinal epithelial barrier,and inflammatory response during the development of gut inflammation.In the first part of the dissertation,a novel microfluidic tumor-vessel co-culture system was established to reproduce the different phases of cancer metastasis(proliferation,migration,intravasation and adherence)individually in vitro for the first time.It was found that both HepG2 and MDA-MB-231 cells migrated in the direction of "blood flow".Furthermore,MDA-MB-231 cells invaded through paracellular mode disrupting the intercellular endothelial junctions,whereas HepG2 cells engaged in transcellular intravasation through transcellular process.Compared with traditional assays,much more potent inhibition of 5-fluorouracil(5-Fu)on different phases of tumor metastasis was observed on the microsystem.In summary,the microfluidic device yielded abundant information about each phase of tumor metastasis,and would provide a powerful platform for use in drug screening,toxicology studies,and personalized medicine in futureIn the second part of the dissertation,a dynamic tumor-vessel microsystem undergoing physiological flow was leveraged.paCOS(FA0.46)significantly inhibited proliferation of HepG2 cells through vascular absorption on the chip,and inhibited migration of HepG2 cells by inhibiting the formation of pseudopod in liver tumor cells.It was also found that paCOS at 10 ?g/mL had a stronger inhibitory effect on liver tumor cells invading into blood vessels than that of paCOS at 100 ?g/mL,which had a significant destructive effect on tumor vascular growth and barrier function.Therefore,the results revealed that paCOS had considerable potential as drugs for anti-tumor metastasis.In the third part of the dissertation,a novel human gut-vessel microfluidic system was established to study the host—microbial interaction.Peristaltic motion of the cells on the chip was driven by a pneumatic pump.When intestinal epithelial cells(Caco2)were co-cultured with vascular endothelial cells(HUVECs)on the peristaltic microfluidic chip,Caco2 showed normal barrier and absorption functions after 5 days cultivation,which generally took 21 days in static Transwell models.Intestinal microvilli and glycocalyx layer were seen after 4 days cultivation,and L.casei was successfully co-cultured for a week in the intestinal cavity.A model for intestinal damage and inflammatory responses caused by E.coli was set up on this chip,which were successfully suppressed by L.casei or antibiotic.In summary,this human gut-vessel microfluidic system showed a good potential for investigating the host-microbial interaction in vitroIn the fourth part of the dissertation,we used a human gut on a chip models to explore the regulatory mechanism of low molecular weight chitooligosaccharides on intestinal barrier injury and inflammatory response.By constructing an in vitro DSS intestinal injury model on the human gut chip,we found that COS can reduce intestinal epithelial injury by promoting the growth of intestinal epithelial cells and promoting the expression of mucous layer.Then,by constructing an E.coli enteritis model on the intestinal chip,we found that under dynamic(Fluid flow plus peristalsis)conditions,COS can significantly reduce the amount of E.coli adhesion and invasion into the vascular cavity.Besides,COS can significantly reduce inflammatory response by reducing the expression of toll-like receptor(TLR)protein and reducing the nuclear DNA binding rate of NF-?B(p65)protein.These results can be concluded that COS can reduce barrier damage caused by enteritis by promoting secretion of mucous layer,and reducing inflammatory response through NF-?B signaling pathway.In summary,the tumor metastasis model and the peristaltic human gut model can be used for the screening of related drugs and the studing of drug action mechanism.paCOS with an acetylation degree of 46%can provide support for the treatment of liver tumor cells metastasis.COS with a degree of 2-8 polymerization can provide more treatment options for enteritis repair.