Construction Of The Concentration Gradient Of Crude Extract Of Peony Seed Meal Based On Microfluidic Chip And Its Application In Tumor Analysis

The technology of microfluidic chip provides a suitable research platform for cell culture and drug screening,with small size,easy operation and good repeatability.As byproducts of Peony seed coat,there are few studies on the potential medicinal value of peony seed,and there are few exploration about its anticancer effect.In this experiment,we focued on developing of a microfluidic chip system and combined with peony seed meal extract to study the inhibitory effect on the growth activity of cancer cells.Calculating of cell death rate by MTT;Through Hoechst33258 staining to observe the morphological changes of cells.The AO/PI double staining was used to detect the death of cells in the induction of drugs.Microfluidic chip which has the comparable scale with the blood issue can imitate the microenviroment in human body.Using this feature,we can prove the authenticity of the test results more fully.The research method realized the real-time monitoring and observation of apoptosis induced by drug concentration gradient by microfluidic chip technology.And it is of great significance on the anti-cancer activity of the oil peony byproducts in the future research.The main experimental results are as follows:1 Using CFD-ACE to monitate the change of velocity in the chip channels.Different flow rates were tested on microfluidic to observe the formation of concentration.We leaened that chip can form stable concentration gradient in the range of 1500 ?L/min.2 Software simulation showed that concentration of culture chamber 1 was 0,the concentration of culture chamber 3 and 5 was 0.5,7 and 11 in the culture room was 0.75,and the concentration of culture chamber 9 was 1.Chip was filled with fluorescein dye and PBS was flowed into the device by syringe pump.The concentration gradient is characterized by the strength of the fluorescence intensity.It was further proved that the mathematical simulation and numerical results were same.3 The results of anti-tumor experiment in vitro showed that water extract(WT),ethanol extract(CT)and ethyl acetate extract(ZT)had anti-tumor activity to Hepg2 and MCF-7.Refering to the results of MTT showed WT.CT.ZT had promoting effect on apoptosis and showed a time-dose dependent manner.4 Hoechst 33342 and AO/PI fluorescence staining,we found that compared with the control group,the number of cells decreased significantly and the cell shape had changes.With increasing action time and drug concentration,the smaller the cell survival rate5 The results of JC-1 fluorescent dye showed that the peony seed coat extract could cause the decrease of cell mitochondrial membrane potentialThe concentration micro fluidic chip used in this study consisted of 16 culture chambers,which can produce a variety of concentration gradient.And also can be realized in the same culture conditions on chip cells were observed;Microfluidic chip which has the comparable scale with the blood issue can imate the microenviroment in human body.The device can makes the drugs to be in full contact with the cells in the pipe and can be used in the cells very well.Microfluidic chip has several advantages including samll reagents consumption,fast analysis and avoid some tedious operations of cancer cells.Under normal culture conditions,cell viability in the culture chamber was not affected,and it could grow normally.It proved that the chips could be used for cell culture,which would not produce great errors in subsequent test results.The syringe pumps can provide continuous perfusion of medium,and remove the waste which is unfavorable for cell attaching.