The Study And Application Of Detection Of Erythrocyte Deformability Based On Microfluidic Chip In Vitro

Human red blood cells(RBCs),as the largest number of host cells in blood,are responsible for the transport of oxygen and also have the immune function.The diameter of red blood cells(RBCs)is about 7-8 μm,so its have to be constantly deformed to squeeze through the capillaries as small as 3 μm in diameter during circulatory system.Good deformability of RBCs is essential for tissue effective perfusion,and it has very important clinical physiology and pathological significance.There have been many clinical diseases related to RBC deformability,and RBC deformability is one of the key pathological indicators in the development of microcirculatory disorders.Severe patients with sepsis are more prone to rheological changes of RBC,and the deterioration of RBC rheology can impair microcirculation and tissue oxygen supply and demand balance,leading to multiple organ failure and death.Therefore,the study of RBC deformability has attracted more and more the attention of the researchers.To date,a variety of techniques have been reported aimed to quantify RBC deformability,but most of these methods have the disadvantage of low throught,time-consuming,expensive instruments and cumbersome operations,hence hindering the utility in the biomedical field.What’s more,these methods cannot simultaneously measure the deformation parameters of a single cell and the bulk population of cells,and thus its cannot be comprehensive provide the information of RBC deformability.The individual differences in drug reactions are widespread in clinical medications.Clinicians hope to achieve individualized drug treatment with the most appropriate drugs at the right time for specific patients,and reduce or avoid adverse drug reactions to achieve maximum therapeutic effect.The selective treatment of coronary heart disease(CHD)and antibiotics is a embodiment of the principle of individualized treatment.Therefore,individualized studies the effects of pentoxifylline(PTX)and lipopolysaccharides(LPS)on the RBC deformability has the potential to provide new indicators for clinical rational drug use.Microfluidic chip technology as a new miniaturized biochemical analysis platform has been widely used in many fields such as disease diagnosis,drug screening and environmental detection.Microfluidic technology has the advantage of less sample and reagents consumption,high throughput,shorter analysis time and high efficiency,integration,the ease of visualization and handling portability.Therefore,this study designed a single-cell microfluidic chip system to explore the deformability of RBCs.Based on the pre-laboratory research,this paper comprehensively studied the individual effects and potential mechanisms of pentoxifylline and endotoxin on RBC deformability by microfluidic chip technology.The main contents are as follows:The first chapter,we discussed the significance of the thesis.The significance and major influence factors of erythrocyte deformability were discussed.The existing methods and their advantages and disadvantages were summarized.The principle of microfluidic technology and its application in the determination of RBC deformability were also introduced;In the second chapter,the composition of microfluidic chip system,the principle of measurement and the production method of PDMS are introduced in detail.In the third chapter,the effects of different concentration of PTX(0,2,20,200μg/mL,clinical dosage of PTX is 20 μg/mL)on single cell deformability in CHD patients and healthy volunteers were explored in vitro using microfluidic chip.To demonstrate the importance of improving RBC deformability,the healthy donor samples were analysis too.Our results support prior conclusion that PTX can improve RBC deformability,reveal simultaneously the fraction of "slow" RBC increased sharply in CHD patients by compared with healthy donors.Various biochemical indicators including Na+-K+-ATPase activity,superoxide dismutase(SOD)activity,malondialdehyde(MDA),cholesterol,sialic acid(SA),hemoglobin(HB)and membrane protein components were assessed in parallel by using available test kits.Varying individual PTX susceptibility and donor-specific variations in dose-dependent deformability shifts were observed.Furthermore,there were strong correlations between RBC deformation,cholesterol levels(r=-0.409),activity of Na+-K+-ATPase(r=0.514)and SOD(r=0.405).The HB content and cytoskeletal proteins content remain relatively unchanged with PTX administration.In the fourth chapter,whole blood were harvested from healthy volunteers and incubated with varying concentrations of LPS(0,20,200,and 2000 pg/mL,clinical cut-off value of LPS is 20 pg/mL),the effect of different concentrations of LPS on RBCs deformability was assessed by microfluid chip.In order to explore the molecular basis of RBC deformation,biochemical indexes of RBCs including Na+-K+-ATPase activity,SOD activity,glutathione peroxidase(GSH-PX),catalase(CAT),adenosine triphosphate(ATP),lipid peroxide(LPO)content,HB and membrane protein components were mearsured simultaneously.The results showed varying individual LPS susceptibility and strong donor-specific variations.We also found that CAT,LPO,SOD,Na+-K+-ATPase,and GSH-PX are closely related to the decrease of erythrocyte deformability.The R values are 0.805,-0.787,0.626,0.531 and 0.509,respectively.In addition,we also explored the effect of LPS on the deformability of washed RBCs.The results showed that LPS had almost no effect on washed RBC deformability,which is consistent with the literature reported.In the fifth chapter,a summary and outlook of the thesis summarized and prospected.