Effect Of Ulinastatin On The Physiological Function Of The Human Umbilical Vein Endothelial Cells And The Study Of The Mechanism Of Action

Ulinastatin (UTI) is a glycoprotein extract refined from human urine. Its molecular weight is 67000 d, is composed of 143 amino acid residues, the bio availability of 100%, the half-life of 40 min,5 min to spike, belong to the body of the endogenous protease inhibitors.It can inhibit a variety of protein, sugar and lipid hydrolysis enzyme activity, inhibit the excessive release of inflammatory mediators, improve microcirculation and tissue perfusion, and other important pharmacological effects, thus reduce the damage in the body such as inflammatory damage factor, maintaining environmental balance in the body. At the same time, ulinastatin stability and lysosome membrane, inhibition of lysosomal enzyme release, inhibiting myocardial suppressor, clear the role of oxygen free radicals. Besides, ulinastatin can also be through a competitive way to dose dependent inhibition of clotting factor Xa, assist, Ⅷ factor, prevent the prothrombin into thrombin, cut inside, extrinsic coagulation pathway, avoid the excessive activation of the coagulation system, thus improving high intravascular coagulation state.It can inhibit the release of plasma kallikrein, V factor release and inhibition, the PT mild prolong APTT and TT in human plasma.It can protect blood coagulation factor XI coagulant activity. Can maintain normal blood vessels integrity and maintenance and diastolic function of endothelial cells, endothelial cell expression of ICAM and CDllb decreased significantly, the neutrophil adhesion, activation can’t instead of.Four dilute acid metabolism, can improve the platelet peanuts can inhibit platelet release of active substances, maintain normal platelet aggregation function, maintain normal diastolic function of vascular endothelial cells and maintaining integrity. Part of the basic and clinical studies have shown that UTI can reduce capillary leakage, reduce tissue edema, have certain vascular endothelium protection, so is often used in severe burns, clinical acute pancreatitis, acute lung injury and systemic inflammatory response syndrome (SIRS), and other treatment. Studies have found that when the body infection, fever, tumor, pregnancy, shock, surgery, or give the stimulation such as corticosteroids, activity of human body in the urine of UTI will rise. Moreover, there are high levels of protease inhibitors in human plasma. When the body suffered serious injuries, the protease inhibitors will be consumed, concentration significantly decline, but along with the gradual recovery of the body, its concentration will gradually rise again. Ulinastatin has, therefore, to resist external stimulation, reduce the injury factor on the body of the damage, maintaining environmental balance in the human body.Vascular Endothelial cells, Vascular Endothelial Cell, VEC) for mounting lined on the inner surface of the cardiovascular system single-layer flat squamous Cell, is the boundary between circulating blood and vessel wall.It’s the shape of the formation of cardiovascular closed system foundation and the vascular permeability of the main barriers, to maintain normal blood flow state, and separate vascular inside and outside, adjust the material exchange of blood vessels inside and outside, barrier effect. Ley, at the same time, such as (1995), the experimental study of vascular endothelial cells or an active metabolic and endocrine organs, can secrete a variety of inflammatory mediators and cytokines, such as prostaglandin (PGs), thromboxane A2 (TXA2), nitric oxide (NO), endothelin (ET), interleukin 1 (IL 1), interleukin 6 (IL-6), tumor necrosis factor (TNF-a), adhesion factor (ICAM-1), E-select element (E-selectin), P (P-selectin)-select element, growth factors, etc., and can activate the nf-kappa B (nuclear factor), in the tissues of blood coagulation and anticoagulation, regulating immune response, and inflammation, etc have played an important role in. Vascular endothelial cells in the human body play an important role in a variety of physio logical and pathological process, is now experiment research hotspot, and one of the most widely used is the human umbilical vein endothelial cells. Relative to the other vascular endothelial cells and human umbilical vein endothelial cells have the potential of stem cells, and have abundant materials easily, and the characteristics of simple operation. At the same time obtain the human umbilical vein endothelial cells in vitro mixed cell is less, and in comparison with the vascular endothelial cells of various sources of experimental animals, closer to the growth characteristics of human endothelial cells, is one of the commonly used in vitro study of vascular endothelial cells.ERK MAPK family is first to be discovered and understood in most of the members, including the two isomers ERK1 and ERK2 (P44 and P42) respectively. Two receptor phosphorylation sites that tyrosine and threonine were glutamic acid residue separated so its phosphorylation site motif is TEY.Now think that P38 lightning and JNK belong to "stress induced" MAPK, and ERK is considered to be related to cell proliferation, transformation and differentiation of MAPK. ERK cascade including typical three levels of MAPKs sequential activation process. The Raf protein phosphorylation (MAPKKK) can activate MEK1/2 (MAPKK), and the latter activation, so that the subsequent ERK1/2 (MAPK) dual phosphorylation and been made. The activation of ERK in the cellular responses induced by the Ras, transcription factors (such as Elkl, cEts1 and c-Ets2) activation and kinase (such as P90rskl, MNK1 and MNK2) activation is crucial.At present, more clinical and basic experiment has proved that ulinastatin can protect vascular endothelial cells, reduce the release of inflammatory mediators. But is it possible that by promoting the proliferation of vascular endothelial cells, accelerate the repair the damaged endothelial cells, shorten the time of the vascular endothelial cell repair, thus reduce the intravascular fluid leakage is still rarely reported. Because in the body in the process of the systemic inflammatory response, vascular endothelial cells of the location and function determines it a ring between stimulus and biological behaviour of the mediation. It’s the role of endogenous and exogenous hormone intervention factor target cells. Therefore this experiment by vascular endothelial cells as the breakthrough point of ulinastatin. By adding different concentration gradient of ulinastatin, using the current commonly used methods for testing, At different concentrations of ulinastatin on human umbilical vein endothelial proliferation, apoptosis, migration, adhesion function. And then further to burn serum, observe whether inflammation in a state of ulinastatin is still has the effect of promoting proliferation. And to explore the effect and the correlation of ERK pathway.Part OneEffect of ulinastatin on the physiological functions of the human umbilical vein endothelial cells and the relation with the pathway of ERK1/2ObjectiveThis study aims to research the Effect of different concentration of ulinastatin on the proliferatton,apotosis,migration,adhesion of the human umbilical vein endothelial cells.MethodsBuy imported identified the original generation of human umbilical vein endothelial cells from liquid nitrogen by 37℃ water bath after the recovery after the human umbilical vein endothelial cells, the cells in the centrifuge, then cell culture in a petri dish, when put in co2 incubator for training, day in liquid, to the microscopic cell fusion by about 80%, to join the pancreatic enzyme digestion,2 minutes after the microscopic observation of cell shrinkage, margin slightly, it become a large patch of separation, separate or show to join complete medium termination of digestion, then the cells of the fourth generation according to 1:2-13 subculture. Microscopically observed cell state, choose better growth state of the fourth generation of human umbilical vein endothelial cells, U groups were randomly divided into control group and experimental group A (625 units/ml) and group B (2500 units/ml), (5000 units/ml) of group C, group D (10000 units/ml). Collect in logarithmic growth phase cells from cell suspension, vaccination within 6 orifice,37℃ carbon dioxide incubator culture. Treated cells grow in 6 orifice to 50-60%, with celltrace experimental test of different concentrations of ulinastatin on human umbilical vein endothelial cell proliferation effects; In accordance with TUN EL experimental steps, detection of different concentrations of ulinastatin on human umbilical vein endothelial cells apoptosis. Scratch experiments, respectively in 0 h,6 h,24 h,48 h the timing of the images and data, calculation of mobility, detection of different concentrations of ulinastatin on human umbilical vein endothelial cell migration effects. Cell adhesion experiments, adherent cells with BSA group OD value of the reference, calculation of matrigel and FN group cell adherent rate, detection of different concentrations of ulinastatin on human umbilical vein endothelial cells adhesion. The data line of single factor analysis of variance (One-way ANOVA), LSD test and t test.ResultsExperiment 1(1) proliferation experiment (celltrace experiment) Results show U group compared with control group, experimental group A, B, C, D group of human umbilical vein endothelial cell proliferation quantity percentage was increased. The group C percentage increases significantly in the A, B and D group (P< 0.05). Experiments in different concentration gradient of ulinastatin can both promote the effect of human umbilical vein endothelial cell proliferation, but the concentration is close to 5000 units/ml, the role of ulinastatin to promote cell proliferation is most obvious. When the concentration of ulinastatin reaches 10000 units/ml its effect on promoting proliferation have weakened.(2) apoptosis experiment(TUNEL experiment)Compared with the control group (U), the experimental group A, B, C groups in different concentration gradient of ulinastatin on apoptosis of human umbilical vein endothelial cells had no obvious effect. Group D human umbilical vein endothelial cells under the mirror appear a large number of apoptotic cells, may indicate high levels (10000 units/ml) of ulinastatin can promote apoptosis of human umbilical vein endothelial cells.(3) migration experiment (scratch experiment) Compared with control group (U), the experimental group A, B, C, D group in different concentrations of ulinastatin foreign cultured human umbilical vein endothelial cells had no significant effect of migration, there was no statistically significant difference (P> 0.05).(4) cell adhesion experimentsCompared with control group (U), the experimental group A and promote adhesion effect is not obvious (P> 0.05). In group B, C, D group shows, high concentration (2500-10000 units/ml) of ulinastatin has to promote the role of human umbilical vein endothelial cells adhesion (P< 0.05). Which group D promote cell adhesion strongest (P< 0.01) and ulinastatin on in vitro cultured human umbilical vein endothelial cell adhesion of and concentration were positively correlated.Experiment 2:The cells were randomly divided into four groups. The control group ck for normal human umbilical vein endothelial cells. The experimental group a is normal human umbilical vein endothelial cells to burn serum. The experimental group b for normal human umbilical vein endothelial cells to burn serum and ulinastatin (5000 units/ml). Group c for normal human umbilical vein endothelial cells join ERK1/2 channel inhibitors, burn serum, ulinastatin (5000 units/ml). The ulinastatin celltrace experiment testing different concentration for each human umbilical vein endothelial cell proliferation effects.Experimental group a in burn serum can inhibit normal human umbilical vein endothelial cell proliferation activity. Ulinastatin can offset in the experimental group b burn serum inhibition, and promote cell proliferation. Group c after adding ERK1/2 pathway inhibitor UO126 ulinastatin is to promote the role of cell proliferation, single role than group b slightly diminished.ConclusionsExperimental result indicates:Experiment 1:(1) Different concentrations of ulinastatin can both promote the in vitro cultured human umbilical vein endothelial cell proliferation, but its effect on promoting proliferation is not enhanced with the increase of concentration. When the concentration is close to 5000 units/ml the strongest effect on promoting proliferation. With the increase concentration, its effect on promoting proliferation have weakened; (2)The low concentration gradient of ulinastatin has no obvious promoting effect of human umbilical vein endothelial cell apoptosis, but high levels of ulinastatin (10000 units/ml) can induce the apoptosis of human umbilical vein endothelial cells; (3) The experiment of different concentration of ulinastatin on human umbilical vein endothelial cells migration activity had no obvious effect; (4) The low concentration of ulinastatin to promote human umbilical vein endothelial cells adhesion effect is no statistical significance,.While the medium and high concentrations of ulinastatin can obviously promote cell adhesion, and positively related to the concentration of the relationship. Experiment 2:Burn serum have to inhibit the action of the human umbilical vein endothelial cell proliferation, and ulinastatin can offset this inhibitory effect, and promote cell proliferation. When join ERK1/2 pathway inhibitor, ulinastatin is to promote the role of cell proliferation, prompt ERK pathway is involved in the process of ulinastatin to promote cell proliferation, but is not the only pathway involved in this process.