The Preliminary Study Of The Relationship Between The Soluble Fractalkine And Acute Graft Versus Host Disease Post Hematopoietic Stem Cells Trasplantation

Background:Allogeneic Hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment on malignant hematological disease but the acute graft versus host disease (aGVHD) is one of its serious complications. at present, the clinicians final diagnose that aGVHD is mainly based on patience's clinical symptoms:like skin rash,damage to the liver,diarrhea, lack of relative stability and reliability of serum markers. Whether there can find a clinical application of serum markers for early diagnosis and early prediction of disease state is the goal of clinical workers.Chemokines(CK) is a kind of cytokine widely existed in leukocytes, monocyte, macrophagocyte, T-cells and B-cells, and is able to mediate leukocyte chemotaxis by integrating the appropriate chemokine. A number of chemokine can combine with numbers of chemokine's receptors, and a chemokine may have multiple high affinity receptors, which constitute a complex network system together to play an important role during the development of many diseases, such as systemic lupus erythematosus. There have been found at least 50 kinds of chemokines so far. All chemokines share the similar structure and function and according to the relative position of the N-terminal cysteines, chemokines are generally divided into four subgroups:C, CC, CXC and CX3C. By now, the defined chemokine receptors are more than 20 kinds, which belong to the G-protein coupled receptor family. Chemokine mainly express all kinds of leukocyte subpopulation originated from bone marrow, meanwhile, they express in epithelial cells, vascular endothelial cells, nerve cells etc. Chemokine was found as the first chemotaxis mediator, however, as more deeper research on chemokine, many experts now have already recognized that chemokine not only mediate chemotaxis to leukocytes, but also play wide range of physical and pathological role in immune cells development and organs development, immunological response, inflammatory reaction, pathogen infection, trauma repair, tumor formation and metastasis. With its extensive cell source and biological effects, chemokine play a vital role in the episode and progress of many inflammations and autoimmune disease.Fractalkine(Fkn) also called irregular chemokine, is a kind of adhering chemokine secreted by several histiocytes which was found to be the unique element of CX3C Fractalkine in 1997 and located in human chromosome 16q13, with membrane-bound type and dissolvable type. These two types of Fractalkine decide the function of dismissing and adhering the peri-leukocytes, which is why they also have the effect as what both chemokine and intercellular adhesion molecule have. Fractalkine is expressed by endothelial cells, fibroblast and microglia. When inflammation appear, the expression will raise. Most Fkns are expressed on the surface of the activated endothelial cells, which is a factor to transfer leukocytes to inflammatory tissue. Because endothelium is the first barricade in leukocyte transmembrane metastasis, Fkn on the surface of endothelial cell is the door to control exudation of leukocytes in inflammation part. When some outer parts of Fkn are hydrolyzed by protease, the hydrolyzed part become dissolvable irregular sFkn. sFkn tend to adhere mononuclear cells, NK cells, CD4+T lymphocytes, CD8+T lymphocytes near endothelium cells in vessel wall. These inflammatory cells are inducted to do transmembrane metastasis and meanwhile, they release IFN-γ, TNF-α, porforin and telomerase to destroy vessel endothelium cells and other histiocytes, causing vasculitis and damaging organ. With the synergism of IFN-γ, defluxion of TNF-α, and the function that IL-4 or IL-3 can completely restrain the defluxion of the outer cell fragment, proving their anticipation in the defluxio regulation process.CX3CR1 is the specificity receptor of Fkn, mainly expressing in NK cells, CD8+T cells, CD14+ mononuclear cells. sFkn molecule manifest the strong adhension activity in adhering these cells to epithelium cells, endothelium cells, arborescent cells. Fkn can also interact with CX3CR1 through the signal transition with G protein to improve the integrin ability to integrate the ability of ligand. Therefore, when integrin and its ligand like ICAM-1 and VCAM-1 coexist with Fkn/CX3CR1 system, together they can greatly improve cells adhesion effect. By now, there are still no exact experiment confirming what response the Fkn/CX3CR1 system do to intracellular. It is researched that CX3CR1 is G-protein coupled receptors. So that when Fkn integrate with CX3CR1, Fkn can couple with G-protein and then start the intracellular signal transition mechanism and promote the expression of nuclear factonκB and TNF-α, which result in relative biology effect. Some scholars apply PBS and Fkn respectively to induct the human peripheral blood monouclear cells. Result shown that both NF-κB masculine cells number expressed by Fkn treatment group and TNF-αexpressing data increase remarkably more than the data the control group shows. During the process of transplanting immune cells, Th1 cells mediate rejection while Th2 cells induct immune tolerance. TNF-αis one of the key cytokine in forming Th1 type subgroup cells. Fkn may lead to tissue damage by changing Th1/Th2 neutralization, and then result in aGVHD.aGVHD is pathologic injury caused by T cells supplied by the substance during transplantation. When T cells indentify host antigen, they attack the host tissues and organs. It's a disease process of causing many of the host viscera damage. aGVHD is also a common grave complication in hemopoietic stem cell transplantation. The study shows that:the internal level of IL-1, soluble IL-2 receptor (sIL-2R), TNF-α,etc, are significantly increased while a aGVHD happening. There have been some researches report that monitoring TNF-α,IL-4,TGF-αvariation after the allogene hemopoietic stem cell transplantation did contribute to diagnosing aGVHD and have certain clinical significance in judging light and heavy press of aGVHD. However, clinicians are still short of a reliable and stable method to prevent aGVHD now. No matter at home or on abroad, research on Fkn is mostly focus on rheumatoid disease, systemic lupus erythematosus, but less relative research literatures are seen on Fkn and aGVHD. Some reports like Robinson said that Fkn expression elevation can greatly increase the rejection to heart transplantation, especially show in the blood vessel endothelium system injury. After using CX3CR1 to obstruct antibody treatment on receptors, the transplant survival time will be prolonged. Satoshi Ueha found that mucous membrane vessel colonization factor and Fkn intervention treatment mitigate damage in intestinal tract transplant anti-host reaction and protect the transplant anti-tumor effect at the meantime. These researches inspire us that Fkn expression elevation may indicate aGVHD. As known above, hemopoietic stem cells transplant patients suffer a large dose of pretreatment and stem cells transplantation, as well as the process of hemotopoiesis restitution and immune restitution. When aGVHD take place, TNF-α,IFN-γ,IL-4 will change accordingly, but sFkn relating to chemotaxis stay invariant in corpore. But in present, reports on this aspect are still rarely. In this research, we did dynamic monitor on blood serum Fkn concentration before and after hemopoietic stem cells transplantation, in order to find another factor to pretest or treat aGVHD for clinician.There are two parts in this study. The partⅠestablished a model to determine sFkn which can also measure the patients' concentration of serum sFkn before and after the allo-HSCT transplantation and 35 cases of health adults. PartⅡcompared the concentration of serum sFkn before and after transplantation at different times with 20 patients who had hematologic malignancies and health adults, which aimed to explore the relationship between sFkn and aGVHD.Objective:This study aimed to establish the measure model of sFkn to determine the the health adults's concentration of serum sFkn. To approach the relationship between sFkn and acute graft-versus-host disease (aGVHD) through observing the difference internal sFkn of the hematopoietic stem cell transplantation (HSCT) patients.Methods:In partⅠ, we built a competitive inhibition ELISA method to measure sFkn in order to draw the standard curve of sFkn and determine the concentration of serum sFkn of health adults.1. Concrete steps for the measure of sFkn1). Prepare all the materials before starting assay procedure. It is recommended that the removal of the standards and samples be conducted by micro pipette at room temperature (20-25℃) and place them for 15-30 minutes. This experiment should be held within room temperature (20-25℃).2). Remove the micro titer plate and add 100μL of standard solution to the blank well according to the order of the standards. Then add 100μL of sample to the blank well and 100μL distilled water to the blank control.3). Add 50μl enzyme marker solution in each hole except the blank control well. The microtiter plates sealed with sealing compound for 1 hour incubation in 37℃which maintains a stable temperature and humidity.4). Wash the micro pipette. Dilute the condensed washing liquid with the distilled water in the proportion of 1 to 100. Wash the enzyme-labeled plate thoroughly for 3 to 5 times and ensure that every well has sufficient water pressure. After final wash, dry the enzyme-labeled plate by hitting it onto absorbent paper or paper towels until no moisture appears.5). Add 50μL substrate A&B to each well except the blank control well.The dark reaction should last for 15 minutes at 20-25℃.6). Add 50μL of stop reagent to each well to terminal the reaction7). Using a enzyme-labeled instrument to read the Optical Density (O.D.) at 450 nm wavelength within 30 minutes.8). The Optical Density (od) and the concentration of sFkn (standard solution) intended to curve, establish the equation. the results are calculated by the computer.In partⅡ, we used the model in partⅠto compared the determine of the concentration of serum sFkn before and after transplantation at different times with 20 patients who had hematologic malignancies and 35 health adults, and analyzed the relationship between leukemia's concentration of serum sFkn before and after transplantation at different times and aGVHD. During this study, all the sFkn antibodies are supplied by Shanghai Lanji Biological Technology Co., Ltd. And all the patients and health adults' venous blood samples were collected immediately after centrifugation (1000 r/min,5min) separating the supernatant stored -20℃under test.2. Statistics and analysis:It used the SPSS 18.0 statistical software for curve fitting to drawn the standard and to analyzed the statistic. Measurement data were expressed with X±S and independent-sample T test was used for significant test with two groups. Differences were significant if P<0.05.Result:1. It is successfully established the method for measuring sFractalkine and the standard curve which had a satisfactory dose-effect relationship among 0.5～5 ng/ml. Intra and inter-coefficients of variation were 14.4% and 9.43%.2. SFkn concentration of health adults' serum was 3.9325±0.8027ng/ml.3. Before the transplantation of all HSCT patients, the concentration of serum sFkn was 4.2806±1.4044ng/ml.4 auto-HSCT patients' concentration of serum sFkn was 3.6800±1.5451ng/ml 15 days after the transplantation.10 allo-HSCT patients' concentration of serum sFkn was 3.6240±0.9980ng/ml 15 days after the transplantation, and didn't appeal with acute GVHD.6 allo-HSCT patients' concentration of serum sFkn was 5.7752±0.6618ng/ml 15 days after the transplantation, and appeal with acute GVHD. Before all the transplantations, the concentration of serum in the auto-HSCT patients and patients who didn't get aGVHD after the allo-HSCT didn't change significantly compared with that of the normal people (p >0.05). Before the the typical clinical symptoms occurred on the allo-HSCT patient with aGVHD, the concentration of sFkn in the blood serum was monitored to increase obviously. Compared to the level of the normal people, the change is striking (p< 0.001)Conclusion:1.competitive inhibition ELISA method can be used to establish sFractalkine Determination;2.health adults' concentration of serum sFkn was between 3.9325±0.8027ng/ml;3.allo-HSCT patients' sFkn tended to increase when acute GVHD happened. But there still needs further study.