| Part1Effect of different transfection methods on human T lymphocyte leukemia cell line Jurkat cellsPurposeExplore the effects of different transfection methods on human T lymphocyte leukemia cell line Jurkat cells from cytotoxicity and transfection efficiencyMethods1. Cell culture, Using the RPMI1640+10%fetal bovine serum dubbed entirely to Jurkat cells were cultured and replace media every2-3days.2. Liposome transfection method:2.1HiperFect transfection reagent method:The transfection complexes is composed of750ng siRNA and12μl Hiperfect transfection reagent. Add the complexes drop-wise onto the Jurkat cells. According to the instructions to incubate the cells24h. The viability of Jurkat cells was observed by the trypan blue dye exclusion assay. Jurkat cells were observed and transfection rate was evaluated under fluorescent microscopy.2.2InstantFECT transfection reagent method:In order to acquire the most effective method of transfection, the cells were divided into three groups:2μl transfection reagents+30pmol siRNA,2μl transfection reagents+60pmol siRNA and3μ1transfection reagents+90pmol siRNA. The transfection complexes drop onto the Jurkat cells, According to the instructions to incubate the cells24h. The viability of Jurkat cells was observed by the trypan blue dye exclusion assay. Jurkat cells were observed and transfection rate was evaluated under fluorescent microscopy.3. Nucleofection methodJurkat cells were transfected with different programs (CL-120, CM-137, CM-137, CM-189, CU-150and DG-150).24hours post Nucleofection, Cell viability were determined by the trypan blue dye exclusion assay. Transfection rate was evaluated by fluorescent microscopy.Result1. In the liposome transfection, the cell activity of HiperFect transfection reagent method and InstantFECT transfection reagent method was (0.90±0.029) and (0.91±0.025), respectively; In the different programs of Nucleofection method, The cell activity of CL-120, CM-137, CM-137, CM-189, CU-150and DG-150program was (0.87±0.018),(0.22±0.29),(0.35±0.04),(0.31±0.015),(0.57±0.025) and (0.32±0.02), respectively. Comparing the cell activity in the different approaches of transfection, This difference between Liposome transfection method and CL-120program of Nucleofection method was not statistically significant (P>0.05).2. In the liposome transfection, the transfection efficiency of HiperFect transfection reagent method and InstantFECT transfection reagent method was (0.22±0.04) and (0.17±0.05), respectively; In the different programs of Nucleofection method, the transfection efficiency of CL-120, CM-137, CM-137, CM-189, CU-150and DG-150program was (0.82±0.008),(0.32±0.02),(0.09±0.04),(0.15±0.025),(0.75±0.057) and (0.33±0.028), respectively. Comparing the transfection efficiency in the different approaches,the difference have significant meaning (P<0.05)ConclusionAccording to the the cell activity and the transfection efficiency, the CL-120 program of Nucleofection method is a new safe and effective transfection.PART2The role of CD47and its signal pathway in the pathogenesis of hemophagocytic lymphohistiocytosisPurposeUsing Jurkat cells and BM-derived macrophages in children with HLH as the object of study. Through siRNA, flow cytometry and other experimental techniques to change the expression level of CD47in the cells, phagocytation of macrophages were observed.Methods1. The cultivation of bone marrow macrophages in vitro:bone marrow cells were from childen with HLH, M-CSF induces monocyte which come from BM, differentiation into macrophages, and then under the stimulation of IFN-γ and LPS, macrophages were differentiated into M1macrophages. Using Microscope to observe the morphological changes, flow cytometry to detect whether the method of culture M1macrophages is successful.2. Transfection method:see CL-120program of Nucleofection method in the part1.3. Flow cytometry:3.1Identification of bone marrow macrophage:Using CD11b-APC, CD14-FITC and F4/80-PE labeled cells, the macrophage differentiation were identified by the flow cytometry at different culture interval (fifth days, eighth days).3.2Using CD47-FITC labeled cells, the expression of CD47in the surfaces of Jurkat cells were observed by flow cytometry after transfection4. The activation of U937cells:Using20ng/ml IFN-γ and10ng/ml LPS to stimulate2×104U937cells48h, washing3times with PBS, spare;5. Detection phagocytosis of cells in vitro:6. Phagocytic index was detected after coincubation with target cells and activated U937cells or bone marrow macrophages from HLH patients by Wright Giemsa staining, phagocytosis index=(phagocytic macrophage/200macrophages)*100%.Result1. Identification of bone marrow macrophage:the typical morphology of macrophages were induced after seventh days by morphological observation;(91.4±4.7)%cells which obtained from BM expressed CD11b and F4/80by flow cytometry; Using20ng/ml IFN-γ and lOng/ml LPS to stimulate the cultured macrophages48H, the typical M1type macrophages which showed spindle shape and elongated pseudopodia were observed by morphology.2. The expression of CD47in the cell membrane was observed by flow cytometry after transfection of different si-CD47after different time,such as24h,48h,72h. The inhibitory effect of si-CD47-3was obvious, the difference was statistically significant (P<0.05); Compared with expression of CD47after transfected24h,48h and72h, found that the inhibitory effect is best at48h, the difference was statistically significant (P<0.05);3. Using the activated U937cells as the object, found that the macrophage phagocytic index difference between si-CD47-1, si-CD47-2and si-CD47-3group was statistically significant (P<0.05)). The expression level of CD47and cell phagocytosis index correlation analysis, results showed that the expression level of CD47and phagocytic index was negatively correlated (r=-0.925, p=0.001)4. Using bone marrow-derived macrophages which come from children with HLH or acute leukemia (AL) as the object, found that there is an inverse correlation between the expression of CD47in the cell membrane and phagocytosis of macrophage, namely the lower expression of CD47, the stronger of phagocytosis of macrophage, but not related to the source of macrophage. In the same expression of CD47, compare the phagocytosis index of macrophage between children with leukemia and children with HLH, but there is no statistically significant. Conclusion1. To establish a stable method for culturing macrophages in vitro;2. Inhibitory effect of si-CD47-3was the best the effect of transcription level. The inhibitory effect was best at48h;3. Using activated U937cells and bone marrow-derived macrophages which come from children with HLH as the object, found that the expression level of CD47and phagocytic index was negatively correlated; there is no obvious relationship with the source of macrophages.PART3The clinical and laboratory features of hemophagocytic syndromeObjectiveTo evaluate the early diagnosis and prognosis factors of HLH by investigating the clinical and laboratory features of HLHMethodsRetrospectively analyzed the laboratory features and treatment factors of19HLH which including death cases and improved cases.Result1. Divided randomly19children with HLH into improved group of8cases and death group of11cases, between the two groups, the difference of fever period, heat peak, lymphadenopathy and hepatosplenomegaly has no statistically significant(P>0.05);2. Laboratory features:Comparison the laboratory features between this two group, found that the blood decreased (P=0.033), abnormal blood coagulation (P=0.020) and prothrombin time (PT) anomaly (P=0.001) was statistically significant; but atypical lymphocytes; activated partial thromboplastin time (APTT), LDH, ALT, AST, IgG, IgA, IgM and β2microglobulin, the level of SF, EBV-DNA loading, EBV antibodies and detected in the bone marrow of hemophagocytic was no statistically significant (P>0.05). 3. Dynamic changes of SF:8cases were improved with the remission in children with HLH, SF also decreased; but in the dead group, there are10patients progressed, SF increased, and the progress of the1cases patients with SF decreased, this children with nervous system involvement as the initial symptom.4. Using19cases of HLH patients and52cases of infectious mononucleosis patients as the object, evaluation SF as the diagnosis of HLH by ROC curve. SF AUC0.931, where SF>10000μg/L, the sensitivity was22.22%, specificity was100%, SF>3000μg/L, the sensitivity was38.9%, specificity was100%, SF>1000μ g/L, the sensitivity was83.33%, specificity was83%, SF>500μg/L, the sensitivity was100%, specificity was73.40%.Conclusion1. Elevated fibrin1blood decrease, decrease the levels of PT and may be associated with adverse outcomes.2. SF>3000μg/L, can be used as a reliable index of preliminary diagnosis HLH.3. The higher of SF, the more with a diagnosis of HLH, but there is no relationship between the levels of SF and the prognosis of the patients, its dynamic changes can be used as prognostic indicators. |
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