| Ankylosing Spondylitis(AS) is a chronic inflammatory disease that mainly affects axial joints. Although the exact pathogenesis of AS remains unclear, but it can be defined that inflammation is the main pathological characteristic of AS during the onset and progress of the disease. The results of some studies about the biopsies of AS local lesions revealed that macrophages are the major cells that invade the local lesions, and it suggested that macrophages may play an important role in the process of inflammation in AS. Macrophages can obtain different phenotypic characteristics and show the different functional state under the stimulus of the local microenvironment. The dynamic changes of the phenotypes of macrophages reflect the pathologies of the body, M1 type activation(Classically Activated Macrophage) means the start and maintenance of inflammation, and M2 type activation(Alternatively Activated Macrophage) suggests the clew of inflammation, the formation of chronic inflammation and tissue repair. ENTPD3(Ectonucleoside Triphosphate Diphosphohydrolase 3) is a member of the ENTPDase families, and the main role of ENTPD3 is to hydrolysis Nucleoside Triphosphate(NTP) and Nucleoside Diphosphate(NDP) as a kind of nucleoside triphosphate diphosphate hydrolytic enzyme, and the the final products of this process are adenosine, Nucleoside Monophosphate(NMP) and inorganic phosphate. As a response to the metabolic disorders, endogenous adenosines are released into the extracellular space and play an important role in a wide range of immune regulation, and the mononuclear phagocyte system is the main target of it. Adenosine regulates the functions of macrophages by G protein coupled receptors, and participates in the processes of inflammation and tissue repair.However, how about the polarization of mononuclear macrophages in AS patients ? Whether ENTPD3 abnormally expresses in the AS patients and what kind of role does it play in the macrophage polarization ? All the questions are still unclear. So, in our research we studied the polarizations of mononuclear macrophages in AS, and the differentially expression of ENTPD3 in AS and it's machanism in regulating the polarization of mononuclear macrophages in vitro studies. And based on these studies we attempt to discover a potential therapeutic target for the treatment of AS. And the research is divided into four parts as follows:PART ? : The Establishment Of Standardized Tissue Bank Of AS And Screening Of Differentially Expressed Genes In AS Hip Ligament TissuesObjective: To develop a standardized and well-rounded tissue bank of AS, and to screen differentially expressed genes through gene chip technology in AS hip ligament tissuses.Methods: The samples such as preoperative whole blood, urine, fresh surgical hip ligament tissue samples were collected according to a standardized operational procedures, and the paraffin-embedded ligament tissues were collected. A well-rounded AS tissue bank was established based on clinical database software and tissue bank management software. The quality of samples was tested regularly. Three cases of AS hip ligament tissue samples were selected from the bank randomly as disease group, and 3 cases of control hip tissue samples that machted in age and gender were selected as control group. The Affymetrix gene chip technology was used to screen differential genes. Based on the result of gene chip screening, and combined with GO-Analysis and bioinformatics research methods, candidate gene was determined.Results: A standard AS tissue bank was established, and a total of 121 AS individuals, 20 femoral neck fracture individuals, 42 osteonecrosis of the femoral head individuals and 20 acetabulum hypoplasia were enrolled as sample donors, respectively. Clinical data, preoperative whole blood, urine, fresh surgical hip ligament tissue samples, paraffin-embedded tissues were collected and preserved. Database access and sample preservation can be managed easily and correctly with the data management platform. The results of quality tests were satisfied. A total of 697 differentially expressed genes were defined, according to the standard of the difference of 1.5 times, the result of gene chip showed that there were 40 up-regulated genes and 91 down-regulated genes. And the ENTPD3 up-regulated significantly(up-regulate 2.12 times, P = 0.003).Conclusion: A standard AS tissue bank using high-quality well-characterized samples was established, which will become the cornerstone for AS research. And the ENTPD3 gene that is associated with NTP's and NDP's decomposition metabolic process was found up-regulated significantly based on the geng chip screening.PART ?: The Validation Of Differentially Expression Of ENTPD3 In AS Hip Ligaments And Analysis Of The Correlation Between ENTPD3 And Clinical DataObjective: To verify the differentially expression of ENTPD3 in AS hip ligaments, and to study the correlation of ENTPD3 and inflammation and structure damage in AS.Methods: A total of 17 cases of AS hip ligament tissue samples and 17 cases of control hip ligament tissue samples were selected randomly from the tissue bank, and thereal-time fluorescent quantitative polymerase chain reaction(QPCR) was used for verifying the differentially expression of ENTPD3 in m RNA level. And immunohistochemical technology was used to detect the manifestation of candidate genes in hip samples(5 AS paraffin-embedded tissues VS 5 control paraffin-embedded tissues). The clinical data were collected, and correlation analysis was performed.Results: In enlarged samples, the expression of ENTPD3 in AS group was obviously higher than in control group either in m RNA level or in protein level(P = 0.015, P < 0.001); the expression of ENTPD3 was positively correlated with modified Stoke Ankylosing Spondylitis Spine Score(m SASSS)(r = 0.639,P = 0.006), and was negatively correlated with ESR(r =-0.521,P = 0.032) in AS.Conclusion: ENTPD3 was significantly higher expression in the AS hip ligament, and was positively correlated with m SASSS, and negatively correlated with ESR. It suggested that ENTPD3 may involed in the processes of inflammation and ossification of ligament.PART ?: The Relationship Between ENTPD3 And Mononuclear Macrophage Polarization In AS Peripheral BloodObjective: To evaluate monocyte polarization occurring in the peripheral blood of patients with AS and the relationship between ENTPD3 and mononuclear macrophage polarization, and to determine the correlations between monocyte polarization and inflammation and structural damage.Methods: A total of 120 AS patients, 50 rheumatoid arthritis(RA) patients and 100 normal healthy controls who were matched by age and gender were enrolled in the study. M1(CD68+CD192+), M2(CX3CR1+CD163+) monocytes and ENTPD3 were characterized by flow cytometry. The levels of TNF-a and IL-10 were determined by ELISA. All the above characters were duplicate detected at baseline and 12 weeks after therapy in 20 AS patients who were recieved Anti-TNF-a treatment. Demographic, clinical, radiographic and laboratory data were collected and analyzed.Results: A large increase in M2(CX3CR1+CD163+) monocytes was observed in AS, and M2/M1 ratio was 7.18 ± 6.12, 2.54 ± 3.14 and 35.61 ± 20.04 in control, RA and AS, respectively. The M2/M1 ratio correlated with m SASSS(r = 0.588; P < 0.001), ESR(r =-0.321; P < 0.001), CRP(r =-0.265; P < 0.001). ENTPD3 mainly expressed in mononuclear cells, and was associated with the M2 polarization closely(r = 0.571, P < 0.001). Anti-TNF-a therapy induced a significant reduction in the percentage of M1 monocyte.Conclusion: M2 type polarized monocytes were predominant in the peripheral blood in AS. The M2/M1 ratio was correlated with structural damage(m SASSS), inflammatory biomarkers(ESR and CRP). ENTPD3 mainly expressed in mononuclear cells, and was associated with the M2 polarization closely.PART ?: The Study Of The Mechanism Of ENTPD3's Effect On Macrophages Polarization In Vitro Cell LevelObjective: The aim of this study was to evaluate the mechanism of ENTPD3's effect on macrophages polarization in vitro.Methods: THP-1 cell was cultivated in vitro, and was stimulated with Lipopolysaccharide(LPS)(100 ng/ml) and IL-4(10 ng/ml) to induce M1 and M2 polarization, respectively. M1(CD68+CD192+), M2(CX3CR1+CD163+) macrophages and ENTPD3 were characterized by flow cytometry. And the levels of IL-10 and TNF-a in culture supernatant were determined by ELISA. ENTPD3 si RNA and ENTPD3 expressing recombinant adenovirus were transfected respectively. All the data were detected repeatedly, and nuclear factor KB(NF-KB), signal transducer and activator of transcription 3(STAT3) and its phosphorylation levels were detected by Western Blot. Inhibited the NF-KB and STAT3 with corresponding inhibitors of NF-KB and STAT3, and repeat the test mentioned above.Results: After stimulated with LPS for 24 h, CD68+CD192+ cells increased significantly, CX3CR1+CD163+ cells reduced, TNF-a increased significantly, phosphorylation IKB increased significantly, IKB was lower, the NF-KBp65 of nucleus increased obviously. After ENTPD3 interference, the change is more apparently. After over expression of ENTPD3, CD68+CD192+ decreased, CX3CR1+CD163+ elevated slightly, TNF-a was lower, phosphorylation of IKB and NF-KBp65 of nucleus decreased, while IKB increased slightly. After inhibited the NF-KB, the effects of LPS were inhibitted significantly. After stimulated with IL-4 for 24 h, THP-1 cell showed higher CX3CR1+CD163+ expression, and CD68+CD192+ reduced significantly, IL-10 rised obviously, the phosphorylized STAT3 and STAT3 in nucleus increased obviously. Theabove phenomenons were more obviously after over expression of ENTPD3. After ENTPD3 interference, CX3CR1+CD163+ expressed slightly lower, CD68+CD192+ elevated slightly; IL-10 levels, the phosphorylized STAT3 and STAT3 in nucleus decreased; After inhibited the STAT3, the effects of IL-4 were suppressed significantly.Conlcusions: ENTPD3 regulates macrophages to M2 polarization by promoting STAT3 pathway and inhibiting NF-KB's activation. |
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