| Objective: Scleroderma is characterized by local or diffuse fibrosis or sclerosis of connective tissue in the skin and other organs with the final results of atrophy. According to the different range of involvement, it is devided into local scleroderma and systemic scleroderma. Although the etiology and pathogenesis is still not clear, it's regarded that multiple factors are involved in it including inheritance, gender, environment, vascular impairment, autoimmune mechanism, fibroblasts activation and so on. The lesion of capillary vessel system is thought to be one of the initial factors which result in development of scleroderma. Under specific conditions, endothelial progenitors could differentiate into endothelial cells and promote formation of neo-blood vessels. Therefore, EPCs play key role in vessel regeneration and recovery at the site of vascular lesions. It's demonstrated that there's angiogenesis disorder to different degree in systemic scleroderma patients, accompanied by decreased EPCs. In our study, number and function of EPCs in peripheral blood from systemic scleroderma patients are compared with that of healthy adults. To disclose relationship between changes of EPCs and pathogenesis of scleroderma could be helpful to establish new theory for treatment of systemic scleroderma. Furthermore, new treatment regimens could be gradually developed with lower dosage of corticosteroids and immunosuppressive agents, which results in remission of the disease and better quality of life. Methods:20 cases diagnosed as systemic scleroderma from outpatients and inpatients department are distributed to experimental group and 20 healthy volunteers are enrolled as control group. 10 ml of blood sample is collected from each case. The number of mononuclear cells is counted immediately isolated by Ficoll density gradient centrifugation. Once mononuclear cells are inoculated into culture bottles, growth of them is then observed under contrast phase microscope and recorded by photography monitor every day. Cells with Dil-Ac-LDL and FITC-UEA-I displayed crocus fluorescence. Cells with double markers of CD133+ (labeled by FITC), CD34+ (labeled by PE) or CD133+, VEGFR2+ (labeled by PerCp/Cy5.5) are identified through flow cytometry. MTT trial is utilized to evaluate proliferation of EPCs with outcomes of OD value under 490 nm wavelength tested by ELIASA. Growth rate is also calculated and equal to ( OD experiment / OD contrast-1 )×100% . Migration and adhesion function of EPCs are assessed by modified millicell chamber assay and adherent activity assay. The experiment data is reported as x±s and study outcomes are analyzed by SPSS 13.0 statistical package. Results : At the beginning of inoculation, mononuclear cells are small and round suspending in the culture solution. 48 hours later, most of them will complete adhesion and become larger. Through 4– 5 days culture proliferation of mononuclear cells accelerates. Some cell clones gradually form with round cells at the central region and spindle cells arranged in radiated shape around the cores. 7 days later spindle cells become predominant growing in line or tending to form reticular configuration at polygon- or pebble-like appearance. Data of flow cytometry indicates that for experimental group the counting of cells with double markers positive including CD133, CD34 or CD133, VEGFR2 are 0.10%±0.09%. The difference is statistically significant if compared with that of control group (0.49%±0.09%). Cell growth rates of experimental and control group are 13.01±4.33% and 23.28±4.34% respectively. Such difference is also statistically significant. Migration assay demonstrates that ability of EPCs migration for experimental group decrease with cells of 7.20±3.67 / HPF, in contrast to cells of 14.15±2.98 / HPF for control group. As to adhesion assay, adherence activity is impaired with cells of 15.20±4.40 / HPF for experimental group while for control group the value is 24.80±3.58 / HPF. Therefore, function of EPCs is identified to be reduced including migration and adhesion activity. The differences between experimental and control group are statistically significant. Conclusions: Endothelial impairment of systemic scleroderma patients is identified in the study. The vascular lesions may be associated with not only decreased number but reduced function of EPCs, including proliferation ability, migration and adhesion activity. Better utilization of EPCs could be developed into a new treatment for systemic scleroderma gradually in the future. Stimulation of proliferation and release from marrow to increase number of EPCs or improvement of the function may be practicable and reasonable approaches to cure systemic scleroderma.
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