| 1. ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disease of the immune dysfunction, to multi-system organ or more autoantibodies characterized by lesions and serum, At present,the pathogenesis of SLE is still unclear. The important clinical of manifestation is damages of blood system,also the mechanism is not very clear.Bone Mesenchymal Stem Cells (BMSCs) are non-hematopoietic stem cells in bone marrow microenvironment, which are of capacity for multi-polarization orientation, a high degree proliferation and self-renew. BMSCs can differentiate into neural cells, osteoblasts, chondrocytes, myocytes and adipocytes in appropriate conditions. BMSCs are important component of hematopoietic microenvironment. They support and regulate the proliferation and differentiation of hematopoietic cells by means of secretion of cytokines. According to the studies on the pathogenesis of bone marrow mesenchymal stem cells of SLE to explain the blood system damage.Apoptosis refers to maintain a stable internal environment, the genetic control of cell autonomous ordered death, is a biological process by some factor regulation of gene and cell inside and outside. BMSCs patients with SLE in disorders of the bone marrow microenvironment, its structure and function also has some defects. Research on SLE in patients with BMSCs apoptosis benefit to elucidate mechanisms of disease, and to explore new therapy.2. MethodsThis experiment was divided into three group:①SLE patients in BMSCs group (10people);②Other autoimmune disease patients in BMSCs group (5people);③healthy (control group) BMSCs group (5people). Two groups of patients before with hematological damage, bone marrow specimens were without the use of hormones and immunosuppressive therapy.All patients were in line with the classification of diagnostic criteria which was enacted by the American College of Rheumatology Society in1997, The patients whose SLE disease activity index(SLEDAI score)≥10points were devided into the active stage group, Age and gender matched control group selection,5healthy human without autoimmune diseases.Aseptic operation extraction marrow liquid10ml, heparin anticoagulation, separation of mononuclear cells using density, suspended in the human mesenchymal stem cell growth medium, placed in an incubator.2.1BMSCs isolation, purification, proliferation and identification2.2P3BMSCs were selected and re-suspended, then cultured with human mesenchymal stem cell growth medium. The biological changes of BMSCs was observed.2.2.1Cell growth curve mapping2.2.2Determination of cell cycle by flow cytometry2.2.3Apoptosis detected by flow cytometry3. Statistical analysisThe data were analyzed by SPSS17software, using single factor analysis of variance analysis between groups was significant, include cell culture time, cell cycle and apoptosis of differences between groups, compared with the LSD-t test,with P<0.05as the difference has statistical significance.4. Results4.1BMSCs were adherent to the bottom of culture dish and displayed long spindle in shape. Cell fusion achieve80%available for subculture. BMSCs populations were positive for several antigens such as CD44, CD90and CD105, negative for the expression of haematopoietic antigens like CD45and CD34. Subculture proliferation of BMSCs after rapid growth period is shortened. After many(6) subcultures, cell phenotype does not change. SLE and other connective tissue diseases group require more time than the control group during primary culture (P<0.05). After subculture, the growth of BMSCs is vigorous, about5-7-day BMSCs subculture again.The difference of three sets was not statistically significant (P>0.05).4.2The biological changes of BMSCs 4.2.1The BMSCs were cultured respectively in three groups and the growth curves were drawed. It was found that the growth curves in the three groups are same without significant differences.4.2.2Determination of cell cycle by flow cytometry.After the subculture, BMSCs are in the relatively static and the original state of the predominance of cells. Further, analyse and compare S+G2/M period. Find SLE and other CTD group, the difference is not statistically significant (P>0.05) in groups comparisons, but this two-group is significantly higher than the control group, the difference statistically significant (P<0.05).4.2.3The apoptosis of BMSCs in three groups was detected by flow cytometry. Compared with healthy control group, SLE group and other autoimmune diseases, the number of apoptotic cells was increased,and the difference determined was statistically significant (P<0.05). The comparison between the two groups, there was no significant difference (P>0.05).5. ConclusionsApoptosis of BMSCs in vitro increased in patients with SLE, It proved BMSCs in patients with SLE itself was defective. Comparison with other autoimmune diseases, there were no significant differences. Maybe autoimmune diseases exist the similar mechanisms of apoptosis regulation. There were defects on the structures and functions. It provided a theoretical foundation for BMSCs transplantation of autoimmune diseases. |
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