| Experimental SectionObjects1. To explore effects and possible mechanisms of bone marrow mesenchymal stem cells on T lymphocyte proliferation in patients with aplastic anemia in vitro experiments.2. To clarify effects and related mechanisms of bone marrow mesenchymal stem cells on generation of CD4+CD25+FoxP3+ T regulatory cells in patients with aplastic anemia in vitro experiments.Methods1. Enriched and purified T lymphocytes in peripheral blood of patients with aplastic anemia in vitro. Then co-cultured them with vitro-amplified BMSC after stained T lymphocytes with CFDA SE fluorescent dye. It was divided into direct contact group and Transwell group on basis of co-culture manner. PHA was added to stimulate T lymphocyte proliferation. Besides, both negative and positive control groups were set.72 hours later, Rates of T lymphocyte proliferation were tested in different groups by flow cytometry.2. Enriched and purified T lymphocytes in peripheral blood of patients with aplastic anemia in vitro. Then co-cultured them with vitro-amplified BMSC. The ratio of CD4+CD25+FoxP3+ T regulatory cells before and after co-culture was both tested by flow cytometry.3. FoxP3 gene expression before and after co-culture was tested by qRT-PCR assay. 4. IL-10 and TGF-β1 content in BMSC cultural supernatant before co-culture were detected by ELISA.5. In the Transwell system, recombinant human IL-10(rhIL-10) with concentration of lng/ml,2ng/ml and 4ng/ml was respectively added into the supernatant.72 hours later, the ratio of CD4+CD25+FoxP3+ T regulatory cells was detected by flow cytometry.6. In the Transwell system, recombinant human TGF-β1(rhTGF-β1) with concentration of 0.025ng/ml,0.05ng/ml and 0.1ng/ml was respectively added into the supernatant.72 hours later, the ratio of CD4+CD25+FoxP3+ T regulatory cells was detected by flow cytometry.Results1. CFDA SE staining showed that the ratio of proliferous T cell in positive control group was 57.25±3.50% and in direct contact group was 12.56±1.49%, which was significantly lower than the positive control group (P <0.01). The ratio of proliferous T cell in Transwell group was 47.75±3.65%, which was also lower than the positive control group (P<0.05). Compared direct contact group with Transwell group, T cell proliferation in direct contact group was much lower (P<0.01).2. Flow cytometry analysis showed, after co-culture, ratio of CD4+CD25+FoxP3+ T regulatory cells in direct contact group were significantly increased (P<0.01); Ratio of CD4+CD25+FoxP3+ T regulatory cells in Transwell group were also increased than before (P<0.05).3. qRT-PCR results showed, after co-culture, the relative expression of FoxP3mRNA in direct contact group was 9.14±0.89 and in Transwell group was 6.03±0.91, which were both higher than before(1). The relative expression of FoxP3mRNA in direct contact group was higher than that in Transwell group.4. ELISA assay results revealed that IL-10 concentration in BMSC supernatant before co-culture was 1038.94±245.23pg/ml. While TGF-β1 concentration in BMSC supernatant was 1.57±0.32ng/ml.5. The ratio of CD4+CD25+FoxP3+ T regulatory cells increased after adding 2ng/ml or 4ng/ml exogenous rhIL-10(P<0.05), and it increased gradually with the increase of the concentration of added cytokines (P=0.022, r=0.741)6. The ratio of CD4+CD25+FoxP3+ T regulatory cells increased after adding 0. 1ng/ml exogenous rhTGF-β1(P<0.05), and it increased gradually with the increase of the concentration of added cytokines (P=0.041, r=0.686) Conclusions1. BMSC could inhibit abnormal proliferation of T cells in patients with AA probably through direct contact manner and secretion of some cytokines. And direct contact mechanism played a major role.2. BMSC could increase CD4+CD25+FoxP3+ T regulatory cells expression through direct contact manner and secretion of some cytokines. And direct contact mechanism played a major role. All of which provided a experimental basis for treatment of AA.3. BMSC could secrete cytokines IL-10 and TGF-β1 and these factors may play a role in increasing the ratio of CD4+CD25+FoxP3+ T regulatory cells through paracrine mechanisms.Clinical PartObjects1. To detect the ratio of CD4+CD25+FoxP3+ Treg in clinical patients with AA and evaluate the immunological abnormalities in patients with AA.2. To observe the effect of exogenous BMSC therapy on CD4+CD25+FoxP3+ T regulatory cells in patients with AA.3. To evaluate the efficacy and safety of BMSC therapy to AA.Methods1. The ratio of CD4+CD25+FoxP3+ Treg was tested by flow cytometry in 15 cases of severe aplastic anemia (SAA) and 15 cases of chronic aplastic anemia (CAA) and were compared with 15 healthy controls.2.26 patients with AA were divided into BMSC treatment group (13 cases) and control group (13 cases) on basis of non-randomized clinical control methods. Patients in control group were treated with conventional IST.while in BMSC treatment group, patients were treated with BMSC infusion therapy in addition to IST.The ratio of CD4+CD25+FoxP3+ Treg in peripheral blood were detected before and after treatment.3. The efficacy of the two groups was assessed to evaluate the efficacy of BMSC treatment to AA.4. Vital signs and infusion reactions were monitored during BMSC transfusion;liver function and kidney function were also tested before and after treatment in BMSC-treated patients to evaluate the safety of BMSC treatment. Results1. Flow cytometry analysis showed the ratio of CD4+CD25+FoxP3+ T regulatory cells in healthy controls was 2.43±0.85%. The ratio of CD4+CD25+FoxP3+ T regulatory cells in SAA group was 0.34±0.27%, which was significantly lower than healthy controls (P<0.01). The ratio of CD4+CD25+FoxP3+ T regulatory cells in CAA group was 0.86±0.63%, which was also lower than healthy controls (P <0.05).2. The ratio of CD4+CD25+FoxP3+ T regulatory cells increased after BMSC treatment (P<0.05). And it increased more significantly in CAA group than SAA group (P<0.01). However, the ratio of CD4+CD25+FoxP3+ T regulatory cells was no significant change in control group (P>0.05).3. The total effective rate in BMSC group was 84.62%, while in control group was 61.54%. BMSC therapy had improved the efficiency. However, probably due to a limited number of cases at present, it had not reached statistical significance (P>0.05).4. The vital signs were stable during BMSC transfusion and rarely infusion reactions occured. Besides, there were no significant changes in liver function and kidney function after BMSC treatment(P>0.05).Conclusions1.The ratio of CD4+CD25+FoxP3+ T regulatory cells in peripheral blood of patients with AA were lower than normal. And its abnormal expression may be involved in the pathogenesis of AA.2. The ratio of CD4+CD25+FoxP3+ T regulatory cells were much lower in patients with SAA which suggesting that its ratio could be used as one of indicators to determine the severity of AA.3. The ratio of CD4+CD25+FoxP3+ T regulatory cell could be up-regulated with BMSC treatment which provided a clinical basis for BMSC correcting the immunological abnormalities in patients with AA.4. The efficacy of BMSC treatment was similar with conventional IST therapy. However, present results showed that it could improve the total efficiency for patients with AA.5. BMSC treatment was safe for patients with AA. Clinical Part of Chinese MedicineObjectTo explore whether there exists differences involving the ratio of CD4+CD25+FoxP3+ T regulatory cell among different TCM syndrome-types.Methods1.The 30 patients with AA in the second part were divided into hepatic and renal yin deficiency, asdthenic splenonephro-yang, asthenia of both yin and yang and urgent-fatigued-epidemic febrile according to main symptoms of Chinese medicine.2. The ratio of CD4+CD25+FoxP3+Treg in peripheral blood was detected among different TCM syndrome-types.ResultThere was no significant difference involving CD4+CD25+FoxP3+ Treg expression among the 4 different TCM syndrome-types.ConclusionThere has no significant value for the ratio of CD4+CD25+FoxP3+Treg prompting TCM syndrome-types in patients with AA. |
PDF Full Text Request