摘要
目的探讨急性特发性血小板减少性紫癜(acuteidiopathicthrombocytopenicpurpura,AITP)患儿外周血T细胞蛋白激酶C(proteinkinaseC,PKC)的活性变化及其与T细胞活化和血小板减少程度之间的关系。方法无菌采集35例急性ITP患儿及30例正常儿童外周抗凝血,用T淋巴细胞分离富集柱法分离纯化T细胞,分别用非同位素标记法检测T细胞PKC的活性变化,用流式细胞仪检测T细胞活化标志FasL蛋白的表达,血细胞计数仪计数血小板。结果急性ITP患儿T细胞PKC的总活性与正常儿童相比明显增强[(0.97±0.21)nmol/(min.ml)、(0.55±0.13)nmol/(min.ml),P<0.01],T细胞活化标志FasL蛋白表达与正常儿童比较显著升高[ThFasL:(32.7±3.4)%、(14.7±4.2)%;TcFasL:(17.3±9.7)%、(11.6±8.5)%,P<0.01],并且T细胞PKC的活性变化与ThFasL、TcFasL的表达均呈显著正相关(r1=0.68,r2=0.53,P<0.05),与血小板计数呈显著负相关(r=-0.75,P<0.05)。结论急性ITP患儿外周血PKC活性增强可引起患儿T细胞的活化,活性T细胞增多可导致患儿血小板大量损伤,并引起临床症状,提示PKC信号传导在急性ITP的免疫病理机制中发挥重要作用。
Objective Acute idiopathic thrombocytopenic purpura （AITP） is a common autoimmune disease in children, Thrombocytes decrease extremely in serious patients, its pathogenesis involves abnormal activation of T lymphocytes and T cell-dependent production of autoantibody, The aim of the present study was to investigate changes of protein kinase C （ PKC ） activity in peripheral blood T lymphocytes in children with AITP and the relationships between PKC activity and T lymphocytes activation and thrombocytopenia. Methods Peripheral blood specimens were collected from children with acute ITP （ n = 35 ） and healthy children （ n = 30 ） , and T lymphocytes were isolated and purified by using T cells Segregation Enrichment Column, PKC activity was detected by using PepTag Assay, a non-radioactive detection method, The reaction mixture, in a final volume of 25 μl, consisted of 5 μl reaction buffer, PepTag C1 5 μl （0.4μg/μl）, PKC activator solution （DG） 5 μl, peptide protection solution 1 μl and sample 9 μl. Phosphorylation reaction was allowed to continue for 30 minutes, then 25 μl reaction mixture was subjected to electrophoresis on a 0. 8% agarose gel at 100 V for about 20 minutes. After electrophoresis, the PepTag C1 peptides which were phosphorylated and non-phosphorylated were separated, phosphorylated PepTag C1 peptide with negative electricity migrated toward the anode （ ＋ ）, but nonphosphorylated PepTag C1 peptide with positive electricity migrated toward cathode （-）, the gel was photographed. Electrophoresis bands on anode represented PKC activity and were analyzed quantitatively. FasL, which is T cell activation marker, was determined by flow cytometer and platelet was counted by cell counting meter. Results Compared with healthy children, children with AITP had significantly higher PKC total activity [ （0. 97 ± 0. 21 ） nmol/（ min· ml ） vs. （0. 55 ± 0. 13 ） nmol/（ min · ml ） , （ P 〈 0. 05 ） ]. Expression of FasL on T cell subpopulation in children with AITP was significantly higher [Th FasL： （32. 7 ±3.4） vs. （ 14. 7 ± 4. 2 ） ; Tc FasL ： （ 17.3 ± 9. 7 ） vs. （ 11.6 ± 8.5 ） %, （ P 〈 0. 05 ） ]. Besides, relationships between the changes of PKC activity, Th FasL and Tc FasL had positive correlation （ r1 = 0. 68, r2 = 0. 53, P 〈 0. 05 ） , However, PKC activity and platelet count had a significantly negnative correlation （r = -0. 75 ,P 〈 0. 05 ） . Conclusion Increased PKC activity was seen in children with AITP, which can cause damage to thrombocytes and reduction of thrombocytes. PKC signal transduction pathway might play an important role in the immunopathogenesis of AITP.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2006年第3期224-227,共4页
Chinese Journal of Pediatrics
基金
广东省自然科学社会发展科技计划资助项目(2004024)
