多房棘球蚴感染BALB/c小鼠转录因子T-bet、GATA-3mRNA表达的实验研究

目的 探讨多房棘球蚴(Em)腹腔感染BALB/c小鼠辅助性T细胞1和2特异性转录因子T-bet mRNA、GA-TA-3 mRNA在脾细胞中的表达及其意义. 方法 选取30只BALB/c小鼠(6-8周龄),随机分为Em腹腔感染组(Em)、Em腹腔感染联合阿苯达唑(ABZ)治疗组(Em+ABZ)、健康HC组(healthy controls HC),每组10只.采用腹腔注射Em头节法建立小鼠继发性腹腔Em感染模型,Em+ ABZ组给予ABZ溶液灌胃,Em组、HC组给予羧甲基纤维素钠(CMC)溶液灌胃,每只小鼠每次灌胃100μl,1次/d,连续35d.采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)法检测实验小鼠脾脏细胞转录因子T-bet mRNA和GATA-3 mRNA的表达. 结果 Em组小鼠脾细胞T-bet mRNA相对表达量为0.057士0.031,HC组为0.057士0.033,差异无统计学意义(F=2.215,P＞0.05);Em组GATA-3 mRNA相对表达量为0.025士0.013,HC组为0.020士0.010,差异无统计学意义(F=0.789,P＞0.05).Em+ ABZ组小鼠脾脏T-bet mRNA相对表达量为0.088士0.038,与HC组和Em组比较差异无统计学意义(F=2.215,P＞0.05);Em+ ABZ组GATA-3 mRNA相对表达量为0.018土0.011,与HC组和Em组比较差异无统计学意义(F=0.789,P＞0.05).T-bet mRNA/GATA-3值Em组为2.853士2.054,HC组为3.160±1.777,差异无统计学意义(F=3.649,P＞0.05).T-bet mRNA/GATA-3值Em+ ABZ组为6.022±3.528,与HC组和Em组比较差异均有统计学意义(F=3.649,P＜0.05). 结论 Em腹腔感染小鼠脾细胞中T-bet/GATA-3值降低,Th1、Th2平衡被打破,Th2成为优势免疫应答模式,经ABZ治疗后T-bet表达升高,GATA-3表达降低,免疫应答模式由Th2占主导向Th1漂移.

儿童急性T淋巴细胞白血病T细胞受体β链基因重排的特点及其在微小残留病定量检测中的意义

Objective To explore the characteristics of T-cell receptor beta (TCRβ) gene rearrangement in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system for quantitative detection of minimal residual disease (MRD) by real-time quantitative PCR (RQ-PCR) targeting the TCRβ gene rearrangement. Methods Multiplex PCR designed by the BIOMED-2 was used to detect TCRβ gene rearrangement in the bone marrow samples of 26 children with T-ALL. Sequences of junctional region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRβ gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient-specific standard curves. Subsequently, TCRβ RQ-PCR was applied to six patients to quantify MRD with germline Jβ primer/probe combinations. To determine the quantity and quality of DNA, we also used RQ-PCR for the N-ras gene.Results Clonal rearrangements were identified in 92.3% of the children with T-ALL ( Vβ-Dβ-Jβ rearrangements in 84.6% and Dβ-Jβ rearrangements in 50% ). Comparative sequence analysis of 42 TCRβ recombination revealed that two downstream Vβ families (BV5, BV6) were preferentially used. The segment Jβ2. 7 was dominant in childhood T-ALL. Jβ1. 3, Jβ2.4, and Jβ2.6 were not detected. The slope of the standard curves was from - 3.54 to -3.37 with the intercepts between 19.35 and 20.51. The correlation coefficients of all the 6 standard curves were ≥0.98. None of the cases had a quantitative range of RQ-PCR lower than 10<'-4>. During the follow-up, an increased incidence of MRD was found before relapse.Conclusions RQ-PCR, which is a highly sensitive and specific method for detection of TCRβ gene rearrangements, will be of high value to study MRD in T-ALL. Close monitoring of MRD is of great importance for prognosis and follow-up of the patients with the disease.

Objective: To determine the sensitivity and significance of B-cell chimerism for the detection of early engraftment, transplant rejection, and disease relapse. Methods: The dynamic monitoring of lineage-specific cell subtypes (B, T, and NK cells) was made in 20 B-cell acute lympho-blastic leukemia (B-ALL) patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the early period after allo-HSCT, the latest establishment of B-cell complete chimerism (CC) was observed in a majority of patients. Results: The percentage of donor cells of B-cell lineage was lower than the percent of T-cell lineage in most of the mixed chimerism (MC) patients. During graft rejection, the frequency of patients with decreasing MC of B-, T-and NK-cell lineage were 5/5, 2/5, and 2/5. When disease relapsed, five patients showed a faster decrease of the donor percent of B-cells than of T-or NK-cells. Only one patient displayed a more rapid decrease in NK-cells than in T-or B-cells. Conclusion: Monitoring of B-cell chimerism after HSCT seems to be valuable for insuring complete engraftment, anticipating graft rejection, and relapse in B-ALL patients. Copyright ? 2015, Chinese Medical Association Production. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).