Measurements of T-cell receptor excision cycles in early stage of immune reconstitution after hematopoietic stem cell transplantation

doi:10.3969/j.issn.2095-4344.0621

Abstract

Abstract:

BACKGROUND: Currently, there is no direct and explicit detection index for immune surveillance after hematopoietic stem cell transplantation. The copy number of T-cell receptor excision circle (TREC or signal joint T-cell receptor excision circle, sjTREC) that reflects the recent function of the thymus is monitored by using constantly improved real-time quantitative PCR technology, thereby assessing the level of immune reconstitution and guiding prognosis treatments.
OBJECTIVE: To analyze the influence of transplantation approach, type of diseases, age, and sex in early stage of immune reconstitution after hematopoietic stem cell transplantation by investigating the level of sjTREC in the peripheral blood.
METHODS: The number of sjTREC copies per 1 000 cells in the peripheral blood in control group (n=29), before-group (n=19), and after-group (n=6) were detected using absolute quantitative real-time PCR with TaqMan probes.
RESULTS AND CONCLUSION: In the 1 000 cells, the number of sjTREC copies in the control group was 26.99±29.3 and it decreased with age but no difference existed between male and female. Compared with the control group, the level of sjTREC was significantly lowered in the before-group (P=0.006). After transplantation, the level of sjTREC was rapidly increased within 1 month, and then decreased at 6 months. No obvious differences were found in the level of sjTREC after half-matched HLA related donor transplantation in two cases and full-matched HLA unrelated donor transplantation in four cases. All the findings indicate that the thymus function in patients is seriously impaired before transplantation, and the changes of sjTREC copies that are transiently increased and then decreased at 1 month after transplantation may be related to the stress response of the thymus in the early immune reconstitution period. Although there are no differences between related and unrelated donors, male and female, or different ages, the increased level of sjTREC reflects the changes of thymus function after transplantation, indicating that a variety of factors contribute to immune reconstitution.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Hematopoietic Stem Cell Transplantation, Gene Rearrangement, T-Lymphocyte, Thymus Gland, Tissue Engineering

CLC Number:

R394.2

Wang Kun, Han Jun-yong, Chen Jin-yan, Liu Shen-min, Xue Shi-jie, Jin Jing-jun. Measurements of T-cell receptor excision cycles in early stage of immune reconstitution after hematopoietic stem cell transplantation[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4643-4649.

Figures/Tables 2

2.1 造血干细胞移植病例植入存活状况收集的6例造血干细胞移植患者样本均获得满意的20个STR基因座基因型和1个性别基因座基因型的分型结果(图2)，患者移植后STR表型均完全表现为供者源型，未检测到供受者嵌合状态。 2.2 标准品制备 2.2.1 构建重组质粒pMD20T-sjTREC-RAG2及鉴定结果对重组质粒pMD20T-sjTREC-RAG2用T1、T2与R1、R2进行PCR扩增，得到的片段大小与文献[10]报道的RAG2片段(244 bp)和sjTREC片段(362 bp) 大小一致(图3)。DNA测序结果显示，sjTREC片段在重组质粒342 bp处插入，RAG2片段在735 bp处插入(图4)，经核对其序列表达与文献[10]报道一致。 2.2.2 标准品曲线在制备实时荧光绝对定量PCR体系时，根据质粒pMD20T-sjTREC-RAG2标准品的7个浓度梯度：0.5×107，0.5×106，0.5×105，0.5×104，0.5×103，0.5×102，5拷贝/μL，每个反应体系加入2 μL标准品，使反应体系中起始DNA模板量分别为1×107，1×106，1×105，1×104，1×103，1×102，10拷贝，根据7个浓度梯度标准品扩增的Ct值，绘制标准曲线(图5，6)。RAG2标准曲线斜率=-2.788 670，截距=36.805 401，R2=0.998 5；sjTREC标准曲线斜率=-3.218 531，截距=41.414 780，R2=0.997 076。两条标准曲线的R2均大于0.99。 2.3 sjTREC水平的比较分析 2.3.1 正常健康人群sjTREC平均水平 29例正常健康对照组外周血每1 000个细胞中的sjTREC拷贝数为26.99±29.33。按年龄将正常健康对照组分为20岁以下(n=14)与21-30岁(n=15)两组，其外周血1 000个细胞中sjTREC拷贝数分别为34.08±34.99与20.37±22.059，进行两独立样本的t 检验，P=0.018，差异有显著性意义(图7)。按性别分组，男性组(n=17)和女性组(n=12) sjTREC拷贝数分别为27.91±33.45，25.68±23.65，男女之间差异无显著性意义。 2.3.2 移植前、移植后外周血中sjTREC平均水平比较移植前患者(n=19)外周血1 000个细胞中sjTREC拷贝数为6.97±9.50，与正常对照组进行两独立样本的t 检验，P=0.006，差异有显著性意义。按年龄分析，其中20岁以下患者(n=7)拷贝数为7.62±11.86；21岁以上患者(n=12)拷贝数为6.59±8.40，进行两独立样本的t 检验，P=0.592，差异无显著性意义。按性别分析，男性(n=14)拷贝数为8.75±10.52；女性(n=5)拷贝数为1.99±1.96，进行两独立样本的t 检验，P=0.071，差异无显著性意义。移植后1个月患者(n=5)外周血1 000个细胞中sjTREC拷贝数为43.49±32.80；移植后6个月患者(n=4)外周血1 000个细胞中sjTREC拷贝数为11.83±13.13 (图8)。按移植方式分析[11]，HLA半相合亲缘移植患者(n=2)移植前拷贝数为17.05±22.28，移植后6个月拷贝数为24.26±25.50；HLA全相合非亲缘移植患者(n=4)移植前拷贝数为6.74±12.85，移植后6个月拷贝数为28.30± 19.12(图9)。按疾病类型分析，6例患者涉及4种疾病分型，移植前的sjTREC水平均很低，移植后1个月时1例3岁急性淋巴细胞白血病亲缘移植患者sjTREC水平最高，为78.362 4拷贝，但其他2例22岁和19岁急性淋巴细胞白血病患者sjTREC水平很低，为1.513 2，8.609 1拷贝；同时1例44岁再生障碍性贫血患者未检出sjTREC，1例急性髓系白血病患者和1例急性单核细胞性白血病患者的sjTREC水平居中，5例检出的sjTREC范围从1.513 2-78.362 4拷贝，差异很大。"

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