| Objectives Naive T cell counts transported from thymus recently could be putatively regarded as a index to measure proliferative ability of T cell and transporting function of thymus regeneration. Two isoforms of CD45 positive T cell, CD45RA+ initiative T cells and CD45RO* T memory cell, have been used as markers of regeneration ability and immunity reconstruction after hematopoietic stem cell transplantation (HSCT) . However, latest researches shows it is not precise to use the proportion of CD45RA"*" positive T cells representing thymus function. Fortunately, Studies recently suggest T cell receptor excision cycle (TREC) is a new marker of nai've T cell and could be indices of regeneration function of thymus. There was a few of the related study about TREC in HSCT. For the purpose of exploring the regeneration function of thymus and associated immunity reconstruction process in patients after HSCT, we intended to develop and optimize the real-time florescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify the TREC levels. It could provide a useful method for immunity reconstruction mechanism study about HSCT in clinical research.Methods The human genomic DNA was extracted from the samples using two methods: UNIQ-10 columns method and SDS-proteinase K-phenol-chloride extraction method. We designed primers and TaqMan-MGB probes using software PCR-Express with reference sequences of TREC and RAG2 downloaded from GENBANK of NCBI web site. Since each normal nucleated cell contains two copies of RAG2 gene, we employed RAG2 as cell number control. The real-time PCR system for amplifying TREC and RAG2 was established on ABI 7000 apparatus using Golden Taq system, the designed primers, TaqMan-MGBprobes and optimized buffer. We firstly optimized PCR conditions with TREC plasmid standard samples. Then, the levels of TREC in 103 cells were calculated in all clinical samples. We collected 90 peripheral blood samples from 49 patients at the pre-transplantation, ISdays, 30days, 90days, and ISOdays after transplantation respectively. Finally, all the obtained data were analyzed with SPSS 10.0 statistical software.ResultsThe UNIQ-10 genomic DNA extracting method is more effective than the conventional proteinase-K-phenol-choride method.Using designed TREC plasmid standard samples with the housekeeping gene RAG2 as cell number control to optimize FQ-PCR conditions. The TREC plasmid standard samples were in concentration of 1x107, 1x106, 1x105, 1x104, 1x103, 1x102,20 and 10 copies/100ugDNA(2ul) as positive control. We have successively established optimized FQ-PCR method to calculate TREC copies in peripheral blood cells, the detailed results were found as following:a. The amplification ability of primer pair TB and T4 is more efficient than that of primer pair T! and TI.b. More specific and efficient amplification in FQ-PCR was observed when using TaqMan-MGB probes compared with general Taq-Man probes.c. Golden Taq is more effective than general Taq in improving specificity, sensitivity, and decreasing artifact because of the heating dependent activation property of Goden Taq.d. We got optimized FQ-PCR buffer component as it consisted of 30 mmol/L of Mg2+ with a pH of 8.9. Furthermore, additives such as DMSO and glycerol could improve efficient amplification.e. We optimized thermal parameter as: 95C 10min, 95C, 5sec, 53 C 30 sec and 40cycles using ABI7000.With the established FQ-PCR protocol, we detected the TREC in the clinical samples and found some clinical results as following:a. The TREC level in the peripheral blood cells of normal health adults is 10.2040+0.98llcopies/103MNC.b. Compared with allo-HSCT patients, auto-HSCT patients hadhigher TREC levels (P<0.01) , and the TREC levels had recovered to normal levels in auto-HSCT patients by 180 day, but not in allo-HSCT patients.c. No significant TREC levels between allo-BMT and allo-PBSCT patients within half a year...
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