实时荧光定量PCR检测造血干细胞移植后患者外周血TREC的方法研究
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摘要
目的 胸腺近期输出的初始T细胞(naive T cell)数量可作为机体T细胞增殖能力和胸腺再生输出功能的评价指标。以往多是应用CD45的同功异构体区分新近生成的初始T细胞(CD45RA~+)和记忆T细胞(CD45RO~+)来研究胸腺再生输出能力及在造血干细胞移植(HSCT)后免疫重建过程中的作用，但近年的研究显示CD45RA~+细胞的含量并不能完全准确地代表胸腺功能。近期研究表明，TCRα键基因重排时形成的T细胞受体删除DNA环(TREC)或称信号结合T细胞受体删除环(sj TRECs)是初始T细胞的标志，可作为胸腺再生输出功能的指标。目前国内尚未见有关造血干细胞移植后TREC研究的报道。为了解移植之后患者的胸腺再生输出功能与相关的免疫功能重建机制，本课题拟研究摸索建立检测移植后患者外周血TREC拷贝数的实时荧光定量PCR(FQ-PCR)的方法及实验影响条件，并以看家基因RAG2为参比对照确定细胞数，从而更准确定量TREC的拷贝数，为临床研究造血干细胞移植的免疫功能重建机制提供了方法学基础。
方法 分别比较用UNIQ-10柱式基因组DNA抽提试剂盒与酚氯仿抽提两种方法提取外周血有核细胞的基因组DNA，以用于PCR模板。以NCBI核酸数据库的TREC和看家基因RAG2参照序列为靶序列，应用PCR-Express软件设计分别特异于TREC和RAG2序列的TaqMan-MGB荧光探针及多种引物，其中看家基因RAG2用于参比对照(每个细胞含2拷贝RAG2，因此RAG2的拷贝数可间接反映外周血的有核细胞数)。分别研究分析金牌Taq酶和普通Taq酶等多个扩增体系影响因素，在ABI7000PCR仪上实时扩增检测构建的TREC质粒
标准品，摸索最佳扩增条件及建立标准曲线，然后用优化的FQ一PCR
方法检测临床样本，计算1000个细胞所含的TREC的拷贝数。临床
样本收集来自南方医院的49例造血干细胞移植病人的移植前、移植
之后巧天、30天、90天和180天等五个不同时间点的90份肝素抗
凝外周血标本。应用SPSS10.O软件统计分析所得数据。
结果
采用uNIQ一10柱式基因组DNA抽提试剂盒提取外周血有核细胞
的基因组DNA，比较酚氯仿抽提法得到的DNA含量高，纯度好，扩
增效率好。
FQ一PCR检测TREc的方法建立:
以TREC标准品[浓度梯度分别为:zxlo7，lxlo6，lxlos，lxlo4，
lxlo3，lxloZ，20和lozloougDN^(Zul)为阳性对照，看家基因R^oZ
作为参比对照，通过定量细胞数检测TREC拷贝数。以标准品为模
板，在相同反应条件下，摸索FQ一PCR的最佳反应体系，结果发现;
1.在相同反应条件下，T3、T4引物和R3、R4引物的扩增效率分别
优于Tl、TZ引物和R;、RZ引物。
2.TaqMan一MGB探针比普通的TaqMan探针扩增效率高，特异
性强，杂交稳定性高。
3.金牌热启动Taq酶，比较普通Taq酶而言，能够最大限度地
减少杂讯的生成，提高了PCR的特异性和敏感性，从而提高扩增效率。
4.Buffer中Mg2+的最适浓度为30 mmoFL，Tris一HcL的最适PH
值为&9。适量加入10%二甲基亚矾(DMSO)和50%甘油，会增
加PCR产量。
5.FQ一PcR的反应条件设定:95℃10分钟后95℃5秒，53℃30
秒40循环结束。
临床检测结果显示:
1.10例正常健康者TREC含量平均为10.2040士0.98lleopies
/103MNC。
2.对49例造血干细胞移植后患者外周血中TREC含量的追踪检
测发现:在移植后半年内，接受自体造血干细胞移植(A uto一HscT)患
者的TREC含量比接受异体造血干细胞移植(Allo一HScT)患者的TREC
含量恢复迅速(P<0.01)。移植后180天，接受Aut。一HSCT患者的
TREC含量恢复到接近正常人水平(P>0.05)。
3.在移植后半年内，接受异体骨髓移植(allo一BMT)与接受异
体外周血干细胞移植(allo一PBSCT)患者的TREC含量没有明显差异
(P=0 .081)。
4.在移植后半年内，非亲缘移植(UD一HSCT)患者与亲缘移植
(RD一HSCT)患者的TREC含量没有明显差异(p=0.278)。
结论通过摸索与优化实验条件，建立了实时荧光定量PCR检
测外周血单个核细胞中TREC的方法。初步的临床研究显示了
Allo一HSCT及Auto一HSCT后TREC的动态变化规律。Allo一HSCT及
Aut。一HSCT后胸腺再生输出功能恢复的不同进程的机制及影响因素
还需要更深一步的研究。
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-MGB
probes 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.
Results
The 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 had
higher 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
方法 分别比较用UNIQ-10柱式基因组DNA抽提试剂盒与酚氯仿抽提两种方法提取外周血有核细胞的基因组DNA，以用于PCR模板。以NCBI核酸数据库的TREC和看家基因RAG2参照序列为靶序列，应用PCR-Express软件设计分别特异于TREC和RAG2序列的TaqMan-MGB荧光探针及多种引物，其中看家基因RAG2用于参比对照(每个细胞含2拷贝RAG2，因此RAG2的拷贝数可间接反映外周血的有核细胞数)。分别研究分析金牌Taq酶和普通Taq酶等多个扩增体系影响因素，在ABI7000PCR仪上实时扩增检测构建的TREC质粒
标准品，摸索最佳扩增条件及建立标准曲线，然后用优化的FQ一PCR
方法检测临床样本，计算1000个细胞所含的TREC的拷贝数。临床
样本收集来自南方医院的49例造血干细胞移植病人的移植前、移植
之后巧天、30天、90天和180天等五个不同时间点的90份肝素抗
凝外周血标本。应用SPSS10.O软件统计分析所得数据。
结果
采用uNIQ一10柱式基因组DNA抽提试剂盒提取外周血有核细胞
的基因组DNA，比较酚氯仿抽提法得到的DNA含量高，纯度好，扩
增效率好。
FQ一PCR检测TREc的方法建立:
以TREC标准品[浓度梯度分别为:zxlo7，lxlo6，lxlos，lxlo4，
lxlo3，lxloZ，20和lozloougDN^(Zul)为阳性对照，看家基因R^oZ
作为参比对照，通过定量细胞数检测TREC拷贝数。以标准品为模
板，在相同反应条件下，摸索FQ一PCR的最佳反应体系，结果发现;
1.在相同反应条件下，T3、T4引物和R3、R4引物的扩增效率分别
优于Tl、TZ引物和R;、RZ引物。
2.TaqMan一MGB探针比普通的TaqMan探针扩增效率高，特异
性强，杂交稳定性高。
3.金牌热启动Taq酶，比较普通Taq酶而言，能够最大限度地
减少杂讯的生成，提高了PCR的特异性和敏感性，从而提高扩增效率。
4.Buffer中Mg2+的最适浓度为30 mmoFL，Tris一HcL的最适PH
值为&9。适量加入10%二甲基亚矾(DMSO)和50%甘油，会增
加PCR产量。
5.FQ一PcR的反应条件设定:95℃10分钟后95℃5秒，53℃30
秒40循环结束。
临床检测结果显示:
1.10例正常健康者TREC含量平均为10.2040士0.98lleopies
/103MNC。
2.对49例造血干细胞移植后患者外周血中TREC含量的追踪检
测发现:在移植后半年内，接受自体造血干细胞移植(A uto一HscT)患
者的TREC含量比接受异体造血干细胞移植(Allo一HScT)患者的TREC
含量恢复迅速(P<0.01)。移植后180天，接受Aut。一HSCT患者的
TREC含量恢复到接近正常人水平(P>0.05)。
3.在移植后半年内，接受异体骨髓移植(allo一BMT)与接受异
体外周血干细胞移植(allo一PBSCT)患者的TREC含量没有明显差异
(P=0 .081)。
4.在移植后半年内，非亲缘移植(UD一HSCT)患者与亲缘移植
(RD一HSCT)患者的TREC含量没有明显差异(p=0.278)。
结论通过摸索与优化实验条件，建立了实时荧光定量PCR检
测外周血单个核细胞中TREC的方法。初步的临床研究显示了
Allo一HSCT及Auto一HSCT后TREC的动态变化规律。Allo一HSCT及
Aut。一HSCT后胸腺再生输出功能恢复的不同进程的机制及影响因素
还需要更深一步的研究。
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-MGB
probes 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.
Results
The 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 had
higher 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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41． Talvensaari K et al.A broad T-cell repertoire diversity and an efficient thymic function indicate afavorable long-termimmunereconstitution after cord blood stem celltransplantation.Blood.2002 Feb 15;99(4) :1458-64．
42． Chao NJ et al.Nonmyeloablative regimen preserves "niches" allowing for peripheral expansion ofdonor T-cells.Biol Blood Marrow Transplant.2002;8(5) :249-56．
43． Hochberg EP et al.Quantitation of T-cell neogenesis in vivo after allogeneic bone marrowtransplantation in adults.Blood.2001 Aug 15;98(4) :1116-21．
44． Sarzotti M et al.T cell repertoire development in humans with SCID after nonablative allogeneicmarrow transplantation.J Immunol.2003 Mar 1;170(5) :2711-8．
45． Accurate SNP Typing by Real-Time PCR Afonina I et al.Proc Natl Acad Sci USA1996,93:3199-312Kutyavin
46． Spatially localized generation of nucleotid sequence-specific DNA damage Afonina I et al.Nucleic Acids Research,1997,25:2657-2660
47． SNP Genotyping With Fluorescence Polarization Detection Kutyavin IV et al.Nucleic Acids Research,1997,25:3718-3723
48． Peptide Nucleic Acid-Assisted Topological Labeling of Duplex DNA Kumar S et al.Nucleic Acids Research,1998,26:831-838
49． USING MODIFIED BASES TO OPTIMIZE HYBRIDIZATION Boger DL et al.J org chem.,1987,52:1521-1530
50． Quantitative Reverse Transcription Polymerase ChainReaction to Study mRNA Decay:Comparisonof Endpoint and Real-Time Methods1 Kutyavin IV et al.Clin Infect Dis,1997:24(Suppll):S121-S130
51． The Nature of Stabilization of the Tandem DNA DuplexpTGGAGCTG ·(PCAGC(Phn-NH-(CH2) 3-NH)pTCCA)Basin g on the UV,CD,andTwo-DimensionalNMRSpectroscopy DataHolland PM et al.Proc Natl Acad Sci USA,1991,88:7276-7280
52． Summary of Thermo-Physical Properties of Sn,And Compounds Lee LG et al.Nucleic Acids Research,1993,21:3761-3766
53． Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology Tyagi S et al.Nat
Biotechnol,1998,16:49-53
54． Domain necessary for DrosophilaELAV nuclear localization:function requires nuclear ELAV Nazerenko IA et al.Nucleic Acids Research,1997,25:2516-2521
55． Quantification of mRNA using real-time RT-PCR:trends and problems S A Bustin Bernard PS et al.Anal Biochem,1998,255:101-107
56． The origin of phonon anharmonicity inMgB2 and related compounds Ririe KM et al.Anal Biochem,1997,244:1-7
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42． Chao NJ et al.Nonmyeloablative regimen preserves "niches" allowing for peripheral expansion ofdonor T-cells.Biol Blood Marrow Transplant.2002;8(5) :249-56．
43． Hochberg EP et al.Quantitation of T-cell neogenesis in vivo after allogeneic bone marrowtransplantation in adults.Blood.2001 Aug 15;98(4) :1116-21．
44． Sarzotti M et al.T cell repertoire development in humans with SCID after nonablative allogeneicmarrow transplantation.J Immunol.2003 Mar 1;170(5) :2711-8．
45． Accurate SNP Typing by Real-Time PCR Afonina I et al.Proc Natl Acad Sci USA1996,93:3199-312Kutyavin
46． Spatially localized generation of nucleotid sequence-specific DNA damage Afonina I et al.Nucleic Acids Research,1997,25:2657-2660
47． SNP Genotyping With Fluorescence Polarization Detection Kutyavin IV et al.Nucleic Acids Research,1997,25:3718-3723
48． Peptide Nucleic Acid-Assisted Topological Labeling of Duplex DNA Kumar S et al.Nucleic Acids Research,1998,26:831-838
49． USING MODIFIED BASES TO OPTIMIZE HYBRIDIZATION Boger DL et al.J org chem.,1987,52:1521-1530
50． Quantitative Reverse Transcription Polymerase ChainReaction to Study mRNA Decay:Comparisonof Endpoint and Real-Time Methods1 Kutyavin IV et al.Clin Infect Dis,1997:24(Suppll):S121-S130
51． The Nature of Stabilization of the Tandem DNA DuplexpTGGAGCTG ·(PCAGC(Phn-NH-(CH2) 3-NH)pTCCA)Basin g on the UV,CD,andTwo-DimensionalNMRSpectroscopy DataHolland PM et al.Proc Natl Acad Sci USA,1991,88:7276-7280
52． Summary of Thermo-Physical Properties of Sn,And Compounds Lee LG et al.Nucleic Acids Research,1993,21:3761-3766
53． Applications of Differential-Display Reverse Transcription-PCR to Molecular Pathogenesis and Medical Mycology Tyagi S et al.Nat
Biotechnol,1998,16:49-53
54． Domain necessary for DrosophilaELAV nuclear localization:function requires nuclear ELAV Nazerenko IA et al.Nucleic Acids Research,1997,25:2516-2521
55． Quantification of mRNA using real-time RT-PCR:trends and problems S A Bustin Bernard PS et al.Anal Biochem,1998,255:101-107
56． The origin of phonon anharmonicity inMgB2 and related compounds Ririe KM et al.Anal Biochem,1997,244:1-7