作者：熊辉霞 陈宝安作者单位：（1．青海大学附属医院；2．东南大学附属中大医院） 8 l& q/ }* c5 B8 D 6 F* B7 N) R- h" s' F" d2 X7 ]6 n % X* a7 m9 m; v8 d6 C 2 p1 @5 z N- s" g& |, d& `. K* w ; y, Y& c$ r. V& L
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* o( X- e( w. Y- v9 t* G , M. n& S5 O& M0 k: }0 j. w 【摘要】 目的 应用STR-PCR半定量嵌合体检测方法监测非清髓异基因造血干细胞移植（non-myeloablative allogeneic stem cell tansplantation，NAST）后嵌合体。方法 采用短串联重复序列-聚合酶链反应（STR-PCR）结合聚丙烯酰胺凝胶电泳和银染色以及凝胶成像分析技术对28例接受NAST的患者进行嵌合体监测。结果 27例患者形成造血细胞嵌合体，完全嵌合（complete chimerism，CC）22例，混合嵌合（mixed chimerism，MC）5例；其中低比例嵌合（low level of mixed chimerism，LMC）3例，高比例嵌合（high level of mixed chimerism，HMC）2例。稳定植入的患者 7天即达HMC，3例患者复发前检测到供体细胞嵌合率（donor cell，DC）下降，1例在CC状态下复发，1例LMC发生早期移植排斥，长期存活者均保持稳定CC。嵌合体监测下早期接受临床干预治疗的MC患者转化为CC并延长了无病生存期。结论 STR-PCR是一种简单、敏感、经济的监测非清髓移植后嵌合体的方法。 熊辉霞（1977～），女，汉族，青海籍，主治医师，硕士 7 E2 _- z! k3 I8 p 【关键词】短串联重复序列 非清髓造血干细胞移植 造血嵌合体 . c4 F3 d& i' q0 i( G3 c( c SEMI-QUANTITATIVE ANALYSIS OF CHIMERISM AFTER. f6 t7 b1 I* b9 _/ q4 E; f
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NON-MYELOABLATIVE ALLOGENEIC STEM CELL8 ~# W$ x; o( H
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TRANSPLANTATION BY STR-PCR ; `0 h% m4 k7 J # K: ^9 ^! S8 [3 U$ AXiong Huixia1，Chen Baoan2 8 L6 n& i- |& H/ s6 x " Z Z0 p* i* w（1. The Affiliated Hospital of Qinghai University; 6 N* K" O4 o. Y; @! P6 j# g1 C1 |+ D; A; b% w, j/ S
2. The Affiliated Zhongda Hospital of Southeast University）# i) g* [$ a' @4 `4 e/ p
. g- {/ N' E9 [AbstractObjectiveTo investigate the value of semi-quantitative analysis of chimerism after non-myeloablative allogeneic stem cell tansplantation(NAST) by short tandem repeat-polymerase chain reaction(STR-PCR).Methods 28 patients received NAST were evaluated. Semi-quantitative assessment of hematopoietic chimerism was performed by STR-PCR, polyacrylamide gels, silver staining and detected by Image Analysis System. Results 27 cases formed hematopoietic chimerism, 22 cases formed complete chimerism(CC) and 5 cases were of mixed chimerism(MC), 3 cases formed low level of mixed chimerism(LMC) and 2 cases were high level of mixed chimerism(HMC). Patients with stable engraftments showed HMC on day 7. Before the time of relapse, STR-PCR indicated decrease of donor cell(DC) in 3 patients. One patient relapsed in CC status and another patient with LMC rejected grafts earlier. Patients with persistent CC were associated with a longer disease-free time. Patients received an early intervention of clinical treatment transferred from MC to CC or got a longer disease-free time. ConclusionSTR-PCR provide a simple, sensitive and economic approach for monitoring of chimerism after transplantation. 7 W7 E0 ?6 R5 ~ i, \" `+ m& }# E5 W# a
Key wordsshort tandem repeatsNon-myeloablative stem cell transplantationNematopoietic chimerism' b p) j; |' N$ g- B }: [9 k! H* l
7 D9 s* a" L' r1 x6 R Z' ^非清髓异基因造血干细胞移植（NAST）由于减量的放、化疗预处理加上强效的免疫抑制，移植后多形成供、受体细胞不同比例的混合嵌合体，并呈动态变化［1, 2］。嵌合体监测对分析植入状态，判断疾病复发及指导移植后续治疗具有重要意义。短串联重复序列（STR）是目前检测嵌合体最常用的遗传标记，我们采用STR-PCR半定量方法对28例接受NAST的患者进行了嵌合体的检测，现报告如下。 % p4 _, a0 h( n f $ e9 A- Y+ o* L v$ z5 g1资料与方法8 a4 `5 [; j2 r1 }) ?& v* x9 a9 s
( i$ S: ^9 f" S7 g, ?+ u, V我们的研究发现植入成功的患者在 14天多数达CC，在之后的随访中，凡长期存活的病例都能保持稳定CC，5例MC均不稳定，有DC进一步下降导致移植排斥和复发的倾向。此结果提示早期供体植入率是影响NAST能否发生移植排斥的重要因素， 100天内更应加强嵌合体监测并及时采取预防措施。STR-PCR技术可在严重粒细胞缺乏阶段实施，嵌合体检测虽然不能直接检测出残留的白血病细胞，但由于受体细胞比例增加与移植排斥和复发的危险性高度相关［8,9］，因此可通过定量检测供体细胞嵌合率，来判断患者体内残留的受体细胞水平。DLI是移植后临床干预治疗的重要组成部分，其诱导的移植物抗白血病效应（graft versus leukemia effect，GVL）在肿瘤细胞负荷低时易发挥根治性作用，把握DLI的时机和数量是取得较好疗效的关键。STR-PCR通过定量检测DC的消长趋势，可以确定实施DLI的时机和强度，判断DLI的疗效，尤其适用于无特异性肿瘤标志基因异常的恶性血液病患者［10］。+ |6 X" O$ f g+ c# Y7 a
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因此，我们认为，在移植后嵌合体的长期监测中，STR-PCR结合聚丙烯酰胺凝胶电泳和银染色以及凝胶成像分析半定量检测嵌合体的方法是一种简单、敏感、经济的监测方法。 % i; U M0 j+ }9 S& p$ j# J 【参考文献】 " i% r1 g; ?& d2 P8 X8 {( s% ^& e A［1］ Le Blanc K, Remberger M, Uzunel M, et al. A comparision of nonmyeloablative and reduced-intensity conditioning for allogeneic stem-cell transplantation［J］.Transplantation， 2004,78(7): 1014-10205 e5 L3 w! Q9 Q; R- u1 _* w. S
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［2］ Michalfet AS, Furst S, Le QH. et al. Impact of chimerism analysis and kinetics on allogeneic haematopoietic stem cell transplantation outcome after conventional and reduced-intensity conditioning regimens［J］. Br J Haematol, 2005, 128(5): 676-689) t T+ k+ J! M* f/ A+ e# E
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% t U' p, a' h, Z; @ b5 n- ? & v' K. B k, `9 P8 m1 r2 O ［3］ 杜志淳, 李莉, 林源, 等.“中国罪犯DNA数据库”模式库——13个STR位点基因在中国人群中的分布［J］. 法医学杂志, 2000, 16(1): 1-5 * M2 \6 E) ~7 s' q w1 q $ `& R1 H4 b4 M8 } {5 ~9 D / U: [/ o' ], s . w8 u; C0 D+ ?/ v7 G1 }. q, m ［4］ 俞卫东, 闵涯邻, 张斌, 等. 江苏地区汉族人群15个STR基因座遗传频率调查［J］. 中国法医学杂志, 2003, 18(4): 245-246 ! b, r6 z% f7 @- L/ F' x ! L# {3 n- ~/ ^9 D: g! m ! Z3 N3 l5 p9 U. P2 U) L, r7 P# a0 v# M- M4 W
［5］ Thiede C, Florek M, Bornha M, et al. Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection［J］. Bone Marrow Transplantation, 1999, (23): 1055-1060/ O& Q8 l6 z( y% F
) @, Y9 T# q: L% T * _" N/ j0 o& ]% z# N- I3 X* n # L' b$ Y+ Y, R* b% \7 {: H ［6］ Nollet F, Billet J, Selleslag D, et al. Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing［J］. Bone Marrow Transplant, 2001, 28: 511-518 , n2 {+ @$ m/ h: M: B" t m; k( c# ^" `, s5 v+ Z1 C9 A$ n
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［7］ Elmaagacli AH. Real-time PCR for monitoring minimal residual disease and chimerism in patients after allogeneic transplantation. Int J Hematol, 2002, 76(Suppl 2): 204-205( r; p9 t) T8 C6 h1 M
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+ e6 ?% K. f; \7 p ［8］ Chalandon Y, Vischer S, Helg C, et al. Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Geneva experience［J］. Leukemia, 2003, 17: 228 - 231" j2 p% S' b4 x6 i" ~& o. p
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［10］ Bader P, Kreyenberg H, Hoelle W, et al. Increasing mixed chimerism is an important prognostic factor for unfarorable outcome in children with acute lymphoblastic leukemia after allogeneic stem cell transplantation: possible role for pre-emptive immunotherapy［J］. J Clin Oncol, 2004, 22: 1696-1705作者: linfeifei 时间: 2009-4-5 13:34