【作者】 邵宇；

【导师】 王健民；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 目的及意义异基因造血干细胞移植(allo-SCT)是目前能够根治多种造血系统良恶性疾病及部分实体瘤的重要及有效的手段之一。然而,移植后的并发症复发排斥及移植物抗宿主病(GVHD)都极大地影响了移植的临床治疗效果。有研究发现精确动态监测移植后患者的供者细胞嵌合率(DC)可以早期预测GVHD或排斥复发,所以移植后检测DC对于判断移植物状态及决定下一步治疗方案非常重要。嵌合率的检测经典方法有：(1)血型；(2)红细胞同工酶；(3)性染色体核型分析；(4)HLA抗原；(5)限制性长度多态性；(6)可变串联重复序列VNTR；(7)微卫星重复序列STR等。大部分病例可用上述方法得到客观的植入证明,但都存在其固有缺点。本研究的第一部分通过实时定量PCR(RQ-PCR)技术对供受者差异单核苷酸多态性(SNP)位点进行检测,建立了一种目前国内未曾报道的定量检测嵌合体的方法,以达到经济、简便、精确的目的；第二部分重点分析了移植后早期(+7d、+14d)CD4+T细胞、自然杀伤(NK)细胞以及T调节细胞(Treg cell)供者嵌合率与临床结局关系,尤其是+7d嵌合率,希望在患者达到完全嵌合(CC)或在出现临床早期事件之前找到一些预测指标,来指导预后、提前干预。第一部分SNP-PCR定量检测异基因移植后嵌合率方法的建立方法1.收集2007年4月至2009年12月长海医院65例行异基因移植的移植前供受者及移植后患者+7d、+14d、+21d、+30d、+60d、+90d、+6m、+1年、+2年等的EDTA抗凝外周血或骨髓标本共650份；2.按试剂盒盐析法或采用DNAzol或Chelex-100试剂提取基因组DNA,分光光度计测取DNA纯度,-20℃冻存备用；3.选取了9条不同染色体上共18个SNP位点作为备选差异SNP位点,通过定量PCR筛选出供受差异SNP位点；4.RQ-PCR扩增筛选出的供受差异SNP位点,基因GAPDH校正误差,5个浓度梯度GAPDH质粒建立标准曲线,最后通过公式计算出供者嵌合率(DC)；5.通过对已知嵌合比例的模拟嵌合标本进行嵌合率检测,以及同时进行XY-FISH、STR-PCR及特殊融合基因检测来验证方法的准确性及敏感度；6.统计学处理：利用SPSS11.0软件进行统计学分析,数据以均数±标准差表示,相关性检验用直线回归分析,批间差及批内差采用单样本t检验,检验水准α=0.05。结果1.标准品扩增的17次标准曲线,平均斜率为-3.39(-3.22～3.65),平均截距为39.97(37.33～43.19),相关系数均大于0.995, RQ-PCR反应体系的扩增效率接近理想水平,实验结果可信；批间差为1.1%,批内差为0.50%,均在可控范围；2.内参GAPDH及各SNP位点所构建的质粒扩增效率基本一致,所以最后仅利用内参一条标准曲线来计算两者的拷贝数；3.95.4%移植前供受者经定量PCR反应后均能找到相互差异的位点。其中阳性率较高的位点有S2、S7b、S10b、Sgst等；4.与模拟混合嵌合相关系数在0.99以上,可重复敏感度可达0.01%,与FISH、STR-PCR结果相比,差异均没有统计学意义,并且与特定白血病融合基因结果比较,供者型100%CC标本,融合基因结果均为0%,相反MC标本,融合基因均为阳性,且同一患者供者细胞的比例越少,融合基因的表达率越高。第二部分异基因移植后早期(+7d、+14d)免疫细胞供者嵌合率的预后意义方法1.收集的标本同前,收集此65名移植患者的一般信息,包括性别、年龄、疾病种类、疾病状态、移植前化疗次数、移植物来源、HLA配型、预处理强度、血型、外周血植入速度、移植后并发症、结局、存活时间；提取基因组DNA(方法同第一部分),-20℃冻存备用；2.采用SNP-PCR方法(同第一部分)对650份标本进行嵌合率检测,分析+7d、+14d嵌合率与移植并发症及结局的关系；3.收集2008年7月至2009年9月长海医院32例异基因移植后+14d、+30d、+60d、+90d、+6m、+1年等的EDTA抗凝骨髓标本共150份,收集患者的一般信息(具体内容同上)；4.免疫磁珠法分选CD3+、CD56+、CD4+CD25+细胞亚群,提取基因组DNA,-20℃冻存备用；3. SNP-PCR对标本行嵌合率检测(方法同第一部分),分析+14d各细胞亚群的嵌合率与移植并发症及结局的关系；5.采用SPSS11.0统计分析软件包进行数据的统计分析,数据以均数±标准差表示,两组计数资料用Fish精确概率法分析,计量资料采取t检验,生存分析采用Kaplan-Meier方法,检验水准α=0.05。结果1.无论清髓性还是减低剂量移植,患者均能很好耐受,在65例患者中,2例未植入,2例移植物被排斥,余均顺利植入；2.中性粒细胞平均恢复时间(NRT)以及血小板平均恢复时间(PRT)分别为14天及16天；NRT(PRT)>14天患者的+7d平均嵌合率要低于NRT(PRT)<14d患者,分别是69.6%vs 77.3%以及69.5%vs 78.4%,但差异没有统计学意义(P>0.05)；+7天嵌合率<65%组与>65%组的平均白细胞、血红蛋白以及血小板计数均没有显著差别,分别是0.38±0.75 vs 0.33±0.65×109/L(P=0.819),83.6±11.8 vs 77.4±17.8g/l(P=0.207)以及22.9±26.9 vs 25.5±37.6x 109/L(]P=0.801),+7d嵌合率水平与外周血象的高低无直接联系；3.在65例患者中,13例复发,复发患者的+7d及+14d平均嵌合率均明显低于非复发患者,分别为51.86%vs 83.02%及82.89%vs 95.37%(P<0.01)；共24例患者发生GVHD,包括14例aGVHD及13例cGVHD(10例局限型和3例广泛型),ⅡtoⅣ级aGVHD共有8例；4.随着+7d嵌合率逐渐增加,复发率逐渐降低(P=0.0001)、GVHD发生率逐渐升高(P=0.001),髓外复发差异不明显；+7d嵌合率<65%组的复发或排斥率显著高于>65%组,为68.8%vs 8.7%(P=0.0001)；+7d嵌合率>85%组GVHD(包括总GVHD及cGVHD)的发生率明显高于<85%组,为46.43% vs 9.52%(P<0.01), aGVHD在两组之间的差异无统计学意义(P=0.091)；5.随着+14d嵌合率水平的逐渐增加,复发率亦逐渐降低(P=0.002),而GVHD发生率上升趋势不明显(P>0.05)；但进一步将嵌合率按85%及95%分组后,>85%组复发率显著降低(P<0.01),>95%组总GVHD及cGVHD的发生率明显升高(P<0.01)；6.共有21名患者在随访的两年半中死亡,24个月总体生存率(OS)为56%,+7d嵌合率在65-85%的这组患者OS明显高于<65%及>85%组,分别是82.6%vs.47.0%(P=0.04),我们将其定义为+7d的“最佳生存区间”。+14d嵌合率85-95%的区间并没有得到同样的结论(P>0.05)。7.+7d嵌合率在65-85%之间与区间之外的患者相比,疾病类型、移植前化疗次数、疾病状态、移植物类型、年龄、HLA配型、血型、干细胞来源均没有显著差别(P>0.05)。8.32例患者T、NK细胞亚群均分选成功,纯度在75-95%之间；在32例拟分选CD4+CD25+细胞亚群的患者中,2例无差异位点,2例未采集标本,3例分选失败,最后成功的有25例,成功率89.3%,纯度在70-80%之间；9.+14d T细胞嵌合率<80%易于复发,40%vs 4.5%(P=0.024),但其比例与GVHD的发生尚无统计学联系；+14d NK细胞的嵌合率与复发及GVHD的发生关系并不密切(P>0.05)；10.+14dCD4+CD25+嵌合率的比例与排斥复发及GVHD的关系无统计学意义(P>0.05),但其与+14d CD4+T细胞嵌合率的差值是否大于零与GVHD的发生有密切联系,如果+14dCD4+CD25+嵌合率高于CD4+T细胞,则不易发生GVHD,反之则GVHD发生明显升高GVHD发生率分别是6.7%vs 50%(P=0.014)。结论1.SNP-PCR方法检测嵌合率方便、准确、可靠,95.4%移植前供受者经定量PCR反应后均能找到相互差异的位点,适宜在中国人群推广应用；与模拟混合嵌合相关系数在0.99以上,可重复敏感度可达0.01%,与FISH、STR-PCR结果相比,差异均没有统计学意义,与特殊融合基因结果相符；2.NRT>14天患者的+7d平均嵌合率要低于NRT<14d患者,但差异没有统计学意义,PRT同样如此；+7天嵌合率<65%组与>65%组的平均白细胞、血红蛋白以及血小板计数均没有显著差别；+7d供体嵌合率水平与外周血象的高低无直接联系；3.在较强预处理作用下,移植后+7d平均嵌合率即达到76.36%,+14d达到CC的患者>50%,植入速度明显快于报道的非清髓移植(NST)；供体T细胞植入相对缓慢,明显慢于NK细胞；4.随着+7d及+14d嵌合率增加,复发率逐渐降低、GVHD发生率逐渐升高；+7d嵌合率<65%组复发或排斥率显著高于>65%组；>85%组GVHD发生率明显高于<85%组；将+14d嵌合率按85%及95%分组后,>85%组复发率显著降低,>95%组总GVHD及cGVHD的发生率明显升高；+7d嵌合率<65%及+14d<85%可以作为排斥或复发的预警标志,+7d嵌合率>85%及+14d>95%可以作为GVHD发生的预警标志；5.+14d T细胞嵌合率<80%易于复发(P=0.024),但其比例与GVHD的发生尚无统计学联系；+14d NK细胞的嵌合率与复发及GVHD的发生关系并不密切；6.可以采用磁珠分选的方法分选CD4+CD25+细胞亚群,分选成功率在90%左右,+14d CD4+CD25+嵌合率高于CD4+T细胞,则不易发生GVHD,反之则GVHD发生明显升高,可以作为+14d达到高比例嵌合患者的补充预警标志；7.本组移植患者24个月OS为56%,+7d嵌合率在65-85%的这组患者OS明显高于<65%及>85%组,定义为+7d的“最佳生存区间”,我们认为+7d嵌合率在65-85%之间是指导长期生存的又一重要指标。更多还原

【Abstract】 Allogeneic stem cell transplantation(allo-SCT) remains an important therapeutic modality for many patients with hematological malignancies and some benign hematological disorders and solid tumors, at the same time, it is also one of the most effective curative method. The major obstacles for achieving long term disease-free survival after allo-SCT are the severe complications graft-verse-host-disease(GVHD) and relapse. Several previous works suggest that an accurate quantitative analysis of chimerism kinetics would permit early detection of patients with a high risk of GVHD or those liable to relapse. So Detection of mixed chimerism(MC) is pre-requisite in order to assess the graft status and decide later therapeutic strategy. The classical methods of chimeric detection includes:(1) red cell phenotypes; (2) RBC isozyme;(3) karyotype; (4)HLA antigens; (5) Restriction fragment length polymorphism(RFLP); (6) variable number tandem repeats(VNTR); (7) Short tandem repeats(STR),et al. Most cases can get the proof of implantation using methods above, but they do have their own inherent drawbacks. We established a new chimerism detection method in China based on real-time quantitative PCR(RQ-PCR) detecting single nucleotide polymorphism(SNP) locis in the first part of our study, trying to make it more economical, simple and accurate and applying more useful prognosis markers; the second part we study the relationship between early chimerism(+7d、+14d) of the CD4+T cell、NK cell and Treg cell subtype and the clinical outcomes, especially the chimerism on day 7. We tried to find some new valuable prognosis markers before reaching complete chimerism(CC) and early events after SCT.Part One:the establish of the chimerism detection method based on RQ-PCR detecting SNP locis after allo-SCT.Motheds:1.650 Peripheral blood or/and bone marrow samples from 65 patients were collected from the donor and recipient before SCT and from the recipient at different intervals after SCT(+7d、+14d、+21d、+30d、+60d、+90d、+6m、+1y、+2y).2.Genomic DNA was extracted using a salting-out method or by DNAzol or Chelex-100 resin. The concentration and purity of DNA were measured by optical density at 260nm and 280nm with a spectrophotometer and preservated at -20℃; 3.18 SNP locis from 9 different chromosomes were selected, donor and recipient samples before SCT were then screened for informative SNP loci using RQ-PCR method (Taqman probe).4. Gene glyceraldehyde-3-phosphate-dehydrogenase(GAPDH) was amplified simultaneously as an internal control for normalizing input DNA. A standard amplification curve was generated from serial dilutions of the GAPDH plasmids; SNP-PCR was performed in an ABi 7500 real-time PCR device on the informative SNP loci and the chimerism were analyzed by the formula. 5. Known artificial serial dilutions (100%～0.01%) of host cells diluted in donor cells were quantified to assess the accuracy and sensitivity of the method. And conventional STR-PCR, FISH assays and special fusion genes detected by the RQ-PCR method were used to validate the SNP-PCR results.6. Statistical analysis:SPSS 11.0 package was used and a p value of 0.05 was considered significant. Results were denoted with mean±standardized deviate and linear regression analysis for the related test, single sample t test for the intra-and interassay variability.Results:1. The average slope of the 17 times amplification of the GAPDH plasmid was-3.39 and the average intercept was 39.97, correlation coefficients were all more than 0.995, which was close to the desired level. The intra-and interassay variability was 0.50% and 1.1 %, respectively, which were both in control; 2. The amplification efficiency of GAPDH is almost the same as that of the 18 selected SNP locis, so one standard curve is sufficient for quantification of both the SNP loci and the internal control; 3. Over 95.4% can find informative markers in 65 donor/recipient pairs; The S1b, S2, S7b and Sgst SNP loci were most informative in our cases.4. The linear correlation with artificial mixed chimerism is >0.99 and a sensitivity of 0.01%; these values have proven to be reproducible; Comparisons with STR-PCR and FISH had no statistical significance(P>0.05); the quantitative results of special fusion gene transcripts of complete chimerism samples were negative, and positive in mixed chimerism samples.Part Two:Prognosis values of the immune cell subsets’early chimerism(day 7 and 14) after allo-SCT.Motheds:1. A total of 650 peripheral blood (PB) or/and bone marrow (BM) samples were collected from donors and recipients before SCT and from recipients at different intervals after SCT(+7d、+14d、+21d、+30d、+60d、+90d、+6m、+1y、+2y, et al). General information were collected, including sex, age, disease category, the number of chemotherapy treatments before SCT, disease stage (CR1/CP1 or not) at SCT, stem cell source, HLA typing, type of regime, ABO type, implantation rate of PB cell, complication, outcome and survival time. DNA were extracted with the mothed in the first part; 2. Chimerism of the 650 samples were detected with the SNP-PCR method descriped in the first part and relationships between outcome after allo-SCT and day 7,14 chimerism level were analyzed.3. A total of 150 bone marrow (BM) samples were collected from recipients at different intervals after SCT(+14d、+30d、+60d、+90d、+6m、+1y, et al). General information was collected(which was the same as part one).4. CD3+、CD56+、CD4+CD25+ cell subsets were achieved through MACS sorting, DNA were extracted with the mothed in the first part; 5. Chimerism of the sorted samples were detected with the SNP-PCR method and relationships between outcome after allo-SCT and day 14 chimerism level were analyzed.6. Statistical analysis:SPSS 11.0 package was used and a p value of 0.05 was considered significant. Results were denoted with mean±standardized deviate; Fisher’s exact analysis for count data, t test for measurement data, Kaplan-Meier method for survival analysis.Results:1. Both RIC and standard regimes were well tolerated. Only 2 patients failed to undergo engraftment and 2 rejected their grafts; all the others achieved engraftment successfully.2. The median periods required for recovery of absolute neutrophil(NRT) and platelet count(PRT) were 14 and 16 days, respectively. Patients whose NRT(PRT) were<14d had higher day 7 chimerism than those with NRT(PRT)>14d, but the difference is not statistically significant,77.3% vs 69.6% and 78.4% vs 69.5%(P>0.05). Counts of white blood cells, hemoglobin and platelets in PB on day 7 were similar in patients whose chimerism was less than and greater than 65%, which were 0.38±0.75 vs 0.33±0.65×109/L(P=0.819), 83.6±11.8 vs 77.4±17.8 g/l(P=0.207) and 22.9±26.9 vs 25.5±37.6×109/L (P=0.801) respectively(P>0.05), the level of chimerism on day 7 had no direct relationship with the count of peripheral blood.3. Of the 65 patients in the study,13 experienced leukemia relapse. The media chimerism level on day 7 and day 14 of relapse patients were much lower than non-relapse patients,51.86% vs 83.02% and 82.89% vs 95.37%(P<0.01); 24 developed varying degrees of GVHD,14 aGVHD and 13 cGVHD (10 localized and 3 extensive). Grades II to IV aGVHD were seen in 8 patients; 4. With increasing chimerism on day 7, the probability of developing GVHD increased(P=0.001) and repalse rate decreased (P=0.0001), the incidence of extramedullary relapse was not significantly different; Day 7 chimerism <65% was strongly associated with increased risk of relapse or rejection (68.8% vs 8.7%,P=0.0001) and>85% was distinctly associated with increased risk of both whole GVHD (aGVHD+cGVHD) and cGVHD(46.43% vs 9.52%, P<0.01), no significant difference was found in the incidence of aGVHD(P=0.091); 5. With increasing chimerism on day 14, repalse rate decreased (P=0.002), but the probability of developing GVHD not increased significantly(P>0.05); When we further devided the day 14 chimerism by 85% and 95%, relapse rate obviously increased in the<85% group(P<0.01), whole GVHD and CGVHD appeared more in the>95% patients(P<0.01).6.21 of the 65 patients in the study died during the two and a half years of follow-up, the 24-month probability of OS for the entire group was 56%. OS of the group whose day 7 chimerism were between 65-85%(which we called "optimal survival chimerism level" on day 7) was significantly higher than that of the other group, with the 24-month probabilities of OS 82.6% vs.47.0%(P=0.04). However, similar results were not reached considering the golden survival chimerism space 85-95% for day 14 samples (P>0.05).7. Disease category, the number of chemotherapy treatments before SCT, disease stage (CR1 or not) at SCT, type of donor, age, HLA typing, ABO type, and stem cell source showed no statistically significant differences between patients whose day 7 chimerism was 65-85% and those out of this region.8.32 cases all got successful T, NK cell sorting with the purity of 75-95%; There were 32 cases ready to sorting CD4+CD25+ cell.2 didn’t have the informative SNP loci; we didn’t acquire samples from 2 patients and 3 failed to get successful cell sorting, finally 25 cases got cell sorted with the purity 70-80%; 9. Day 14 T cell chimerism<80% had the tendency to relapse(40% vs 4.5%, P=0.024), but had no significant relationship with developing GVHD(P>0.05), neither for the NK cell.10. Day 14 chimerism of the CD4+CD25+ cell subset had no significant relationship with relapse and GVHD, but the margin between CD4+T and CD4+CD25+ cell do have close relationship, which means if the chimerism of day 14 CD4+CD25+ is higher than CD4+ T cell, the incidence of GVHD reduced, or reverse, the incidence of GVHD was 6.7% vs 50%(P=0.014).Conclusion:1. SNP-PCR method detecting chimerism after SCT is easy, accurate and reliable; Over 95.4% can find informative markers in 65 donor/recipient pairs, this system is suitable for popularization and application in Chinese people; the linear correlation with artificial mixed chimerism is>0.99 and a sensitivity of 0.01% and proved to be reproducible; Comparisons with STR-PCR and FISH had no statistical significance(P>0.05); and the results were consistent with the quantitative results of special fusion gene transcripts in AL patients.2. Patients whose NRT(PRT) were<14d had higher day 7 chimerism than those with NRT(PRT)>14d, but the difference was not statistically significant (P>0.05). Counts of white blood cells, hemoglobin and platelets in PB on day 7 were similar in patients whose chimerism was less than and greater than 65%, respectively(P>0.05), the level of chimerism on day 7 had no direct relationship with the count of peripheral blood;3. Under strong conditioning regimen, the median chimerism level on day 7 could reach 76.36%, and over 50% patients acquired CC on day 14, the implant rate was significant faster than the non-myeloablative stem cell transplantation(NST) reported; the implantation rate of donor T cells was significantly slower than NK cells;4. With increased chimerism on day 7, the probability of developing GVHD increased and repalse rate decreased; Day 7 chimerism<65% was strongly associated with increased risk of relapse or rejection and>85% was distinctly associated with increased risk of both whole GVHD(aGVHD+cGVHD) and cGVHD; When we devided day 14 chimerism by 85% and 95%, relapse rate obviously increased in the<85% group, whole GVHD and cGVHD appeared more in the>95% patients;Day 7 chimerism<65% and day 14 chimerism<85% can be used as rejection or recurrence early-warning markers; Day 7 chimerism>85% and day 14 chimerism>95% can be applied as GVHD early-warning markers;5. Day 14 T cell chimerism<80% had the tendency to relapse(P=0.024), but had no significant relationship with GVHD, neither for the NK cell;6. We can use magnetic beads to sort CD4+CD25+ cell subsets, about 90% achieved successful cell sorting; if the chimerism level of day 14 CD4+CD25+ cell is higher than CD4+ T cell, the incidence of GVHD reduced, or reverse. It can be used as an additional prognostic marker for patients who achieved high level chimerism on day 14;7.24-month probability of OS of patients in our study was 56%. OS of the group whose day 7 chimerism were between 65-85%(which we called "optimal survival chimerism leve"on day 7) was significantly higher than those out of the region. We believe that day 7 chimerism between 65% and 85% is an important indicator for the long-term survival.更多还原