间充质干细胞治疗糖皮质激素耐药性慢性移植物抗宿主病临床疗效观察

Objective To assess whether treatment with mesenchymal stem cells (MSCs) is an effective adjunct therapy for refractory extensive chronic graft-versus-host disease (GVHD) resistant to conventional therapy. Methods 12 patients with steroid-resistant extensive chronic GVHD were treated with MSCs. One patient received one dose, 10 received two doses, and the remaining three doses. The MSCs were obtained from HI,A-identical sibling donors (n = 14), haploidentical donors (n = 2), unrelated mismatched donor (n = 1) and third-party HLA-mismatched donors (n = 7). Of the 11 patients treated with multiple infusions, 5 received cells derived from two donors. The median first dose of MSCs was 1.0 (0. 4-2. 1) × 106/kg , the median second dose was 1.2(0. 8-1.9) × 106/kg , and the third dose in one patient was 1.1 × 106/kg. Meanwhile the proportion of CD3+ ,CD4+,CD8+ ,CD19+,CD4+ CD25+ ,FOXP3+,FOXP3+CD4+ and FOXP3+ CD25+ was determined with double fluorescent-labeled antibodies and flow cytometry before and 4 weeks after the MSCs infusion. Results No patients had side-effects during or immediately after the infusions of MSCs. After a treatment course of one to three doses, 3 patients had complete response(CR), 6 showed partial response(PR) and 3 did not respond; the total effective rate was 75% (9/12). Complete resolution was seen in the involvement of skin (3/12), lung (1/3), joints (1/5), liver (3/10), oralcavity (4/12) and eye (2/7). Response rate was not related to donor HLA-match. 3 CR patients discontinued all of the immunosuppressive agents without relapse 100 to 292 days after the MSC infusion and 6 PR patients taped all immunosuppressive agents after 60 to 79 days. Mean follow-up period was 1152(795-1914) days, leukemia free survival rate was 91.7% (11/12) and the overall survival rate was 75% (9/12). The ratio of CD4/CD8 and the proportion of regulatory T cells were significantly higher than that before MSCs treatment. Conclusion Third-party MSCs were as effective as HLA-identical or haploidentical cells. This finding has practical implications and suggests that third-party cells can be prepared and stored frozen to be used for steroid-resistant extensive chronic GVHD therapy. It is concluded that MSCs may prevent the lethal cGVHD after allogeneic hematopoietic stem cell transplantation and raise the survival rate by increasing the ratio of CD4/CD8 and proportion of regulatory T cells in vivo.     

儿童急性淋巴细胞白血病分型及其不同治疗方案与预后的临床研究

目的：研究儿童急性淋巴细胞白血病(ALL)的细胞形态学、免疫表型分型及不同方案治疗与预后的相关性。方法：采用骨髓涂片染色进行细胞形态学检查，采用单克隆抗体(McAb)和流式细胞仪(FCM)进行免疫表型检测。结果：103例儿童ALL患者中，67例(65．05％)为L1型，33例(32．04％)为L2型，无L2型，3例(4．91％)为其他。行免疫分型的87例中，58例(66．67％)为B系表达，12例(13．79％)为T系表达，10例(11．49％)为B系、髓系混合表达，3例(3．45％)为T系、B系混合表达，1例(1．15％)为T系、髓系混合表达，1例(1．15％)为B系、T系、髓系混合表达，2例(2．30％)为髓系表达。采用XH-88方案治疗25例，缓解卒92％，复发率52．17％，死亡率43．48％；协作组方案治疗41例，缓解率92．68％，复发率36．84％，死亡率55．26％；SUM-99方案治疗29例，缓解率93．10％，复发率18．52％，死亡率14．81％。结论：结合免疫分型与细胞形态学分型，将儿童ALL患者分为标危(SR)、中危(IR)和高危(HR)三组，按型采用不同的治疗方案对提高儿童ALL缓解率和降低其复发率有重要意义。     

68例儿童重型再生障碍性贫血强化免疫抑制治疗的疗效分析

目的探讨强化免疫抑制治疗（immunosuppressive therapy,IST）与单用环孢菌素A（cyclosporine A,CSA）治疗儿童重型再生障碍性贫血（severe aplastic anemia,SAA）间疗效差异,并研究强化免疫抑制治疗前,患儿对粒细胞集落刺激因子（granuloeyte colony stimulating factor,G-CSF）反应程度与疗效的关系。方法2000年1月至2007年11月在中山大学附属第二医院儿科确诊的重型再生障碍性贫血患儿为68例,其中55例采用强化免疫抑制治疗（IST组）,13例因经济原因采取单用环孢菌素A治疗（CSA组）,比较两组疗效差异（本研究遵循的程序符合中山大学附属第二医院人体试验委员会所制定的伦理学标准,得到该委员会批准,分组征得受试对象的知情同意,并与试验患儿监护人签署临床研究知情同意书）。24例重型再生障碍性贫血患儿在强化免疫抑制治疗前做粒细胞集落刺激因子试验性治疗,根据对粒细胞集落刺激因子的反应程度,将IST组分为G-CSF反应IST亚组（n=13）与C-CSF无反应IST亚组（n=11）,比较两亚组强化免疫抑制治疗后6个月及长期疗效差异。结果IST组和CSA组患儿治疗的平均显效时间分别为3．4个月（1～13个月）和4．5个月（1～15个月）。IST组和CSA组患儿患儿治疗后第1、第3、第6、第9及第12个月末的有效率分别为70．9％,58．2％,79．5％,87．2％和89．7％VS．46．2％,38．5％,46．2％,61．5％和61．5％。随访1年以上,IST组有效率为89．7％（35／39）,CSA组患儿有效率为61．5％（8／13）,两组比较,差异有显著意义（Х^2=5．419,P〈0．05）。G-CSF反应IST亚组及其无反应IST亚组患儿治疗6个月后,有效率分别为76．9％（10／13）和45．6％（5／11）,两亚组比较,差异有显著意义（P〈0．05）。G—CSF反应IST亚组患儿总有效率（92．3％,12／13）较无反应IST亚组（54．5％,6／11）高,且差异有显著意义（P〈0．05）。结论强化免疫抑制治疗重型再生障碍性贫血的疗效明显优于单用环孢菌素A。强化免疫抑制治疗前,对粒细胞集落刺激因子有反应的患儿,强化免疫抑制治疗的预后相对较好。     

间充质干细胞治疗糖皮质激素耐药性慢性移植物抗宿主病临床疗效观察

Objective To assess whether treatment with mesenchymal stem cells (MSCs) is an effective adjunct therapy for refractory extensive chronic graft-versus-host disease (GVHD) resistant to conventional therapy. Methods 12 patients with steroid-resistant extensive chronic GVHD were treated with MSCs. One patient received one dose, 10 received two doses, and the remaining three doses. The MSCs were obtained from HI,A-identical sibling donors (n = 14), haploidentical donors (n = 2), unrelated mismatched donor (n = 1) and third-party HLA-mismatched donors (n = 7). Of the 11 patients treated with multiple infusions, 5 received cells derived from two donors. The median first dose of MSCs was 1.0 (0. 4-2. 1) × 106/kg , the median second dose was 1.2(0. 8-1.9) × 106/kg , and the third dose in one patient was 1.1 × 106/kg. Meanwhile the proportion of CD3+ ,CD4+,CD8+ ,CD19+,CD4+ CD25+ ,FOXP3+,FOXP3+CD4+ and FOXP3+ CD25+ was determined with double fluorescent-labeled antibodies and flow cytometry before and 4 weeks after the MSCs infusion. Results No patients had side-effects during or immediately after the infusions of MSCs. After a treatment course of one to three doses, 3 patients had complete response(CR), 6 showed partial response(PR) and 3 did not respond; the total effective rate was 75% (9/12). Complete resolution was seen in the involvement of skin (3/12), lung (1/3), joints (1/5), liver (3/10), oralcavity (4/12) and eye (2/7). Response rate was not related to donor HLA-match. 3 CR patients discontinued all of the immunosuppressive agents without relapse 100 to 292 days after the MSC infusion and 6 PR patients taped all immunosuppressive agents after 60 to 79 days. Mean follow-up period was 1152(795-1914) days, leukemia free survival rate was 91.7% (11/12) and the overall survival rate was 75% (9/12). The ratio of CD4/CD8 and the proportion of regulatory T cells were significantly higher than that before MSCs treatment. Conclusion Third-party MSCs were as effective as HLA-identical or haploidentical cells. This finding has practical implications and suggests that third-party cells can be prepared and stored frozen to be used for steroid-resistant extensive chronic GVHD therapy. It is concluded that MSCs may prevent the lethal cGVHD after allogeneic hematopoietic stem cell transplantation and raise the survival rate by increasing the ratio of CD4/CD8 and proportion of regulatory T cells in vivo.     

Transwell培养体系中人AGM区基质细胞支持脐血CD34+细胞造血

[目的]探讨Transwell系统中人胚主动脉-性腺-中肾(AGM)区基质细胞对脐带血造血干/祖细胞的造血能力长期维持及扩增的作用.[方法]采用免疫磁珠方法分离人脐带血CD34 细胞,接种于底层铺有人AGM区基质细胞的Transwell培养板的Inserts中,非接触共培养7～35 d,每星期取样检测细胞总数,用半固体培养基分析CFU-C及HPP-CFU集落形成数,流式细胞术检测CD34 、CD34 CD38-细胞百分率.[结果]在Transwell中非接触共培养条件下,人AGM区基质细胞培养体系较胚胎躯干基质细胞和无饲养层培养体系对有核细胞总数、CFC和CD34 细胞均具有明显的扩增作用,共培养14 d的CD34 、CD34 CD38-造血干/祖细胞均获得峰值扩增(2.62±0.8和2.15±1.04,P<0.05),而MNC总数和CFC均在21 d获得最大扩增(32.5±4.3和4.2±0.6倍,P<0.05).克隆分析发现CFU-Mix、CFU-GM、BFU-E在共培养4～5星期后均仍然可见.原始祖细胞HPP-CFC在3星期也得到2.23倍的扩增,较空白及hFT对照组均有显著性差异(P<0.05),并在共培养35 d后仍可见HPP-CFU集落形成.[结论]人AGM区基质细胞hAGM-S3/hAGM-S4均具有造血支持作用,在非接触共培养条件下中可长期维持脐血中造血干/祖细胞的多系造血能力和高增殖潜能达21～35 d,对脐血CD34 /CD34 CD38-细胞数也有一定程度的扩增作用.     

含脐血MSCs体系扩增后的脐血重建NOD/SCID小鼠造血的研究

[目的]探讨含人脐血(umbilical cord blood,UCB)来源的间充质干细胞(mesenchymal stem cells,MSCs)联合造血生长因子(hematopoietic growth factors,HGFs)体系扩增后的脐血造血细胞在NOD/SCID小鼠体内植入及重建小鼠造血的能力.[方法]①用含女性胎儿脐血MSCs(UCBMSCs)及HGFs组合的无血清扩增体系体外扩增男性新生儿UCB CD34 细胞.②收获扩增第6天的UCB细胞,经尾静脉移植给亚致死量照射后的雌性NOD/SCID小鼠.③动态观察移植后小鼠生存情况及外周血象的恢复.④移植后8周,流式细胞仪检测存活小鼠骨髓中人CD45 、CD45 CD33 、CD45 CD41 、CD45 CD3 和CD45 CD19 细胞含量;PCR法检测移植小鼠外周血中人Y染色体表达.[结果]①非扩增组小鼠存活率为60%,扩增组存活率为90%.②动态观察发现:扩增组白细胞和血红蛋白于移植后第20天恢复,PLT于移植后第40天恢复,明显短于非扩增组.③移植后8周,两组小鼠外周血中人Y染色体检测均为阳性,扩增组和非扩增组小鼠骨髓中人CD45 细胞的含量分别为18.5%±8.3%和16.5%±5.7%,差异无统计学意义(P＞0.05).④移植后8周,扩增组小鼠体内CD45 CD33 和CD45 CD41a 细胞的百分率均高于非扩增组,但CD45 CD3 、CD45 CD19 细胞百分比均低于非扩增组.[结论]①含人UCBMSCs体系扩增后的UCB细胞具有NOD/SCID小鼠体内植入并重建小鼠造血的能力,但不具有提高小鼠体内植入率的作用.②扩增后的UCB移植可显著促进移植后小鼠造血恢复,提高小鼠存活率.③扩增后的UCB移植主要促进小鼠髓系及巨核系的植入,但对淋巴系的植入有抑制作用.     

同种异基因小鼠骨髓腔内脐血移植模型的建立及其对造血干细胞植入的影响

[目的]建立同种异基因小鼠骨髓腔内脐血移植模型,研究该技术对造血干细胞(HSC)归巢、植入率、移植后免疫造血重建、移植物抗宿主病(GVHD)等方面的影响.[方法]用C57BL/6胎鼠及新生鼠外周血(FNPB)作供体,以单侧胫骨骨髓腔内注射(IBMI)和尾静脉注射(Ⅳ)两种途径移植经亚致死量60Coγ射线辐照预处理的BALB/c鼠.200只受鼠随机分为4组:骨髓腔内注射组(IBM组);尾静脉注射组(Ⅳ组);放疗对照组;正常对照组,每组50只.用组织冰冻切片和流式细胞术动态了解CFSE标记FNPB在受体内的分布变化,并观察移植后受鼠存活状况、植入水平、造血与免疫功能恢复及GVHD情况.[结果]①荧光标记FNPB体内动力学显示直至输注后72 h,供体FNPB经IBMI后主要积聚于注射侧骨髓腔内,肺部滞留很少.而Ⅳ组及IBM组非注射侧骨髓中的FNPB细胞数显著少于IBM组注射侧,Ⅳ组非造血组织器官如肺部有明显供体细胞滞留.②同种异基因小鼠CBT模型中IBM组造血、免疫功能的恢复明显快于Ⅳ组,无GVHD,移植后90 d存活率达到90%,注射侧胫骨植入水平(29.53±6.64)%,较Ⅳ组有显著改善.③IBM输注侧骨髓中供体HSCs植入水平及造血重建速度明显优于非注射侧骨髓.[结论]成功建立同种异基因小鼠骨髓腔内脐血移植模型,并证实IBMI途径对促进HSCs植入,造血免疫功能重建,提高UCBT效果方面优于Ⅳ途径,多部位IBMI可能更有利于HSC归巢骨髓.     

CD158分子表达在他克莫司、霉酚酸酯联合甲基强的松龙治疗顽固性慢性移植物抗宿主病中的追踪观察

目的：探讨慢性移植物抗宿主病（graft-versus-host disease，GVHD）症状期及缓解期CD158分子的表达变化。方法： 观察异基因造血干细胞移植后发生广泛性慢性GVHD的儿童患者，应用他克莫司（FK506），霉酚酸酯（MMF）和甲基强的松龙（MP）联合治疗的毒副作用和疗效；同时采用流式细胞仪检测外周血中CD158在NK细胞和T细胞的表达，比较治疗前后CD158的表达。结果： 移植前、慢性GVHD症状期和治疗后CD4+CD158a+和CD4+CD158b+表达水平极其低下，但无明显差异。5例急性GVHD患者CD3+CD158b+和CD3+CD8+CD158b+的表达水平分别为4.97%±2.36%和4.58%±2.90%，与移植前相比差异显著(P＜0.05)，CD3-CD16+CD158b+的表达也明显高于移植前(P＜0.05)。6例慢性GVHD患者症状期，CD3+CD158b+和CD3+CD8+CD158b+的表达均高于移植前的水平(P＜0.05)；在缓解期，CD3+CD158b+、CD3+CD8+CD158b+和CD3-CD16+CD158b+的表达水平逐渐下降，与慢性GVHD症状期相比差异显著(P＜0.05)，与移植前无明显差异。移植后6-12个月CD3-CD16+CD158b+ 表达水平与移植前无明显差异。结论： CD158b分子在T细胞表达水平增加与慢性GVHD的发生有关，FK506，MMF和MP联合有效治疗慢性GVHD的作用机制之一可能是下调T细胞CD158b+的表达，从而抑制cGVHD。     

雷公藤多甙片治疗小儿哮喘疗效及其免疫作用机理的研究

作者对37例小儿哮喘病患者经用雷公藤多甙片(Tabellon Multiglycosidorum Triptergii)治疗12周后,随访8个月,其中有效者33例,总有效率为89.18%,各类型病者间有效率无显著差异(P>0.05);对其中27例病儿服药前后外周血T细胞亚群及抑制性细胞活性进行检测,结果显示:病儿CD_?~+亚群从25.36±5.16%上升为28.96±3.03,(P<0.05),CD_3~+、CD_4~+亚群改变不大(P>0.05),而抑制率(SR)从-20.86±39.10上升为11.93±30.83(P<0.001),同时发现,抑制率的恢复与临床疗效间有一定关系。由此,作者认为,该药对小儿哮喘的治疗机理可能与机体免疫抑制功能恢复有关。