体外阻断CD40-CD40L共刺激途径减轻小鼠异基因骨髓移植中移植物抗宿主病的研究

目的在异基因骨髓移植移植物抗宿主病(GVHD)小鼠模型中,观察抗CD40L单克隆抗体(单抗)体外预处理供鼠T细胞输注,观察其减轻GVHD的作用并探讨其作用机制.方法供鼠(C57BL/6H-2b)脾T细胞作为反应细胞,受鼠(BALB/cH-2d)脾细胞作为刺激细胞,分别加抗CD40L单抗或不加单抗进行混合培养,培养第5天的细胞作为经体外诱导后的脾T淋巴细胞,分别混合供鼠骨髓细胞后移植给接受8.0 Gy全身照射预处理的受鼠.比较受鼠GVHD发生和造血重建.在移植后不同时间点采用流式细胞仪检测受鼠脾细胞中T细胞表型的改变,用ELISA法检测外周血血清中Th1和Th2类细胞因子的水平.结果移植后对照组受鼠均于25 d内死于GVHD,实验组GVHD的发生率为20%,与对照组小鼠相比,存活率和存活时间明显增高和延长(P<0.01);存活的实验组小鼠(8只)第40天时骨髓细胞中H-2Db阳性细胞为(93.54±2.32)%.实验组小鼠CD3+CD4+、CD3+CD8+、CD4+CD25+、CD4+CD69+、CD8+CD25+、CD4+CD40L+和CD8+CD69+T细胞比例明显低于对照组(P<0.05),CD8+CD40L+和CD4+CD45RA+T细胞比例在两组中变化差异无显著性(P>0.05).实验组受鼠血清中细胞因子水平明显低于对照组(P<0.05).结论应用抗CD40L单抗体外孵育的脾T细胞与骨髓细胞混合移植,可明显减少GVHD的发生率,且不影响供鼠造血干细胞的植入,CD4+和CD8+T细胞对异体抗原发生耐受,耐受化T 细胞的活化障碍发生于活化的早期和成熟阶段,同时抑制了Th1和Th2类细胞因子分泌,为抗CD40L单抗应用于临床移植预防GVHD提供了实验依据.     

体外阻断CD137-CD137L途径控制小鼠移植物抗宿主病的研究

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径控制小鼠异基因骨髓移植（allo-BMT）后急性移植物抗宿主病（aGVHD）及其机制。方法供、受鼠的淋巴细胞体外混合培养，分别加入抗CD137L单抗或不加抗CD137L单抗培养后与供鼠骨髓细胞混合移植给受鼠。采用流式细胞术检测移植后受鼠T细胞亚群的变化，RT-PCR法检测细胞因子mRNA表达水平的变化，观察移植后受鼠GVHD的临床及病理改变。结果与未用抗CD137L单抗组相比，应用抗CD137L单抗组CD3^＋CD8^＋T细胞明显降低（P＜0．01）；IFN-γ表达水平明显减低（P＜0．01），IL-10的表达水平明显升高（P＜0．01）。移植后未预防GVHD组（A组）小鼠移植后15d内均死于aGVHD。采用甲氨蝶呤＋环孢素预防GVHD组（B组）小鼠100％发生aGVHD，但临床及病理改变程度较A组轻，平均存活时间[（9．5±2．5）d]较A组[（7．5±1．5）d]略有延长。采用抗CD137L单抗预防GVHD组（C组）受鼠aGVHD的发生率为70％，程度比A、B两组轻，与A、B两组相比生存率明显提高（P＜0．01），平均存活时间[（16．0±2．5）d]明显延长（P＜0．01），30％的小鼠生存时间大于30d。结论抗CD137L单抗体外阻断CD137-CD137L共刺激途径能有效控制小鼠GVHD，可能与影响Ⅰ类和Ⅱ类T细胞因子平衡有关。     

CD4+CD25+Foxp3+T调节细胞与小鼠异基因造血干细胞移植GVHD相关性研究

目的探讨CD4 CD25 foxp3 调节性T细胞与小鼠GVHD发生的相关性。方法取8～10周龄的SPF级CB6F1小鼠(H-2b/d,♀)30只,随机分为正常对照组、同基因移植组和GVHD组,每组10只,正常对照组不接受照射和移植,同基因移植组小鼠照射后接受CB6F1小鼠骨髓细胞(1×107)和脾细胞(3×107),GVHD组小鼠照射后接受C57BL/6(H-2b,♂)小鼠骨髓细胞(1×107)和脾细胞(3×107)。在发生GVHD的时间点检测3组小鼠脾细胞CD4 CD25 T细胞和foxp3 mRNA的表达。结果正常对照组、同基因移植组和GVHD组小鼠脾细胞中CD4 CD25 T细胞的比例分别为(8.47±1.03)%、(15.40±0.80)%和(24.03±0.85)%,均有显著性差异(P<0.01)。GVHD组小鼠脾细胞foxp3 mRNA的表达水平明显低于正常对照组和同基因移植组,有显著性差异(P<0.001)。结论CD4 CD25 foxp3 调节性T细胞与小鼠GVHD发生呈负相关,发生GVHD时小鼠脾细胞中CD4 CD25 foxp3 调节性T细胞明显减低。     

同种异基因Th2细胞移植对GVHD和GVL效应的作用

为探讨同种异基因Th2细胞移植是否可以减低移植物抗宿主病(GVHD)的发生和保留移植物抗白血病(GVL)效应的作用,在体外用rmIL-4,Con A和离子霉素把供鼠(C57BL/6H-2b)T细胞优势化诱导培养为Th2细胞,再把Th2细胞与供鼠骨髓细胞混合移植给红白血病受鼠模型(EL9611H-2d).结果表明未处理的对照组,受鼠平均存活时间为(10.6±1.3)天,10只均死于白血病;环磷酰胺化疗组,小鼠平均存活(18.7±4.2)天,10只小鼠最终均死于白血病;骨髓细胞和脾T淋巴细胞移植组,小鼠平均存活(22.7±7.4)天,9/10只均死于GVHD;骨髓细胞和Th2细胞移植组,3/10只在28天内死于GVHD,其余7/10只无GVHD发生,白血病发病时间明显推迟,平均存活(36.9±10.8)天,在50天时检查有2只鼠无白血病证据.研究证明体外极向化诱导培养的Th2细胞移植可减低GVHD发生的同时保留了抗白血病的能力.     

IL-2和IL-15激活供者自然杀伤细胞在异基因造血干细胞移植应用的实验研究

目的 探讨IL-2和IL-15激活的供者自然杀伤(NK)细胞在异基因造血干细胞移植(allo-HSCT)中减轻移植物抗宿主病(GVHD)发挥移植物抗白血病效应方面的作用.方法 采用免疫磁珠分选小鼠脾NK细胞,采用添加IL-2和IL-15的培养基扩增NK细胞并测定NK细胞杀伤活力.C57BL/6小鼠作为供鼠,BALB/c小鼠作为受鼠,部分小鼠移植前8天静脉接种EL9611白血病细胞.异基因移植小鼠输注骨髓细胞5×106 和脾细胞5 × 106.NK细胞治疗组输注骨髓细胞和脾细胞各5×106 以及激活的NK细胞1 × 107,并且给予腹腔注射IL-2和IL-15.移植后观察GVHD发生、生存期、嵌合度、免疫重建.结果 分选NK细胞纯度为95.7%～97.1%.培养后NK细胞杀伤活力较静息时增加3倍.单纯异基因骨髓细胞输注组小鼠未见GVHD发生,异基因骨髓及脾细胞移植对照组移植后1周起开始出现GVHD表现.实验组小鼠发生GVHD的严重程度明显低于脾细胞输注小鼠(P＜0.05).单纯全身照射预处理小鼠生存期9.5～14.0 d.白血病模型中对照组移植后100 d生存率10%,其余死于白血病;实验组80%生存期超过100 d,实验组生存期明显长于对照组(P＜0.01).实验组小鼠移植后2周外周血NK细胞占4.8%,对照组外周血NK细胞占2.8%,实验组NK细胞恢复早于对照组(P＜0.05).实验组TRBV基因重建比对照组快,而且TRBV家族基因表达数比对照组多,对照组小鼠多见单克隆及寡克隆表达.结论 IL-2和IL-15在体外可以有效促进NK细胞增殖与激活.allo-HSCT时给予激活的供者NK细胞输注以及相关细胞因子处理可以促进免疫重建、减轻GVHD发生、降低白血病复发.     

GVHD靶器官中浸润CD3~+T细胞动态变化与组织损伤关系

目的:探讨T细胞在小鼠异基因造血干细胞移植术后急性移植物抗宿主病(aGVHD)靶器官中的浸润及其变化趋势,并研究其与靶组织病理损伤和a GVHD进展的相关性。方法:以8-10周龄雄性C57BL/6(H-2K~b)小鼠为供鼠,获取骨髓细胞、脾单个核细胞,将10-12周龄雄性BALB/c(H-2K~d)小鼠经7.5 Gy全身照射后作为受鼠进行移植。受鼠随机分为异基因骨髓移植(BMT)组和异基因骨髓移植联合脾细胞输注诱导aGVHD(BMT+T)组。移植后每天观察小鼠状态,动态检测各组小鼠体重变化及临床评分;HE染色观察aGVHD靶器官的病理学改变,并做病理评分;免疫组织化学方法动态检测移植后d 7、14、28、40、47靶器官中CD3+T细胞的数量并做数据统计。结果:与BMT组相比,BMT+T组小鼠a GVHD靶器官肝脏、肺以及各段肠道中浸润T细胞数量自移植后d 7开始升高。移植后d 14、28、40、47 BMT+T组小鼠靶组织中浸润的CD3+T细胞较BMT组明显增高(P0.05)。GVHD小鼠的靶器官病理评分和临床评分均明显升高(P0.05)。肝脏中浸润T细胞数量与病理损伤呈正相关,肠道中浸润CD3+T细胞数量与aGVHD临床病理评分亦呈正相关。结论:CD3+T细胞迁移至aGVHD靶组织介导靶器官的病理损伤,T细胞浸润数量是评价aGVHD严重程度的重要指标。     

供体Treg细胞对小鼠异基因骨髓移植后GVHD和GVL效应的影响

本研究探讨供体CD4+CD25+调节性T细胞(regulatory T cells,Treg cells)输注对异基因骨髓移植(allo-BMT)后小鼠移植物抗宿主病(GVHD)及移植物抗GVL)效应的影响。建立BALB/c→C57BL/6小鼠EL4白血病骨髓移植模型,体外磁珠分离纯化供鼠CD4+CD25+T细胞,在骨髓移植的同时分别予尾静脉输注CD4+CD25+T细胞、CD4+CD25-T细胞。以移植后生存期、GVHD临床评分、组织病理形态等为观察指标并进行组间比较。结果显示:白血病对照组小鼠平均生存时间为(17.9±0.7)天,均存在白血病细胞浸润;移植对照组及效应T细胞组平均生存时间为(23.2±1.6)天和(22.3±1.9)天,肝脏、皮肤和小肠病理切片显示均存在GVHD病理改变;Treg细胞组小鼠平均生存时间为(47.3±6.5)天,70%的受鼠获得长期存活,病理显示无GVHD及白血病细胞浸润,其GVHD评分较移植对照组及效应T细胞组明显降低(p0.05)。结论:在小鼠allo-BMT中联合输注供体CD4+CD25+T细胞可减少及减轻GVHD,并保留GVL效应。     

不同照射剂量建立非清髓性移植物抗宿主病模型小鼠外周血T细胞消长性研究

目的探讨不同照射剂量建立非清髓性移植物抗宿主病(GVHD)模型小鼠外周血T细胞消长性变化,了解GVHD发病中T细胞的作用。方法 50只BALB/C小鼠随机分成5组(10只/组),包括对照组:不作任何处理;8Gy单纯照射(IRD)组:仅接受8Gy全身照射(TBI);7.5 GyIRD组:仅接受7.5 GyTBI;8 GyGVHD组:接受8 GyTBI后24 h移植供鼠(10只C57BL/6)1×107个骨髓细胞和2.5×107个脾细胞;7.5 GyGVHD组:除接受7.5 GyTBI,其他实验条件同8 GyGVHD组。观察2个GVHD组小鼠移植后GVHD临床症状,肝、肺与肠病理变化,嵌合率以及T细胞亚群变化。结果 8与7.5 GyGVHD组小鼠移植后均为供鼠H-2Kb阳性细胞90%。10、17、24 d的GVHD评分,8与7.5 GyGVHD组分别为7.25±0.29 vs 5.63±0.25(P0.01),5.00±0.41 vs 4.00±0.41(P0.05),6.50±0.41 vs 8.00±0.41;GVHD评分组内两两比较,除8 GyGVHD组10与24 d比较有较大变化(P0.05)外,其余变化更明显(P0.01)。CD4+/CD8+比值,8与7.5 GyGVHD组分别为0.42±0.03 vs 0.31±0.03,0.27±0.02 vs 0.19±0.01,0.43±0.03vs 0.32±0.03(P0.01);CD4~+/CD8~+比值组内两两比较,除10与24 d相近外,10较17 d和17较24 d都变化明显(P0.01)。CD4~+CD25~+T比例,8与7.5 GyGVHD组分别为0.028 4±0.009 1 vs 0.020 9±0.004 9(P0.05),0.014 0±0.004 3 vs 0.006 6±0.001 2(P0.05),0.009 4±0.000 8 vs 0.005 1±0.000 7(P0.01);组内两两比较,均为17与24d相近,10较17 d和10较24 d变化明显(P0.01)。结论非清髓性移植物抗宿主病模型小鼠发病过程中CD4+T细胞起更重要的作用;随着小鼠移植后生存时间的延长,其体内的CD4~+CD25~+T细胞呈阶梯式下降,下降幅度与照射剂量呈负相关。     

应用扩增的Treg预防异基因骨髓移植后移植物抗宿主病的实验研究

目的：探讨扩增后的CD4+CD25+调节T细胞(regulatory T cell ,Treg)输注对异基因骨髓移植(allo-BMT)后小鼠移植物抗宿主病(GVHD)的影响。方法：利用免疫磁珠法分选小鼠Treg细胞； 以抗鼠 CD3ε 单抗、抗鼠CD28 单抗 、鼠重组 IL-2及辐射过的Balb/C小鼠脾细胞为共刺激因子，扩增TREG细胞；建立C57BL/6→BALB/c小鼠allo-BMT模型,接受移植小鼠随机分为3组,移植骨髓后6-8小时后分别予尾静脉输注扩增的Treg细胞、CD4+CD25-T细胞和RPMI 1640培养液(空白对照)。以移植后GVHD表现,肝、脾、小肠组织病理形态、生存期为观察指标并进行组间比较。结果：经免疫磁珠法分选可获得高纯度(91.80±2.15)%及具较强活力(97.58±1.23)%的Treg细胞；移植后(四周左右)的扩增后Treg细胞移植组小鼠肝、脾、小肠GVHD病理损害较同期CD4+CD25-T细胞移植组与空白对照组小鼠有一定程度的减轻。3组小鼠移植后平均存活时间分别为（61. 45±27. 88）天、(18.58±12. 39)天和(26. 37±15. 65)天, 扩增后Treg细胞移植组小鼠平均存活时间较CD4+CD25-T细胞、空白对照组小鼠延长(P<0.05)。结论：allo-BMT后输注增殖的Treg细胞可以减轻移植后GVHD程度,延长小鼠生存时间。     

地塞米松联合IL-2对供鼠体内调节性T细胞选择性扩增作用及对急性移植物抗宿主病反应的抑制作用

目的 通过地塞米松(Dex)联合IL-2处理小鼠,建立体内扩增调节性T细胞(Treg细胞)的方法 .观察用该方法 处理的小鼠脾细胞移植后受鼠急性移植物抗宿主病(aGVHD)的发生与转归.方法 使用Dex和IL-2处理雄性C57BL/6N供鼠3 d后提取脾单个核细胞(MNC),用流式细胞术(FCM)分析CD4~+ CD25~+、CD25~+ FOXP3~+细胞的变化.,以以上方法 处理的雄性C57BL/6N小鼠为供者,对雌性BALB/c受鼠进行非清髓异基因淋巴细胞移植.移植后观测受鼠的生存率、生存时间、组织病理学变化,以及用PCR和FCM分析测定受鼠嵌合体等来评价aGVHD的发生.结果 经过Dex和IL-2联合处理,供鼠脾CD4~+ CD25~+ FOXP3~+ Treg细胞数量[(24.2±7.6)%]明显高于对照组[(4.0±0.8)%](P=0.01).Dex联合IL-2组供鼠Treg细胞与效应T细胞(Teff)的比值(0.43±0.15)显著高于对照组(0.14±0.01)(P=0.01).经过Dex联合IL-2处理供鼠后进行异基因淋巴细胞移植,受鼠aGVHD明显减轻,中位生存时间＞60 d,与对照组的中位生存时间12 d相比明显延长(P=0.0045).对照组出现典型的aGVHD表现,移植后2周Dex联合IL-2组和对照组的总体死亡率分别为29.4%和71.4%(P＜0.05).结论 经IL-2和糖皮质激素处理供鼠后进行主要组织相容性抗原复合物完全不相合脾淋巴细胞移植能明显减轻aGVHD,显著延长生存时间,可能与供者体内诱导扩增的Treg细胞增加有关.     

Clinical Science (40)

Longitudinal quantitative measures of the natural course of low back pain. (Ohio State University, Columbus, OH) Spine 2000;25:1950–1956. This prospective study compared traditional self‐report measures of low back pain recovery with quantitative measure of recovery. Sixteen occupational and 16 non‐occupational patients with low back pain were recruited. Recovery was monitored prospectively every 2 weeks for 3 to 6 months, using subjective work status, pain symptoms, activities of daily living, and objective functional performance probability (trunk kinematics). Return to work underestimated the percentage of subjects impaired, as compared with all other outcome measures. Symptoms, activities of daily living, and functional performance probability all showed similar patterns of recovery for 0 to 12 weeks. At 14 weeks, there was a lag in functional performance recovery. Both symptoms and activities of daily living indicated that 80% of the population was recovered, whereas functional performance indicated the figure to be 68%. Conclude that the objective kinematic functional performance measure of recovery quantifies a different aspect of impairment not evaluated by traditional subjective measures. Use of several outcome measures may lead to a better understanding of low back pain recovery or residual impairment, which may minimize the risk of recurrent injury. Comment by Hemmo A. Bosscher, MD. This study evaluates several traditional and not so traditional quantitative measures in the recovery of low back pain. The authors hypothesize that a better understanding of the natural course of low back pain (LBP) recovery may provide the knowledge necessary to prevent high cost recurrent low back injuries. But the relationship between acute low back pain, its recovery, and the subsequent recurrence of LBP is not studied here at all. However the study offers some other interesting information. The traditional outcome measure “back to work” is a very insensitive one. It depends on the job rather than the severity of the LBP. A relatively new functional outcome measure was introduced: the lumbar motion monitor, a device that evaluates trunk kinematics. This measure seems more important in later stages of recovery.     

激发型抗CD40单克隆抗体对CD40+B细胞恶性肿瘤的生长抑制和促凋亡作用

目的　研究CD40 抗原在恶性B淋巴细胞肿瘤中的表达及其CD40 抗原激发所致的生物学效应。方法　将激发型CD40 单克隆抗体 (单抗 ) 5C11加入肿瘤细胞的体外培养体系 ,采用细胞计数、流式细胞仪分析等方法分析单抗 5C11对不同肿瘤细胞株的作用。结果　 5C11能引起CD40 表达强阳性的多发性骨髓瘤 (MM)细胞XG2发生同型聚集 ,抑制其生长并介导细胞凋亡 ;5C11可引起CD40 的恶性B淋巴瘤细胞株Daudi发生同型聚集 ,抑制其生长并使细胞发生G2 M期阻滞 ,但并不介导细胞凋亡。结论　激发型CD40 单抗通过诱导恶性B淋巴瘤细胞的凋亡或使G2 M期细胞阻滞 ,抑制肿瘤细胞的体外生长     

Sensitivity and responsiveness of the health-related quality of life in stroke patients-40 (HRQOLISP-40) scale

Abstract

Purpose: To investigate the sensitivity and responsiveness of the Health-Related Quality of Life in Stroke Patients-40 (HRQOLISP-40) scale in evaluating stroke patients from onset to 12 months. Methods: Fifty-five patients with first-incidence stroke were followed-up for 12 months. The HRQOLISP-40 scale was used to assess health-related quality of life (HRQOL) while stroke severity was assessed with the Stroke Levity Scale. Sensitivity to change was assessed by analyzing changes in the HRQOLISP-40 scores between pairs of months with paired samples t-test. Standardized effect size (SES) and standardized response mean (SRM) were used to express responsiveness. Results: Overall HRQOL and domains in the physical sphere of the HRQOLISP-40 were sensitive to change at different time intervals in the first 12 months post-stroke. Marked responsiveness (SES and SRM >0.7) was demonstrated by the overall scale, and the physical, psycho-emotional and cognitive domains at varying time intervals. For instance, SRM was greater than 0.7 between 1 and 6, 3 and 12, 1 and 9, and 1 and 12 months for both the physical and psycho-emotional domains. Conclusion: The HRQOLISP-40 is a sensitive and responsive stroke-specific quality of life measure that can be used to evaluate the outcome of stroke rehabilitation.

• Implications for Rehabilitation

• Enhancing the health-related quality of life (HRQOL) of stroke survivors can be regarded as the ultimate goal of stroke rehabilitation.

• Sensitive and responsive stroke-specific HRQOL measures are required for use in evaluative studies, and clinical trials and practice.

• The Health-Related Quality of Life in Stroke Patients-40 (HRQOLISP-40) is a sensitive and responsive stroke-specific scale.

     

Peginterferon-alpha2a (40 kDa) (Pegasys) for hepatitis B

Chronic hepatitis B virus is a serious and life threatening disease afflicting 350 million people worldwide, despite the availability of effective vaccines. Thus far, current monotherapy with conventional interferon-alpha, lamivudine and adefovir dipivoxil remains unsatisfactory. In addition, the use of conventional interferon-alpha needs to be administered subcutaneously three-times weekly and is associated with frequent adverse events. Although nucleoside/nucleotide analogs such as lamivudine and adefovir dipivoxil are well tolerated and can normalize serum alanine aminotransaminase rapidly, 1-year therapy with either lamivudine or adefovir dipivoxil results in low hepatitis B e antigen seroconversion rates. In hepatitis B e antigen-negative patients, most would relapse after lamivudine has been discontinued. Peginterferon-alpha2a, an immunomodulatory agent, is a new drug that has just completed Phase III clinical trials for the treatment of both hepatitis B e antigen-positive and -negative chronic hepatitis B virus infection. The advantage of peginterferon-alpha2a in achieving sustained virologic response over nucleoside/nucleotide analogs is particularly obvious in the hepatitis B e antigen-negative group. In both studies, sustained off-treatment response is superior to the use of monotherapy with lamivudine, and concomitant use of lamivudine and pegnterferon-alpha2a does not have advantages over the use of peginterferon-alpha2a alone. These recent data put peginterferon-alpha2a as the antihepatitis B virus therapy of choice, especially in young and motivated patients with chronic hepatitis B virus infection. However, despite the superiority of peginterferon-alpha2a over currently licensed nucleoside/nucleotide analogs, more research needs to be conducted in order to find the most optimal treatment regimen in our fight against chronic hepatitis B virus infection.     

肺泡癌的CT诊断(附40例分析)

目的：收集我院10年来经手术或／和病理证实的细支气管肺泡癌40例,对其CT表现及病理切片进行分析,发现肺泡癌的CT表现多种多样,是肿瘤不同发展阶段的反映。文中以CT表现结合病理所见进行了分析,解释了不同CT表现的病理基础。结合文献,笔者对细支气管肺泡癌的CT诊断与鉴别诊断进行了讨论。