T淋巴细胞清除促进脐血造血细胞的体外扩增

采用抗CD3或抗CD8单克隆抗体的补体细胞毒方法清除脐血单个核细胞 (MNC )中T淋巴细胞 ,CD34免疫亲和柱纯化MNC中CD34 +细胞 ,流式细胞技术 (FACS )分析细胞表面标志。将CD34 +细胞中加入含多种造血生长因子的培养基进行体外液态扩增 ,并观察粒 巨噬集落 (CFU GM )和多向祖细胞集落 (CFU GEMM )形成能力。结果CD3+或CD8+细胞清除组和MNC经CD34免疫亲和柱纯化后 ,CD34 +细胞分别为 5 9 5 2 %、 5 6 70 %和 5 0 72 % ,比未纯化组 (1 0 7% )纯度大幅度提高。抗CD3单抗清除 +CD34纯化组和单纯CD34纯化组经造血生长因子刺激培养第 14天 ,细胞总数分别扩增 110 40倍和 87 0 0倍。抗CD3单抗清除 +CD34纯化组、抗CD8单抗清除 +CD34纯化组和单纯CD34纯化组的CFU GM产率分别为 2 86 5 0± 12 0 2、2 88 5 0± 17 68和 2 19 5 0± 5 3 0 3,前者虽高于后者 ,但差异无显著性。CFU GEMM产率分别为 376 67± 43 2 4、 438 33± 36 73和 311 0 0± 40 11,抗CD3单抗清除 +CD34纯化组无显著差异 ,抗CD8单抗清除 +CD34纯化组显示出显著性作用 (P <0 0 5 )。     

淋巴细胞和巨噬细胞去除对人骨髓造血细胞体外扩增效应的影响

为了探讨淋巴细胞和巨噬细胞去除对人骨髓造血细胞体外扩增效应的影响 ,采用抗CD3、抗CD5 6和抗CD14单克隆抗体介导的补体细胞毒方法 ,去除骨髓单个核细胞 (MNC )中的T淋巴细胞、NK细胞和巨噬细胞 ,流式细胞技术 (FACS )分析细胞表面标志。将经不同单抗处理的骨髓MNC加入含有多种造血生长因子的培养基中进行体外有核细胞扩增 ,并观察多向祖细胞集落 (CFU GEMM )的形成能力。结果显示 ,经抗CD3、抗CD5 6、抗CD14单抗分别处理和用上述三种单抗共同处理的骨髓MNC中相应的靶细胞与MNC对照组相比均有明显的降低。经多种造血生长因子刺激培养 2 0d ,骨髓MNC对照组有核细胞总数扩增了 6 5倍 ,T细胞、NK细胞和巨噬细胞去除组分别扩增了 90倍、 77 2倍和 6 7 4倍 ,而三种细胞共同去除组仅为 2 86倍。骨髓MNC对照组CFU GEMM产率为 16 1 5 0± 12 5 4 ,T细胞去除组CFU GEMM产率为 370 0 0± 5 8 2 2 ,明显高于前者 ,差异显著 (P <0 0 0 5 )。NK细胞去除组为 2 0 5 5 0± 30 73,与MNC对照组相比也有显著差异 (P <0 0 5 )。巨噬细胞去除组为 16 3 2 5± 18 5 4 ,三种细胞共同去除组仅为 4 9 75± 9 98。本文结果提示 ,单纯去除骨髓MNC中的T淋巴细胞或NK细胞可明显促进人骨髓造血细胞的体外扩增效应。     

CD34~+细胞免疫亲合柱的研制

本文建立了利用免疫亲合柱纯化CD34+细胞的方法。将亲合素交联于聚丙烯酰胺凝胶Bio Gel上 ,装于塑料柱制成免疫亲合柱 ;将脐血单个核细胞与生物素化抗CD34单克隆抗体作用后 ,加到该免疫亲合柱上进行纯化 ,得到纯化的CD34+细胞。纯化后CD34+细胞纯度达 48 8%± 18 2 % ,细胞回收率为 5 5 6 %± 14 5 % ,台盼蓝染色法显示活细胞达 98%。利用该方法纯化CD34+细胞具有纯度高、回收率高、细胞活性好等优点 ,是一种方便有效的细胞纯化方法。     

rhTPO对巨核系的定向分化效应

目的 :探讨重组人促血小板生成素 (rhTPO)对外周造血干细胞中巨核系祖细胞的定向诱导分化能力。方法 :经化疗及G CSF动员后分离的外周造血干细胞 ,进行液体和集落培养 ,研究rhTPO单独及与IL 3、IL 6、SCF的协同作用。结果 :液体培养后 ,TPO组单个核细胞数 (MNC)扩增了 ( 1.73± 0 .49)倍 ,IL 3 IL 6 SCF组MNC比种植时增加 ( 4.2 0± 1.14)倍 ,IL 3 IL 6 SCF TPO组MNC增加了 ( 4.5 3± 1.2 7)倍。单独应用TPO及TPO与IL 3、IL 6、SCF合用产生高比例的CD41a 细胞 ,TPO组培养前CD41a 细胞为 11.70 %± 5 .2 3% ,培养后CD41a 细胞为 19.17%± 6 .2 6 % ( P <0 .0 5 )。CD41a 细胞数在TPO组扩增了 ( 3.5 2± 1.18)倍 ,IL 3 IL 6 SCF组扩增了 ( 5 .32± 1.79)倍 ,IL 3 IL 6 SCF TPO组扩增了 ( 6 .94± 2 .19)倍。结论 :TPO可以定向诱导巨核系细胞的分化 ,IL 3、IL 6、SCF可以协同TPO的作用 ,这在造血调控研究 ,体外定向扩增外周血干细胞 ,促进外周造血干细胞移植患者血小板的恢复具有应用前景     

TAT-HOXB4蛋白对脐血CD34~+细胞的体外扩增实验研究

目的:研究TAT-HOXB4蛋白对脐血CD34+细胞的体外扩增效果。方法:采用磁珠阳性分离(MiniMARCS)纯化法分离纯化脐血CD34+细胞;用造血干细胞体外悬浮培养法以及流式细胞术检测法,观察应用TAT-HOXB4蛋白对脐血CD34+细胞的体外扩增能力。结果:加入TAT-HOXB4蛋白组的CD34+细胞数为对照组的13倍,MNC为对照组的6倍,并且在实验的第4天体外扩增效果最明显。结论:TAT-HOXB4蛋白对脐血CD34+细胞的体外扩增效果显著。     

人正常骨髓CD34~ 造血细胞体外扩增形成粒单系造血祖细胞的能力

CD34 造血细胞以其独特的生物学功能正成为造血调控、造血于细胞移植和基因治疗最理想的靶细胞。本文探讨人正常骨髓CD34 造血细胞在体外扩增形成单个核细胞（MNC）及粒单系集落形成单位（CFU－GM）的能力，采用CIMS－100新型免疫磁性无菌分离术可获得纯度＞90％的CD34 造血细胞，其直接形成CFLJ－GM的能力要较骨髓MNC提高的倍，并且在含EGIIS组合造血生长因子的无基质液培养系统中，CD34 造血细胞在四周内可持续扩增产生大量MNC及CFU－GM，最高分别可达1770倍和48倍，但不同个体问CD34 造血细胞的这种能力差别较大。上述结果提示，CD34 造血细胞在最佳组合HGFs的无基质液培养条件下，能扩增产生大量成熟及晚期造血祖细胞，可以适用于临床治疗的需求，     

细胞因子短期扩增对造血干/祖细胞黏附和迁移能力的影响

目的：探讨干细胞因子（SCF）+白细胞介素-6（IL-6）短期扩增对CD34+造血干/祖细胞黏附和迁移能力的影响。方法:用密度剃度离心的方法分离脐血CD34+细胞，经SCF和IL-6孵育48 h，用CCK-8方法检测CD34+细胞增殖能力；用流式细胞仪检测处理前后的CD49d（VLA-4）、CD11a（LFA-1）、CD62L（L-selectin）及CD184（CXCR4）的表达。用纤连蛋白（FN）包被96孔板，检测经或未经因子扩增的CD34+细胞的黏附能力。扩增的CD34+细胞悬浮于transwell培养板的上层，下层添加基质细胞衍生因子（SDF-1），流式细胞仪检测迁移细胞数，计算迁移率。结果:经SCF+IL-6处理48h后CD34+细胞扩增近3倍；表达CD49d、CD11a、CD62L及CD184的CD34+细胞的百分数分别由原来的26.34%±5.37%、17.63%±4.57%、46.38%±6.61%和9.58%±1.56%增加到65.67%±8.72%、56.67%±6.34%、84.76%±9.57%和19.32%±3.64%（P<0.01）。扩增后的CD34+细胞对FN的黏附能力及在SDF-1诱导下的迁移作用都显著增强（P<0.01）。结论:SCF+IL-6短期扩增CD34+ 造血干/祖细胞显著增加细胞的黏附能力，增加SDF-1诱导的迁移作用，可能是SCF+IL-6促进归巢的主要机制之一。     

Ad-hOSM转基因饲养层细胞对脐血CD34~+造血干/祖细胞增殖及归巢能力的影响

目的:建立转人抑瘤素M(hOSM)基因腺病毒载体的饲养层细胞,观察转基因细胞对脐血CD34+造血干/祖细胞扩增的影响,比较扩增前、后造血干/祖细胞体外迁移能力的变化。方法:建立转hOSM基因腺病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术(FCM)检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,FCM检测各组增殖效果;扩增后的造血干/祖细胞用跨膜迁移实验(Transwell实验)检测自发迁移率和SDF-1诱导迁移率以鉴定体外扩增的造血干/祖细胞的归巢能力。结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实有目的基因表达,免疫磁珠法分离的脐血CD34+造血干/祖细胞纯度可达(96.8±2.28)%,与Ad-hOSM转基因饲养层细胞共培养7 d后CD34+造血干/祖细胞可扩增15.73倍,表面黏附分子CXCR4和CD54表达量仍较高,培养后的细胞用Tran-swell板做体外迁移实验,与转基因饲养层细胞共培养的干细胞,其诱导迁移率为(40.68±1.35)%,明显高于对照组,可以较好的保持其归巢能力。结论:转hOSM基因腺病毒载体的饲养层细胞可有效扩增脐血CD34+造血干/祖细胞,延缓其分化,并且体外扩增后仍保持较高的归巢能力。     

脐血CD34+CD19-造血干/祖细胞体外B细胞分化条件研究

目的:研究体外脐血造血干/祖细胞向B细胞分化的条件.方法:体外免疫磁珠分离纯化脐血CD34+CD19-造血干/祖细胞;在小鼠S-17基质细胞支持下,脐血CD34+CD19-造血干/祖细胞、T3、各种细胞因子共培养建立体外B细胞分化发育培养体系,诱导脐血CD34+CD19-造血干/祖细胞向B细胞分化;用流式细胞仪检测培养的B细胞.结果:T3、IL-7与小鼠S-17基质细胞共培养诱导CD34+CD19-造血干/祖细胞28天时,分化形成的B细胞数可达初始培养细胞的198倍,诱导细胞大部分表达CD10、CD19.结论:在选用的实验条件下,T3、IL-7与小鼠S-17基质细胞体外能诱导脐血造血干/祖细胞的B细胞分化.     

Ad-hLIF转基因饲养层细胞对CD34+造血干/祖细胞增殖和分化的影响

目的 用腺病毒载体介导的人白血病抑制因子基因(Ad-hLIF)感染WI-38人胚肺成纤维细胞制备饲养层细胞,体外观察转基因细胞对CD34+造血干/柑细胞增殖和分化的影响,体内研究对辐射损伤SCID小鼠造血功能恢复的效果.方法 用RT-PCR和ELISA法鉴定Ad-hLIF转基因饲养层细胞目的 基因的表达后,将经免疫磁珠法分离和流式细胞术检测后的CD34+细胞在饲养层和/或细胞因子培养体系中扩增28 d,检测不同时间点的单个核细胞(MNC)数量及CD34+细胞阳性率;扩增后的MNC经CFDA SE荧光标记后移植入辐射损伤SCID小鼠体内,RT-PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的门巢情况.结果 感染50MOI(multiplicity of infection)Ad-hLIF的饲养层细胞均有绿色荧光,RT-PCR和ELISA法结果 显示hLIF目的 基因能在WI-38饲养层细胞中表达,免疫磁珠法分离的CD34+造血干/祖细胞经流式细胞术检测其纯度可达95.60%±2.58%,MNC在Ad-hLIF转基因饲养层培养体系持续扩增,最高可达356.95±0.87倍,其中CD34+细胞仪在0～14 d能维持较高水平,最高可扩增52.11±1.13倍,以后逐渐降低.将其移植辐射损伤SCID小鼠后,可明显提高小鼠存活率,4周内小鼠骨髓中不仅可观察到CFDA SE荧光标记的细胞,而且经RT-PCR法搭定后.还可检测到表达Alu人源基因的人脐血造血归巢细胞.结论 成功建立的Ad-hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34+造血干/祖细胞,并延缓其分化.扩增的CD34+细胞对辐射损伤SCID小鼠具有造血功能恢复的功能.     

Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs

1. Rates of oxygen uptake and of anaerobic glycolysis were estimated in slices from the renal cortex and medulla (a) of adult rats and guinea-pigs, (b) of new-born (1-, 5- and 21-day-old) rats and of guinea-pigs of 1, 12, 21, 24 and 120 hr age.2. In the adult rat, Q(O2) values for the cortex were 12.55 +/- 0.20 (22) and for the medulla: 8.56 +/- 0.17 (22) mul./hr.mg dry weight, while in the new-born rat (24 hr old) they were 10.99 +/- 0.46 (12) and 9.33 +/- 0.18 (9) mul./hr.mg dry weight respectively.3. Values for Q(CO2) (N2) (anaerobic glycolysis) in the 14 hr old new-born rat were in the renal cortex 9.65 +/- 0.35 (5) and in the medulla 7.39 +/- 0.43 (5) mul./hr.mg dry weight; while in the adult they were 2.25 +/- 0.08 (16) and 5.76 +/- 0.14 (16) mul./hr.mg dry weight, respectively.4. In the adult guinea-pig values for Q(CO2) (N2) were of the same order as in the adult rat, though the rate of O(2) uptake was for the cortex 8.12 +/- 0.22 (12) and for the medulla 5.02 +/- 0.23 (11) mul./hr.mg dry weight.5. Though the Q(O2) values in the renal cortex and medulla were smaller in the 1 hr old new-born guinea-pig, they were already increasing in the 12 hr old neonate.6. The results are discussed in the light of enzyme changes occurring during the process of maturation of the nephron as indicated by histochemical observations.     

Modes of reinforcement and gender and culture of experimenter and subject: effects of "alien" and external reinforcement for Japanese and American men and women

In this study, we examined external and "alien" reinforcement (ER and AR. respectively) as a factor in social learning, and studied the combined effects of culture and reinforcement mode. A female (Experiment 1) and a male (Experiment 2) experimenters conducted experimental sessions. Both men and women, who grew up in the same culture as the experimenter, participated and performed the experimental task. A three-way interaction effect of experimenter gender, culture, and reinforcement mode was found on task performance. And the effect was more pronounced for a Japanese experimenter. A female and a male experimenters conducted Experiments 3 and 4, respectively; however participants this time were men and women who grew up in different cultures than the experimenter. Results indicated that the pattern of the subject gender and reinforcement mode interaction effect, when the experimenter was Japanese with American subjects, was exactly opposite to that when the experimenter was American. These experiments showed that AR was as effective for social learning as ER, and that the cultural backgrounds of experimenter and subject influenced AR and ER effectiveness.     

Timing and intensity of early fevers and the development of allergies and asthma

BACKGROUND: Early childhood fevers appear to protect against later allergies and asthma. What is not known is the time in which fevers exert this effect and whether the degree of temperature increase is important. OBJECTIVE: We sought to examine the relationship between the time and degree of early fevers and later allergies and asthma. METHODS: Eight hundred thirty-five children from southeast Michigan were enrolled at birth. Clinic records from their first 2 years were abstracted for episodes of fever. At age 6 to 7 years, children underwent allergy testing. We examined fevers occurring within 6-month intervals in the first 2 years of life and outcomes at age 6 to 7 years. The primary outcome measures were allergic sensitization, asthma, asthma with allergic sensitization, and asthma without allergic sensitization. RESULTS: In the unadjusted analysis each episode of fever between 7 and 12 months of age was associated with a lower odds of allergic sensitization (odds ratio [OR], 0.71; 95% CI, 0.54-0.93) and asthma with allergic sensitization (OR, 0.43; 95% CI, 0.21-0.90) at age 6 to 7 years. Likewise, every 1 degrees C increase in the maximum temperature between 7 and 12 months was associated with a lower odds of allergic sensitization (OR, 0.77; 95% CI, 0.61-0.96) and asthma with allergic sensitization (OR, 0.62; 95% CI, 0.40-0.94). After adjusting for potential confounders, each episode of fever between 7 and 12 months was associated with a lower likelihood of asthma with allergic sensitization (adjusted OR, 0.33; 95% CI, 0.11-0.94) at age 6 to 7 years. CONCLUSIONS: Both the timing and intensity of childhood fevers appear to be important factors in the development of allergies and asthma.     

纳米水平分子成像与诊疗一体化研究进展与挑战

分子成像能以非侵入性的方式重现活体细胞的生理功能和生物学过程,提高疾病的早期和特异性诊断水平。纳米颗粒/材料具有物理性质可控性高、易于表面修饰、血液循环时间长和可功能化等优点,在疾病诊断与治疗中显示出巨大潜力。但如何阐明纳米材料多功能间的内在联系、解决其代谢及安全性等关键机制难题、实现纳米颗粒/材料多功能性到临床多功能...