人胎盘组织造血干/祖细胞的分离富集

为了探索从胎盘组织中分离富集造血干/祖细胞(HSPC)的标化流程,采用机械法加胶原酶消化法制备人胎盘组织单个细胞悬液,用羟乙基淀粉(6%HES)法从中分离出单个核细胞(MNC),再经免疫磁珠分选法分选出CD34-、CD34+CD38-、CD34+CD38+3个细胞亚群,用流式细胞术对各阶段分选细胞进行表型分析并计算分选细胞的富集度和回收率。结果表明:机械法加胶原酶消化法制备的人胎盘组织单个细胞悬液中单个核细胞(MNC)数达(12.30±3.51)×108,与脐血初始样品所含的MNC数(8.86±5.38)×108比较差异无统计学意义,而其CD34+细胞所占百分率[(3.93±2.31)%]则明显高于脐血[(0.44±0.29)%]。胎盘组织单个细胞悬液经6%HES分离后MNC和CD34+细胞的回收率分别为(45.3±11.7)%和(51.1±9.8)%;MNC经免疫磁珠分选后,其CD34+细胞的纯度和回收率分别为(73.4±14.1)%和(52.7±11.7)%。结论:本实验所建立的"机械法加胶原酶消化法-HES分离MNC-MACS分选目标细胞"的分离纯化方法可从胎盘组织获得高丰度、高富集度、高活性的HSPC,为进一步研究胎盘HSPC提供了比较经济、效果较好的分离富集方案。     

Prospective Study of Attitudinal and Relationship Predictors of Sexual Risk in the Multicenter AIDS Cohort Study

We examined the influence of attitudes concerning HIV transmission, safe sex, and sexual sensation seeking, as well as negotiated risk reduction with primary partners, on the proportion of unprotected sexual partners (%UASP) among men who have sex with men (MSM). Participants were 263 HIV-seropositive and 238 HIV-seronegative MSM in the Multicenter AIDS Cohort Study between 1999 and 2003 who completed a 20-item attitude survey twice. Behavioral data were collected concurrently and 6–12 months after each survey. Among seropositives, decreased HIV concern and increased safer sex fatigue were associated with higher %UASP at 6 and 12 months. Among seronegatives, increased %UASP at 12 months was associated with safer sex fatigue. At 6 months and 12 months, risk reduction agreements were associated with increased %UASP among seronegatives in seroconcordant monogamous relationships, reflecting their abandonment of condoms in such partnerships. We conclude that HIV prevention efforts should target modifiable attitudes (reduced concern about HIV and safer sex fatigue) and increases in sexual risk-taking of MSM, particularly among HIV+ men having sex with serodiscordant partners.     

从分化胚胎干细胞中分选血管构建种子细胞的实验研究

目的本研究主要比较了应用免疫磁珠(MACS)和流式细胞仪(FACS)两种分选手段,从ES分化细胞中获得血管平滑肌细胞和内皮细胞的效率。方法利用MACS和FACS从ES分化细胞中分选出FLK-1阳性细胞,分别在VEGF和PDGF诱导培养下获得血管平滑肌细胞和血管内皮细胞,运用MACS和FACS进行细胞性质和纯度鉴定,并对两种方法所获细胞的活力和细胞得率进行比较研究。结果利用MACS分选获得的FLK-1阳性细胞经过PDGF诱导培养后可以得到108～109量级和纯度超过90%的血管平滑肌细胞,可满足构建血管的需要,而利用FACS分选难以获得构建血管肌层所需的大量细胞。利用FACS分选的FLK-1阳性细胞经过VEGF诱导培养后的纯度约60%,表达vWF和吸收低密度脂蛋白(Ac-LDL)的血管内皮细胞;而利用MACS从ES细胞中分选获得的血管内皮细胞纯度较低,两种分选方法获得的细胞活力无明显差别。结论因组织工程血管构建所需要的平滑肌细胞数量巨大,宜选用MACS从ES分化细胞中分选获得;而血管内皮细胞宜选用FACS分选获得。     

人与犬牙周膜干细胞的生长特性比较

目的：培养、分离人与犬牙周膜干细胞（PLSCs）,比较其生长特性,为相关体内研究提供理论和实验依据。方法：采用免疫磁珠分选系统分别从原代培养的人与犬牙周膜细胞中分离PLSCs,通过倒置相差显微镜、细胞计数、集落形成检测、流式细胞仪等方法比较人与犬PLSCs的细胞形态、生长曲线、集落形成率（CFR）及STRO-1^＋表达情况。结果：人与犬PLSCs形态相似,均为梭形,生长曲线也都呈“S”形。但CFR人PLSCs显著高于犬PLSCs（P〈0．05）,而且人牙周膜中STRO-1^＋细胞的表达率高于犬牙周膜中STRO-1^＋细胞（P〈0．05）。结论：STRO-1^＋免疫磁珠分选系统可用于分离人和犬PLSCs,所分离的两种PLSCs的形态和体外生长模式相似,但细胞亚群增殖能力和表型有差异。     

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: A putative role for progesterone deficiency

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCRαβ+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCRαβ+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, naïve cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-β mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation.     

MACS方法分选CD133 /CD44 前列腺癌干细胞的初步鉴定

【摘要】 目的：通过磁珠细胞分选（Magnetic bead cell sorting, MACS）方法从人前列腺癌细胞系PC-3中分选CD133 /CD44 干细胞,为进一步功能性研究奠定基础。方法：运用流式细胞仪检测MACS分选前后PC-3细胞膜上CD133和CD44表达情况;观察无血清培养成球情况,免疫荧光表达情况;比较细胞在分选前后的形态学、增殖能力方面的差异;免疫组化和Western blot检测诱导分化前后PAP蛋白情况。结果：流式细胞术检测PC-3细胞CD133和CD44的阳性表达分别是(1.33?0.05)%和(0.87?0.06)%,而MACS分选后分别为(84.82?0.07)%和(99.91?0.03)%;免疫荧光检测CD133 /CD44 细胞培养后继续呈阳性表达;CD133 /CD44 细胞增殖能力高于PC-3细胞(t=11.0, P=0.008)以及高于诱导后的CD133 /CD44 细胞(t=40.1, P=0.001);CD133 /CD44 细胞经过TGF-β诱导后免疫组化和Western blot检测PAP表达呈阳性,而未诱导的CD133 /CD44 细胞表达呈阴性。结论：MACS从PC-3细胞株中分选的CD133 /CD44 细胞经过初步功能性鉴定具有干细胞的某些特性,可为前列腺癌干细胞的进一步探索充当铺垫。     

U251细胞系中脑肿瘤干细胞耐药性的初步研究

目的 探讨脑肿瘤干细胞对抗肿瘤药物的耐药性及其机制.方法 通过磁珠分选(MACS)将U251细胞中的CD133+与CD133-细胞进行分选,用MTT法和流式细胞技术比较CD133+与CD133-细胞对抗肿瘤药物替尼泊苷(Vm-26)、卡莫司汀(BCNU)和顺铂(DDP)的敏感性、细胞凋亡率及细胞内药物蓄积的差异性,通过RT-PCR检测CD133+与CD133-细胞耐药基因谱的差异.结果 U251细胞系中,CD133+细胞约占5.4%.分选后的CD133+细胞的阳性率可达92.4%,CD133-细胞对抗肿瘤药物替尼泊苷、卡莫司汀和顺铂的敏感性明显高于CD133+细胞;流式细胞仪试验显示,在中等浓度的替尼泊苷作用下,CD133-细胞的凋亡明显,并出现明显的"亚G1峰",而CD133+细胞并未受到明显抑制和杀灭;流式细胞仪检测显示CD133+细胞内的药物蓄积明显低于CD133-细胞;RT-PCR实验显示MDR1、BCRP、MRP、MGMT和Bcl-2表达在CD133+明显高于CD133-细胞.结论 CD133+是U251细胞系中的耐药细胞亚群,高表达ABC转运体,DNA修复和抗凋亡基因可能是CD133+细胞耐药的重要机制.     

MACS检测胃癌腹腔冲洗液游离癌细胞的研究

目的 探讨胃癌腹腔微转移情况的检测方法及意义。方法 手术中切除肿瘤前收集腹腔冲洗液，采用磁激活细胞分离术（MACS）对不同病理分期胃癌患者腹腔冲洗液中癌细胞进行富集并检测。分别标记带磁珠的细胞角蛋白（CK）抗体，经磁柱富集CK^＋上皮细胞，用流式细胞仪检测其含量，并比较胃癌组与胃平滑肌瘤组（对照组）以及胃癌不同分期之间、磁富集前后CK^＋上皮细胞含量的差异。结果 在未经MACS富集的标本中较少发现CK^＋ CD45^-细胞；在富集后的标本中其含量在胃癌组与对照组有显著差异（41／50，1／10，P〈0．001）；pTNMⅠ～Ⅱ期与Ⅲ～Ⅳ期之间（0．67％，3．42％，P〈0．001）差异有非常显著性。结论 MACS能有效地富集上皮来源细胞，提高上皮源细胞的检出率，并能反映腹腔游离癌细胞数量；上皮细胞数量与胃癌的存在及临床病理分期有关，其有利于判断肿瘤转移和预后并指导治疗。