Sensitive Detection of p53 Gene Mutations in Esophageal Endoscopic Biopsy Specimens by Cell Sorting Combined with Polymerase Chain Reaction Single-strand Conformation Polymorphism Analysis

For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis. Mutations in exons 5–8 of the p53 gene were investigated by FCR-SSCF analysis using 10³ sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR-SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR-SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.     

免疫磁珠法分离白血病转基因小鼠骨髓造血干细胞

本文分离转基因小鼠骨髓造血干细胞。运用针对小鼠干细胞表面特异表达的干细胞抗原 1(stemcellantigen 1,Sca 1)的单克隆抗体和包被于磁颗粒表面的第二抗体 ,采用磁吸附细胞分选方法 (MACS )分离小鼠骨髓造血干细胞 ,用流式细胞术 (FACS )检测MACS分离后骨髓细胞中干细胞 (Sca 1阳性细胞 )比例 ,监测细胞分选效果。结果显示MACS分离后骨髓细胞中Sca 1阳性细胞所占比例达 85 %以上 ,涂片观察发现细胞组成和细胞形态学特征与FACS所得结果一致。MACS可以从转基因小鼠全骨髓细胞中分离出Sca 1阳性的骨髓造血干细胞 ,细胞纯度可达 85 %以上。     

人胎盘CD133+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133⁺细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析，证明人胎盘存在早期造血干/祖细胞（HSPC）。 方法 采用机械法制备人胎盘组织（PT）单细胞悬液，用Histopaque-1007分离出单个核细胞(MNC)，经磁式分选（MACS）富集CD133⁺细胞，培养28 d后观察HPP-CFC集落形成能力，用流式细胞仪（FCM）对分选的细胞组份和HPP-CFC进行表型分析，实验全程用脐带血（UCB）作平行比较分析。 结果 培养28 d后，PT-CD133⁺与UCB-CD133⁺细胞组份分别扩增了266和362倍，前者低于后者（P<0.01）；PT-CD133⁺与UCB-CD133⁺细胞中HPP-CFC分别为(32.4±11.2)/5×10³、(17. 7±5.7)/5×10³，前者形成的HPP-CFC数量明显高于后者（P<0.01）；PT-CD133⁺、UCB-CD133⁺细胞培养至28 d时，除UCB-CD133⁺组的CD133⁺CD34^-亚群比例无明显改变外，CD133⁺CD34⁺、CD133^-CD34⁺和CD133⁺CD34^-（PT-CD133⁺组）亚型均比培养前减少。 结论 人胎盘组织CD133⁺细胞中存在HPP-CFC，说明胎盘CD133⁺细胞群中存在早期HSPC。     

An interleukin-4-dependent precursor clone is an intermediate of the differentiation pathway from an interleukin-3-dependent precursor clone into myeloid cells as well as B lymphocytes

An interleukin-3-dependent progenitor clone LyD9 and its interleukin-4-dependent derivative clone K-4 were shown to differentiate into myeloid cells as well as B lymphocytes by coculture with bone marrow stroma cells. The K-4 clone is an intermediate between myeloid/lymphoid cells and the LyD9 clone that requires interleukin-4 for differentiation into B lymphocytes and myeloid cells. Granulocyte-macrophage colony stimulating factor-dependent derivatives (LS-1 and K-GM) were also established from induced LyD9 cells. LS-1 and K-GM were myeloid-committed cells.     

神经营养素受体在人外周血T细胞中表达的研究

目的 研究人外周血CD4^ /CD8^ T细胞4种神经营养素受体基因的转录。方法 应用尼龙手法分离出T细胞，磁式细胞分离法（MACS）分离CD4^ /CD8^ T细胞亚群，再以RT－PCR法研究4种神经营养素受体在两种T细胞亚群上的表达。结果 未经刺激的CD4^ /CD8^ T细胞亚群不表达任何神经营养素受体。经PHA或PPD刺激后，CD4^ /CD8^ T细胞亚群表达trkA,CD8^ T细胞亚群表达trkC；而在各种状态下的T细胞上均未见表达trkB及p75^NGFR。结论神经营养素受体在两种T细胞亚群中有不同的表达格局，提示不同T细胞亚群受神经营养素调节的模式可能各不相同。     

H-Y Antigen Expression Patterns in Human X- and Y-Chromosome-Bearing Spermatozoa

PROBLEM: Restricted expression of H-Y antigen on Y-chromosome-bearing sperm has been reported in some species, although such preferential expression for H-Y antigen in human sperm has yet to be described. In this study, an immunomagnetic approach was used to characterize antigen expression patterns as a function of sex-chromosome content. METHOD OF STUDY: Human sperm was treated with monoclonal immunoglobulin (Ig) M antibodies directed against H-Y antigen. This preparation then was incubated with sheep antimouse IgM antibody affixed to paramagnetic beads, which then were exposed to a magnetic field and sorted. X- and Y-chromosome frequencies in the two subgroups of sperm were assayed by multiprobe fluorescent in situ hybridization (FISH). RESULTS: Sperm were immunomagnetically separated into two populations: a reactive group (presumably, H-Y Ag+); and a nonreactive group (presumably, H-Y Ag-). Triple-color FISH analysis of 1,600 spermatozoa (800 in each group) showed the antigen's expression to be somewhat more prevalent among Y-chromosome-bearing sperm (54.1%), but a large proportion of Y-chromosome-bearing sperm (49.0%) did not express this antigen. The difference was not significant (P = 0.43). CONCLUSIONS: The expression of H-Y antigen has a slightly higher frequency in human sperm containing the Y-chromosome, but its expression among X-chromosome-bearing sperm also is considerable. Current immunologic techniques relying on this antigen are unlikely to effect the sex selection of human sperm.     

Activation and Sorting of Human White Blood Cells

We demonstrate a novel activation behavior of human leucocyte adhesion under physiological flow conditions in a microfabricated silicon array of channels with length scales similar to those of human capillaries. Vital nuclei stains and cell specific, flourochrome labeled antibodies reveal that the equilibrium distribution of stuck cells in the arrays displays a strong dependence on cell type and nuclear morphology, and there is eventual separation of the two cell types in the array. The distortion of the cells is the same as they experience in vivo and the response of the granulocytes is consistent with a model describing adhesion as a function of the distortion of the cell by its environment; in other words, activated adhesion. We propose that this complex non-random behavior is due to a deformation activated change in the cells relevant to observed in vivo behavior.     

基于免疫磁珠法的不同筛选方案对胎儿有核红细胞富集效果的比较

目的：将密度梯度离心(density gradient centrifugation,DGC)与免疫磁珠法(magnetic-activated cell sorting,MACS)相结合,应用4种不同的筛选方案,对脐血中的胎儿有核红细胞(fetal nucleated red blood cells,FNRBC)进行分离,评估不同策略富集FNRBC的效果。方法：对10例脐血标本(12 mL/例)按4种不同方案进行筛选：DGC;DGC⁺MACS阴性筛选(CD45^-);DGC⁺MACS阳性筛选(CD71⁺);DGC⁺MACS阴性/阳性筛选(CD45-/CD71⁺)。通过细胞计数板计数,K-B染色后显微镜下人工FNRBC计数,从获得总细胞量、FNRBC量和FNRBC比例经t检验后评估不同方案富集FNRBC的效果。结果：(1)与其他3组相比,DGC方案可获得的总细胞量(0.95×10⁷/mL)最多,FNRBC数量(39.87/mL)最多,F...