Hemoglobin aggregation in single red blood cells of sickle cell anemia

A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.     

Regulation of erythrocyte cation and water content in sickle cell anemia

The pathophysiological events in sickle cell disease are critically dependent on the intracellular concentration of hemoglobin S, which varies inversely with cell cation and water content. Erythrocytes of SS homozygotes exposed to oxygen or carbon monoxide decrease their potassium and water content through a pathway for potassium transport that is activated by both cell swelling and decrease in internal pH. This pathway is not inhibited by ouabain either with or without bumetanide. When SS erythrocytes were separated according to density, the pH- and volume-dependent potassium transport was greatest in the least dense fraction and was reduced in the densest cells. This pathway, which does not depend on polymerization of sickle hemoglobin, may be important in regulating the cation and water content of SS erythrocytes.     

组织块贴附法原代培养小鼠皮肤细胞方法的改进

试验研究一种简易的小鼠皮肤细胞组织块原代培养方法.从幼龄小鼠获得皮肤组织块,用D-Hanks缓冲液和DMEM培养液洗涤组织块,将其剪至1mm3左右大小,胶头滴管吸取组织块悬液1～2 ml,注入培养瓶,然后匀速而缓慢地翻转培养瓶,于CO2培养箱中培养24 h后,再翻转培养瓶继续培养.结果表明,该方法接种的组织块有15%能迁移牛长出细胞单层.并对细胞长期培养的行为进行了观察.     

Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and germ cells in vivo. Cloning by transfer of ntES cell nuclei could result in normal development of fertile adults. These studies demonstrate the full pluripotency of ntES cells.     

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.     

Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.     

Derivation of oocytes from mouse embryonic stem cells

Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.     

Human T-cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells

The thyroid glands of patients with autoimmune diseases such as Graves' disease and certain forms of goiter contain infiltrating activated T lymphocytes and, unlike cells of normal glands, the epithelial follicular cells strongly express histocompatibility antigens of the HLA-DR type. In a study of such autoimmune disorders, the infiltrating T cells from the thyroid glands of two patients with Graves' disease were cloned in mitogen-free interleukin-2 (T-cell growth factor). The clones were expanded and their specificity was tested. Three types of clones were found. One group, of T4 phenotype, specifically recognized autologous thyroid cells. Another, also of T4 phenotype, recognized autologous thyroid or blood cells and thus responded positively in the autologous mixed lymphocyte reaction. Other clones derived from cells that were activated in vivo were of no known specificity. These clones provide a model of a human autoimmune disease and their analysis should clarify mechanisms of pathogenesis and provide clues to abrogating these undesirable immune responses.     

Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing

Persistence of human fetal hemoglobin (HbF, α(2)γ(2)) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.     

利用成年母兔耳部皮肤上皮细胞生产克隆胚胎的研究

以成年母兔耳部皮肤上皮细胞为核供体,对家兔体细胞核移植技术中融合、激活以及不同来源受体卵母细胞的选择等环节进行了研究,比较了不同电场强度对重构胚胎融合率、分裂率的影响。结果表明,200V/mm电场强度时重构胚胎的融合率(82.53%)显著高于160V/mm(59.73%),但与240V/mm(79.56%)无显著差异;200V/mm时重构胚胎的分裂率(71.43%)显著高于160V/mm(49.43%)和240V/mm(57.80%)。来源于输卵管卵母细胞组成的重构胚胎的融合率(84.71%)和裂解数与来源于卵巢大卵泡(78.75%)的无显著差异;但来源于输卵管的重构胚胎分裂率(73.68%)显著高于来源于卵巢大卵泡的(56.35%)。将646枚2~4细胞期重构胚移植到38只同期化及非同期化的受体后无幼仔出生。     

洛美沙星透皮吸收制剂对小鼠皮肤表皮结构的影响

以含氮酮的洛美沙星透皮吸收制剂为模型药物,昆明种(KM)雄性小鼠在体皮肤为研究材料,设计6个试验时间段(涂药后2.25、4.5、9、18、36和72 h),综合运用光镜(LE)、扫描电镜(SEM)和透射电镜(TEM)技术,观测经透皮吸收制剂作用后小鼠皮肤表皮及毛囊结构的变化,探讨氮酮促进洛美沙星透皮吸收的机理.结果表明,氮酮通过以下途径促进药物透皮吸收:作用于表皮角质层细胞间脂质,使角质层细胞间距增大,从而引起角质层外层细胞易于脱落,皮肤角质层变薄,减弱表皮对药物的屏障作用;作用于细胞内基质,引起基底角质层肿胀,增加了角质细胞的水化程度和药物的存储空间;作用于毛囊,使毛囊口附近角质疏松、脱落,使药物易于经毛囊途径吸收,但毛囊在本研究中为非主要渗透途径.研究还表明,单次应用该制剂对小鼠皮肤结构的影响超过72 h;由该制剂引起小鼠皮肤结构的改变是个非损伤性可复过程.     

珍珠贝外套膜酶解产物促进小鼠皮肤创伤愈合作用研究

为探讨珍珠贝Pinctada martensii外套膜酶解产物(enzymatic hydrolysis from mantle of pearl oyster,EHM)对小鼠皮肤创伤的促愈合效果,采用小鼠(体质量20 g±2 g)背部全皮层创伤模型,连续灌胃给药14 d,隔天观察伤口愈合情况,计算创伤愈合率和瘢痕缩小率,并于造模后的第3、7和14天分批处死动物,测定伤口边缘皮肤组织中白介素-6(interleukin 6,IL-6)、白介素-10(interleukin 10,IL-10)、转化生长因子β(transforming growth factor-β,TGF-β)、碱性成纤维细胞生长因子(fibroblast growth factor 2,FGF-2)、表皮细胞生长因子(epidermal growth factor,EGF)、细胞生长周期素1(cyclin D1,CCND1)和羟脯氨酸含量,对EHM的作用效果和作用阶段进行研究。结果表明:各试验组动物在第2天时皆已结疤,第6、10天时,各EHM给药组创伤愈合率均高于阴性对照组,第14天时,低剂量组的愈合率达100%;EHM可提高瘢痕收缩率,其对炎症因子IL-6生成无显著性影响(P>0.05),但能极显著促进IL-10分泌(P<0.01);与阴性对照组相比,高剂量EHM给药组的TGF-β、CCND1和EGF含量均显著增加(P<0.05),而FGF-2含量则无显著性差异(P>0.05);给药后第14天,各EHM给药组均能显著提高皮肤组织中的羟脯氨酸含量(P<0.05)。研究表明,EHM具有抑制炎症作用并能促进生长因子分泌及胶原蛋白的生成,从而加快小鼠软组织开放性创伤愈合,EHM还对浅表瘢痕增生具有一定的抑制作用。     

珍珠贝外套膜酶解产物促进小鼠皮肤创伤愈合作用研究

为探讨珍珠贝Pinctada martensii外套膜酶解产物(enzymatic hydrolysis from mantle of pearl oyster,EHM)对小鼠皮肤创伤的促愈合效果,采用小鼠(体质量20 g±2 g)背部全皮层创伤模型,连续灌胃给药14 d,隔天观察伤口愈合情况,计算创伤愈合率和瘢痕缩小率,并于造模后的第3、7和14天分批处死动物,测定伤口边缘皮肤组织中白介素-6(interleukin 6,IL-6)、白介素-10(interleukin 10,IL-10)、转化生长因子β(transforming growth factor-β,TGF-β)、碱性成纤维细胞生长因子(fibroblast growth factor 2,FGF-2)、表皮细胞生长因子(epidermal growth factor,EGF)、细胞生长周期素1(cyclin D1,CCND1)和羟脯氨酸含量,对EHM的作用效果和作用阶段进行研究。结果表明:各试验组动物在第2天时皆已结疤,第6、10天时,各EHM给药组创伤愈合率均高于阴性对照组,第14天时,低剂量组的愈合率达100%;EHM可提高瘢痕收缩率,其对炎症因子IL-6生成无显著性影响(P>0.05),但能极显著促进IL-10分泌(P<0.01);与阴性对照组相比,高剂量EHM给药组的TGF-β、CCND1和EGF含量均显著增加(P<0.05),而FGF-2含量则无显著性差异(P>0.05);给药后第14天,各EHM给药组均能显著提高皮肤组织中的羟脯氨酸含量(P<0.05)。研究表明,EHM具有抑制炎症作用并能促进生长因子分泌及胶原蛋白的生成,从而加快小鼠软组织开放性创伤愈合,EHM还对浅表瘢痕增生具有一定的抑制作用。     

孕鼠子宫内膜上皮细胞分离、培养及活力检测

为探讨昆白系孕鼠子宫内膜上皮细胞的分离与体外培养条件、生长特点及其增殖规律,采用2．5 mg／mL胰蛋白酶+0．4 mg／mL EDTA分别以20,30,40 min消化子宫内膜,结合刮取法,对昆白系孕鼠子宫内膜上皮细胞的分离培养进行了研究。结果表明,随着消化时间的增加,所获得的细胞数目也增多,但细胞活力下降(噻唑兰法检测);消化30min所获得的细胞活力高、数目多,细胞比较单一;子宫内膜上皮细胞的活力随着细胞培养代数的增加而下降。     

Human monocytic cell lines derived from cord leukocytes by co-cultivation with irradiated CM-S cells

The CM-S cell line was established from the bone marrow of a child with congenital hypoplastic anemia and resembles its monocyte-macrophage lineage. Lethally x-irradiated CM-S cells from various passages and clones, representing different stages in the progression of the transformed growth phenotype, were tested for their ability to affect the survival and proliferation of normal human cord or adult blood leukocytes in co-culture. One clone, CM-SM, which is tumorigenic in athymic mice, consistently immortalized umbilical cord mononuclear cells but did not immortalize adult peripheral blood leukocytes. Six autonomous monocyte-like diploid cell lines were obtained and all were found to be of cord origin. Three lines were tumorigenic in athymic mice. Attempts to immortalize human leukocytes with cell-free supernatants from CM-S cells were unsuccessful.     

Human-mouse cell hybrids: a suggestion of structural mutation for dipeptidase-2 deficiency in mouse cells

The dipeptidase-2 enzyme is inactive in certain cultured cell lines from the mouse. In somatic cell hybrids between such deficient cells and diploid human fibroblasts, the mouse deficiency was complemented when the homologous human peptidase-A was retained. The results suggested that the murine peptidase deficiency was the result of a structural mutation, rather than a regulatory one.     

携带CD自杀基因NDV重组病毒对小鼠肝癌细胞转移模型的治疗效果

采用反向遗传操作技术构建新城疫病毒基因组NP/P位点表达胞嘧啶脱氨酶(Cytosine deaminase,CD)基因弱毒力重组新城疫病毒rClone30-CD和中毒力重组新城疫病毒rAnhinga-CD。MTT检测结果表明,在协同5-氟胞嘧啶(5-Fluorocytosine,5-FC)条件下,两株重组新城疫病毒可在体外有效杀伤人肝癌细胞HepG2。通过Balb/c鼠尾静脉注射小鼠肝癌细胞H22构建小鼠肝癌细胞转移模型,经重组病毒协同5-FC联合治疗,荷瘤小鼠转移肿瘤体积显著缩小、肿瘤组织出现明显凋亡与坏死。体内试验结果表明,中毒力重组新城疫病毒rAnhinga-CD抑瘤效果优于弱毒力重组新城疫病毒r Clone30-CD。     

电池废液对小鼠骨髓细胞染色体的影响

电池废液主要来源于废旧电池的内容物,有关报道曾指出电池废液对染色体有一定影响,文章对此做进一步的研究和讨论。利用遗传毒理学原理,采用“小鼠骨髓细胞染色体畸变分析”方法,探讨电池废液对小鼠骨髓细胞染色体畸变率的影响。实验结果表明,0.4,0.6实验组的小鼠与阴性对照组的小鼠细胞染色体畸变率存在极显著差异(P<0.01)。从而证明了电池废液对染色体有畸变作用,并且发现雌雄小鼠对电池废液的反应程度不同,两者间的差异显著(P<0.05),由此证明雌雄个体在对药物的耐受上存在差异。