骨髓间充质干细胞的分化潜能及临床应用价值

目的:观察人的骨髓间充质干细胞多分化潜能及在糖尿病治疗领域的价值。方法:实验于2005-07/2006-07在青岛大学医学院附属医院内分泌科完成。骨髓来源于非造血系统疾病的16岁儿童胸骨骨髓血(查体以排除造血系统疾病,结果显示为健康体质),经得家属同意。Percoll淋巴细胞分层液分离骨髓间充质干细胞,取第3代细胞,等密度接种于培养瓶中,经CD44抗体、CD45抗体、CD34抗体鉴定。取其第4代细胞,诱导其向脂肪细胞及神经细胞分化,利用碱性成纤维细胞生长因子预处理先获得巢蛋白阳性细胞,分别用两种方法诱导其向胰岛祖细胞的转化:化学物质诱导和共培养法诱导,免疫荧光检测胰岛祖细胞标志-胰十二指肠同源异型盒基因(蛋白的表达,电化学发光法检测其是否表达胰岛素)。胶原酶消化法获取胰腺间充质干细胞,鉴定,用添加碱性成纤维细胞生长因子的无血清低糖DMEM使其增殖。将胰腺间充质干细胞接种于底层已接种骨髓间充质干细胞的6孔板共培养,共培养法诱导骨髓间充质干细胞向胰岛祖细胞的初步转化。结果:成脂诱导及成神经诱导可获得油红O染色阳性细胞以及巢蛋白阳性细胞。化学法向胰岛祖细胞诱导后可检测到PDX-1免疫反应阳性细胞。共培养法诱导也可获得PDX-1免疫反应阳性细胞。新生儿胰腺具有巢蛋白、CK-19阳性的胰腺间充质干细胞,体外高糖诱导可形成胰岛样细胞团。结论:骨髓间充质干细胞在体外具有诱导分化为脂肪细胞、神经元样细胞及胰岛祖细胞的潜能。新生儿胰腺间充质干细胞向胰岛细胞分化过程中所分泌的一些物质对骨髓间充质干细胞向胰岛祖细胞的转化具有促进作用。     

Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid-->lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.     

Commentary on the safety of red cells preserved in extended-storage media for neonatal transfusions

Red cells preserved in extended-storage media are the standard product dispensed by many regional blood centers. When the red cells are intended for neonatal transfusion, concern exists about the safety of the relatively high quantities of additives present in these media. Definitive studies to address these concerns are not available. Therefore, to estimate the effects of additives and to delineate circumstances in which they might be harmful, the quantities transfused in defined clinical settings were calculated, and the following recommendations are offered for transfusing infants less than 4 months of age. First, red cells preserved in extended-storage media should present no substantive risks when used for small-volume (approximately 10 mL/kg) transfusions of premature infants and can be used without additional processing. Second, the risks of the most premature neonatal patients or those with severe renal and/or hepatic insufficiency cannot be defined clearly, and, because data are not available to ensure safety for these infants, removal of the additive medium and resuspension of the red cells in saline or albumin solution immediately before transfusion are recommended. Third, following a similar rationale, it seems prudent to avoid using entire units of red cells preserved in extended-storage media in massive transfusion settings (e.g., exchange transfusion, cardiac surgery, and extracorporeal membrane oxygenation). In these settings, the preservative medium should be removed and the red cells resuspended in the fluid that is most appropriate for the procedure that is planned. It must be emphasized that these recommendations are based on calculations and hypothetical settings, not actual data. Accordingly, they are tentative and should be altered as definitive information becomes available.     

Oral pilocarpine HCl stimulates labial (minor) salivary gland flow in patients with Sjögren's syndrome

Pilocarpine HCl has been shown to stimulate parotid and submandibular gland salivary flow. The purpose of this study was to determine whether this cholinergic-muscarinic drug also stimulates labial (minor) salivary gland (LSG) flow and to relate that with whole unstimulated salivary (WUS) flow rateS. Subjects diagnosed with primary Sjögren's syndrome (SS-1; n = 9) or secondary Sjögren's syndrome (SS-2; n = 9) were enrolled in this study after meeting stringent enrollment criteria. An age-gender matched control group was also enrolled. The labial saliva was collected in a standardized manner on Per-iopaper® for 5 min and the volume was analysed by the Periotron®.Whole unstimulated salivary samples were collected for 5 min by the method of Mandel and Wot-man (1976).Each subject was dosed with pilocarpine HCl (5 mg; tablets; p.o.).After 60 min the LSG flow as well as the WUS flow was determined again as previously. The results indicated a significant (>180%) increase in both labial salivary gland flow as well as whole salivary flow in the SS-1 and SS-2 subjects (mean ± S. e.m.): [SS-1: WUS = 0.1080 ± 0.03 vs 0.2242 ± 0.03 ml per 5 min; LSG = 93.1 ± 22.2 vs 167.8 ± 15.9 μl/5 min; P < 0.001; SS-2: WUS = 0.1384 ± 0.02 vs 0.2775 ± 0.09 ml per 5 min; LSG = 97.7 ± 20.2 vs 182.8 ± 17.9 μl per 5 min; P < 0.001]. These results indicate a significant increase in labial salivary gland flow as well as whole salivary flow as stimulated by pilocarpine HCI in Sjögren's syndrome patients.     

Detection of meticillin‐resistant staphylococcus aureus (MRSA) colonization in newborn infants using real‐time polymerase chain reaction (PCR)

Objective: Meticillin‐resistant staphylococcus aureus (MRSA) colonization on neonatal units is a common and important clinical problem. Effectiveness of polymerase chain reaction (PCR) for detecting MRSA nasal colonization of infants was evaluated and compared to culture‐based methods. The effect of skin decolonization in affected infants was studied. Methods: Paired nasal swabs were collected from infants in our neonatal unit over a 12‐month period (September 2007–2008). Colonization with MRSA was determined with a commercially available PCR method and compared to culture. Results: A total of 696 paired nasal swabs were taken. Three infants were colonized at the beginning and were included. There were positive PCRs in 12 infants. Five infants cultured MRSA from a nasal swab at the same time. No infants were culture‐positive when PCR was negative (sensitivity 100%, specificity 99% compared to culture). PCR results were available within 24 h. Five infants were PCR+ and isolated meticillin‐sensitive Staphylococcus aureus. This organism gave a false‐positive PCR result. Two infants transferred in on broad‐spectrum antibiotics were PCR+ and negative by culture. Decolonization led to negative nasal PCR and culture in 4/5 infants to discharge. Conclusions: PCR methods are sensitive and specific for detection of MRSA colonization in newborn infants of all gestations with results 1–2 days before culture.     

Thymic cysts in mediastinal Hodgkin disease

Three cases of proved thymic cysts associated with mediastinal Hodgkin disease are presented. Two illustrate regression of lymphoma with chemotherapy but persistence of thymic cysts. The third case demonstrates a thymic cyst in untreated Hodgkin disease. These cases suggest that such cysts are probably neither coincidental with nor a consequence of therapy but are probably related to initial thymic involvement by Hodgkin disease.     

Pulmonary lymphangiomyomatosis: correlation of CT with radiographic and functional findings

In 14 patients with biopsy-proved lymphangiomyomatosis, disease extent at computed tomography (CT) was correlated with findings at chest radiography and pulmonary-function testing. The CT scans and chest radiographs were read independently by two chest radiologists. Disease extent was assessed on CT scans by using a visual score (0%-100% involvement of the lung parenchyma) and on radiographs by using an adaptation of the International Labour Office classification of the pneumoconioses. There was good concordance between the two observers for CT and radiographic scores (Kendall tau greater than or equal to .86, P less than .01). A significant but relatively low correlation was present between CT findings and radiographic severity of disease (r = .59, P less than .05). Impairment in gas exchange as assessed with the diffusing capacity correlated better with disease extent seen on CT scans (r = .69) than with chest radiographic findings (r = .59). Three patients had evidence of parenchymal disease on the CT scans but not on the radiographs. In one patient CT findings were negative despite a positive finding on chest radiographs. The authors conclude that CT is superior to chest radiography in the assessment of patients with lymphangiomyomatosis.     

Isolation of an abnormal protein C molecule from the plasma of a patient with thrombotic diathesis

Protein C has been purified from the plasma of a patient with thrombotic diathesis. Both before and after isolation, the protein showed reduced capacity to hydrolyze synthetic substrates and to anticoagulate plasma. Proteolysis with the soluble thrombin- thrombomodulin complex proceeded normally and to completion as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Approximately one-third of the protein is functional, indicating a heterozygous defect. Indirect studies suggest that the abnormal component can bind to protein S and phospholipids. Both forms of activated protein C can also incorporate radiolabeled diisopropylfluorophosphate.     

Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.