5-氮胞苷诱导骨髓间充质干细胞向心肌细胞的分化

目的:目前在5-氮胞苷诱导骨髓间充质干细胞向心肌细胞分化领域中,对用于诱导的细胞代次选择不一。实验选择第2,9代骨髓间充质干细胞,观察比较经5-氮胞苷体外诱导后其各自向心肌细胞分化过程中心肌特异性肌钙蛋白cTnT和早期分化基因的表达。方法:实验于2005-07/2007-07在天津中医药大学病理三级实验室完成。①实验方法:清洁级Wistar雄性大鼠,脱颈处死后取双侧股骨、胫骨,采用密度梯度离心法分离骨髓单个核细胞进行原代培养。去掉原代细胞瓶内的培养液,D-Hank’s液冲洗去除血清,胰蛋白酶 乙二胺四乙酸消化,制备单细胞悬液,按1∶3传代。用10μmol/L的5-氮胞苷分别诱导第2,9代骨髓间充质干细胞,诱导4周,并设立未加诱导剂的阴性对照。②实验评估:倒置相差显微镜下观察细胞的生长情况及形态变化;免疫组织细胞化学法检测心肌特异性肌钙蛋白cTnT的表达,胞浆出现棕黄色颗粒状物质为阳性;嵌合荧光法检测心肌早期基因NKx2.5、GATA-4、Desmin、α-actin的相对表达量。结果:①细胞形态学观察:5-氮胞苷诱导前细胞生长较快,诱导后死亡细胞较多且生长较慢。诱导后2,3,4周,第9代骨髓间充质干细胞无论在形态和细胞活力方面均优于第2代。②心肌特异性肌钙蛋白cTnT的表达:5-氮胞苷诱导4周后,第9代骨髓间充质干细胞心肌特异性肌钙蛋白cTnT阳性表达率明显高于第2代(22.42±9.97),(11.22±5.62)%,P<0.05,阴性对照无阳性表达。③心肌早期基因的表达:反转录-聚合酶链反应结果显示,5-氮胞苷诱导4周后,第9代骨髓间充质干细胞心肌早期基因NKx2.5、GATA-4、Desmin、α-actin的相对表达量均明显高于第2代(P<0.05)。结论:经5-氮胞苷诱导后,骨髓间充质干细胞表达心肌特异性肌钙蛋白cTnT及4种心肌早期基因,证实其能够向心肌细胞分化,且传至第9代时向心肌细胞分化的能力要强于第2代。     

T lymphocytes specific for immunoglobulin allotype. II. Cloned Igh-1(b)- specific cytotoxic T cells

This study describes long-term-cultured lines and clones of cytotoxic T cells (Tc) with specificity for determinants of the Igh-1(b) immunoglobulin allotype. These Tc clones were initiated by repeated stimulation of immune spleen cells from BALB/c mice with an Igh-1(b)-producing myeloma, and then they were maintained in medium supplemented with mitogen-induced growth factors in the absence offurther antigenic stimulation . The lytic potency of these clones was 30-100-fold greater than the primary cultures from which they were derived, and specificity studies showed them to be lytic for Igh-1(b) targets and not for targets expressing Igh-1(a) or Igh-4(b), nor the lipopolysaccharide blasts . Finally, soluble preparations of Ig were tested for their ability to block lysis of labeled Igh-1(b)-expressing targets. The results showed that Igh-1(b) and not other immunoglobulin allotypes or isotypes could block lysis, and that the mechanism of lytic inhibition is due to Igh-1(b)-induced autolysis of the killer cells.     

Regulation of human monocyte surface antigen expression. I. Up- modulation of Mo3e antigen expression on U-937 and HL-60 cells stimulated by pharmacologic activators of protein kinase C

Mo3e is a protein (p 50,80) that is expressed on the surface of human monocytic cells after exposure in vitro to soluble activating factors that include bacterial lipopolysaccharide, muramyl dipeptide, and phorbol myristate acetate (PMA). The surface expression of Mo3e may represent a cellular event that occurs in response to the formation of "secondary messengers" that include diacylglycerol, inositol trisphosphate, and calcium ions. This postulate is based on the stimulatory effect of agents that can mimic the activity of endogenous diacylglycerol (PMA and other biologically active phorbol compounds, mezerein, and L-alpha-1,2 dioctanoylglycerol) and inositol trisphosphate (ionomycin) on Mo3e expression by U-937 and HL-60 cells. The inhibitory effect of phospholipid-active calmodulin inhibitors (trifluoperazine, chlorpromazine, and dibucaine), calcium antagonists (nicardipine and TMB-8), and EGTA further support the involvement of phospholipid- and calcium-dependent protein kinase (protein kinase C) and calcium ions in the up-modulation of Mo3e surface expression.     

Mitochondrial dysfunction in skeletal muscle of children with cardiomyopathy

OBJECTIVES: This study sought to examine skeletal muscle of children with cardiomyopathy (CM) for changes in mitochondrial enzyme activities and in mitochondrial DNA (mtDNA). BACKGROUND: Heart mitochondrial enzymatic activity defects have been often found in dilated and hypertrophic CM. The defects primarily involve the activities of the electron transport system and oxidative phosphorylation pathway including respiratory complexes I, III, IV, and V. METHODS: Skeletal muscle biopsies of 8 children with CM were examined for specific mitochondrial enzyme activities, mtDNA copy number and the presence of pathogenic mutations and deletions in mtDNA. RESULTS: A marked deficiency in specific mitochondrial enzyme activities was found in 6 of 8 patients in skeletal muscle as well as in 2 of 3 hearts of those in whom cardiac tissue was available. Specific activity defects were found in complex I (2 cases), complex III (5 cases), complex IV (3 cases), and complex V (4 cases). Complex II and citrate synthase activities were unaffected. None of the previously reported pathogenic mutations associated with CM were detected, nor was there any evidence of mtDNA depletion. The incidence of defective respiratory complex activities in skeletal muscle was similar to the incidence of defective complex activities previously reported in cardiac tissue. CONCLUSIONS: Mitochondrial analysis of skeletal muscle is warranted in the overall clinical evaluation of children with CM, and particularly before consideration for cardiac transplantation.     

Six-month follow-up of children with obstructive sleep apnoea

OBJECTIVES: This study examined prospectively changes in development, temperament and sleep related behaviour in children referred for obstructive sleep apnoea (OSA) and polysomnographic sleep study, some of whom had surgical intervention. METHODOLOGY: Using a prospective cohort study design, parents of 56 children referred for OSA completed sleep and temperament questionnaires and their child was assessed developmentally at the time of the polysomnographic sleep study. Forty (72%) of the children were neurologically normal. At 6 months, 42 children were reassessed using sleep and temperament questionnaires and a developmental assessment. After excluding the primary snorers, subjects were categorised as having had intervention (n = 24) or not (n = 15), and differences over the 6-month period in Griffiths scores, temperament and sleep related behaviour were examined. RESULTS: Regardless of intervention status, there was an improvement in night-time and day-time sleep behaviour for the total group, though the extent of improvement was more marked in the intervention group. For the neurologically normal children, improvement in the sleep behaviour was only significant for the intervention group (P < 0.05). Intervention did not result in any significant changes in Griffiths developmental score or temperament. CONCLUSION: Surgical intervention improves sleep behaviour in children though not temperament or development.     

Effect of vitamin E on chromosomal aberrations in lymphocytes from patients with Down's syndrome

A possible protective effect of vitamin E (DL-alpha-tocopherol) on chromosomal damage was evaluated in lymphocytes from patients with Down's syndrome (DS) and from controls. This included the analysis of the basal and G2 chromosomal aberration frequencies in lymphocytes cultured with and without 100 microM vitamin E. The chromosomal damage in G2 was determined by scoring the number of chromosomal aberrations in lymphocyte cultures treated with 5 mM caffeine, 2 h before harvesting. Vitamin E treatment decreased the basal and G2 chromosomal aberrations both in control and DS lymphocytes. In DS cells, this protective effect, expressed as a decrease in the chromosomal damage, was greater (50%) than in controls (30%). These results suggest that the increment in basal and G2 aberrations yield in DS lymphocytes may be related to the increase in oxidative damage reported in these patients.