鼠胚神经干细胞体外培养方法的优化

目的:神经干细胞能够分化为神经元和神经胶质细胞,但其纯化、培养技术尚不完善通过不同接种密度和传代方法,筛选优化神经干细胞的体外培养技术。方法:实验于2006-05/12在四川大学移植免疫实验室完成。①动物:选取清洁级孕12～16 d雌鼠,实验过程中对动物的处置符合动物伦理学标准。②实验方法:取孕鼠胚胎脑皮质,胰蛋白酶 乙二胺四乙酸消化组织,分离神经干细胞,加入含B27、碱性成纤维细胞生长因子、表皮生长因子的DMEM/F12培养基.传至第3代时.调整细胞密度至1×10~7L~(-1),1×10~8L~(-1),1×10~9L~(-1),1×10~(10)L~(-1)进行培养。另取原代培养7～10 d后的神经干细胞,收集形成的细胞球,机械吹打法传代是离心后用由粗到细的吸管轻柔吹打.或用带5号针头的无菌注射器吹打分离细胞.操作中避免产生气泡.使用注射器时吹打的次数保持5次。胰酶消化联合机械吹打法传代是离心后加入胰蛋白酶,37℃水浴10min,用由粗到细且经火焰抛光的吸管轻柔吹打.或用带5号针头的无菌注射器吹打.以胎牛血清终止消化。③实验评估:观察第3代神经干细胞生长状态.MTT法检测各密度下培养1,3,5,7 d时的增殖情况。计数两种传代方法亚代神经干细胞形成的克隆球数目,比较增殖效果。免疫荧光染包检测神经球巢蛋白、BrdU标记、神经元特异性烯醇化酶、胶质细胞原纤维酸性蛋白的表达。结果:①神经干细胞的培养和鉴定:从胚胎大鼠脑皮质分离培养出的细胞可聚集成球状.并在体外大量扩增和长期存活,巢蛋白、神经元特异性烯醇化酶、胶质细胞原纤维酸性蛋白均呈阳性表达,免疫荧光染色可见大量BrdU标记的阳性细胞。②不同接种密度对神经干细胞增殖的影响:按1×10~9L~(-1)密度培养的细胞分裂增殖最为迅速.于第7天达高峰,且克隆球生成数量明显多于1×10~7L~(-1),1×10~8 L~(-1),1×10~(10)L~(-1)密度(P均<0.05)。③不同传代方法对亚代神经干细胞克隆增殖效果的影响:采用机械吹打法传代的神经干细胞所增殖形成的细胞球结合紧密,抛光玻璃管 注射器吹打后细胞球能较好地分开.但不能完全分散成单细胞.传代后细胞能够继续生长,并形成亚代神经干细胞球。经胰酶消化的神经球在机械吹打后也不能完全形成单细胞,且其亚代克隆的形成能力明显低于机械吹打法(P<0.05)。结论:①从胚胎大鼠脑皮质分离培养出的细胞具有神经干细胞特性,可诱导分化为神经元和星形胶质细胞②1×10~9L~(-1)为最佳细胞接种密度,机械吹打法亚代克隆的活性和增殖能力明显优于胰酶消化联合机械吹打法。     

On improvement in ejection fraction with iron chelation in thalassemia major and the risk of future heart failure

Background

Trials of iron chelator regimens have increased the treatment options for cardiac siderosis in beta-thalassemia major (TM) patients. Treatment effects with improved left ventricular (LV) ejection fraction (EF) have been observed in patients without overt heart failure, but it is unclear whether these changes are clinically meaningful.

Methods

This retrospective study of a UK database of TM patients modelled the change in EF between serial scans measured by cardiovascular magnetic resonance (CMR) to the relative risk (RR) of future development of heart failure over 1 year. Patients were divided into 2 strata by baseline LVEF of 56-62% (below normal for TM) and 63-70% (lower half of the normal range for TM).

Results

A total of 315 patients with 754 CMR scans were analyzed. A 1% absolute increase in EF from baseline was associated with a statistically significant reduction in the risk of future development of heart failure for both the lower EF stratum (EF 56-62%, RR 0.818, p < 0.001) and the higher EF stratum (EF 63-70%, RR 0.893 p = 0.001).

Conclusion

These data show that during treatment with iron chelators for cardiac siderosis, small increases in LVEF in TM patients are associated with a significantly reduced risk of the development of heart failure. Thus the iron chelator induced improvements in LVEF of 2.6% to 3.1% that have been observed in randomized controlled trials, are associated with risk reductions of 25.5% to 46.4% for the development of heart failure over 12 months, which is clinically meaningful. In cardiac iron overload, heart mitochondrial dysfunction and its relief by iron chelation may underlie the changes in LV function.     

Recurrent recurrent gallstone ileus

We describe the second reported case of three consecutive episodes of gallstone ileus and ask the question whether recurrent gallstone ileus justifies definitive surgery to the fistula itself or can be safely managed by repeated enterotomies.     

Autologous bone marrow transplantation in acute leukemia: a phase I study of in vitro treatment of marrow with 4- hydroperoxycyclophosphamide to purge tumor cells

This phase I study was conducted to determine the maximal safe concentration of 4-hydroperoxycyclophosphamide (4HC) that could be used for in vitro treatment of bone marrow from patients with acute leukemia undergoing autologous bone marrow transplantation. Concentrations of 40 to 120 micrograms/mL of 4HC were used in 30 patients with relapsed or high-risk acute leukemia and in six patients with nonleukemic malignancies. All patients received marrow-lethal cytoreductive therapy followed by infusion of the 4HC-treated marrow. Complete inhibition of granulocyte and macrophage colony-forming cells was obtained at 80 micrograms/mL. Nevertheless, only one transplant-related death and otherwise full hematologic recovery was observed at concentrations of 4HC up to 100 micrograms/mL. At 120 micrograms/mL, there were three transplant-related deaths, including two of the three patients who required the infusion of reserve marrow. Among the acute leukemia patients, three remain in complete remission at 1,337, 1,017, and 967 days after transplant. Among the nonleukemic patients, two remain in complete remission at 1,081 and 1,017 days after transplant. At the maximum safe concentration of 4HC (100 micrograms/mL), satisfactory hematologic recovery can be obtained, despite elimination of detectable hematopoietic progenitors.     

NITRIC OXIDE-DEPENDENT ENDOTHELIAL FUNCTION IS UNAFFECTED BY ALLOPURINOL IN HYPERCHOLESTEROLAEMIC SUBJECTS

1. Hypercholesterolaemia is associated with abnormal endothelium-related vasodilator function, possibly due to increased destruction .NO by superoxide anions (.O2-). Oxypurinol, a xanthine oxidase (XO) inhibitor with anti-oxidant properties and the active metabolite of the commonly used drug allopurinol, reduces .NO quenching in vitro and has been reported to acutely improve endothelial function in hypercholesterolaemic subjects. 2. The purpose of the present study was to determine whether oral allopurinol improves .NO dilator function in hypercholesterolaemic subjects. 3. A randomized double-blind, placebo-controlled cross-over design evaluated the effect of allopurinol (300 mg daily for 4 weeks) on forearm blood flow (FBF) responses to intrabrachial infusion of acetylcholine (ACh), sodium nitroprusside (SNP) and NG-monomethyl-L-arginine (L-NMMA) in nine hypercholesterolaemic subjects. 4. Endothelium-dependent vascular responses to ACh and L-NMMA were not significantly altered by allopurinol. The endothelium-independent vasodilator response to SNP was similarly unchanged. 5. These results indicate that allopurinol does not influence basal or stimulated activity of the .NO dilator system in hypercholesterolaemic subjects. If intracellular .O2- inactivation .NO is responsible for endothelial dysfunction in hypercholesterolaemia, the source may be other than XO dependent. However, generation of .O2- during the conversion of allopurinol to oxypurinol could offer an alternative, and probably more likely, explanation for the ineffectiveness of allopurinol in vivo.     

Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality.

The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.     

A Review of Immunotherapy for Stage III and Metastatic Non-Small Cell Lung Cancer and the Rationale for the ECOG-ACRIN EA5181 Study

ECOG‐ACRIN EA5181 is a phase III prospective, randomized trial that randomizes patients undergoing chemo/radiation for locally advanced non‐small cell lung cancer (LA‐NSCLC) to concomitant durvalumab or no additional therapy, with both arms receiving 1 year of consolidative durvalumab. Radiation dose escalation failed to improve overall survival in RTOG 0617. However, conventionally fractionated radiation to 60 Gy with concomitant chemotherapy is associated with a high risk of local failure (38%–46%). It is hoped that concomitant immunotherapy during chemo/radiation can help decrease the risk of local failure, thereby improving overall survival and progression‐free survival with acceptable toxicity. In this article, we review conventional chemo/radiation therapy for LA‐NSCLC, as well as the quickly evolving world of immunotherapy in the treatment of non‐small cell lung cancer and discuss the rationale and study design of EA5181.Implications for PracticeThis article provides an up‐to‐date assessment of how immunotherapy is reshaping the landscape of metastatic non‐small cell lung cancer (NSCLC) and how the impact of this therapy is now rapidly moving into the treatment of patients with locally advanced NSCLC who are presenting for curative treatment. This article reviews the recent publications of chemo/radiation as well as those combining immunotherapy with chemotherapy and chemo/radiation, and provides a strategy for improving overall survival of patients with locally advanced NSCLC by using concomitant immunotherapy with standard concurrent chemo/radiation.     

MR coronary angiography: 2001 update

Cardiovascular magnetic resonance (CMR) has developed multiple techniques that have made it possible to overcome the substantial difficulties in imaging coronary arteries. Tortuous small coronary arteries are imaged in 3D-volume data sets. Cardiac motion is reduced by diastolic gating with ultra-fast sequences. Respiration is suppressed by breath-holding or respiratory gating. Signal-to-noise can be increased with contrast agents. In clinical trials CMR has been successfully used to assess coronary artery stenoses, coronary artery bypass grafts, and anomalous coronary arteries. Recent developments in steady state imaging, volume selective imaging with tracking, parallel imaging techniques, vessel wall imaging, and intravascular contrast agents may soon enable CMR of the coronary arteries to become an effective and widespread clinical tool.     

Acute activation of eNOS by statins involves scavenger receptor-B1, G protein subunit Gi, phospholipase C and calcium influx

Background and purpose:

Statins (HMG CoA reductase inhibitors) have beneficial effects independent of reducing cholesterol synthesis and this includes their ability to acutely activate endothelial nitric oxide synthase (eNOS). The mechanism by which this occurs is largely unknown and thus we characterized the pathways by which statins activate NOS, including involvement of scavenger receptor-B1 (SR-B1), which is expressed in endothelial cells and maintains cholesterol concentrations.

Experimental approach:

Nitric oxide production was monitored in bovine aortic endothelial cells (BAECs) exposed to lovastatin (LOV) or pravastatin (PRA) for 10–20 min, alone or following pre-exposure to the end product of HMG-CoA reductase (mevalonate), G protein inhibitors (pertussis/cholera toxins), phospholipase C (PLC) inhibitor (U-73122), or intracellular and extracellular calcium chelators – BAPTA-AM and EGTA (respectively), or a function blocking antibody to SR-B1.

Key results:

Both statins increased NO production in a rapid, dose-dependent and HMG-CoA reductase-independent manner. Inhibiting Gi protein or PLC almost completely blocked statin-induced NO generation. Additionally, removing extracellular calcium inhibited statin-induced NO production. COS-7 cells co-transfected with eNOS and SR-B1 increased NO production when exposed to LOV or high-density lipoprotein (HDL), an agonist of SR-B1. These effects were not observed in COS-7 cells with eNOS alone or co-transfected with bradykinin receptor 2, indicating specificity for SR-B1. Further, pretreatment of BAEC with blocking antibody for SR-B1 blocked NO responses to statins and HDL.

Conclusions and implications:

LOV and PRA acutely activate eNOS through pathways that include the cell surface receptor SR-B1, Gi protein, phosholipase C and entry of extracellular calcium into endothelial cells.