改性羟基磷灰石骨修复纳米复合材料的制备及生物学评价

目的:制备羟基磷灰石/聚乳酸聚乙醇酸骨修复材料,并对其进行生物学评价。方法:实验于2006-06/2007-02在中科院长春应用化学研究所完成材料制备,在吉林大学基础医学院实验动物中心完成动物实验。将低聚乳酸的羧基与羟基磷灰石表面的钙原子用化学键连接,得到表面接枝聚左旋乳酸的羟基磷灰石,将其与聚乳酸聚乙醇酸共混,得到复合材料PLLA-g-HA/PLGA。溶于氯仿后铺膜(厚0.2mm),用DMEM培养液浸泡材料膜制备浸提液。首先,进行材料生物安全性实验:①细胞毒性实验:将浸提液与培养液混合,接种兔成骨细胞,培养24h,MTT法检测细胞增殖,计算细胞增殖率和细胞毒性级(细胞毒性级0或1级为合格)。②全身毒性实验:小鼠以50mL/kg的剂量静脉注射浸提液,观察72h内小鼠中毒症状。③皮肤刺激实验:兔脊柱两侧皮内注射材料浸提液,观察72h内皮肤有无异常反应。④热原实验:自兔耳缘静脉注入浸提液(10mL/kg)。注射后每0.5h测肛温1次,共6次,以6次中最高的1次减去正常体温,计为升高度数。其次,对复合材料进行细胞黏附性检测:将复合材料制成1%氯仿溶液,涂于硅化的盖玻片上,置于6孔板,每孔接种1×105个成骨细胞,培养3d,在2,24,72h行FITC荧光染色,数码摄像系统拍摄细胞荧光照片。结果:制备了新型PLLA-g-HA/PLGA复合材料。①生物安全性实验结果:MTT实验检测复合材料细胞增殖率为94.8%,细胞毒性级为1级;全身毒性实验中动物无死亡、惊厥、瘫痪、呼吸抑制、腹泻和体质量下降等不良反应;热原实验中兔体温最大的变化值是0.25℃(国家标准为<0.6℃);皮肤刺激实验中未见任何刺激反应,无红斑、焦痂、水肿表现。②细胞黏附性实验结果:细胞接种后2h可见少量细胞开始贴壁;24h时可见贴壁细胞明显增多,并呈聚集生长;培养3d后可见细胞逐渐融合,细胞状态良好。结论:新型PLLA-g-HA/PLGA复合材料符合生物材料细胞毒性要求,按毒性剂量分级属无毒级,无致热原性、对皮肤无刺激作用,具有良好的生物相容性和细胞黏附性。     

Graft outcomes in pediatric kidney transplantation: focus on the role of race

While significant racial disparities in graft outcome persist among adult and pediatric kidney transplant recipients in the US, some international studies do not show these differences. The aim of this study is to examine predictors of graft outcomes and the impact of race in our pediatric kidney transplant cohort. Records of 109 pediatric kidney transplant recipients performed at our institution between 7/99 and 4/07 were studied. Patients were grouped based on race: African-American (AA) vs. non-AA. Fifty-five AA (12 ± 5 years) and 54 non-AA patients (11 ± 6 years) were studied. There were more females, pre-emptive transplants and living donors in the non-AAs. Survival analysis showed significantly higher rejection rates in AAs, P = 0.02, and lower unadjusted graft survival (P = 0.09). Cox Proportional Hazards Survival Regression Analysis revealed biopsy-proven acute rejection and delayed graft function contributed to worse graft survival, while pre-emptive transplantation had a favorable effect. Race was not an independent risk factor for decreased graft survival in the final model. In conclusion, our cohort showed several modifiable risk factors that can partially account for poorer graft survival in pediatric AA kidney transplant recipients.     

Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.     

The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active "phospholipid replacing" activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.     

Drug-induced thrombocytopenia is associated with increased binding of IgG to platelets both in vivo and in vitro

Thrombocytopenia is a common serious adverse effect of drug treatment. A variety of in vitro diagnostic techniques to confirm the diagnosis are available, but the majority lack sufficient sensitivity to detect all cases of drug-induced thrombocytopenia. We studied 19 patients with suspected drug-induced thrombocytopenia and demonstrated that platelet- associated IgG (PAIgG) was elevated in all at the time of thrombocytopenia, and PAIgG returned to normal levels as the thrombocytopenia resolved. In the majority of patients, the platelet count rapidly returned to normal after the drug was discontinued; however, in six patients, the thrombocytopenia persisted well beyond the period of time that the offending drug would be expected to be cleared from the blood. In 13 patients, serum obtained after recovery was used to identify the drug responsible for the thrombocytopenia in an in vitro assay. In all cases, the addition of the drug historically associated with the thrombocytopenic episode was associated with an increased binding of IgG to control platelets. For uncertain reasons, the concentration of drug required to increase the in vitro binding of IgG to test platelets was often more than the concentration usually achieved in vivo. Wider application of these techniques may provide better understanding of the clinical characteristics and mechanisms responsible for drug-induce thrombocytopenia.     

Combined use of nonmyelosuppressive nitrosourea analogues with hydroxyurea in the induction of F-cell production in a human erythroleukemic cell line

OBJECTIVE: Although hydroxyurea (HU) has been used clinically to treat patients with sickle cell disease (SCD), not all patients benefit from HU treatment due to its toxicity. The objective of this study was to investigate the effectiveness of the use of two new Hb F-inducing nitrosourea analogues, 2-[3-(2-methyl, 2-nitroso) ureido]-2-deoxy-D-glucopyranose (MNGU) and 2-[3-(2-chloroethyl) ureido]-2-deoxy-D-glucopyranose (CGU), in combination with HU in K562 cells or erythroid progenitors. MATERIALS AND METHODS: After K562 cells were cultured with different concentrations of HU with CGU or MNGU, aliquots of the cells were obtained to determine the total (benzidine-positive) hemoglobin level, number of F cells, and Hb F level. Erythroid progenitor cells of SCD patients and healthy donors were cultured with the optimal drug concentrations, and the number of BFU-E and Hb F level were determined. RESULTS: Our results showed that the combined use of HU with CGU or MNGU increased the number of both benzidine-positive normoblasts and F cells in a synergistic manner. Further, a lower concentration of HU was required to induce a significant level of Hb F synthesis when combined with either of the two compounds in comparison with treatment with HU alone. On day 4, the number of benzidine-positive cells was 4.5- to 6.5-fold and the number of F cells was 5.0- to 8.0-fold higher than the respective numbers in the untreated K562 cells. Similarly, a 3.2- to 14.3-fold induction of Hb F was obtained when human erythroid progenitors from SCD patients were treated with the same drug combinations. CONCLUSION: Based on these results, the use of CGU or MNGU in combination with HU might offer substantial benefits to patients with SCD and other hemoglobinopathies.     

Inflammatory myofibroblastic tumor of the liver

Inflammatory myofibroblastic tumor is a rare benign entity formerly known as inflammatory pseudotumor. Involvement of the liver is extremely rare. There are controversies about the optimal treatment of this benign entity. Newer reports suggest an association with autoimmune sclerosing pancreatitis and primary sclerosing cholangitis. We present a case of an 18-year-old patient with biliary obstruction from a perihilar mass of the liver requiring hepatic resection. Division of the hepatic bile duct resulted in drainage of yellow, thick, gelatinous material in the presence of benign margins and absence of cholangitis. Histological examination showed a mass with fibroblastic and myofibroblastic cells set in a loose myxoid matrix containing scattered lymphocytes, consistent with an inflammatory myofibroblastic tumor. One-year recovery was uneventful. This report discusses the presentation, diagnosis, and controversies in management of this disease.