凋亡素原核表达载体的构建和表达

目的 构建凋亡素原核表达系统，以制备抗原物质凋亡素融合蛋白。方法 通过PCR方法，以pcDNA-VP3质粒为模板，扩增出凋亡素VP3基因。将其克隆到原核表达载体pET-DsbA的多克隆位点，构建成凋亡素的高效原核表达栽体pET-DsbA-VP3，将该质粒转化到大肠杆菌E．coliBL21(DE3)plysS中，以异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside，IPTG)对其进行诱导表达，聚丙烯酰胺凝胶电泳分析目的蛋白。结果 转化有凋亡素原核表达载体pET-DsbA-VP3的大肠杆菌E．coliBL21(DE3)plysS经IPTG诱导后，聚丙烯酰胺凝胶电泳出现38300的目的蛋白条带。结论 凋亡素原核表达栽体pET-DsbA-VP3能高效表达出凋亡素融合蛋白。     

Epidermal growth factor receptor inhibition modulates the nuclear localization and cytotoxicity of the Auger electron emitting radiopharmaceutical 111In-DTPA human epidermal growth factor.

(111)In-DTPA-human epidermal growth factor ((111)In-DTPA-hEGF [DTPA is diethylenetriaminepentaacetic acid]) is an Auger electron-emitting radiopharmaceutical that targets EGF receptor (EGFR)-positive cancer. The purpose of this study was to determine the effect of EGFR inhibition by gefitinib on the internalization, nuclear translocation, and cytotoxicity of (111)In-DTPA-hEGF in EGFR-overexpressing MDA-MB-468 human breast cancer cells. METHODS: Western blot analysis was used to determine the optimum concentration of gefitinib to abolish EGFR activation. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled hEGF were evaluated by confocal microscopy in MDA-MB-468 cells (1.3 x 10(6) EGFRs/cell) in the presence or absence of 1 microM gefitinib. The proportion of radioactivity partitioning into the cytoplasm and nucleus of MDA-MB-468 cells after incubation with (111)In-DTPA-hEGF for 24 h at 37 degrees C in the presence or absence of 1 microM gefitinib was measured by cell fractionation. DNA double-strand breaks caused by (111)In were quantified using the gamma-H2AX assay, and radiation-absorbed doses were estimated. Clonogenic survival assays were used to measure the cytotoxicity of (111)In-DTPA-hEGF alone or in combination with gefitinib. RESULTS: Gefitinib (1 microM) completely abolished EGFR phosphorylation in MDA-MB-468 cells. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled EGF were not diminished in gefitinib-treated cells compared with controls. The proportion of internalized (111)In that localized in the nucleus was statistically significantly greater when (111)In-DTPA-hEGF was combined with gefitinib compared with (111)In-DTPA-hEGF alone (mean +/- SD: 26.0% +/- 5.5% vs. 14.6% +/- 4.0%, respectively; P < 0.05). Induction of gamma-H2AX foci was greater in MDA-MB-468 cells that were treated with (111)In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL) plus gefitinib (1 microM ) compared with those treated with (111)In-DTPA-hEGF alone (mean +/- SD: 35 +/- 4 vs. 24 +/- 5 foci per nucleus, respectively). In clonogenic assays, a significant reduction in the surviving fraction was observed when (111)In-DTPA-hEGF (5 ng/mL, 6 MBq/microg) was combined with gefitinib (1 microM ) compared with (111)In-DTPA-hEGF alone (42.9% +/- 5.7% vs. 22.9% +/- 3.6%, respectively; P < 0.01). CONCLUSION: The efficacy of (111)In-DTPA-hEGF depends on internalization and nuclear uptake of the radionuclide. Nuclear uptake, DNA damage, and cytotoxicity are enhanced when (111)In-DTPA-hEGF is combined with gefitinib. These results suggest a potential therapeutic role for peptide receptor radionuclide therapy in combination with tyrosine kinase inhibitors.     

尺神经腕背支营养血管逆行皮瓣的临床应用

目的探讨应用尺神经腕背支营养血管逆行皮瓣修复小指皮肤缺损的临床效果及手术操作要点。方法以尺神经腕背支营养血管远端为蒂,选择小指尺侧分支为皮瓣轴心血管,将皮瓣向小指远端转移,修复小指掌背侧皮肤缺损8例。结果8例全部成活,其中1例因创面止血不彻底,皮瓣受压导致远端部分坏死,经换药后痊愈。结论尺神经腕背支走向较恒定,本组未发现变异。沿皮神经干有纵行的皮神经旁血管网及皮神经干内血管网,此皮瓣血供可靠的,皮瓣切取容易,对供区影响小,是修复小指皮肤缺损的理想方法。     

X线引导下经皮通用脊柱系统内固定治疗胸腰椎骨折

目的探讨X线引导下经皮通用脊柱系统(USS)内固定治疗胸腰椎骨折的可行性.方法 2002年10月～2004年5月对无须减压胸腰椎骨折8例采用后路经皮USS椎弓根螺钉植入,在椎旁肌深层置入固定棒行伤椎复位固定.结果手术时间120～240 min,平均140 min.术中出血量20～90 ml,平均40 ml.后凸Cobb's角术前(21.5±4.3)°、术后(2.7±1.5)°(t=17.541,P=0.001),椎体塌陷术前18.5%±4.1%、术后2.4%±1.0%(t=16.504,P=0.001).术后3周佩戴腰围支具下床,复查Frankel分级,C级恢复至D级2例,D级恢复至E级1例,保持D级1例,保持E级4例.结论经皮USS内固定治疗无须减压的胸腰椎骨折创伤小,恢复快,但技术难度高,X线暴露时间长.     

湿热环境下密闭性功能敷料护理伤口对β—内啡肽的影响

OBJECTIVE: To study the changes of plasma beta-endorphin (beta-EP) in pigs with traumatic injury after occlusive wound dressing supplemented with antiphlogistic and analgesic agents in hot and humid environments (HHE). METHODS: Traumatic models were established in 10 pigs, 5 of which received antiphlogistic- and analgesic-supplemented occlusive dressings of the wounds (experiment group, EG), while the rest pigs were assigned to control group (CG) to receive routine wound management. The pigs in both groups were then exposed to artificial HHE and at different time points during the exposure, the plasma beta-EP level, respiratory frequency and heart rates were measured respectively. RESULTS: The plasma beta-EP concentration of EG was significantly lower than that of CG (P <0.01) after the injury, but in both groups, the levels before the injury were similar to those measured at hour 8 during HHE exposure and at hour 24 following the injury. The variation range of the respiratory frequency and heart rates during HHE exposure were significantly smaller in EG than CG (P <0.01). CONCLUSION: This supplemented occlusive wound dressing can help restrain the peak of plasma beta-EP level and the variation range of respiratory frequency and heart rates of pigs exposed to HHE.     

血清铁和铁蛋白与肝病患者肝纤维化指标的关系

OBJECTIVE: To study the relationship of serum iron and ferritin with the indicators for hepatic fibrosis and hepatic iron overload. METHODS: Liver tissue specimens were obtained from 41 patients with benign (16) or malignant (25) liver diseases by 1 second liver biopsy, and routine microscopic examination was performed after haematoxylin-eosin (HE) and Perl's Prussian staining. Atomic absorption spectrum, radioimmunoassay and enzyme-linked immunosorbent assay were respectively employed to examine the serum levels of iron, ferritin, hyaluronic acid, laminin, human procollagen type , and collagen type . RESULTS: Between patients with benign and malignant liver diseases, significant differences were found in the serum ferritin levels (P < 0.05), but not in serum iron levels (P > 0.05). It was also noted that the levels of the indicators for hepatic fibrosis in patients with benign and early-stage malignant diseases varied significantly from the levels in normal subjects, but these differences were not observed between normal subjects and patients with end-stage hepatic malignancies. Serum iron and ferritin were found to be associated with serum laminin levels (serum iron: r=0.439, P=0.031; serum ferritin: r=0.476, P=0.016), and no iron granules detected in the tissue specimens of hepatocellular carcinoma. CONCLUSIONS: Most of the patients with hepatocellular carcinoma have elevated serum ferritin levels. The serum levels of iron and ferritin are statistically correlated with serum laminin level. Obvious reduction of iron content is typical of hepatic malignant tissues in comparison with the benign tissues, and the reduction in the levels of the indicators for hepatic fibrosis might involve the inhibition of collagen synthesis in the tumor tissues from patients with end-stage hepatocellular carcinoma. Most of the cases of alcoholic fatty liver are complicated by liver iron overload, often marked by serum iron and ferritin levels.     

膀胱出口梗阻后逼尿肌收缩蛋白表达和膀胱重量的改变

目的探讨膀胱出口梗阻(BOO)后逼尿肌收缩蛋白表达和膀胱重量的改变及意义。方法BOO组病人16例,筛选条件为入院诊断良性前列腺增生症(BPH)并经尿动力学压力-流率检查证实为高压低流型;对照组5例,为外伤等情况入院并排除有下尿路梗阻病史者。BOO组所有病例均行耻骨上经膀胱前列腺摘除术,术前B超检查测定膀胱重量和前列腺内外径比值,术中切取膀胱上壁组织2cm×1cm×1cm大小,标本行RT-PCR反应,检测膀胱逼尿肌中肌动蛋白和肌球蛋白mRNA的表达,并比较其与膀胱重量间的线性相关性。结果BOO组与对照组膀胱重量分别为(92.15±34.89)g和(56.08±20.35)g,(P<0.05);前列腺内外径比值分别为(0.57±0.16)和(0.18±0.06),(P<0.01);与对照组相比,BOO组肌动蛋白和肌球蛋白mRNA的表达量均有显著增加,分别为(40.32±59.67)×106和(6.59±5.62)×106,(P值均<0.01);且两者表达量与膀胱重量之间均有明显线性正相关性(P<0.05)。结论逼尿肌中肌动蛋白和肌球蛋白的表达与膀胱逼尿肌的功能状态密切相关。     

Extremely High Association Between Appearance of HLA Antibodies and Failure of Kidney Grafts in a Five-Year Longitudinal Study

Longitudinal studies were conducted over a five-year period for HLA antibodies on 493 sera tested from 54 kidney transplant patients. HLA single antigen beads were employed to establish donor specificity of the antibodies. Only 3 of 22 patients without antibodies rejected a graft in contrast to 17 out of 32 patients with posttransplant antibodies (p = 0.003). Using a serum creatinine value of 4.0 mg/dL as the cut-off for a failed graft, 4 of 22 patients without antibodies failed compared to 21 of 32 with antibodies (p = 0.0006). Among patients with donor-specific antibodies (DSA) 13 of 15 failed (p = 0.000004). Even among patients with non-donor specific antibodies (NDSA), 8 of 17 failed (p = 0.05). Among patients who could be identified as making de novo antibodies (since they developed antibodies while not having antibodies for more than six months after transplantation), 6 of 11 failed (p = 0.03). Sequential testing for HLA antibodies shows that antibodies appear prior to a rise in serum creatinine and subsequent graft failure. The very strong association between the production of HLA antibodies after transplantation and graft failure indicates the importance of monitoring for posttransplant HLA antibodies.