骨形态发生蛋白4转基因治疗大鼠的胫骨缺损

目的:观察基因工程技术构建人骨形态发生蛋白4复制缺陷腺病毒的成骨效果。方法:实验于2006-03/08在安徽医科大学第一附属医院实验动物中心完成。实验分组:选取普通级雄性SD大鼠30只,体质量(200±10)g,全部动物胫骨上端部分分别造成8mm×5mm长方形缺损。采用自身对照法,右侧骨缺损为实验组,左侧骨缺损为对照组。实验组植入人骨形态发生蛋白4复制缺陷腺病毒复合明胶海绵,对照组植入单纯明胶海绵。实验评估:术后分别于4,6,8周麻醉后处死10只动物,取材行X线、组织病理、免疫组织化学、透视电镜检查,观察成骨情况。结果:纳入30只大鼠,全部进入结果分析。①大鼠胫骨缺损X线、组织病理学检查结果:术后8周实验组和对照组骨缺损均得到修复,但实验组无论从成骨时间、成骨效果、新生骨量等方面都要优于对照组。其中各时间点实验组骨密度明显高于对照组,差异有显著性意义[4周:(95.91±16.33),(87.93±11.52);6周:(128.34±10.64),(102.41±9.81);8周:(138.36±10.49),(121.56±9.63);P<0.01]。各时间点实验组新生骨占骨缺损面积比明显高于对照组,差异有显著性意义[4周:(41.39±5.65)%,(26.58±5.62)%;6周:(80.35±7.25)%,(65.41±6.52)%;8周:(96.45±2.76)%,(82.22±7.30)%;P<0.01]②术后4周免疫组织化学染色结果:实验组软骨及骨痂内呈强阳性反应,而对照组骨痂内骨形态发生蛋白4表达微弱。结论:人骨形态发生蛋白4重组腺病毒具有良好的成骨活性,骨形态发生蛋白4直接转基因治疗能够加快骨缺损的修复。     

两种荧光显微成像系统亚细胞定位的对比

目的:应用高分辨率荧光显微成像系统采集细胞器探针图像,并与激光共聚焦显微成像系统进行对比。方法:实验于2003-05/2004-01在解放军总医院完成。①实验材料:鼠肺毛细血管内皮细胞株(1H11)由上海复旦张江生物公司提供;荧光探针Rhodamine-123,Lucifer Yellow,DiOC6[3],BODIPY(美国Sigma公司)。②细胞培养及荧光探针染色:细胞培养采用含体积分数为0.2胎牛血清的低糖DMEM培养基,密度5×107L-1。选择Rhodamine-123作为细胞线粒体特异性荧光探针,选择DiOC6[3]作为细胞内质网特异性荧光探针,选择BODIPY作为细胞高尔基体特异性荧光探针,选择Lucifer Yellow作为细胞溶酶体探针。前3个探针在完全避光条件下与培养的细胞共同孵育0.5h,后者则共同孵育15h。③高分辨率荧光成像系统的图像采集:线粒体荧光图像采集,选取经Rhodamine-123共孵育完成的细胞,选择激发滤色镜为BP460-490,吸收滤色镜为BA515,分光镜为DM500,另加一绿通道液晶滤光片,激发出Rhodamine-123的荧光。电荷耦合器件采集图像并送入计算机。重复上述步骤,采用DiOC6[3]标记内质网,BODIPY标记高尔基体,Lucifer Yellow标记细胞溶酶体,激发条件同Rhodamine-123。分别采集同一视野靶细胞DiOC6[3]、BODIPY或Lucifer Yellow的荧光图像,完成全部图像采集并储存在计算机中。④激光共聚焦显微成像系统的图像采集:选择经4种探针染色的靶细胞,使用氩离子激光器在488nm激发Rhodamine-123,Rhodamine-123荧光通过配置有530/60-G发射滤光片的通道1探测。重复上述步骤,在488nm激发DiOC6[3]和BODIPY,在457nm激发Lucifer yellow,3种荧光均由通道1探测,后2个探针的发射滤光片的配置为515/30-G,DiOC6[3]选择530/60-G。由光电倍增管接收信号并传输入计算机成像。结果:①高分辨率荧光成像系统所采集图像,靶细胞中由荧光探针Rhodamine-123染色的线粒体呈多个典型的小棒状或卵圆状,聚集在核周;Lucifer yellow染色的溶酶体呈多个非对称球型,在胞浆内随机分布,颗粒尺寸通常大于线粒体;荧光探针DiOC6[3]着色的内质网占据胞浆的很大空间,以囊状聚集为特征;BODIPY特异性地结合在高尔基体上,荧光图像显示围绕在细胞核周围呈条索状。②与高分辨率荧光成像系统比较,激光共聚焦显微成像系统所采集的图像其荧光强度基本相同,但分辨率低、细节显示模糊、胞浆中细胞器的准确分布信息和形态特征显示效果欠佳。结论:两种荧光显微成像系统均可采集到细胞器探针的荧光图像。但高分辨率荧光成像系统采集的荧光图像具有细节清晰、分辨度高、准确显示胞浆中细胞器的分布信息和形态特征等优点。     

β-锗代-α-氨基酸衍生物的合成及抗肿瘤活性

自六十年代初期发现羧乙基锗倍半氧化物(Ge—132)广泛的生物活性以来,已有许多相应的有机锗化合物被发现,在抗肿瘤活性方面,因有毒性较低、抗瘤谱广等一系列优点而引起人们广泛的兴趣,但由于活性较低影响了应用及发展,据文献报道,有机锗倍半氧     

Improved outcome in adult B-cell acute lymphoblastic leukemia

A total of 68 adult patients with B-cell acute lymphoblastic leukemia (B-ALL) were treated in three consecutive adult multicenter ALL studies. The diagnosis of B-ALL was confirmed by L3 morphology and/or by surface immunoglobulin (Slg) expression with > 25% blast cell infiltration in the bone marrow (BM). They were characterized by male predominance (78%) and a median age of 34 years (15 to 65 y) with only 9% adolescents (15 to 20 y), but 28% elderly patients (50 to 65 y). The patients received either a conventional (N = 9) ALL treatment regimen (ALL study 01/81) or protocols adapted from childhood B-ALL with six short, intensive 5-day cycles, alternately A and B. In study B-NHL83 (N = 24) cycle A consisted of fractionated doses of cyclophosphamide 200 mg/m2 for 5 days, intermediate-dose methotrexate (IdM) 500 mg/m2 (24 hours), in addition to cytarabine (AraC), teniposide (VM26) and prednisone. Cycle B was similar except that AraC and VM26 were replaced by doxorubicin. Major changes in study B-NHL86 (N = 35) were replacement of cyclophosphamide by ifosphamide 800 mg/m2 for 5 days, an increase of IdM to high-dose, 1,500 mg/m2 (HdM) and the addition of vincristine. A cytoreductive pretreatment with cyclophosphamide 200 mg/m2, and prednisone 60 mg/m2, each for 5 days was recommended in study B-NHL83 for patients with high white blood cell (WBC) count (> 2,500/m2) or large tumor burden and was obligatory for all patients in study B-NHL86. Central nervous system (CNS) prophylaxis/treatment consisted of intrathecal methotrexate (MTX) therapy, later extended to the triple combination of MTX, AraC, and dexamethasone, and a CNS irradiation (24 Gy) after the second cycle. Compared with the ALL 01/81 study where all the patients died, results obtained with the pediatric protocols B-NHL83 and B-NHL86 were greatly improved. The complete remission (CR) rates increased from 44% to 63% and 74%, the probability of leukemia free survival (LFS) from 0% to 50% and 71% (P = .04), and the overall survival rates from 0% to 49% and 51% (P = .001). Toxicity, mostly hematotoxicity and mucositis, was severe but manageable. In both studies B-NHL83 and B-NHL86, almost all relapses occurred within 1 year. The time to relapse was different for BM, 92 days, and for isolated CNS and combined BM and CNS relapses, 190 days (P = .08). The overall CNS relapses changed from 50% to 57% and 17%, most probably attributable to the high-dose MTX and the triple intrathecal therapy. LFS in studies B-NHL83 and B-NHL86 was significantly influenced by the initial WBC count < or > 50,000/microL, LFS 71% versus 29% (P = .003) and hemoglobin value > or < 8 g/dL, LFS 67% versus 27% (P = .02). Initial CNS involvement had no adverse impact on the outcome. Elderly B- ALL patients (> 50 years) also benefited from this treatment with a CR rate of 56% and a LFS of 56%. It is concluded that this short intensive therapy with six cycles is effective in adult B-ALL. HdM and fractionated higher doses of cyclophosphamide or ifosphamide seem the two major components of treatment.     

Ambiguous phenotypes and genotypes in 16 children with acute leukemia as characterized by multiparameter analysis

Ambiguous phenotypes and genotypes were observed in 16 children with acute leukemia. Surface marker, cytogenetic, molecular genetic, and DNA flow cytometric analyses as well as standard morphologic and cytochemical studies were used to divide the patients into three groups. The first group comprised five children with acute leukemia whose blast cells were morphologically lymphoid, while immunophenotyping disclosed simultaneous expression of early pre-B cell and myeloid features. Molecular genetic studies showed evidence of heavy-chain immunoglobulin (Ig) gene rearrangements in all patients. Cytogenetic data, available in three of these children, revealed t(4;11). In five of the 16 patients, morphologic and surface marker analyses indicated the coexistence of two separate cell populations, one with myeloid and the other with early pre-B cell features. Further evidence of B cell commitment in these patients was provided by demonstration of Ig heavy-chain gene rearrangements in all five patients. Surprisingly, one of the five patients showed oligoclonal Ig heavy-chain as well as monoclonal gene rearrangement for the beta chain of the T cell receptor (beta-TCR). The last group consisted of four cases with otherwise typical acute lymphoblastic leukemia (ALL), early pre-B cell phenotype, and coexpression of myeloid or T cell-associated antigens, and two children with unequivocal acute myeloid leukemia (AML) and coexpression of T cell antigens. Gene rearrangement of Ig heavy-chain could be demonstrated in five of six patients, additional Ig light-chain gene rearrangement in two children with ALL, and bigenotypic features (Ig heavy-chain and beta-TCR gene rearrangement) in one patient. In none of the 16 patients did flow cytometry disclose clonal abnormalities of leukemic cell DNA content. Based on these findings, we suggest that malignant transformation in the first and second group of patients took place at a stage ontogenetically close to the pluripotent stem cell, whereas ambiguous phenotypes in the third group resulted from aberrant gene expression or insufficient reagent specificity.     

Selective targeting of human lymphokine-activated killer cells by CD3 monoclonal antibody against the interferon-inducible high-affinity Fc gamma RI receptor (CD64) on autologous acute myeloid leukemic blast cells

The potential of the CD3 monoclonal antibody (MoAb) OKT3 to selectively target lymphokine-activated killer (LAK) cells and T-cell clones in vitro against autologous tumor cells was studied using material from patients with acute leukemias (19 acute myeloid leukemias [AML], and 3 acute lymphoblastic leukemias [ALL]). Cytotoxicity mediated by patient LAK cells against AML blasts, but not against ALL cells and autologous Epstein-Barr virus-transformed B cells, was enhanced 1.5-fold to 9.3- fold by OKT3 in all AML patients studied. The following findings suggest that the major target molecule on AML cells for OKT3-coated LAK cells is the high-affinity Fc receptor for IgG (Fc gamma RI; CD64): (1) susceptibility to killing by OKT3-coated effector LAK cells segregated with target cell expression of CD64; (2) preincubation of AML blasts with monomeric OKT3 (murine IgG2a), the Fc portion of which is known to have preferential binding affinity to CD64, resulted in lysis by autologous T cells that were not spontaneously cytotoxic; (3) OKT3- dependent increase in lysis of primary and relapsed AML cells by autologous T-cell clones correlated with the amount of target cell expression of CD64; (4) anti-leukemic cytotoxicity of OKT3-coated T cells could partially be inhibited by monomeric human Ig, the natural ligand of CD64; and (5) expression of CD64 (Fc gamma RI) on fresh AML cells could be increased by interferon-gamma (IFN-gamma) and IFN-alpha translating into further enhancement of lysis by autologous OKT3-coated LAK cells. Nonmalignant CD34+ cells sorted from peripheral blood were found to lack expression of CD64 and hence were not affected by OKT3- triggered T-cell targeting, as detected by colony formation assays. In conclusion, the in vitro data presented provide a rationale for the combined clinical use of recombinant interleukin-2, IFN-gamma, and low doses of CD3 MoAb to eliminate AML cells while sparing nonmalignant hematopoietic progenitor cells, for example, in the setting of purging procedures for autologous bone marrow transplantation.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Biochemical markers of bone turnover in seronegative spondylarthropathy: relationship to disease activity

To investigate bone turnover in patients with seronegative spondylarthropathy, a bone formation marker, type 1 procollagen carboxy- terminal propeptide (P1CP), and resorption markers, the pyridinium cross-links of collagen [urinary free (f) PYR and DPYR], were measured. The median f-PYR, f-DPYR and P1CP (+/-interquartile range) were 15.8 (6.00) nmol/mmol creatinine, 3.8 (2.2) nmol/mmol creatinine and 101.5 (38) micrograms/1, respectively. There was a positive correlation between resorption markers and acute-phase reactants such as C-reactive protein (r = 0.42 for PYR, r = 0.42 for DPYR, P < 0.05), and a negative correlation observed between P1CP and the erythrocyte sedimentation rate (r = -0.64, P < 0.05). In the subgroup of patients with an elevated CRP concentration, the concentration of PYR and DPYR was significantly increased (f-PYR 25.7 vs 15.8 and f-DPYR 6.6 vs 3.8, P < 0.01 for f-PYR, P < 0.05 for f-DPYR). This study suggests than an elevation in acute-phase response in patients with seronegative spondylarthropathy is associated with increased concentration of bone resorption markers with a tendency for reduction in bone formation markers. This may represent uncoupling of bone formation and resorption, leading to bone loss in such patients.      

Digital and conventional chest images: observer performance with Film Digital Radiography System

The Film Digital Radiography System (FilmDRS) is a device with a laser optical film digitizer, 2,000 X 2,000 X 12-bit memory, and a 1,000-line video display. To evaluate the adequacy of this device for general radiography of the chest, four readers independently analyzed both radiographs and the corresponding video display of the digitized chest images of 150 patients, consisting of 100 images of abnormalities and 50 normal images. The overall results indicate equal sensitivity for the two systems. The FilmDRS, with interactive windowing, proved superior in the detection of hilar and mediastinal disease. X-ray film was superior in allowing detection of hyperlucent states. There was equivalent sensitivity for other disease categories. Superior specificity was achieved with conventional radiographs.     

Orbital dermoids: features on CT

The computed tomographic (CT) features of orbital dermoids were retrospectively reviewed in 17 patients; 15 of the lesions were proved histologically. On the basis of clinical and CT features, the tumors were classified as superficial or deep. All but one were extraconal in location. Seven lesions appeared cystic; only six showed typical fat density. The presence of a margin or rim, often partially calcified, was identified in ten lesions. Irregular scalloping of adjacent bone was a highly suggestive feature, occurring with 11 dermoids. Other bone changes, such as linear defects, thinning, or sclerosis, also occurred. Superficial dermoids showed less apparent bone changes. An extraconal orbital lesion associated with adjacent bone thinning or notching should raise the possibility of a dermoid, especially if a rim with calcification is seen. The appearance is pathognomonic if fat density is also present.