A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.     

跟骨定量超声骨质测量参数与骨密度及骨组织形态计量学指标的关系

目的:分析跟骨定量超声骨质测量中各参数与骨密度及形态计量学指标的相关性。方法:选择2004-01/2005-12广州市第六人民医院和中山大学三院骨科小腿以上截肢患者38例,将其跟骨定量超声测定的超声振幅衰减平均值与健康青年人骨峰值进行比较,>-2.5 SD者为骨量正常组(12例),<-2.5 SD者为骨质疏松组(26例)。分别进行跟骨定量超声、双能X线骨密度测量仪及骨形态计量学测量,应用直线相关分析法分析跟骨定量超声测定中各参数与骨密度及骨组织形态计量学各指标的相关性。结果:38例全部进入结果分析。①骨质疏松组跟骨超声振幅衰减平均值和骨硬度指数值均小于骨量正常组(P<0.01)。②骨量正常组跟骨骨密度值显著高于骨质疏松组[(352±16),(233±14)mg/cm2,P<0.01]。③骨量正常组跟骨平均骨小梁间距或弥散度低于骨质疏松组而松质骨体积高于骨质疏松组(P<0.05)。④超声振幅衰减平均值和骨硬度指数与骨密度呈直线正相关(r=0.814,0.326,P<0.01,0.05)。⑤超声传播速度与骨小梁游离末端、平均骨小梁间距呈直线负相关(r=-0.688,-0.712,P<0.01),与小梁间连点数、松质骨体积呈直线正相关(r=0.672,0.794,P<0.01);骨硬度指数与平均骨小梁间距呈直线负相关(r=-0.358,P<0.05),与松质骨体积呈直线正相关(r=0.513,P<0.01)。结论:跟骨定量超声测量中,超声振幅衰减平均值能较好地反映骨的密度,超声传播速度能较好地反映骨的质量,而骨硬度指数能较综合地反映骨强度的改变。     

Clinical and operative experience with non-caucasian patients with Crohn's disease

Fifteen black patients with Crohn's disease were seen during a ten-year period (1975-1985). They represented 11 percent of our experience with Crohn's disease during that time. These patients had an earlier age of onset of Crohn's symptoms than our white patients, and correct diagnosis was delayed for an average of four years. All 15 patients required abdominal surgery, and seven (47 percent) suffered recurrences necessitating additional abdominal operations. The five-year actuarial estimate of probability of reoperation was 77 percent. Extraintestinal manifestations were present in all patients, and six (40 percent) had multiple manifestations. These disease manifestations are more severe than those noted in series that studied predominantly caucasian Crohn's populations, and suggest that Crohn's disease in the black patient is a distinctly aggressive form.     

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 μg of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.     

碱性成纤维细胞生长因子在激素性股骨头缺血性坏死模型兔体内变化及桃红四物汤的干预

目的:已有研究证实,活血化瘀中药可对抗激素的作用,对激素性股骨头缺血性坏死早期有明显的防治作用。观察桃红四物汤对激素性股骨头缺血性坏死碱性成纤维细胞生长因子表达的影响,进一步阐明活血化瘀法防治激素性股骨头缺血性坏死的机制。方法:实验于2005-04/2006-10在福建中医学院中心实验室完成。①实验材料:健康成年新西兰大白兔60只,雌雄各半,体质量1.8～2.2kg,清洁级,由福建中医学院实验动物中心购自上海市松江区松联实验动物场。桃红四物汤的组成成分为:桃仁、红花、熟地、当归、白芍、川芎。②实验方法:60只健康成年新西兰大白兔,随机分为正常组6只和实验组54只。实验组采用贺氏造模法建立股骨头缺血坏死模型,6周后处死正常组和实验组各6只,确定造模成功。将造模剩余动物随机分为中药治疗组和激素对照组,每组21只:中药治疗组以桃红四物汤7mL/kg(浓度为0.75g/mL)灌胃治疗,1次/d,激素对照组以生理盐水7mL/kg灌胃治疗,1次/d。③实验评估:于治疗后1,2,4周进行股骨头组织病理学观察,并采用放射免疫法检测血清碱性成纤维细胞生长因子含量变化。结果:纳入新西兰大白兔60只,实验过程中因心脏、脑血管梗死死亡18只,进入结果分析42只。桃红四物汤灌胃治疗1,2,4周后,中药治疗组股骨头局部空骨陷窝数明显减少,与激素对照组比较,差异有显著性意义(P<0.05),中药治疗组碱性成纤维细胞生长因子表达增强,与激素对照组相比,差异有显著性意义(P<0.01)。结论:桃红四物汤能促进激素性股骨头缺血性坏死模型兔体内碱性成纤维细胞生长因子的表达,明显减少股骨头局部空骨陷窝数,促进坏死股骨头的修复,这可能是活血化瘀法防治激素性股骨头缺血性坏死的机制之一。     

丘脑卒中患者认知功能损害及其临床判别

目的:探讨丘脑卒中后认知损害的特点,尝试应用回归函数方程推测和判别这种损害程度。方法:①选择2004-04/12大连市第二人民医院和大连市海港医院收治,并经CT确诊的丘脑卒中患者21例为丘脑卒中组;按1∶3匹配原则,将63例年龄与性别与丘脑卒中组匹配的非脑部疾病者入选为对照组。②丘脑卒中组住院后进行影像学和生理、生化学指标检查,对所有入组人员进行神经心理学测查其认知功能,内容包括4个纬度(智能纬度、执行功能纬度、注意机能纬度和记忆纬度)13个认知因子,以韦氏成人智力量表、韦氏记忆量表和威斯康星卡片分类实验等为测查工具。③将对照组每项认知因子均数根据其在认知中的作用不同,加或减1.64标准差为界值,以判断丘脑卒中组认知损害程度,并建立回归方程和认知损害程度表达式。结果:84例均进入结果分析。①丘脑卒中组在13个认知指标中有9个指标与对照组相比差异显著(P<0.05),4个纬度均有涉及。②建立丘脑组认知损害程度函数表达式,计算出每例认知损害程度函数值(F0)。以丘脑组每例认知损害程度函数值为应变量,以临床影响因子为自变量建立了回归方程Y=43.679-2.304*β。以Y值推测F0值。当Y<0.98,可考虑为轻度认知损害,当Y=0.98～8.15时可考虑为中度认知损害,当Y>8.15时可考虑为重度认知损害。结论:①丘脑卒中可引起广泛的认知损害。②临床可以根据影响因素建立回归方程,计算出Y值,以Y值推测F0值。