转染癌基因的骨髓基质干细胞体外分化特征

目的:观察转染癌基因的骨髓基质干细胞在体外分化情况,为肝细胞癌的细胞源研究提供实验依据。方法:实验于2003-05/2004-06在南方医科大学药理教研室实验室完成。①两步法获取大鼠肝细胞,梯度离心法分离大鼠骨髓基质干细胞。②单基因转染是单独将c-myc或K-ras癌基因瞬时转染大鼠骨髓基质干细胞,6孔培养板中培养,24h后荧光显微镜下观察骨髓基质干细胞转染结果。双基因转染步骤相同,只是将c-myc和K-ras癌基因同时转染大鼠骨髓基质干细胞。③c-myc癌基因转染组、K-ras癌基因转染组、双癌基因转染组常规培养,加入含体积分数为0.1胎牛血清的DMEM培养基,于37℃、体积分数为0.05的CO2孵箱培养,每24h半量更换培养液。④c-myc癌基因转染 肝细胞组、K-ras癌基因转染 肝细胞组、双癌基因转染 肝细胞组将已转染癌基因的骨髓基质干细胞,置于叠加的培养板半透膜的上方(细胞密度均为1×105个/cm2),再将肝细胞置于半透膜的下方(每孔细胞密度为3×105/cm2)进行共培养,其余步骤同常规培养。⑤通过反转录聚合酶式反应和细胞免疫组化检测骨髓基质干细胞分化情况。结果:①癌基因转染24h骨髓基质干细胞检测结果:单独转染c-myc或K-ras癌基因的细胞,其绿色荧光蛋白呈均匀一致分布;双基因转染的细胞,绿色荧光蛋白呈点片状分布。②各组骨髓基质干细胞向肿瘤细胞分化检测结果:c-myc癌基因转染组、K-ras癌基因转染组、双癌基因转染组的骨髓基质干细胞,均未向肿瘤细胞分化;c-myc癌基因转染 肝细胞组、K-ras癌基因转染 肝细胞组、双癌基因转染 肝细胞组的骨髓基质干细胞,均向肝细胞癌发展;空白对照组骨髓基质干细胞细胞均为阴性。此外,双癌基因转染 肝细胞组的骨髓基质干细胞分化增殖迅速,反转录聚合酶式反应和免疫组化检测发现,培养第7天出现甲胎蛋白表达,并迅速增加,而第7天出现的白蛋白和细胞角蛋白18表达迅速减弱,第14天消失。结论:转染癌基因的骨髓基质干细胞,在向肝细胞诱导的条件下,部分癌基因可以使干细胞分化为肝癌细胞;多癌基因转染时,更易于使干细胞分化为肝癌细胞。     

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

Serologic test for syphilis as a surrogate marker for human immunodeficiency virus infection among United States blood donors

BACKGROUND: This study evaluated the usefulness of the serologic test for syphilis (STS) in preventing the transmission of human immunodeficiency virus (HIV), hepatitis B and C viruses, and human T- lymphotropic virus via the transfusion of seronegative, infectious window-period blood. STUDY DESIGN AND METHODS: Demographic and laboratory information on blood donations made between January 1992 and June 1994 in 18 American Red Cross regions was analyzed. It was assumed that the same proportion of HIV-positive and HIV-infectious window- period donations reacted on STS and were negative on other screening tests (hepatitis B and C viruses and human T-lymphotropic virus). This proportion multiplied by the estimated number of HIV-infectious window- period donations is the number of post-screening HIV-infectious donations removed by STS. RESULTS: Of 4,468,570 donations, 12,145 (0.27%) were STS positive and 377 (0.008%) were HIV positive. Among donations that were negative on other screening tests, STS-reactive donations were 12 times more likely to be HIV positive (odds ratio = 11.9; 95% CI = 5,26). However, of an estimated 13 infectious window- period donations, 0.2 would have been removed because of a reactive STS, at a cost of over $16 million. CONCLUSION: STS is a poor marker and a costly strategy for preventing post-screening HIV infections and other blood-borne diseases.     

Polycystic Kidney Disease Re-evaluated: A Population-based Study

A genetic register of all known cases of autosomal dominantpolycystic kidney disease occurring in South and Mid-Wales hasbeen established. In a population of 2.1 million, 209 familieswith affected members were identified, 303 of whom are currentlyalive, 70 on renal replacement therapy. An additional 551 caseswould be predicted amongst family members at 50 per cent and25 per cent risk, giving an apparent prevalence of 1:2459 inthe general population. Five possible new mutations were seenwhere adults with phenotypic autosomal dominant polycystic kidneydisease had both parents alive, age > 55 years with no cystsvisible on ultrasound. The take-on rate for renal replacementtherapy increased during 1970–79 but has apparently reacheda plateau of 4.8 cases per million population per year overthe last 8 years, despite a rapidly increasing acceptance ofuraemic patients as a whole (72/10⁶/year in 1988–89).Considerably more patients with autosomal dominant polycystickidney disease aged over 50 years were started on treatmentin 1980–89 than in 1970–79, but the survival overallimproved with time. All cases of autosomal dominant polycystickidney disease reaching end-stage renal disease are now beingtreated, but the apparent clinical prevalence of this conditionin our region is less than half the supposed gene frequency,suggesting that undiagnosed cases have a benign prognosis.     

Inhalational anesthetics inhibit spreading depression: relevance to migraine

Cortical spreading depression (SD) has not been shown in the human neocortex by direct cortical recordings. However, animal studies suggest that cortical injury, such as that occurring during neurosurgical procedures, should result in the initiation of SD. It is possible that inhibition of SD by volatile anesthetic agents may partially explain the failure to observe SD in the human neocortex during surgery. This study examines the effect of the anesthetic agents α-chloralose, halothane, nitrous oxide and isoflurane on the initiation of cortical SD in the cat neocortex. SD was seen in 100% of cats anesthetized with α-chloralose ( n = 15), in 3 of 7 (42%) animals anesthetized with isoflurane ( p < 0.05, χ² with Yates correction) and none of the animals ( n = 4, 6 hemispheric preparations) anesthetized with halothane ( p < 0.005, χ² with Yates correction, halothane vs α-chloralose group). In all cases this inhibitory effect was reversible. In four animals the administration of nitrous oxide (66%) reduced the inspired concentration of isoflurane required to inhibit SD by 0.75%. This study suggests that halothane, and to a lesser extent isoflurane and nitrous oxide, protect against the initiation of cortical SD. This observation may partially explain why SD has not been demonstrated in human neocortex during surgery. Further studies are needed to determine if SD may occur under pathological conditions, such as during migraine with aura, where the cortex may be predisposed to SD.     

文献计量学分析我国双相情感障碍的发展趋势

目的通过分析我国双相情感障碍的文献分布,探讨我国双相情感障碍的发展趋势。方法采用文献计量学方法和MicrosoftExcel技术,通过利用中国生物医学文献数据库中收录的有关以双相情感障碍为主题的文献,从时间分布、单位分布、地域分布、期刊分布、第一作者分布和主要内容等方面,进行多角度文献分析。结果双相情感障碍文献的时间分布、单位分布、地域分布与地区经济发展密切相关;浙江常州全军精神卫生中心解放军第102医院、深圳市精神卫生研究所（深圳市精神卫生中心）、南京医科大学附属脑科医院及在以上单位工作的作者可视为该病的核心研究机构和研究者;《临床精神医学杂志》（6．67％）、《四川精神卫生》（5．14％）、《上海精神医学》（4．72％）、《中国神经精神疾病杂志》（4．16％）可视为该病研究的核心期刊;国内学者对该病的研究主要集中于临床药物治疗、临床诊断、临床病例报告（约占临床研究的61．98％）、流行病学调查（约占预防医学研究的63％）等方面,对于双相情感障碍预防方面（约占论文总数的28．97％）及合并外科疾病围手术期（约占临床研究的4．04％）的专题研究报道较少。结论双相情感障碍研究在国内尚处于初步研究阶段,各地区发展不平衡,对该病的预防和治疗认识不足。各地区应重视该病的防治工作,建立预警机制,制定相应防治措施,防止该病的流行和突发事件的发牛。     

儿童颈动脉内-中膜厚度与肥胖类型相关性的超声评价

目的应用超声手段探测儿童颈动脉内膜-中层厚度（IMT）,探讨其与儿童肥胖类型的关系。方法依体质指数（BMI）、腰围身高比（WHtR）标准,入组外周性肥胖组160名（A组）,内脏性肥胖150名（B组）,正常体重儿童160名（正常对照组）;应用超声手段探测各组儿童内脏脂肪厚度（VFT）和颈动脉内膜-中层厚度,比较3组间各项检测参数。结果内脏肥胖组VFT、IMT均高于外周性肥胖组及正常组,差异具有统计学意义（P〈0.05）;外周性肥胖组VFT、IMT与正常组相似,差异没有显著性。结论IMT与肥胖类型相关,内脏性肥胖儿童VFT、IMT增加。实时超声检查技术为研究儿童肥胖类型提供了一种新的检测手段。     

应用非每日锥形束CT校位减少摆位误差对鼻咽癌调强放疗剂量影响

目的 探讨应用非每日千伏级锥形束CT(KVCBCT)校位能否改善摆位误差对鼻咽癌调强放疗(IMRT)剂量分布影响。方法 对14例行根治性IMRT的鼻咽癌患者治疗开始后连续5次用KVCBCT检测摆位误差,并将其均值作为系统误差预测值,若其＞1.5 mm则在第6次离线校位。假设通过移床能完全校正系统误差,那么从第6次起实际各方向摆位误差值加上离线校位值可得到未行校位时的摆位误差值,在治疗计划系统中通过等中心移位重新计算剂量来模拟应用非每日校位策略前后摆位误差所致的剂量变化。结果 对10例系统误差预测值＞1.5 mm者摆位误差明显降低了靶区剂量:98％大体肿瘤体积(GTV)所接受剂量(GTV-D98)平均减少3.8Gy(Z=-2.81,P=0.005),原发灶临床靶体积(CTVns) D95( CTVns-D95)平均减少4.8Gy(Z=-1.96,P=0.050),高危CTV1 -D95平均减少1.0Gy(Z=-2.82,P=0.005),低危CTV2-D95减少不明显(Z=-0.13,P=0.900)。应用非每日校位后明显减少了摆位误差的三维方向位移总量,均值从3.6 mm减少为2.6mm(t=2.00,P=0.000),GTV-D98平均增加3.8 Gy(Z=-2.70,P=0.007),CTVns-D95平均增加5.0Gy(Z=-2.15,P=0.030),CTV1 -D95平均增加0.9Gy(Z=-2.80,P=0.005),减少了危及器官剂量增加＞3％、5％患者比例。结论 应用非每日KVCBCT校位能有效减少摆位误差对鼻咽癌IMRT剂量分布的不利影响。     

食管癌切除胸腹二区淋巴结清扫手术疗效的研究

目的：分析总结食管癌切除胸腹二区淋巴结清扫的手术疗效。方法：回顾分析1986年2月～2007年12月我院对中下段食管癌和上段食管癌分别采用Ivor—Lewis术式,即上腹正中、右胸后外侧二切口切除及Akiyama术式．即右胸后外侧、上腹正中、左颈部三切口切除,并作胸腹二区淋巴结清扫治疗胸段食管癌1690例的临床资料,总结胸腹二区淋巴结转移的发生率并随访1、3、5年的生存率。结果：全组手术切除率为97．86％（1690／1727）。全组有淋巴结转移782例,占46．27％,其中胸部淋巴结转移占38．93％（658／1690）,腹部淋巴结转移占25．92％（438／1690）,胸部淋巴结转移发生于最上纵隔位于气管食管沟及喉返神经旁占20．47％（346／1690）,术后共有178例发生230例次各种并发症,总的并发症的发生率为13．6％（230／1690）,其中肺部并发症为第一位,占34．3％,心律失常占17．4％,喉返神经损伤发生率为8．7％,吻合口瘘发生率为1．7％。术后1、3、5年的生存率分别为88．2％（1161／1316）、63．5％（634／998）和51．8％（331／639）。无淋巴结转移的5年生存率为65．2％（219／336）,有淋巴结转移的5年生存率为32．3％（102／316）。结论：Ivor-Lewis术式和Akiyama术式胸腹腔有良好的显露,淋巴结清扫彻底、方便,尤其对右侧最上纵隔沿喉返神经旁淋巴结清扫便利。特别对有淋巴结转移的食管癌患者行胸、腹二区淋巴结清扫十分必要,能明显提高术后5年生存率。