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.     

酶联免疫吸附试验中HBsAg室内质控参考品的制备和应用

目的建立室内质控参考品以鉴别诊断试剂的质量和控制操作误差,提高乙型肝炎病毒表面抗原的检测准确度。方法以国家标准品为标准制备灵敏度系列参考品、特异性参考品及精密性参考品。检测其稳定性,并进行同厂家、不同批号试剂的比较及不同厂家、三批试剂的比较。结果制备的室内质控参考品在-20℃与4℃保存3～4周检测结果无统计学差异。采用同一厂家、不同批号的HBsAg诊断试剂检测室内质控参考品,结果无统计学差异。采用不同厂家、同一批号的HBsAg诊断试剂检测结果有统计学差异。结论我们建立的室内质控参考品适宜作HBsAg室内质控,提醒我们在更换试剂厂家时应特别注意室内质控参考品的变化。     

Complex and simple coronary artery stenoses: a new way to interpret coronary angiograms based on morphologic features of lesions

For many years, atherosclerotic coronary artery lesions have been described by angiographers only in terms of location and degree of narrowing. However, it has become apparent that coronary stenoses generally have distinct morphologic features that can be recognized at angiography and that allow them to be classified as either "simple" or "complex" plaques. Complex plaques are those characterized by ulcerated or ruptured surfaces, subintimal hemorrhage, superimposed partially occluding thrombi, recanalized thrombi, or some combination. Pathologic studies have shown a very high frequency of these lesions at sites of total thrombotic occlusion of coronary arteries. Clinical and angiographic studies have demonstrated a high frequency of such lesions in living patients with both unstable angina and acute myocardial infarction. The presence of complex stenoses has also been found to increase the risk of future myocardial infarction. Plaque morphology thus appears to significantly affect the prognosis of patients with coronary disease and should be carefully evaluated in interpretation of all coronary angiograms.