PSNAV2.0-TNFα重组质粒的构建及其在体外的表达

目的:在前期微囊化基因工程细胞制备平台的基础上,构建分泌型人肿瘤坏死因子α的真核表达载体PSNAV2.0-TNFα重组质粒,并鉴定其蛋白的体外瞬时表达,为进一步利用该基因进行微囊化细胞移植治疗和改善疾病奠定基础。方法:实验于2006-06/2007-05在解放军总医院老年医学研究所细胞生物学实验室完成。①以含有人肿瘤坏死因子αcDNA序列的质粒为模板,通过PCR扩增获得人肿瘤坏死因子α基因片段;将其定向插入真核表达载体PSNAV2.0中,获得重组质粒PSNAV2.0-TNFα。采用SalⅠ和EcoRⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。②利用阳离子脂质体介导法,将其转染到人胚胎肾细胞HEK-293细胞中,构建可持续分泌人肿瘤坏死因子α的基因工程细胞,采用RT-PCR法和Western blot法检测转染细胞培养上清液中人肿瘤坏死因子蛋白的体外瞬时表达。结果:①通过SalⅠ和EcoRⅠ双酶切、PCR及测序鉴定证明:在HEK-293中插入片段正确。②采用RT-PCR和Western blot法检测表明HEK-293细胞培养上清中有人肿瘤坏死因子α蛋白,Mr17000。结论:成功构建了重组质粒PSNAV2.0-TNFα真核表达载体,转染HEK-293细胞后可有效分泌人肿瘤坏死因子α蛋白,并能分泌到细胞外。     

Use of the gel agglutination technique for determination of fetomaternal hemorrhage

BACKGROUND: Adequate administration of Rh immune globulin requires an accurate determination of the number of D-positive cells in the circulation of D-negative women. Although several tests have been described for the detection of fetomaternal hemorrhage, there is still a need for a rapid, simple, and clinically relevant screening test. STUDY DESIGN AND METHODS: Serial dilutions of a monoclonal anti-D were incubated with stock solutions (0.2 mL) of adult D-negative red cells in the absence or presence of various amounts of fetal D-positive cells (0.1, 0.2, 0.3, 0.4, and 0.5%). After incubation, the supernatants were tested against D-positive red cells by using the new, gel agglutination technique (GAT). After the GAT was adapted to detect D-positive cells at concentrations of > or = 0.2 percent, unselected postpartum samples from D-negative women (n = 420) who delivered D-positive infants were analyzed by both the new test and the Kleihauer-Betke test (KBT). RESULTS: Three of a total of 420 postpartum samples were positive (> or = 0.4% fetal cells), and 406 were negative in both tests. One had 0.5- percent fetal cells in the KBT and gave negative results in the GAT. The latter test was, however, performed after administration of Rh immune globulin. The KBT gave false-positive results in two cases, because of hereditary persistence of hemoglobin F, and the GAT gave a false-positive reaction in one case because of a maternal weak D variant. In the remaining seven cases, the KBT results were only weakly positive (0.2%) and could not be attributed solely to D positive red cells. CONCLUSION: The GAT is suited for routine screening. It provides rapid and specific detection of D-positive red cells at clinically relevant concentrations. The test may (rarely) yield false-negative results due to insufficient administration of Rh immune globulin before testing.     

Epidemiologic background and long-term course of disease in human immunodeficiency virus type 1-infected blood donors identified before routine laboratory screening. Transfusion Safety Study Group

BACKGROUND: The long-term course of human immunodeficiency virus type 1 (HIV-1)-related disease among seropositive blood donors has not been described. The enrollment and epidemiologic background of HIV-1- infected donors in the Transfusion Safety Study and their immunologic and clinical progression are described. STUDY DESIGN AND METHODS: Through the testing of approximately 200,000 sera from donations made in late 1984 and early 1985, 146 anti-HIV-1-positive donors and 151 uninfected matched donors were enrolled. These two cohorts were followed with 6-month interval histories and laboratory testing. RESULTS: Seropositive donors detected before the institution of routine anti-HIV-1 screening disproportionately were first-time donors and men with exclusively male sexual contacts. The actuarial probability of a person's developing AIDS within 7 years after donation was 40 percent; the probability of a person's dying of AIDS was 28 percent. AIDS developed more often when the donor was p24 antigen-positive at donation. Over a 3-year period, significant decreases occurred in CD4+, CD2+CD26+, CD4+CD29+, and CD20+CD21+ counts, but not in CD8+ subsets, CD20+, or CD14+. CONCLUSION: The high proportions of first-time donations and exclusively homosexual men among seropositive donors suggest that test-seeking may have contributed to the high HIV-1 prevalence in the repository. Implementation of alternative test sites when routine donor screening began in 1985 may have averted many high- risk donations. The disease course in HIV-1-infected donors had the same wide spectrum of immunologic and clinical manifestations as were reported for other cohorts.     

Cerebral Tuberculoma

SUMMARY Intracranial tuberculoma has become a rarity. It remains a curable lesion that responds well to medical therapy. Although diagnosis in developed countries is often made only postoperatively, early and effective treatment can be instituted if a high index of suspicion is maintained and diagnostic criteria are looked for. A case is presented which illustrates the difficulties in reaching a diagnosis, and a review of the literature is given.     

Regional changes in monoamine metabolism in the aging constant estrous rat

Studies were undertaken to determine levels of monoamines and their metabolites in brain regions in young (3–4 months) normally cycling and old (25–26 month) constant estrous female rats. Dopamine (DA) concentrations were reduced in old rats in the median eminence (ME), medial basal hypothalamus (MBH), preoptic area-anterior hypothalamus (POA-AH) and the striatum. Similarly, concentrations of dihydroxyphenylacetic acid (DOPAC), the major acid metabolite of DA, were reduced significantly in all 4 regions. In the ME, a strong positive correlation was observed between DA and DOPAC concentrations in both young and old rats. Concentrations of norepinephrine (NE) were reduced in old rats in the MBH and POA-AH but not in the ME or striatum. Concentrations of serotonin (5HT) and its major metabolite, 5-hydroxyindoleacetic acid (5HIAA) were generally unchanged with age in all of the regions examined. These studies indicate the age-related regional alterations in DA and 5HT metabolism can be monitored by methods which quantitate monoamines and their metabolites.     

Dose-dependent effects of chronic treatment with estradiol or progesterone on LH secretion in ovariectomized rats

Ovariectomized (OVX) Sprague-Dawley rats bearing atrial cannulae were implanted subcutaneously with fused pellets containing estradiol (E2) or progesterone (P4). Variable doses of E2 (0.1, 0.5, 1 and 5%) or P4 (10, 50, 75, 100%) were achieved by varying the ratio of the hormone to cholesterol (CHOL) in the pellet. Control groups were treated with CHOL containing pellets. Blood samples were collected in the morning and afternoon the day before and 1, 2, 5, 8, 11 and 14 days after pellet implantation. The concentrations of E2, P4 and LH were measured by RIA. Throughout the sampling period, plasma concentrations of both steroids were proportional to pellet composition. On days 1 and 2, high concentrations of E2 and P4 in plasma were obtained, but between days 5 and 14 stable levels at E2 and P4 were observed. The effectiveness of chronic replacement with E2 and P4 on the negative feedback on LH secretion was assessed from morning samples and positive feedback on LH from afternoon samples. E2 implants suppressed the morning LH levels in plasma in a time and dose-dependent manner. The afternoon concentrations of LH were significantly elevated on each sampling day, except for day 1. P4 pellets had no effect on morning-afternoon difference in LH level, but low doses suppressed LH shortly after the implantation and high doses suppressed LH level after the 8th day of implantation. These results indicate that fused pellets of E2 and P4 are effective in chronically maintaining plasma E2 and P4 at levels observed during various normal and pathological reproductive states. Further, these studies indicate that E2 can stimulate afternoon hypersecretion of LH for at least 14 days in ovariectomized rats.     

Estradiol protects against cerebellar damage and motor deficit in ethanol-withdrawn rats.

On the basis of findings obtained from this study, we hypothesize that the female sex steroid 17beta-estradiol (E(2)) protects against cerebellar neuronal damage and behavioral deficit in rats withdrawn from chronic ethanol exposure. Ovariectomized rats implanted with E(2) or an oil pellet received liquid ethanol (7.5% [wt./vol.]) or dextrin diet for 5 weeks, followed by 2 weeks of ethanol withdrawal. On termination of diet administration, rats were tested for both overt withdrawal signs and latency (seconds) to fall from an accelerating rotarod in six consecutive sessions (the longer the latency, the better the performance). The initial latency was measured separately to assess motoric capacity before learning occurred. Rats were then killed, and cerebella were prepared for accessing of Purkinje cell damage. The study revealed three specific findings. (1) In the absence of E(2), the ethanol withdrawal group showed higher total ethanol withdrawal sign scores than those for the dextrin group, whereas the score for the ethanol withdrawal group was lower in the presence of E(2). (2) In the absence of E(2), the ethanol withdrawal group showed shorter rotarod latency than that for the dextrin group, whereas the latency for the ethanol withdrawal group increased in the E(2)-treated group. In ethanol withdrawal groups, E(2) treatment also resulted in a longer latency than that observed with oil treatment in the initial session and in subsequent sessions. (3) Purkinje cell numbers in the ethanol withdrawal group without E(2) were lower than those in dextrin groups and in the ethanol withdrawal group with E(2) treatment. These findings support the suggestion that E(2) exerts protective effects against withdrawal signs, cerebellar neuronal damage, and motoric impairment in subjects exposed to, and withdrawn from, chronic ethanol exposure.