抗菌肽基因在E.Coli JM109中的克隆表达

合成5条50～70 bp的DNA片段,用"片段拼凑"的方法通过三步PCR扩增得到目的基因,天蚕素基因被克隆到PUC19载体上,再转化大肠杆菌JM109.电泳分析重组质粒,确定天蚕素基因已转化到JM109上.摇床发酵转化子,IPTG诱导表达目的肽,最后提纯表达产物.SDS-PAGE电泳分析表明有特异性带出现,荧光色谱分析表明表达肽与目的肽组成一致,最后确定天蚕素在JM109中得到表达.抑菌圈活性实验表明,表达肽具有一定抗菌活性.     

Validation and reproducibility of computerised cell-viability analysis of tissue slices

Background

The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results.

Methods

To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups – unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes.

Results

Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77).

Conclusions

This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

     

金雀异黄素及二个结构改造物与抗肿瘤、抗血小板作用的关系

目的;合成金雀异黄水溶性衍生物并研究它们在抗肿瘤,抗血小板作用中的影响。方法：通过浓硫酸酯化金雀异黄素（Ｇ）,加入饱和ＮａＣｌ溶液,获得二个水溶性衍生物;金雀异黄素－４’－硫酸酯钠（Ｇ１）的金雀异黄素－７,４‘－二硫酸酯二钠（Ｇ２）。通过     

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid cell-specific monoclonal antibodies

Second or third chemotherapy-induced remissions in acute myelogenous leukemia (AML) are limited by early relapse of the leukemia. We developed monoclonal antibodies (MoAbs) that are cytotoxic to myeloid leukemia cells to treat bone marrow from these patients ex vivo for autologous transplantation. In this pilot study, bone marrow was harvested from ten patients with AML in remission, treated with one or two complement-fixing MoAbs, PM-81 and AML-2-23, which react with myeloid differentiation antigens, incubated with rabbit complement, and cryopreserved. These MoAbs were chosen because they have broad reactivity with AML cells but not with pluripotent progenitor cells. At the time of transplant, 6 patients were in second complete remission, 1 each was in third complete or partial remission, and 2 were in early first relapse. The patients were treated with cyclophosphamide (60 mg/kg a day for 2 days) and total body irradiation (200 cGy twice a day for 3 days) and given infusions of MoAb-treated bone marrow. Full bone marrow reconstitution was observed in eight patients; two patients did not recover platelets. Seven of the ten patients are surviving and disease-free at 21.0, 15.0, 13.0, 10.0, 6.0, 3.0, and 2.0 months posttransplant. Treating bone marrow with MoAbs to myeloid differentiation antigens does not interfere with pluripotential stem cell engraftment. Longer follow-up and a controlled study are necessary to prove that the apparent efficacy of this therapeutic approach in some patients is attributable to MoAb-mediated killing of leukemia cells.     

SYBR Green Ⅰ联合TaqMan荧光定量PCR检测HBV-DNA的意义

目的探索SYBR GreenI联合TaqMan荧光定量PCR检测HBV-DNA的意义。方法选择浓度为108.48、105.70和103.70copies/ml的3种HBV-DNA阳性血清和<1×103.0copies/ml的阴性血清各1份,在TaqMan-PCR混合反应体系中加入SYBR Green I组成双荧光PCR(TaqMan SYBR Green I组),同时进行TaqMan和SYBR Green I的单荧光PCR(分别为TaqMan组和SYBR Green I组),设置同一PCR和熔解曲线的循环参数,检测HBV-DNA含量及其Tm,每种方法一次检测每份血清5次。结果TaqMan SYBR Green I组检测的HBV-DNA阳性血清均为阳性,其平均含量为108.55±、105.79±、103.81±,与TaqMan组的108.49±、105.69±、103.72±copies/ml0.320.290.300.310.300.25对应浓度值取10对数比较,无统计学意义(t=0.31、0.54和0.27,P>0.05);与SYBR Green I组的108.41±、0.35105.21±和103.26±copies/ml(不含未检出的两次血清)比较,除高浓度外,中低浓度有统计学意义(t=2.90和0.340.262.62,P<0.05)。TaqMan SYBR Green I组和SYBR Green I组阳性血清均出现明显熔解曲线,熔解温度(Tm)分别为71.8℃、72℃和79.8℃,阴性血清未出现扩增曲线和Tm值。结论SYBR Green I联合TaqMan-PCR检测HBV-DNA时,具有能维持TaqMan-PCR的高灵敏度、特异性更强,并能同时检测HBV-DNATm的特点,为HBV的DNA多态性分析,尤其是在HBV基因分型方面提供了新的检测思路。