富血小板血浆对人骨髓间充质干细胞成骨分化的抑制效应

目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。     

Does the publication of NICE guidelines for venous thromboembolism chemical prophylaxis influence the prescribing patterns of UK hip and knee surgeons?

IntroductionWe assessed the practice of surgeons regarding venous thromboembolism (VTE) chemical prophylaxis for total hip replacement (THR) and total knee replacement (TKR), before and after issuing of updated National Institute for Health and Care Excellence (NICE) guidance in 2018.MethodsA survey, circulated through the British Hip Society and regional trainee networks/collaboratives, was completed by 306 UK surgeons at 187 units. VTE chemical prophylaxis prescribing patterns for surgeons carrying out primary THR (n=258) and TKR (n=253) in low-risk patients was assessed after publication of 2018 NICE recommendations. Prescribing patterns before and after the NICE publication were subsequently explored.ResultsFollowing the new guidance, 34% (n=87) used low-molecular-weight heparin (LMWH) alone, 33% (n=85) aspirin (commonly preceded by LMWH) and 31% (n=81) direct oral anticoagulants (DOACs: with/without preceding LMWH) for THR. For TKR, 42% (n=105) used aspirin (usually monotherapy), 31% (n=78) LMWH alone and 27% (n=68) DOAC (with/without preceding LMWH). NICE guidance changed the practice of 34% of hip surgeons and 41% of knee surgeons, with significantly increased use of aspirin preceded by LMWH for THR (before=25% vs after=73%; p<0.001), and aspirin for TKR (before=18% vs after=84%; p<0.001). Significantly more regimens were NICE guidance compliant after the 2018 update for THR (before=85.7% vs after=92.6%; p=0.011) and TKR (before=87.0% vs after=98.8%; p<0.001).ConclusionOver one-third of surveyed surgeons changed their VTE chemical prophylaxis in response to 2018 NICE recommendations, with more THR and TKR surgeons now compliant with latest NICE guidance. The major change in practice was an increased use of aspirin for VTE chemical prophylaxis.     

Reduced levels of glutathione in liver and tumour tissue of the mouse after administration of eupatoriopicrin

In this study the effect of the cytostatic acting sesquiterpene lactone eupatoriopicrin, isolated from Eupatorium cannabinum L., on gluthathione (GSH) levels in liver and tumour tissue of the mouse is described. C57Bl mice, bearing a solidly growing Lewis Lung carcinoma or a FIO 26 fibrosarcoma, with a volume between 500–1000 μL, were injected with eupatoriopicrin 20 or 40 mg/kg, intraperitoneally (i.p.) or intravenously (i.v.). At different time points, between 0–48 h after administration, the GSH content in liver and tumour tissue was determined. A dose dependent reduction of the GSH content was found. No difference in reduction was seen between i.p. and i.v. administration, but the route of administration appeared to be of great importance regarding acute toxicity: 40 mg/kg i.p. was lethal within 48 h, whereas mice receiving the same dose i.v. survived for over three months.     

Cisplatin sensitivity and thermochemosensitisation in thermotolerant cDDP-sensitive and -resistant cell lines.

Development of thermotolerance is an important phenomenon that must be considered when thermochemotherapy with multiple heat treatments is used clinically. To study the effect of thermotolerance on cellular cisplatin (cDDP) sensitivity at 37 degrees C and 43 degrees C in cell lines with different cDDP sensitivities, two Ehrlich ascites tumour cell lines (one with high cDDP sensitivity and one with in vitro acquired cDDP resistance) were used. The results indicate that in both cell lines the state of thermotolerance per se did not affect the cDDP sensitivity at 37 degrees C. Thus, general elevations in ''all'' heat shock protein levels as found in thermotolerant cells apparently do not influence cDDP sensitivity to a considerable extent. The sensitising effect of a (second) heat treatment given simultaneously with a cDDP treatment was less in thermotolerant cells. Thermal enhancement ratios (TERs) at the 10% survival level for heat doses of 43 degrees C for 30 min or 43 degrees C for 60 min were reduced by a factor of 1.6 and 2.1 in cDDP-resistant and -sensitive thermotolerant cells respectively, as compared with control cells. Thus, protection against heat damage in thermotolerant cells seems to be paralleled by diminished thermal chemosensitisation. Although the effect of thermotolerance on the cDDP-sensitising effect was less pronounced in the resistant cells, a modifying effect on the resistance factor was not achieved.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study on the relationship of serum fetuin-A concentration with aortic stiffness in patients on dialysis.

BACKGROUND: An increase in aortic stiffness, as reflected by an increase in pulse wave velocity (PWV) or aortic augmentation index (AI) is an important predictor of cardiovascular mortality in dialysis patients. Dysregulation of calcification inhibitors, such as fetuin-A, is involved in vascular pathology in dialysis patients and fetuin-A is inversely related to mortality in dialysis patients. In this study, the relation between serum fetuin-A concentration and parameters of aortic stiffness was investigated in patients with end-stage renal disease. METHODS: In a cross-sectional study we included 131 dialysis patients, aged 62+/-14 years (33 on peritoneal dialysis and 98 on haemodialysis), and 41 controls, aged 60+/-8 years. Time-averaged pre-dialysis values of serum albumin, Ca, P and intact parathyroid hormone were included in multiregression analysis, as were high-sensitivity C-reactive protein (hsCRP), fetuin-A, age, mean arterial pressure (MAP) and dialysis modality. PWV and AI were measured with the SphygmoCor device. RESULTS: Mean fetuin-A concentration in dialysis patients (0.63+/-0.16 g/l) did not differ from controls (0.63+/-0.11 g/l). Median hsCRP levels in dialysis patients were higher compared with controls (4.0 vs 1.9 mg/l; P<0.0001). PWV but not AI was higher in dialysis patients than in controls (9.9 vs 7.9 m/s; P<0.0001). In univariate analysis in dialysis patients, fetuin-A levels were inversely related to both PWV (r = - 0.25, P = 0.007) and AI (r = - 0.26, P = 0.006), respectively. However, after correction for age, gender, MAP and diabetes mellitus, this relation lost statistical significance. CONCLUSIONS: In a dialysis population with a relatively low level of inflammatory activity, the soluble calcification inhibitor fetuin-A could not be identified as an independent predictor of aortic stiffness as measured with PWV and AI.     

Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.     

Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

The crystal structure of the dimeric gene V protein of bacteriophage f1 was determined using multiwavelength anomalous diffraction on the selenomethionine-containing wild-type and isoleucine-47-->methionine mutant proteins with x-ray diffraction data phased to 2.5 A resolution. The structure of the wild-type protein has been refined to an R factor of 19.2% using native data to 1.8 A resolution. The structure of the gene V protein was used to obtain a model for the protein portion of the gene V protein-single-stranded DNA complex.     

Radiotherapy planning: direct tumor location on simulation and port films using CT. Part I. Principles

Although there have been great advances in cancer diagnosis in recent years, it remains difficult to transfer tumor location information from cross-sectional computed tomographic (CT) scans or magnetic resonance images to the simulation and verification films used in planning radiotherapy. A newly developed system uses radioopaque markers attached to the patient as reference points. These markers are identified on both CT scans and simulation films and their locations entered into the treatment planning computer. The tumor and any desired normal structures are then outlined manually on each CT section. Transparent overlays produced by the computer show the position of the reference markers and tumor outlines for any combination of gantry angles and source-film distance. Because the overlays are scaled to the simulation films, the reference points enable precise alignment of overlay and film. The tumor outline thus appears on the simulation or verification films exactly as it is "seen" by the therapy beam, making field verification straightforward and accurate, even on oblique films.