人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能

目的:人羊膜间充质细胞具有比骨髓间充质干细胞更强的扩增能力和免疫原性低等优势。建立体外适宜的诱导培养条件,观察人羊膜间充质细胞定向分化为软骨细胞和成骨细胞的能力。方法:实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①材料来源:经产妇知情同意,无菌采集健康足月分娩新生儿胎盘6份,实验经医院医学伦理委员会批准。②实验方法:采用机械法剥离羊膜组织,二步酶消化法分离收获人羊膜间充质细胞,按2.2×10~8L~(-1)密度接种,传至第1～2代用于诱导分化实验。向软骨细胞诱导分化时,人羊膜间充质细胞按3×10~8L~(-1)密度接种,诱导培养液为含体积分数0.01的胎牛血清、10 mg/L转化生长因β1、100 nmol/L地塞米松、50 mg/L抗坏血酸、1%培养基添加物。向成骨细胞诱导分化时,人羊膜间充质细胞按6×10~7L~(-1)密度接种,诱导培养液为含体积分数0.1的胎牛血清、100 nmol/L地塞米松、50 mg/L抗坏血酸、5 mmol/Lβ-甘油磷酸。③实验评估:原代细胞用流式细胞仪分析表型,免疫细胞化学染色进行波形蛋白表达鉴定。分别于体外诱导第7,14,21,28天采用免疫细胞化学法检测软骨特异性Ⅱ型胶原的表达,细胞化学法检测蛋白聚糖的表达,钙-钴法检测成骨细胞特异性碱性磷酸酶的表达,茜素红S检测钙盐沉积情况。结果:①免疫组化与表型特征:人羊膜间充质细胞高表达间充质干细胞表面标志CD29、CD44和间充质细胞标志波形蛋白。②向软骨细胞诱导分化:诱导14 d后,人羊膜间充质细胞由长梭型逐渐变为多角形,可检测到Ⅱ型胶原蛋白表达及软骨细胞特异性细胞外基质蛋白聚糖。③向成骨细胞诱导分化:诱导21 d后,可观察到人羊膜间充质细胞的胞浆内有碱性磷酸酶表达,且可见钙盐沉积。结论:人羊膜间充质细胞具有分化成软骨细胞和成骨细胞的特性,可作为骨及软骨组织工程种子细胞的新来源。     

Pharmacokinetics of cyclosporine in monkeys after oral and intramuscular administration: relation to efficacy in kidney allografting

In cynomolgus and rhesus monkeys, the dose-normalized exposure of cyclosporine administered orally as microemulsion preconcentrate (Neoral) was lower than that upon intramuscular administration. For oral administration, mean values ( ± SD) of C_max, 24-h area-under-the curve (AUC) and 24-h trough level, all normalized for a 1 mg/kg dose, were 20 ± 9 ng kg/mg ml, 210 ± 70 ng h kg/mg ml and 2.6 ± 0.9 ng kg/mg ml, respectively. For intramuscular administration, levels were about 5.5-fold, 9-fold and 22-fold higher. Based on pharmacokinetic data, the efficacy of oral cyclosporine treatment (without any other immunosuppressant) was evaluated in life-supporting cynomolgus monkey kidney allotransplantation. Rejection-free kidney allograft survival could be achieved using oral cyclosporine monotherapy with average 24-h trough concentrations above 100 ng/ml during maintenance treatment. Typically, daily oral doses of 100 mg/kg–150 mg/kg during the first two weeks post-transplantation, followed by daily 30 mg/kg–100 mg/kg dose levels during subsequent maintenance can result in long-term allograft survival, with 24-h average trough levels in individual animals during maintenance between 110 ng/ml and 700 ng/ml. Received: 1 October 1997 Revised: 20 April 2001 Accepted: 7 June 2001     

Surprisingly poor correlation among pain scores prior to total hip arthroplasty

Pain is the main indication for total hip arthroplasty but is a subjective experience and can therefore be difficult to assess. Using completed forms from six hundred consecutive patients who were to undergo a total hip arthroplasty, we investigated the level of correlation between three widely used pain rating scales. These are: visual analogue pain scale, Merle d’Aubigne pain score and the first question on the Oxford Hip Score. We demonstrate here that there is a surprisingly poor correlation between the scores obtained from the three rating scales, albeit statistically significant. The use of more than one of the above pain scores is indicated due to the slightly different ways in which they ask the patients about their pain experience. This undoubtedly contributes to the modest correlation levels observed. Drawing extensive conclusions from a single pain rating scale may have significant clinical implications.     

Assessment of a daily online implanted fiducial marker‐guided prostate radiotherapy process

The aims of this study were to investigate whether intrafraction prostate motion can affect the accuracy of online prostate positioning using implanted fiducial markers and to determine the effect of prostate rotations on the accuracy of the software‐predicted set‐up correction shifts. Eleven patients were treated with implanted prostate fiducial markers and online set‐up corrections. Orthogonal electronic portal images were acquired to determine couch shifts before treatment. Verification images were also acquired during treatment to assess whether intrafraction motion had occurred. A limitation of the online image registration software is that it does not allow for in‐plane prostate rotations (evident on lateral portal images) when aligning marker positions. The accuracy of couch shifts was assessed by repeating the registration measurements with separate software that incorporates full in‐plane prostate rotations. Additional treatment time required for online positioning was also measured. For the patient group, the overall postalignment systematic prostate errors were less than 1.5 mm (1 standard deviation) in all directions (range 0.2–3.9 mm). The random prostate errors ranged from 0.8 to 3.3 mm (1 standard deviation). One patient exhibited intrafraction prostate motion, resulting in a postalignment prostate set‐up error of more than 10 mm for one fraction. In 14 of 35 fractions, the postalignment prostate set‐up error was greater than 5 mm in the anterior–posterior direction for this patient. Maximum prostate rotations measured from the lateral images varied from 2° to 20° for the patients. The differences between set‐up shifts determined by the online software without in‐plane rotations to align markers, and with rotations applied, was less than 1 mm (root mean square), with a maximum difference of 4.1 mm. Intrafraction prostate motion was found to reduce the effectiveness of the online set‐up for one of the patients. A larger study is required to determine the magnitude of this problem for the patient population. The inability in the current software to incorporate in‐plane prostate rotations is a limitation that should not introduce large errors, provided that the treatment isocentre is positioned near the centre of the prostate.     

Pharmacokinetics and pharmacodynamics of midazolam administered as a concentrated intranasal spray. A study in healthy volunteers

AIMS: To investigate the pharmacokinetic and pharmacodynamic profile of midazolam administered as a concentrated intranasal spray, compared with intravenous midazolam, in healthy adult subjects. METHODS: Subjects were administered single doses of 5 mg midazolam intranasally and intravenously in a cross-over design with washout period of 1 week. The total plasma concentrations of midazolam and the metabolite 1-hydroxymidazolam after both intranasal and intravenous administration were described with a single pharmacokinetic model. beta-band EEG activity was recorded and related to midazolam plasma concentrations using an exponential pharmacokinetic/pharmacodynamic model. RESULTS: Administration of the intranasal spray led to some degree of temporary irritation in all six subjects, who nevertheless found intranasal administration acceptable and not painful. The mean (+/-s.d.) peak plasma concentration of midazolam of 71 (+/-25 ng ml-1) was reached after 14 (+/-5 min). Mean bioavailability following intranasal administration was 0.83+/-0.19. After intravenous and intranasal administration, the pharmacokinetic estimates of midazolam were: mean volume of distribution at steady state 1.11+/-0.25 l kg-1, mean systemic clearance 16.1+/-4.1 ml min-1 kg-1 and harmonic mean initial and terminal half lives 8.4+/-2.4 and 79+/-30 min, respectively. Formation of the 1-hydroxymetabolite after intranasal administration did not exceed that after intravenous administration. CONCLUSIONS: In this study in healthy volunteers a concentrated midazolam nasal spray was easily administered and well tolerated. No serious complications of the mode of administration or the drug itself were reported. Rapid uptake and high bioavailability were demonstrated. The potential of midazolam given via a nasal spray in the acute treatment of status epilepticus and other seizure disruptions should be evaluated.     

Intestinal paracellular permeation enhancement with quaternised chitosan: in situ and in vitro evaluation

Previous studies have shown that N-trimethyl chitosan chloride (TMC) is a potent absorption enhancer for hydrophilic and macromolecular compounds across mucosal surfaces. TMC proved to be effective in neutral and basic pH environments where the absorption enhancing ability of chitosan is severely hampered by its insolubility in these environments. The absorption enhancing characteristics of TMC polymers with different degrees of quaternisation were investigated in vitro and in situ to identify the most effective polymer in a neutral pH environment. Different degrees of quaternisation were obtained by varying the number and duration of the reaction steps in the synthesis process of TMC. The TMC polymers were characterised with 1H-NMR spectroscopy and the degrees of quaternisation were between 22.1 and 48.8%. Everted intestinal sacs (rats) were used to determine the effect of the polymers (0.0625-0.5% w/v) on the permeation of the hydrophilic model compound, [14C]mannitol, at a pH value of 7.4. A single pass intestinal perfusion method was also used to evaluate the permeation enhancing properties of the TMC polymers under the same conditions. The results obtained from both methods clearly showed a pronounced enhancement of [14C]mannitol permeation when administered with the different TMC polymers. It was shown that the permeation enhancing effects depend on the degree of quaternisation of TMC. In both models the best permeation enhancing results were obtained with the highest degree of quaternisation of TMC (48.8%) at a concentration of 0.5% w/v.     

CD154 is expressed during treatment with calcineurin inhibitors after organ transplantation

BACKGROUND: CD154 (CD40 ligand) monoclonal antibody prevents allograft rejection in rodents and monkeys. Inasmuch as calcineurin inhibitors (CI) inhibit CD154 expression by pharmacologic agents in vitro, we investigated whether CD154 is also inhibited by CI in vivo and in vitro during allogeneic stimulation. METHODS: CD154 expression was determined by immunohistochemistry and flow cytometry in human lymph nodes and spleen sections from rhesus monkeys with or without CI treatment. The effect of CI on induction of CD154 expression was studied by stimulating lymphocytes with phorbol 12-myristate 13-acetate (PMA) and ionomycin or with allogeneic monocyte-derived mature dendritic cells. RESULTS: Lymph nodes from patients with or without CI cyclosporine (CsA) or FK506 (FK) treatment showed comparable CD154 expression, which was present on the cell surface of T cells. CD154-expressing cells were also present in spleens from monkeys treated with CsA in comparable numbers to those in the nontreated group. Moreover, in several liver transplant rejection biopsies taken during CI therapy CD154-expressing cells were observed. In vitro, CsA and FK strongly inhibited induction of CD154 expression on peripheral blood mononuclear cells by pharmacologic stimuli. Maximum inhibition was found at 50 ng/ml CsA and 20 ng/ml FK. CD154 expression induced by dendritic cells in peripheral blood mononuclear cells or spleen cells was also almost completely inhibited by CsA. CONCLUSION: Although CI strongly suppressed pharmacologic and allogeneic induction of CD154 expression on T cells in vitro at concentrations at approximately clinical trough levels, CD154 is prominently expressed during CI therapy in lymphoid tissue and (sporadically) in liver allografts. This suggests that the CD154-CD40 pathway remains functional during CI therapy, which may contribute to allograft rejections in the clinical setting.     

移植肾功能延迟恢复的护理体会

1临床资料我院1997-11/2004-11共行肾移植562(男386,女176)例,期中87例患者出现移植肾功能延迟恢复(DGF),男性58例,女性29例,年龄16～62(平均38.5)岁.术后常规服免疫抑制剂:环孢素,强的松,骁悉.发生DGF最早术后第4日,最晚20 d,均采用综合治疗措施,纠正水、电解质紊乱,控制血压,预防感染等,同时对不同原因引起的DGF采用相应的治疗、护理.85例患者均在术后3wk左右肾功能恢复,2例因并发症死亡.