Cell-based fluorescence assay for evaluation of new-drugs potential for phospholipidosis in an early stage of drug development

To evaluate new-drugs potential for phospholipidosis (PL), we developed a cell-based fluorescence assay using a fluorescent-labeled phospholipid analogue (NBD-PE). CHL/IU cells derived from newborn hamster lung were exposed to positive reference compounds (amiodarone, imipramine, chloroquine, propranolol, chlorpromazine and amantadine) in the presence of NBD-PE, and the level of PL, as indicated by accumulation of fluorescent inclusions in the cytoplasm, was evaluated using fluorescence microscopy and fluorometry. All positive reference compounds induced accumulation of fluorescent inclusions in a concentration-dependent manner with an increase in fluorescence intensity. Fluorescence microscopically, the positive dose of test compound was determined as the concentration with a grade equivalent to or above that of 3.13 μM of amiodarone. Based on this criterion, 8 of 20 test compounds including PL-positive or -negative compounds were judged positive that were concurrent with the pathological results from rat toxicity studies. Furthermore, a positive criterion for fluorometry was decided as equivalent to or above 25% of maximum intensity induced by 1.56–25.0 μM amiodarone. In comparison of fluorometry methods with fluorescence microscopy method, 19 of 20 compounds were judged same. From these findings, we concluded that the assay developed in this study is a rapid and reliable method to predict new-drugs potential for PL at an early stage of drug development.     

Lag screw fixation of the premaxilla during bilateral cleft lip repair

In synchronous primary premaxillary setback and cleft lip repair for bilateral cases with severely protruding premaxilla, stabilization of the premaxilla is mostly achieved by gingivoperiosteoplasty. This kind of repair carries risk of impairment of blood supply to the premaxilla and/or prolabium, and at the same time it cannot ensure adequate stabilization of the premaxilla postoperatively. To overcome these problems, we have developed a unique technique of fixation of the premaxilla. In this paper, we discussing this technique, its advantages, and potential complications associated with it. From 2016, 10 patients aged 4–10 months, with bilateral cleft lip and palate with premaxillary protrusion (≥10 mm) underwent premaxillary setback and cheilorhinoplasty in the same stage. Instead of gingivoperiosteoplasty, a ‘lag screw’ fixation technique was used to stabilize the premaxilla. The follow-up period ranged between 5 and 32 months. In all the cases, we achieved adequate stabilization of the premaxilla. None of the patients had any issue related to the vascularity of the premaxilla or prolabium. There was no impairment in the eruption process of deciduous teeth in the premaxillary segment. Overall aesthetic outcomes of the lip and nose were acceptable. This technique of premaxillary fixation with lag screw gives us the liberty to perform primary cheilorhinoplasty along with premaxillary setback in the same stage, without risking the vascularity of premaxilla and prolabium. It ensures adequate stabilization of the premaxilla, but evaluation of regular growth of the midface and, if needed, corrective orthodontic and surgical treatment in the follow-up periods are advisable.     

VIDAS CHL诊断法检测男性尿液中沙眼衣原体试验研究

[目的]为研究能否用全自动快速免疫（VIDAS CHL）诊断法检测尿液中沙眼衣原体（CT）.[方法]采用VIDAS CHL检测262例男性患者尿道拭子及尿液沉渣中CT抗原.[结果]与扩大金标准相比,VIDAS CHL检测尿道拭子、尿沉渣敏感性分别为89.09%（P＞0.05）、81.82%（P＞0.05）;特异性均为99.52%（P＞0.05）.[结论]ⅥDAS CHL诊断法检测男性尿液中的沙眼衣原体具有高度敏感性和特异性.     

VIDAS CHL检测泌尿生殖道沙眼衣原体感染

目的探讨VIDAS CHL法用于性病患者尿道拭子标本以及男性首段尿(FCU)标本沙眼衣原体(CT)检测的可行性.方法使用组织培养法(TC)、VIDAS CHL检测法和聚合酶链反应(PCR)平行检测男性和女性拭子标本中CT,以组织培养为金标准,对VIDAS CHL和PCR进行评价.结果 232例男性标本TC阳性49例;VIDAS CHL和PCR法用于男性尿道拭子标本的敏感性分别为95.9%、93.9%,特异性分别为95.6%、94.5%,差异无显著性;VIDAS CHL法用于男性FCU的敏感性和特异性分别为85.7%和96.7%,与拭子标本作自身配对比较,检测结果无差异.151例女性拭子标本,TC法阳性23例,VIDAS CHL和PCR法的敏感性分别为100%和95.7%,特异性分别为96.1%、93.8%,差异无显著性.结论 VIDAS CHL法用于性病患者男性和女性拭子标本的CT检测,具有很高的敏感性和特异性,用于男性患者FCU的CT检测也是可行的;对于CT阳性率较高的性病人群,VIDAS CHL可以不做阻抑证实试验.     

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.     

盐酸左旋咪唑和甲苯咪唑的致突变性试验研究

盐酸左旋咪唑和甲苯咪唑均为抗肠道蠕虫类药物。本实验根据我国新药审批的有关要求来检测这二类药物的致突变作用,该实验通过诱发CHL 细胞染色体畸变的体外试验和小鼠骨髓细胞染色体的体内试验这两条途径来检测。实验结果表明:在体内试验中,甲苯咪唑(400-1600mg/kg)与阴性对照组(N.S)相比,有非常显著性差异(p<0.01);在体外试验中,甲苯咪唑(0.39-0.78μg/ml)培养24h 和48h 对CHL 细胞所诱发的染色体变化率显阳性。说明甲苯咪唑具有损伤小鼠骨髓细胞染色体和诱发CHL 细胞染色体畸变的遗传物质存在。而盐酸左旋咪唑的体内、体外试验显示阴性。     

雄黄致体内外染色体畸变

目的:用仓鼠体内染色体畸变试验和中国仓鼠肺细胞CHL细胞染色体畸变试验评价雄黄的遗传毒性.方法:体内染色体畸变试验用毛足属仓鼠连续ig5 d给予33.25,66.5,133 mg·kg-1剂量雄黄悬浊液,处死前2 h ip秋水仙素.处死后取骨髓细胞制备染色体.油镜下观察每只动物骨髓细胞的有丝分裂指数和100个中期分裂相细胞的畸变类型.CHL细胞染色体畸变试验用雄黄浸出液终质量浓度为0.15,0.3,0.6g.L-1作用于CHL细胞,培养24 h或48 h,终止培养前4h加入秋水仙素.收获细胞,制备染色体,油镜下观察CHL细胞有丝分裂指数和200个中期分裂相细胞的畸变类型.结果:①雄黄265.0 mg· kg -1组和雄黄530.0 mg·kg-1组ig给药和雄黄浸出液(1.2,2.4g·L-1)作用于CHL细胞显示有明显的抑制有丝分裂作用.②与阴性对照组比较,雄黄ig给药仓鼠体内染色体畸变率和雄黄体外给药CHL细胞染色体畸变率均显著升高,差异有极显著意义,且有明显的量效关系.但体内试验的畸变率低于体外试验.③雄黄给药后CHL细胞染色体畸变试验中可见较多的染色体断片,而仓鼠体内染色体畸变试验中未发现,这可能与体内外药物作用的方式不同.结论:①雄黄能致体内外细胞染色体畸变,具有遗传毒性.②仓鼠体内染色体畸变试验可作为中药新药遗传毒性评价试验组合的方法之一.     

寿胎丸对CHL细胞株染色体畸影响的研究

目的探讨寿胎丸对CHL细胞株染色体畸变的影响,对其遗传安全性进行研究。方法采用CHL细胞培养技术,设寿胎丸不同剂量组,并对CHL细胞株进行体外染毒,观察寿胎丸致CHL细胞株染色体数目及结构变化。结果收集24 h及48 h细胞,在5 000、2 500、1 250μg/mL不同测试剂量浓度下,并在 S9与-S9两种测试条件下进行染色体畸变分析,各剂量组、阴性对照染色体畸变率均在正常范围。结论寿胎丸在本试验所确定的5 000μg/mL测试剂量浓度以下,初步认为无诱发CHL染色体畸变作用。     

Altered expression of CHL1 by glial cells in response to optic nerve injury and intravitreal application of fibroblast growth factor-2

The close homologue of L1 (CHL1) is a member of the L1 family of cell recognition molecules. The protein is expressed by a variety of nerve cell types and subpopulations of glial cells in vivo and promotes elongation of neurites and survival of nerve cells in vitro. Here we demonstrate that glial cells up-regulate expression of CHL1 in response to an intraorbital crush of the adult mouse optic nerve. We also demonstrate that a single intravitreal application of fibroblast growth factor-2 (FGF-2) increases expression of CHL1 in retinal astrocytes and Müller cells. Elevated expression of CHL1 by glial cells in injured optic nerves and astrocytes and Müller cells in FGF-2-treated retinas suggests a role of the protein in the lesioned central nervous system. Results also suggest that trophic factors might exert part of their biological function by modifying expression of cell recognition molecules.     

Hep G2培养条件的摸索及其与CHL、3T3细胞代谢能力的比较

目的：以MTT试验方法计算环磷酰胺（cyclophosphamide,CP）对Hep G2细胞和体外安全性评价常用细胞系CHL、3T3细胞的LC50,间接比较几种细胞系的代谢能力并确定Hep G2细胞适宜的培养试验条件.方法：用需代谢活化的阳性物CP以含血清和不含血清的培养液配制染毒液,按10.0、7.21、5.19、3.73、2.69、1.93、1.39、1.00 mg/ml浓度对CHL、3T3细胞和以不同培养液培养的Hep G2细胞染毒,在24 h时间点以MTT方法对细胞存活情况进行分析,计算LC50,比较各种细胞和不同培养条件下Hep G2细胞对CP的代谢活化水平,并观察不同培养条件下Hep G2细胞的生长状态.结果：MTT试验结果表明Hep G2细胞对CP的代谢能力最强,CHL细胞次之,而3T3细胞对CP的代谢能力最弱;3种不同培养液培养的Hep G2细胞无论染毒液是含血清的还是不含血清的均观察到CP对细胞的LC50：MEM＞DMEM＞RPMI 1640,Hep G2细胞在DMEM、RPMI 1640两种培养液中生长状态均良好,尤其是RPMI 1640培养的Hep G2细胞,在无血清的染毒液培养下既能获得较多的细胞又能表现出较强的代谢CP的能力.结论：Hep G2细胞对CP的代谢活化能力较CHL和3T3细胞强,以RPMI 1640培养的Hep G2细胞生长状态良好且较DMEM和MEM培养的细胞表现出更强的代谢活化CP的能力,能获得较理想的培养和试验结果.