阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

辛伐他汀对人低密度脂蛋白氧化修饰影响的实验研究

目的研究辛伐他汀在体外对低密度脂蛋白氧化修饰的影响.方法以低密度脂蛋白氧化修饰为模型,以硫代巴比妥酸反应物质(TBARS)生成量及相对电泳迁移率(REM)为指标,研究了辛伐他汀对铜离子(Cu2 )诱导低密度脂蛋白氧化修饰的抑制作用.结果随辛伐他汀浓度从1～10μmol/L的增加,TBARS值分别减少67.3%,73.9%,81.9%,REM值减少48.3%,55.2%,58.6%,呈浓度及时间依赖关系.其中10μmol/L辛伐他汀可几乎完全抑制低密度脂蛋白氧化.结论辛伐他汀在体外能明显抑制Cu2 诱导的低密度脂蛋白氧化修饰.     

ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

关于我国体外诊断试剂监督管理改革的建议

本文综述了美国、欧盟、日本、中国目前对体外诊断产品（试剂）监督管理的现状以及我国存在的问题。并根据我国具体情况提出了对体外诊断试剂监督管理的改革建议。     

Improved Performance of Hip DXA Using a Novel Region of Interest in the Upper Part of the Femoral Neck: In Vitro Study Using Bone Strength as a Standard of Reference

We tested the hypothesis that bone mineral density (BMD) and bone mineral content (BMC) in proximal human femur specimens in the upper neck region of interest (ROI) and femoral neck axis length (FNAL) provide a significantly better prediction of femoral bone strength than standard ROIs in vitro. BMD and BMC were measured in 110 proximal femur specimens using a standard dual-energy X-ray absorptiometry (DXA) scanner. The analysis included a new ROI in the upper neck as well as the standard ROIs. FNAL was obtained from the scan images. The specimens' failure-load was measured in a mechanical loading device, simulating a fall on the greater trochanter. For the standard ROIs, correlations between failure-load and BMD ranged from R2 = 0.64 (shaft ROI) to R2 = 0.70, p < 0.001 (femoral neck). Prediction of strength by BMD did not significantly differ from those of BMC (R2 ranging from 0.65 to 0.75, p < 0.001). In the upper neck ROI, for both BMD and BMC correlations with failure-load were higher (R2 = 0.76 and 0.81, respectively; p < 0.001). A lower, yet still significant, correlation was found between FNAL and bone strength (R2 = 0.23, p < 0.001). Normalization of failure-load with respect to FNAL did not significantly increase the correlations with densitometric measures. This study provides in vitro evidence indicating that among the ROIs of the proximal femur the newly defined upper neck ROI provides the best prediction of bone strength. Only a weak association was observed between failure load and FNAL.     

In vitro/in vivo correlation of prolonged release dosage forms containing diltiazem HCl

Six preparations were considered: three multiple unit dosage forms (micropellets in capsules) (D, E and G) and one matrix tablet (B) were experimental prolonged release formulations, two non-disintegrating tablets (A and C) were commercial products. The in vitro dissolution behaviour of the differing formulations was investigated using the USP XXII paddle apparatus. The in vivo study was effected on a panel of 12 healthy volunteers. The two commercial tablets (A and C) showed mean dissolution time (MDT) of 1.34 and 1.44 h and t_d of 91 and 92 min, respectively; for prolonged release formulations (B, E, D, and G) MDT ranged between 2.28 and 4.23 h and t_d between 149 and 291 min. The mean residence time (MRT) was 8.68 and 6.47 h for tablets A and C, respectively; it ranged between 9.62 and 10.24 h for the multiple unit formulations E, D, and G and was 11.27 h for matrix B. Formulation B also showed the higher apparent elimination half-life t_1/2 (7.12 h), while apparent t_1/2 for all the other formulations were very similar, ranging between 5.04 and 5.28 h. High variability between the various formulations was found for C_max and AUC values, and no relationships could be established with the type of formulation. An in vitro/in vivo correlation was found for all the formulations examined on the basis of analogous parameters (MDT and MRT); (r = 0.83, p <0.05). In a few cases the Wagner-Nelson deconvolution method was applied to individual plasma level versus time curves and the corresponding absorption curves were obtained. In these cases the in vitro/in vivo correlation was tested on the basis of the comparison of the in vivo absorption curves with the in vitro dissolution profiles. This was accomplished using the ‘Levy's plot’ (per cent released versus per cent absorbed) approach and provided further support for the correlation found.     

大鼠气管上皮细胞体内—体外转化模型的研究

本研究建立了大鼠气管上皮细胞体内－体外转化模型，大鼠气管内滴注苯并芘，三天后处死大鼠，消化气管上皮细胞，接种于无血清完全培养基。细胞形成集落后，换为选择培养基继续培养五周，统计转化率。结果显示，２５ｍｇ／ｋｇ和５０ｍｇ／ｋｇ的苯并芘可诱导大鼠气管上皮细胞转化及微核增加，用同样方法研究了煤焦沥青提取物，结果表明，剂量为８ｍｇ／ｋｇ和２５ｍｇ／ｋｇ的煤焦沥青提取物能明显诱导大鼠气管上皮细胞转化。     

Splicing of the mitochondrial group-II intron rl1: conserved intron-exon interactions diminish splicing efficiency

The mitochondrial intron rI1 is a self-splicing group-II intron of algal mitochondria that can be transferred into chloroplasts from the green alga Chlamydomonas reinhardtii for in vivo investigations (Herdenberger et al. 1994). Thus, rI1 is a suitable system to compare in vitro and in vivo RNA processing. Interestingly, rI1 shows correct RNA splicing, although typical cis-acting exon-sequences (IBS2, δ) of group-II introns are lacking. In order to examine the effect of these exon-intron interactions on splicing, we introduced the endogenous mitochondrial IBS2 sequence in order to produce optimal IBS2-EBS2 base pairing. In addition, the first nucleotide of the 3′exon (δ′) was substituted to create an optimal δ-δ′ interaction. Neither of the two mutations, nor a combination of both, had any effect on the precision of the splice-site selection. Unexpectedly, introduction of IBS2 led to a reduction in the efficiency of the second splicing step in vitro but not in vivo. These findings lead us to conclude that trans-acting factors are present in vivo to optimize splicing efficiency. The possibility is discussed that these factors may, for example, stabilize tertiary intron structures that are a prerequisite for correct RNA processing. Furthermore, our data indicate that similar trans-acting factors promote correct intron splicing in chloroplasts and mitochondria. Received: 18 October / 4 December 1997     

Endocrinology: High incidence of embryo transfer cancellations in patients with polycystic ovarian syndrome

This study was aimed at assessing the outcome of in-vitro fertilization(IVF) and embryo transfer in patients with polycystic ovariansyndrome (PCOS). The results of IVF and embryo transfer in PCOSpatients (PCOS group, 78 cycles of 26 patients) were comparedwith those of a control group (423 cycles in 202 patients withoutmale factor; age and ovarian stimulation protocol were matched).Although the pregnancy rate per transfer was not different inthe two groups of patients (25 versus 34%, PCOS versus controlgroup), the PCOS group had a significantly lower pregnancy rateper follicle aspiration (19 versus 31%, P < 0.05). A notableresult was a significantly higher incidence of embryo transfercancellations in the PCOS group (22 versus 8%, P < 0.01),which resulted from unpredictable failure of either oocyte recoveryor fertilization. The incidence of unexplained complete failureof fertilization was significantly higher in the PCOS group(18 versus 5%, P < 0.01). These results may reflect a reducedquality of the oocytes in the PCOS group, and there was a subgroupof PCOS patients who repeatedly produced poor results of treatment.Although the ovarian stimulation regimen best suited to PCOSpatients remains to be determined, special care should be takenduring ovarian stimulation, especially when the PCOS patientshad experienced unexplained failure of oocyte recovery or fertilizationin the previous treatment cycle(s).     

Comparative bactericidal exposures for selected oral bacteria using carbon dioxide laser radiation

Although relatively high CO2 laser energies have been shown to sterilize root canals, the response of several bacterial strains to decreasing exposures of CO2 laser energy remains unknown. Freshly grown bacterial cells were irradiated on glass microscope coverslips. A comparison of equivalent energy exposures with differing parameters was made on the bacterial viability. No statistically significant difference was found in the energy required to kill closely related bacterial species. However, the energy density required to kill greater than 99.5% of the bacteria is less than 200 J/cm2, much less than that shown to sterilize in a previous study.