细胞培养操作中常见问题及处理对策

细胞培养技术广泛用于科研、教学及临床研究实验,这项技术看似简单又不太容易操作.首先从概念上说,细胞是生命的小单位,离体以后失去了生命体给予的一切复杂的机体内环境,在实验室内能够生长并形成一定生长规律,是研究者们经过长期研究摸索总结得到的科研成果.

丙型肝炎病毒细胞培养研究进展

丙型肝炎病毒(hepatitis C virus,HCV)是引起人类丙型肝炎的病原体.全球HCV的感染率约为3%,约1.7亿人感染HCV.HCV感染易慢性化,其慢性化率可高达85%,部分慢性丙型肝炎患者终可发展为肝纤维化、肝硬化,甚至肝细胞癌.目前主要采用干扰素和利巴韦林联合治疗丙型肝炎,但并非对所有慢性丙型肝炎患者均有效.

烧伤皮肤再生的核心技术——原位培植干细胞复制皮肤

烧伤是一种常见的灾害性疾病.说它是灾害有两个原因,一是几乎所有烧伤病人都由意外伤害所致,重者直接危及生命;二是因治疗不当可导致病人死亡,或造成终身残废,尤其是大面积烧伤和深度烧伤.今天,原位培植干细胞复制皮肤技术已在我国获得成功,较好地解决了长期困扰烧伤医学发展的难题.顾名思义,原位培植干细胞是让干细胞在烧伤创面原部位活化、分裂、增殖及分化;复制皮肤是说按原来受伤之前正常的皮肤样式重新制作.由于其机理十分复杂,为使广大读者对该项技术有所了解,根据我们的临床经验,特作如下讲解.

微重力培养提高大鼠胰岛冻存质量的研究

本研究应用三维组织细胞旋转培养系统(the rotary cell culture system, RCCS)对冻存前后的大鼠胰岛细胞进行三维微重力组织培养,以期能提高胰岛复苏率,从而为人类胰岛的建库、进行胰岛移植奠定基础.

After hypoxia, ischemia, or inlfammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, lfow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy ifrst appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

三维细胞培养在牙科材料生物相容性实验中的应用

三维细胞培养(three-dimensional cell culture,TDCC)自上世纪初以来已广泛运用到生物医学科研的各个领域.TDCC做为体外无细胞系统及单层细胞系统的研究与组织器官及整体研究的桥梁,显示了它既能保留体内细胞微环境的物质及结构基础,又能展现细胞培养的直观性及条件可控制性的优势.

OBJECTIVE To study the structural and preliminary functional characterization of the hepatitis B virus(HBV) enhancer Ⅰ in patients with chronic hepatitis B treated with interferon (IFN). METHODS The characteristics of the HBV enhancer Ⅰ in 12chronic carrier who were treated with alpha interferon was detected by the methods of molecular biology including PCR, cloning of PCR products, sequencing and cell culture.RESULTS Four of 6 patients cleared viral DNA; all 6 in this group also seroconverted from e antigen to antibody. Prior to therapy, the HBV enhancer Ⅰ region demonstrated many point mutations in all 6 patients who became nonresponders, compared to patients who responded to interferon. The mutated sequences, many of which were within regions of transcription factor binding, were significantly more active than the corresponding wild type sequences in reporter gene assays. CONCLUSION These results imply that the mutations found in nonresponders appear to render the virus less sensitive to interferon.

E型沙眼衣原体临床株热休克蛋白60含量变化及传代衣原体培养与疗效的关系

目的 探讨E型标准株和细胞培养成功的沙眼衣原体E型临床株随着传代次数的增加其热休克蛋白60(HSP60)含量的改变.方法 经McCoy细胞培养法检测出的40例E型沙眼衣原体临床株,分为首次传代出现和未出现包涵体两组,通过RT-PCR方法检测1～4次传代的HSP60含量,x2检验其与对应患者疗效的关系.结果 经细胞培养成功的E型沙眼衣原体临床株传代未出现包涵体时其HSP60含量较出现包涵体时高(P＜0.05).18例首次传代出现包涵体的标本1～4次传代HSP60/16SrRNA分别为0.38±0.06、0.39±0.03、0.38±0.04、0.39±0.03；12例2次传代出现包涵体的标本1～4次传代HSP60/16SrRNA分别为1.18±0.10、0.28±0.06、0.30±0.03、0.29±0.05；10例3次传代出现包涵体的标本1～4次传代HSP60/16SrRNA分别为1.20±0.04、1.20±0.04、0.28±0.04、0.28±0.05.患者疗效对1次传代是否出现包涵体有影响,20例治疗失败患者16例1次传代未出现包涵体,1个疗程治疗后转阴患者20例中14例在1次传代出现包涵体.结论 80％治疗失败患者1次传代未见包涵体,临床株传代培养未出现包涌体时沙眼衣原体可能处于持续感染状态,其CHSP60合成增加,临床采集的衣原体标本至少应进行3次盲传以避免漏检.

模拟微重力影响EPO诱导K562细胞向红系分化的机制研究

研究发现,人体暴露于微重力状态中会出现血液流变学改变和溶血现象,细胞膜对抵抗重力引起的细胞破坏起到关键作用,同时空间环境可以使细胞膜的某些成分改变,从而影响红细胞生成[1].模拟微重力对红系分化的影响及其机制尚不清楚.本研究采用人白血病细胞株K562细胞为模型,应用美国国家航天局(NASA)研发的第四代旋转细胞培养系统(Rotary Cell Culture System, RCCS-4)模拟微重力环境,与地面环境同时用EPO诱导培养K562细胞,并分别用联苯胺染色法、流式细胞术、Western-blotting等观察和分析模拟微重力对促红细胞生成素(EPO)诱导的K562细胞向红系分化的影响,并初步探讨其可能机制.

The dynamic level of immunosuppressants such as alpha-fetoprotein(α-FP), gestation associated protein, progesterone, estradiol, chorionic gonadotropin, etc. of peripheral sera and the regulatory mechanism of cellular immunity in the peripartum stages in sows were determined by advanced radioimmunoassay (RIA) and immunochemistry methods in this study. The effects on lymphocyte cultivated in vitro by the immunosuppressants mentioned above were tested by cell culture technology, respectively. The results were as follows: (1) α-FP: The level of α-FP of peripheral sera in the stage of ante-parturition was slightly higher than that of post-parturition in sows, but it didn't show statistical difference. The concentration of α-FP of peripheral sera in conceptus sows had no obvious effect on lymphocyte cultivated in vitro, and it indicated that α-FP was not the major immunosuppressant in the pregnancy of sows. However, the α-FP, whose concentration was equal to that of amniotic fluid, had obvious inhibition on lymphocyte cultivated in vitro. (2) Gestation associated protein: The level of gestation associated protein was maintained highly (100-162 mg/L) in peripheral sera and amniotic fluid in the stage of ante-parturition in sows, then descended gradually after parturition and disappeared step by step 5-10 d after parturition. The gestation associated protein, whose concentration were equal to that of peripheral sera and amniotic fluid, were added to lymphocyte culture in vitro, and the effect on lymphocyte activity was not observed yet. This showed that gestation associated protein was not the major immunosuppressant in the peripartum stage in sows. (3) Estradiol and progesterone: Estradiol or progesterone at the level of sera in pregnant sows, could lower the rate of lymphocyte transformation and the level of cellular immunity. (4) Chorionic gonadotropin: The level of chorionic gonadotropin of peripheral sera in the stage of ante-parturition was slightly higher than that of post-parturition in sows, but it didn't have statistical difference. The chorionic gonadotropin, whose concentration was equal to that of sera, was not yet observed obvious effect on lymphocyte cultivated in vitro. (5) Added the five above-mentioned immunosuppressants to lymphocyte culture in vitro, whose concentration were equal to those of sera in pregnant sows, they could drop the rate of lymphocyte resette formation and lymphocyte transformation, lower the level of cellular immunity and reduce the concentration of IL-2 which synthesized and secreted by lymphocyte in culture remarkably. Their inhibitory intensity of cellular immunity was evidently higher than that of any single immunosuppressant.

Effects of hydrogen peroxide (H2O2)on Cryptosporidium parvum infection in vitro were studied in this paper, using an oocyst excystation assay, a cell culture model, and a free radical inhibition technique. H2O2 treatment at 500 and 1 000 μmol/L significantly inhibited excystation of bleach-treated oocysts (P＜0.01). Concentrations of H2O2 at 500 and 750 μmol/L resulted in a significant decrease in C. Parvum infection at 35.77% and 58.16% respectively, when compared with the untreated control at 48 hours postinoculation. Surprisingly, C. parvum infection were significantly increased by 22.21% to 39.33% following treatment with 50 (P＜0.01), 100 (P＜0.01) or 200 (P＜0.05) μmol/L H2O2, respectively. Stimulatory effect with treatment of 100 μmol/L H2O2 was most obvious, compared with the untreated control at 48 hours postinoculation. Effects of H2O2 on C. parvum at 24, 48 and 96 hours postinoculation were similar, the highest infection being infection at 48 hours postinoculation, with the maximum inhibitory effect being seen at 96 hours postinoculation. The stimulatory and inhibitory effects of H2O2 treatment on C. parvum infection were, to a certain extent, abolished in the presence of free radical scavengers, reduced glutathione or mannitol. These observations indicate that reactive oxygen species (ROS), such as H2O2, may play a role in the course of C. parvum infection. This is the first time to demonstrate a significant action of ROS in C. parvum infection in vitro.