Modulators of growth in primary culture of rat proximal tubular cells II

Summary: The present studies assessed the effects of manipulating extracellular sodium (Na) concentration and Na transport on cellular hypertrophy and hyperplasia in primary culture of rat proximal tubular cells. A concentration-dependent effect on thymidine incorporation and protein content was observed with cell culture media Na concentration of 130, 140 and 150 mmol/L. This effect was independent of osmolality (matched with mannitol) and no stimulatory effect occurred if choline was substituted for Na. Cells derived from sham-operated (Sx) animals exposed to a higher media concentration of Na (150 vs 140 mmol/L) had both stimulated thymidine incorporation to 186.8 ± 35.41% (P<0.05) and enhanced cell protein content to 134.7 ± 135% (P<0.05). This effect was more pronounced in cell cultures derived from unilaterally nephrectomized (Nx) animals, being 212.8 ± 31.5% (P<0.01) for thymidine incorporation (P<0.05 vs cells from sham-operated animals grown in high Na media) and 114.4 ± 3.2% (P<0.001) for protein content (P=0.11 vs sham-operated cells grown in similar conditions). the addition of 10^?4 mmol/L ethylisopropyl amiloride hydrochloride (EIPA) to Nx cells in a normal or high Na concentration media resulted in a decrease in cellular protein content to 82.6 ± 6.8% (P<0.05) and 85.5 ± 0.2% (P<0.0001) compared to respective controls. 10^?4 mol/L EIPA in media supplemented with insulin-like growth factor (IGF-1) blocked the proliferative response normally seen in response to this growth factor from 156.6 ± 13.7 to 27.5 ± 3.1% (P<0.0001) compared to control. However, the presence of EIPA did not abrogate the hypertrophic response elicited by IGF-1 (cell protein content 128.1 ± 13.1% of control with IGF-1 vs 124.9 ± 12.5 with IGF-1 and EIPA; P= n.s.). Addition of 10^?4 mol/L EIPA to 10% serum derived from either Sx or Nx animals blocked the growth response to the sera, limiting the cellular protein content to 76.6 ± 5.5% (P<0.0001) and 89.7 ± 4.4% (P<0.0001) and thymidine incorporation to quiescent levels of 0.2 ± 0.1% (P<0.0001) and 0.4 ± 0.1% (P<0.0001) compared to respective controls. In summary, rat renal proximal tubular cell growth is influenced by Na concentrations in the cell culture environment and inhibited in the presence of EIPA. This supports a role for altered epithelial transport in the cellular growth response to a number of stimuli.     

Reciprocity between tissue calmodulin and cAMP levels: modulation by excess zinc

Signal transduction of many intracellular events is initiated by a minute influx of calcium ions into the cells, resulting in the formation of calcium-calmodulin complex and cAMP. Because zinc appears to have an inhibitory effect on a number of tissue reactions, it is postulated that this occurs through modulation of intracellular calcium influx. To test the hypothesis that the inhibitory effects of zinc are mediated through the calcium calmodulin-cAMP pathway, zinc was administered by various routes to five groups of nude mice (control, intragastric, intraperitoneal. intradermal and oral groups), and calmodulin and cAMP concentrations were measured in the cytosol of epidermal cells. Calmodulin levels decreased significantly in the groups given intraperitoneal zinc (P>0.025) and intradermal zinc (P>0.001) injections. Significant elevations of cAMP levels were noted with intradermal zinc (P>0.025). Overall, the relationship between calmodulin and cAMP appeared to be inversely logarithmic, with the lowest calmodulin levels associated with the highest cAMP concentrations. In addition, there was a significant trend towards a smaller calmodulin/cAMP ratio in all zinc-treated groups, except the mice fed dietary zinc. These results appear to correlate with tissue zinc levels obtained with these various forms of zinc administration. Our results therefore indicate that there is a reciprocity between epidermal calmodulin and cAMP levels, which may be modulated by external factors such as zinc.     

蛇足石杉内生真菌2F09P03B产石杉碱甲发酵条件的研究

 目的 研究发酵条件对蛇足石杉支顶孢属内生真菌2F09P03B 石杉碱甲生成的影响，并探讨发酵条件的优化。 方法 通过测定 2F09P03B 生长曲线、石杉碱甲积累曲线和培养液 pH 值随时间的变化情况，了解该菌株的培养特性；考察不同碳源和氮源对石杉碱甲积累的影响。在单因素试验的基础上，利用正交试验优化培养基碳源、氮源组成；考察 MgSO4、KH2PO4、NaCl、CaSO4 对石杉碱甲生成的影响，确定无机盐组成；优化接种量、发酵时间、培养温度、摇床转速等发酵工艺条件。 结果 菌株 2F09P03B 的最佳培养基组成为：马铃薯 150 g/L，玉米粉 30 g/L，麦芽糖 25 g/L，酵母膏 10 g/L，麸皮 30 g/L，KH2PO4 1.5 g/L，MgSO4 0.5g/L，NaCl 0.3 g/L，CaSO4 0.3 g/L，pH 自然；最佳发酵工艺条件为：接种量 8%，发酵时间 10 d，培养温度 28 ℃，摇床转数 140 r/min。在此条件下，发酵液中石杉碱甲含量为 8.17 μg/L，最高达到 8.32 μg/L，约为其干重的 0.07% 左右。 结论 发酵条件对菌株 2F09P03B 石杉碱甲产量有明显影响。优化后的发酵条件可使该菌株石杉碱甲产量明显提高，有望成为石杉碱甲新药源。     

真核表达人呼吸道合胞病毒F蛋白的纯化方法

目的 真核表达人呼吸道合胞病毒(human respiratory syncytial virus,SV)融合蛋白(fusion protein,),并完成蛋白纯化及纯度测定.方法 根据编码F蛋白的基因序列设计引物,CR方法扩增出3'端带His标签的F基因序列,克隆入pGEM-T-easy载体,经核酸序列分析后,进一步克隆到pcDNA3.1( )真核表达载体,限制性内切酶鉴定,用脂质体Lipofectamine2000转染COS-7细胞,2 h后再用Westem blot检测目的蛋白的表达.Ni柱亲和层析纯化COS-7细胞表达的F蛋白,高效毛细管电泳分析纯化后蛋白纯度.结果 核酸序列分析证实获得带His标签的RSV F基因序列,没有发生无义突变.转染COS-7细胞后,利用Western blot方法检测到F蛋白的特异性条带,纯度达99%以上.结论 初步建立了真核表达RSV F蛋白的纯化方法,为进一步优化RSV F蛋白制备条件及单克隆抗体及诊断试剂等研究奠定了基础.     

使用碱性蛋白酶去除人体骨骼标本软组织的技术研究

<正>未防腐的人体骨骼标本的常用制备方法包括自然腐蚀法、水煮法和药液浸泡法[1]。1989年开始有动物解剖学者尝试使用蛋白酶分解蛋白的方式来去除(仓鼠和家兔)肌肉组织来获取骨骼标本,他们使用蛋白酶的浓度为0.3%～0.5%[2～4]。然而这一方法是否适     

2017—2019年攀枝花市某医院心血管疾病住院患者疾病谱及住院费用分析

目的 分析心血管疾病住院患者疾病谱及住院费用,了解疾病构成的规律特点及经济负担,为心内科疾病诊治工作提供参考依据。方法 回顾性分析攀枝花市某三级医院2017—2019年心血管疾病住院患者资料,从医院相关管理信息系统中导出诊断为心血管疾病的病例条目、详细病历及病例住院费用等数据,采用描述流行病学分析方法对心血管疾病住院患者资料进行分析。结果 共纳入心血管疾病住院患者11 376例,2017—2019年分别有3 492、3 815、4 069例,分别占30.7%、33.5%、35.8%,病例数逐年上升。3年间心血管疾病住院患者的性别分布差异无统计学意义（P>0.05）,年龄分布差异有统计学意义（P<0.01）,心血管疾病住院患者发病呈年轻化趋势。病种均以冠心病和高血压所占比例最大,合计分别占当年心血管疾病住院患者的57.6%、55.5%,2019年则上升至62.8%。3年间心血管疾病住院患者有伴发疾病比例（27.6%、26.1%、25.4%）差异无统计学意义（P>0.05）。心血管疾病住院患者住院天数分布差异有统计学意义（P<0.01）,2019年的患者住院时间<7 d的比例有所上升。2017、2018、2019年该医院心血管疾病住院患者诊治自费费用分别为（5 925.6±285.7）、（5 904.2±264.3）、（5 890.9±230.8）元/人,差异有统计学意义（P<0.01）。结论 2017—2019年攀枝花市某医院心血管疾病住院患者病例数增加,住院患者发病年龄有年轻化趋势,主要病种仍以冠心病和高血压为主,住院天数及自费费用呈下降趋势。     

关于中药新药用饮片取样法的探讨

从药材及饮片（植物药）的包件取样、包件内取样及试验样品制备等方面对《中国药典》（Chinese Pharmacopoeia,ChP）、《欧洲药典》（European Pharmacopoeia,EP）、《英国药典》（British Pharmacopoeia,BP）及《美国药典》（United States Pharmacopoeia,USP）进行对比,结合新药用饮片检验的要求,就中药新药用饮片取样法存在的问题进行探讨,并提出以下建议：（1）相关部门组织专家起草中药新药用饮片取样的指导原则或技术要求。（2）暂时参照EP、BP的取样法对中药新药用饮片进行研究,并关注饮片取样对检验结果的影响。（3）根据试验样品的特点研究确定其处理方法。如饮片的不同药用部位或位置的质量差异大,可采用分层抽样的方法,减少取样误差。（4）加强药材的源头管理,减少药材质量差异。（5）在新药用饮片的生产过程中采用混合步骤提高同批饮片的同质性。（6）在ChP“中药材及饮片取样办法”中增加：待检批次饮片应具有较好的同质性;有些样品或试验需要采用更严格的取样方法,包括更多取样包件数或包件内取样量;此外,可对包件内抽取的位...     

藏汉不同骨密度人群肠道菌群及粪便短链脂肪酸差异研究

目的 比对研究四川红原藏族与成都汉族不同骨密度人群肠道菌群及粪便短链脂肪酸差异,为通过调节肠道菌群预防不同人群骨质疏松积累资料。方法 选取红原藏族与成都汉族居民,测定其足跟骨骨密度,收集粪便与人口学信息,以藏族低骨密度个体为基础,依据年龄、性别、民族、是否服用抗生素四个维度进行倾向性评分获得研究人群,并按骨密度水平分组,测定粪便16S rRNA序列及短链脂肪酸的浓度与构成比,对比分析各组的差异。结果 红原藏族肠道菌群的物种丰度高于成都汉族。与汉族人群相比,藏族人群拟杆菌门相对丰度较高,变形菌门相对丰度较低（拟杆菌门:z = - 4.156,P＜0.001;z = - 3.226,P = 0.001;z = - 2.990,P = 0.002;变形菌门:z = - 4.409,P＜0.001;z = - 3.287,P = 0.001;z = - 2.392,P = 0.016）。柯林斯菌属是藏族中骨密度组优势菌属,瘤胃球菌属2和乳球菌属分别是汉族中、高骨密度组优势菌属。两组人群粪便短链脂肪酸比较,藏族三组骨密度人群乙酸、异戊酸浓度均低于汉族人群（乙酸:t = - 3.119,P = 0.003;t = - 3.056,P = 0.003;t = - 5.104,P＜0.001;异戊酸:z = - 3.822,P＜0.001;z = - 3.497,P＜0.001;z = - 2.158,P = 0.031）,但高、中骨密度组异丁酸、异戊酸、戊酸和己酸构成比高于汉族人群（异丁酸:z = - 3.693,P＜0.001;z = - 3.388,P = 0.001;异戊酸:z = - 3.748,P＜0.001;z = - 3.844,P＜0.001;戊酸:z = - 3.778,P＜0.001;z = - 3.966,P＜0.001;己酸:z = - 2.535,P = 0.011;z = - 3.570,P＜0.001） 结论 红原藏族和成都汉族不同骨密度组的肠道菌群及粪便短链脂肪酸含量和构成比不同,其与骨密度的关系值得进一步探讨。     

Nucleostemin特异性RNA干扰对HeLa细胞体内外增殖能力的影响

目的：检测肿瘤细胞Nucleostemin（NS）的表达，研究NS特异性RNA干扰对HeLa细胞体内外增殖的影响。方法：提取6种肿瘤细胞总RNA，用 RT-PCR和Northern blot方法检测NS的表达。用NS特异性siRNA表达载体转染HeLa细胞，观察转染的HeLa细胞（简称NS-siRNA-HeLa细胞）体内外增殖的变化。结果：6种肿瘤细胞中NS明显高表达。体外培养的NS-siRNA-HeLa细胞中NS表达显著低于对照组，G0/G1期细胞百分率显著升高。体内致瘤实验显示，NS-siRNA-HeLa细胞在裸鼠体内增殖显著低于对照组。结论： NS在肿瘤细胞中高表达具有普遍性。NS特异性RNA干扰使HeLa细胞进入S期受阻，并可明显降低HeLa细胞体内外的增殖能力。     

人工心脏圆盘泵的流动特性研究

人工心脏（血泵）一直存在泵体对血细胞剪切力过大和流速过快容易引起溶血的问题。为了研究人体正常血压情况下,血泵内部剪切力和速度场的分布情况,选择圆盘泵叶轮代替传统离心泵叶轮,对两种模型进行数值计算,分析不同叶轮内部剪切力和速度场的分布规律。研究表明传统离心泵内部流速高,叶片表面剪切力大,对血细胞的伤害大。圆盘泵相比传统离心泵,剪切力更小,流场速度分布均匀,流速更小。和传统离心泵相比,不同转速下圆盘泵能降低溶血的发生率。圆盘泵叶片数为6片时,抗溶血性能更好。研究结果为血泵的优化提供理论依据。