支气管哮喘患儿中性粒细胞胞外诱捕网水平与患者免疫功能及临床预后的关系

目的探讨支气管哮喘患儿中性粒细胞胞外诱捕网水平与患者免疫功能及临床预后的关系。方法选取本院2017年9月-2018年9月支气管哮喘患者80例作为观察组,并以同期健康体检的30例儿童作为对照组,采用pico Green dsDNA荧光染色定量检测支气管哮喘患者NETs水平,采用T淋巴细胞亚群评价患者免疫功能,对比不同NETs水平患者免疫功能及临床特点,采用Logistic回归分析探讨小儿支气管哮喘急性发作的风险因素。结果观察组白细胞、中性粒细胞、单核细胞、Cf-DNA/NETs水平均高于对照组(P <0. 05),而淋巴细胞水平低于对照组,差异均有统计学意义(P <0. 05)。Logistic回归分析显示,哮喘家族史、呼吸道感染、Cf-DNA/NETs水平上升、有害气体接触、被动吸烟均是支气管哮喘急性发作的独立危险因素(P <0. 05)。结论中性粒细胞胞外诱捕网水平与支气管哮喘患者免疫功能及预后密切相关,值得临床重点关注。     

Extracellular vesicles transmitted miR-31-5p promotes sorafenib resistance by targeting MLH1 in renal cell carcinoma

Sorafenib provides survival benefits in patients with advanced renal cell carcinoma (RCC), but its use is hampered by acquired drug resistance. It is important to fully clarify the molecular mechanisms of sorafenib resistance, which can help to avoid, delay or reverse drug resistance. Extracellular vesicles (EVs) can mediate intercellular communication by delivering effector molecules between cells. Here, we studied whether EVs are involved in sorafenib resistance of RCC and its possible molecular mechanisms. Using differential centrifugation, EVs were isolated from established sorafenib-resistant RCC cells (786-0 and ACHN), and EVs derived from sorafenib-resistant cells were uptaken by sensitive parental RCC cells and thus promoted drug resistance. Elevated exogenous miR-31-5p within EVs effectively downregulated MutL homolog 1 (MLH1) expression and thus promoted sorafenib resistance in vitro. Mice experiments also confirmed that miR-31-5p could mediate drug sensitivity in vivo. In addition, low expression of MLH1 was observed in sorafenib-resistant RCC cells and upregulation of MLH1 expression restored the sensitivity of resistant cell lines to sorafenib. Finally, miR-31-5p level in circulating EVs of RCC patients with progressive disease (PD) during sorafenib therapy was higher when compared to that in the pretherapy status. In conclusion, EVs shuttled miR-31-5p can transfer resistance information from sorafenib-resistant cells to sensitive cells by directly targeting MLH1, and thus magnify the drug resistance information to the whole tumor. Furthermore, miR-31-5p and MLH1 could be promising predictive biomarkers and therapeutic targets to prevent sorafenib resistance.     

液相色谱-电喷雾串联质谱分析人全血中环孢素的基质效应评估及其机理探究

目的： 评估液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）分析人全血中环孢素的基质效应，并探究其发生机理。方法： 全血裂解物的蛋白沉淀上清液用作全血基质。分别用乙腈水溶液和全血基质配制等浓度环孢素A（CyA）、环孢素C（CyC）的质控样品。分别在添加H⁺、NH₄⁺和Na⁺的流动相中，用LC-ESI-MS/MS的MRM模式检测质控样品中CyA，CyC的[M+H]⁺、[M+NH₄]⁺和[M+Na]⁺（M=CyA，CyC）峰面积，以其计算基质效应因子；用MS Scan模式检查质控样品中CyA、CyC色谱流出峰的质谱离子组成情况，用于判断基质效应原因。结果： 在添加H⁺、NH₄⁺和Na⁺的流动相中，全血基质中CyA、CyC基质效应因子分别为-76.6%~-54.0%、-86.0%~-55.3%和-42.8%~-34.1%。在加H⁺、加NH₄⁺流动相中，标准液加全血基质的CyA、CyC色谱峰的质谱中出现丰度很高的[M+Na]⁺和[M+K]⁺，在加Na⁺流动相中它们出现丰度很高的[M+K]⁺，而对应的定量离子[M+H]⁺、[M+NH₄]⁺或[M+Na]⁺的丰度显著降低。结论： 全血基质对环孢素的LC-ESI-MS/MS分析产生明显的基质效应，其原因与基质中Na⁺、K⁺同添加在流动相中用于辅助电离的H⁺、NH₄⁺或Na⁺竞争与环孢素加合，导致定量目标加合离子丰度显著降低有关。     

Matrix metalloproteinases-2 and -9 in cervical cancer: different roles in tumor progression

The incidence of uterine cervical cancer has increased slightly in Western countries, with an increase in relatively young women. Overexpression of matrix metalloproteinases (MMPs)-2 and -9 has turned out as a prognostic factor in many cancers. We compared the expression of the proteins MMP-2 and MMP-9 in cervical primary tumors with clinical outcome and risk factors of cervical cancer. One hundred sixty-one patients with cervical cancer treated in Ume? University Hospital or Sahlgrenska University Hospital, Sweden, between 1991 and 1995 were included in the study. Paraffin-embedded tissue samples obtained prior to treatment were examined immunohistochemically by specific antibodies for MMP-2 and MMP-9. Forty-two percent of the tumors were intensively positive for MMP-2 and 31% for MMP-9. Nineteen percent of the samples were intensively positive for both proteinases and 47% negative or weak for both. Overexpression of MMP-2 seemed to predict unfavorable survival under Kaplan-Meier analysis and in the multivariate analysis. Early sexual activity and low parity seemed to correlate to overexpression of MMP-2. MMP-9 was not associated with survival or sexual behavior. Intensive MMP-9 was noted in grade 1 tumors. We conclude that MMP-2 and MMP-9 have different roles in uterine cervical cancer. MMP-2 could be associated with aggressive behavior, but MMP-9 expression diminishes in high-grade tumors.     

An analysis of astrocytic cell lines with different abilities to promote axon growth

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin andN-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.     

输尿管无细胞基质移植物的制备和评价

背景：与小肠黏膜下层等材料相比，脱细胞血管具有天然管状结构，与输尿管形态结构相近，替代输尿管时仅需端端吻合即可，手术操作简单，血管外壁光滑，获取及制备方法简便等优点。 目的：拟应用同种颈动脉血管无细胞基质体外构建输尿管。 设计、时间及地点：观察性实验，于2006-09/2008-06在上海交通大学医学院附属新华医院动物实验中心完成。 材料：长风杂交白猪由上海松联实验动物公司提供。上海交通大学医学院实验动物中心提供的健康成年大鼠8只，用于血管无细胞基质的动物毒性实验。 方法：剥去猪颈动脉血管外膜，PBS冲洗若干遍，然后将血管置于pH 7.1 的PBS中4 ℃下振荡清洗，0.5%十二烷基硫酸钠振荡24 h，后使用双蒸水4 ℃下反复振荡洗涤1周，每日换双蒸水2次。对于解剖过程中肌肉残留相对稍多的血管，在双蒸水洗涤前以混合消化液37 ℃下振荡消化约2 h，再行双蒸水洗涤。制备的无细胞基质置于青、链霉素溶液中，4 ℃保存，完成脱细胞支架制备。 主要观察指标：光镜及电镜观察脱细胞后管壁无细胞基质片的主要成分。将同种异体来源内皮祖细胞培养增殖后植入血管无细胞基质，观察细胞生长情况，并进行血管无细胞基质动物毒性实验。拉力实验了解血管无细胞基质材料的收缩性能。 结果：颈动脉血管无细胞基质中已无细胞成分，无细胞基质主要由胶原成分组成。扫描电镜未见该材料表面存在细胞及细胞碎片，同时发现该无细胞基质存在孔隙样结构。体外同种异体来源的内皮祖细胞培养增殖后植入血管无细胞基质，细胞黏附于无细胞基质。血管无细胞基质按毒性分级属于无毒级。拉力实验说明该无细胞基质具有一定的韧性和牵张性。 结论：采用胰酶和十二烷基硫酸钠制备的颈动脉血管无细胞基质材料无细胞残留，具有一定的韧性和牵张性，种植于其中的种子细胞具备一定的生长能力。