Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

OBJECTIVE:In the present study,we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine,celastrol and pristimerin,to enhance recombinant adeno-associated virus(rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro,and in murine hepatocytes in vivo.METHODS:Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin.The transgene expression,percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment.In addition,nonobese diabetic/severe combined immunodeficient gamma(NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide,celastrol,pristimerin or a positive control,bortezomib.The transgene expression in liver was detected and compared over time.RESULTS:We observed that treatment with pristimerin,at as low as 1 umol/L concentration,significantly enhanced rAAV2 vector-mediated transgene expression in vitro,and intraperitoneal coadministration with pristimerin at 4 mg/(kgd) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo.The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin.The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.CONCLUSION:These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.     

HPLC测定烈香杜鹃中6种黄酮成分的含量及主成分分析

目的: 建立HPLC测定烈香杜鹃中芦丁、金丝桃苷、槲皮苷、槲皮素、山柰酚和异鼠李素6种黄酮成分的方法,同时应用主成分分析评价其质量。方法: 样品经75%甲醇超声,采用C₁₈色谱柱(4.6 mm×250 mm,5 μm),乙腈-0.2%磷酸水为流动相梯度洗脱,流速1.0 mL·min^-1,检测波长365 nm。结果: 芦丁,金丝桃苷,槲皮苷,槲皮素,山柰酚,异鼠李素在0.009 7～0.194 9,0.007 1～0.141 6,0.188 0～3.752,0.027 1～0.434 0,0.010 8～0.216 0,0.005 8～0.116 6 μg线性范围良好,加样回收率均在100%～105%,RSD都 < 3%。结论: HPLC操作简便、重复性好、结果准确。根据不同化学指标,采用主成分分析可以进行烈香杜鹃质量评价。     

不同茬口对当归根际土壤酶活性及其产量和品质的影响

目的研究当归正茬、迎茬、连茬对根际土壤主要酶活性和产量品质的影响。方法采用比色法测定不同茬口当归根际土壤中脲酶、磷酸酶、多酚氧化酶活性,采用高锰酸钾滴定法测定过氧化氢酶活性。结果苗期和收获期连茬土壤中的脲酶和磷酸酶活性显著低于正茬,脲酶活性分别降低16.53%和37.17%,中性磷酸酶活性分别降低28.01%和30.69%;多酚氧化酶活性在整个生育期均是连茬显著高于正茬,苗期、根膨大期和收获期分别比正茬增加15.10%、83.67%、38.53%。连茬当归产量、挥发油和浸出物量均显著低于正茬,分别比正茬下降49.00%、25.26%、12.58%。结论连茬栽培中当归产量、挥发油和浸出物量显著低于正茬;连茬栽培当归根际土壤脲酶和磷酸酶活性显著降低,而过氧化氢酶和多酚氧化酶活性呈升高趋势。     

HPLC法测定十五味龙胆花丸中龙胆苦苷和丁香酚

目的建立十五味龙胆花丸(白花龙胆、檀香、诃子等)中龙胆苦苷和丁香酚的定量测定方法。方法采用ShiseidoC18色谱柱(250 mm×4.6 mm,5μm);甲醇-0.2%磷酸水溶液梯度洗脱为流动相;体积流量1 mL/min;柱温为室温;检测波长270 nm。结果龙胆苦苷和丁香酚质量浓度分别在0.141 4～1.060 8μg/mL(r=0.999 9)及0.156 6～1.174 8μg/mL(r=0.999 7)范围内与峰面积呈良好的线性关系;加样回收率(n=6)分别为99.85%(RSD为1.77%)和99.72%(RSD为1.85%)。结论方法简便可行、灵敏度高,可用于十五味龙胆花丸的质量控制。     

基于气味分析系统的不同产地秦艽的区分及相关性研究

目的：基于电子鼻PEN3系统结合多元统计分析对不同产地秦艽进行鉴别。方法：先通过电子鼻系统动态采集秦艽芳香成分并得到响应值,通过单因素试验确定最佳试验条件,再应用主成分分析（PCA）、线性判别分析（LDA）及负荷加载分析法（Loadings）对结果进行分析。结果：秦艽电子鼻最佳试验条件为样品称样量3.0 g、进气量250 mL·min–1、样品室温放置时间15 min、颗粒度60目。PCA和LDA均对不同产地的秦艽有一定的区分能力,并且LDA优于PCA。结论：电子鼻对秦艽的种类和产地鉴别准确性好且简便可行,能更准确、客观地对不同种、不同产地、不同栽培方式的秦艽进行鉴别。     

急进高原缺氧对大鼠肝脏孕烷X受体表达的影响

目的: 研究急进高原缺氧环境对大鼠肝脏孕烷X受体（PXR）表达的影响及相关机制。方法: Wistar雄性大鼠40只,随机分为对照组和急进高原缺氧12、24、36、48 h组,每组8只。待预定缺氧时间完成,采集各组大鼠腹主动脉血,全自动血气分析仪进行血气分析;HE染色观察肝组织病理变化状况;实时定量PCR测定各组大鼠肝组织中PXR mRNA水平;蛋白质印迹法测定各组大鼠肝组织中PXR和蛋白酶SUG1的表达。结果: 与对照组比较,急进高原缺氧12 h后大鼠血液酸碱度值降低;缺氧24 h后动脉血氧饱和度（SaO₂）低于80%,动脉氧分压（PaO₂）低于60 mmHg（1 mmHg=0.133 kPa）;缺氧48 h后动脉二氧化碳分压（PaCO₂）增加（P < 0.05）。对照组大鼠肝组织结构完整,急进高原缺氧12、24 h组肝组织中央静脉有明显水肿,缺氧36、48 h组大鼠肝组织见炎性浸润。大鼠肝脏内PXR mRNA表达从急进高原缺氧12 h时即显著下调,缺氧12、24、36、48 h组分别降低了63%、96%、86%、85%（均P < 0.05）;其蛋白表达从缺氧36 h后显著上调,缺氧36、48 h后分别上调93%、99%（均P < 0.05）。蛋白酶SUG1的表达从缺氧24 h开始降低,缺氧24、36、48 h后分别下降14%、34%、46%（均P < 0.01）。结论: 急进高原缺氧可对大鼠肝脏中核受体PXR表达产生影响,蛋白酶SUG1可能是影响急进高原缺氧下PXR表达的调控因素。     

超滤-纳滤集成技术纯化、浓缩当归水提取液的工艺考察

目的:为提高当归水提取物的品质,降低生产成本,研究超滤-纳滤集成技术用于当归水提取液纯化和浓缩的可行性。方法:以阿魏酸保留率为评价指标,以陶瓷膜孔径、工作压强、滤过温度为考察因素,通过正交试验优化当归水提取液的超滤纯化工艺。以阿魏酸泄露率为评价指标,以截留相对分子质量、料液温度为考察因素,通过全面试验确定当归水提取液的纳滤浓缩工艺。结果:当归水提液最佳超滤工艺为膜孔径50 nm,滤过压强0.10 MPa,滤过温度50℃;阿魏酸保留率>90%。当归水提液纳滤浓缩工艺为膜截留相对分子质量200 Da,滤过温度45℃;阿魏酸泄漏率0.1%。结论:超滤-纳滤集成技术纯化、浓缩当归水提取物时,有效成分保留率高、料液受热时间短、能耗低,可提高当归水提物的品质并降低其生产成本。     

Comparative study of the outcomes of the second kidney transplantation from the young deceased donors versus living-unrelated donors

PurposeTo compare the kidney graft function and survival in patients who had second kidney transplantation from living donors versus those who had a second transplant from young deceased donors.MethodsIn this retrospective cohort study, a total of 86 patients who underwent second kidney transplantation in Shariati hospital from 2001 until 2017 were enrolled. Baseline clinical data on the age, sex, type of kidney donor (living unrelated or deceased), duration of pretransplant dialysis, and the length of hospitalization were recorded. As the indicators of the graft function, we used the serum creatinine level and estimated glomerular filtration rate (eGFR) at time intervals during the study. The 1, 5, and 10-year graft survival rates were reported using life tables and the relative hazard ratios of the graft failure were calculated using the forward stepwise Cox proportional hazard model.ResultsForty-six of our patients were men (53.5%), with a mean ± SD age of 44.3 ± 12.3 years at the time of transplantation. The majority of the enrolled patients received the kidney from living unrelated donors (50 vs. 36 patients). In terms of serum creatinine and eGFR, at time intervals, no significant difference was found between the two recipient groups. In the living donor group, the 1, 5, and 10-year graft survival rates of the second transplant were 91% (95%CI: 73–96%), 87% (95%CI: 69–95%), and 82% (95%CI: 59–92%), and for the deceased donor group were 95% (95% CI: 69–99%), 95% (95%CI: 69–99%), and 79% (95%CI: 31–95%), respectively.ConclusionConsidering the long-term outcomes of the second kidney transplantation, in our experience, the graft function and survival, either from the living or deceased donors, were favorable; and the type of organ donation had no significant effect on the risk of graft failure.     

Th17/Treg cell balance in stable liver transplant recipients

BackgroundImmune monitoring of transplanted patients may provide a reliable basis for the individualization of immunosuppressive therapy. In addition, it might be applied for realizing the early and non-invasive diagnosis of acute allograft rejection.MethodsPercentages of TCD4 + IL-17+ (Th17) and TCD4 + CD25 + CD127^dim/− (Treg) cells, as well as serum levels of interleukin (IL)-17 and transforming growth factor (TGF)-β1, were evaluated in 30 stable patients using flow cytometry and ELISA techniques before and six months after liver transplantation. Besides, the same cells and cytokines were quantified in 10 recipients with acute allograft rejection.ResultsSix months post-transplant, the percentage of Th17 and Treg cells in the peripheral blood of stable liver transplant recipients reduced significantly, but the Th17/Treg ratios were comparable to the pre-transplant period (1.24 vs. 1.56); however, Th17/Treg ratios in the rejection group was significantly higher than in the stable recipients (4.06 vs. 1.56, P-value = 0.001). Stable patients showed decreased amounts of serum IL-17 which was remarkably lower than in the rejection group (P-value = 0.01). Moreover, there was a significant correlation between the serum level of IL-17 and the percentage of Th17 cells (P-value <0.001). Th17 frequency was negatively associated with the liver allograft function. Notably, TGF-β1 levels differed neither between pre-and post-transplant samplings nor between stable and rejection groups.ConclusionSix months after liver transplantation, the mean Th17/Treg ratio in stable recipients remained comparable to the pre-transplant values; however, it was significantly elevated in patients with acute allograft rejection, suggesting the Th17/Treg ratio as a probable predictor of acute rejection.     

定西市栽培当归生态适宜性研究

目的:了解定西市栽培当归生态适宜性,筛选影响其分布的主要生态因子。方法:通过走访和实地调查,从定西市当归栽培县区获取857个采样点的经度、纬度、海拔等地理信息,结合全国生态因子数据,利用Maxent模型和ArcGIS软件的空间分析功能进行分析。结果:定西市栽培当归适宜分布区主要集中在定西市南部岷县、漳县,渭源县、临洮县、陇西县也有分布,但适宜区域较小。影响定西市当归适宜性分布的主要生态因子为海拔高度、降水量、温度。结论:本研究对定西市栽培当归生态适宜区进行了等级划分,生成了当归生态适宜性区划图,可为定西市当归合理引种栽培提供理论依据。