葡萄VvIQD10果形相关基因定位及其互作研究

以葡萄6个不同果形品种盛花期花序为材料,通过荧光定量PCR方法分析比较葡萄IQ67结构域(IQ67 domain, IQD)基因家族成员VvIQD10的表达;从椭圆形葡萄品种‘天山’中克隆出VvIQD10基因编码区序列,对其进行生物信息学分析,采用亚细胞定位瞬时表达载体转化烟草的方法研究VvIQD10蛋白在细胞中的分布情况,并通过酵母双杂交实验和双分子荧光互补试验进行蛋白互作研究。结果表明:(1)VvIQD10基因在长果形葡萄品种中的表达量高于近圆形葡萄品种。(2)VvIQD10基因开放阅读框长度为1 401 bp,编码466个氨基酸。(3)VvIQD10蛋白为不稳定亲水性蛋白,无信号肽和跨膜区,但含保守的IQ67结构域,主要结构为α螺旋和随机卷曲,与猕猴桃IQD家族成员(PSS09955.1)亲缘关系最为接近。(4)VvIQD10蛋白主要定位于微管和质膜,并且直接与细胞骨架组织相关蛋白——钙调素类蛋白(VvCML)相互作用。研究认为,VvIQD10基因可能参与葡萄果形调控,葡萄VvCML蛋白从胞质到微管的募集依赖于VvIQD10,推测VvIQD10蛋白可能通过和钙调素类蛋白相结合来调控细胞骨架运动,进而参与葡萄果实形状的变化。     

Mast cells expressing interleukin 17 in the muscularis propria predict a favorable prognosis in esophageal squamous cell carcinoma

The proinflammatory cytokine interleukin 17 (IL-17) is considered to play a crucial role in diverse human tumors; however, its role in disease progression remains controversial. This study investigated the cellular source and distribution of IL-17 in esophageal squamous cell carcinoma (ESCC) in situ and determined its prognostic value. Immunohistochemistry, immunofluorescence and immunoelectron microscopy were used to identify IL-17-expressing cells in ESCC tissues, paying particular attention to their anatomic localization. Kaplan–Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival in 215 ESCC patients with long-term follow-up (>10 years). The results showed that mast cells, but not T cells or macrophages, were the predominant cell type expressing IL-17 in ESCC tissues. Unexpectedly, these IL-17⁺ cells were highly enriched in the muscularis propria rather than the corresponding tumor nest (p < 0.0001). The density of IL-17⁺ cells in muscularis propria was inversely associated with tumor invasion (p = 0.016) and served as an independent predictor of favorable survival (p = 0.007). Moreover, the levels of IL-17⁺ cells in muscularis propria were positively associated with the density of effector CD8⁺ T cells and activated macrophages in the same area (both p < 0.0001). This finding suggested that mast cells may play a significant role in tumor immunity by releasing IL-17 at a previously unappreciated location, the muscularis propria, in ESCC tissues, which could serve as a potential prognostic marker and a novel therapeutic target for ESCC.     

单纯疱疹病毒2型潜伏相关转录体ORF1的表达及其抗凋亡作用

旨在研究单纯疱疹病毒2型潜伏相关转录体 (LAT) 开放读码框1 (ORF1) 对放线菌素D诱导的凋亡作用的影响。以HSV-2 333基因组为模板PCR扩增ORF1片段,构建重组质粒pEGFP-ORF1,转染Vero细胞,RT-PCR鉴定ORF1的表达。放线菌素D诱导Vero细胞凋亡,通过荧光显微镜观察凋亡小体,Hochest33258荧光染色观察细胞形态变化,MTT检测细胞活性,流式细胞术检测细胞凋亡率。双酶切和测序确认pEGFP-ORF1构建成功,RT-PCR表明该真核表达载体能在Vero细胞中高效表达。转染了pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后,Hochest33258染色显示细胞形态正常。MTT结果表明转染了重组质粒pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后Vero细胞活性与未经任何处理的正常对照组相比,无显著差异 (P＞0.05),但高于放线菌素D诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2且放线菌素D诱导凋亡的Vero细胞组,差异具有统计学意义 (P＜0.05)。流式结果表明,转染重组质粒pEGFP-ORF1且经放线菌素D诱导凋亡组与正常对照组凋亡率差异不显著 (P＞0.05),而显著低于放线菌素D诱导凋亡组和转染空质粒pEGFP-C2且经放线菌素D诱导凋亡组 (P＜0.05)。HSV-2 LAT ORF1具有抗放线菌素D诱导的Vero细胞的凋亡作用。     

NO体外供体硝普钠诱导蚕豆气孔保卫细胞死亡机理研究

为分析NO在植物细胞死亡过程中的作用,以蚕豆表皮条和NO体外供体硝普钠(SNP)及NO信号途径抑制剂为材料,采用表皮条生物法,探讨SNP对蚕豆叶面保卫细胞的毒性机理.结果表明:(1)0.5～9 mmol· L-1的SNP可使蚕豆气孔保卫细胞活性降低,部分细胞死亡,且随着SNP浓度的增高细胞死亡率增高.(2)凋亡抑制剂Z-Asp-CH2-DCB或TLCK可显著降低SNP诱发的保卫细胞死亡率.(3)抗坏血酸(AsA)、过氧化氢酶(CAT)、Ca2+螯合剂EGTA或Ca2+通道抑制剂LaCl3与SNP共同作用时,细胞死亡率显著降低.(4)NO清除剂c-PTIO、MAPK激酶抑制剂PD98059和鸟苷酸环化酶抑制荆ODQ亦能有效阻止SNP诱发的细胞死亡.研究发现,较高浓度的SNP可诱导蚕豆保卫细胞程序性死亡,SNP诱发植物细胞死亡与胁迫组保卫细胞内NO、ROS和Ca2+水平升高有关,cGMP和MAPK参与了SNP诱发的细胞死亡.     

‘阳光玫瑰’雄性不育株花蕾发育期抗氧化酶活性和内源激素含量的变化北大核心CSCD

以葡萄品种‘阳光玫瑰’及其实生后代雄性不育株‘Y-14’为试材,研究花蕾发育期抗氧化酶活性和内源激素含量的变化,阐明葡萄雄性不育新种质的生理生化特征。结果表明:(1)与‘阳光玫瑰’相比,雄性不育株‘Y-14’花蕾的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著降低,而丙二醛(MDA)含量显著增高。(2)‘Y-14’花蕾的生长素(IAA)含量高于‘阳光玫瑰’,而脱落酸(ABA)含量显著低于‘阳光玫瑰’;‘Y-14’花蕾的赤霉素含量(GA_(3))在花蕾发育前期低于‘阳光玫瑰’,而其茉莉酸甲酯(MeJA)在后期显著低于‘阳光玫瑰’;‘Y-14’花蕾的反式玉米素核苷(TZR)含量较‘阳光玫瑰’偏低。(3)在单核早期,‘Y-14’花蕾的ABA含量变化平缓,而‘阳光玫瑰’的ABA含量急剧升高,与此同时GA_(3)含量开始高于‘阳光玫瑰’,而MeJA含量则在单核早期低于‘阳光玫瑰’且差异逐渐显著。研究认为,‘阳光玫瑰’实生后代不育株‘Y-14’花蕾发育期SOD、POD、CAT活性和MDA、IAA、ABA、GA_(3)、MeJA含量异常变化可能导致了雄性不育的发生,而且单核早期可能是‘Y-14’雄性不育产生的关键时期。     

Genotoxicity of hydrated sulfur dioxide on root tips of Allium sativum and Vicia faba

Genotoxicity of sulfur dioxide (SO(2)) and its hydrates (bisulfite and sulfite) in human lymphocytes and other mammalian cells have been found earlier in our laboratory. In the present studies, we used Allium stavium and Vicia faba cytogenetic tests, which are the highly sensitive and simple plant bioassays. A mixture of sodium bisulfite and sodium sulfite (1:3), at various concentrations from 1 x 10(-4) to 2 x 10(-3)M was used for the treatment. Genotoxicity was expressed in terms of anaphase aberration (AA) frequencies in the Vicia-AA test and in terms of micronuclei (MCN) frequencies in both Vicia-MCN test and Alllium-MCN test. On average, the results showed a 1.7-3.9-fold increase of AA frequencies and a 3.5-4.5-fold increase of MCN frequencies in Vicia root tips as compared with the negative control. Similarly, results of Allium-MCN test also showed a significant increase in MCN frequencies in the treated samples. In addition, pycnotic cells (PNC) appeared in Allium root tips of treated groups. The frequencies of MCN, AA and PNC increased dose-dependently and the cell cycle delayed at the same time in bisulfite treated samples. Results of the present study suggest that the Vicia and Allium cytogenetic bioassays are efficient, simple and reproducible in genotoxicity studies of bisulfite.     

单纯疱疹病毒Ⅱ型潜伏、激活细胞模型建立

【目的】建立单纯疱疹病毒Ⅱ型(HSV-2)潜伏感染人神经母细胞瘤细胞株SH-SY5Y及激活的细胞模型。【方法】分别加入20,40,60,80,100,120,140μmol/L的阿昔洛韦(ACV),观察对SH-SY5Y细胞生物性状的影响;在ACV存在的情况下,分别将含有0.1、1、10、100MOI的病毒液接种SH-SY5Y细胞,运用相差显微镜观察病毒对细胞的影响,确定潜伏的建立;分别用41℃、42℃、43℃、44℃、45℃加热0.5h、1.0h、1.5h、2.0h、2.5h,观察加热时间及温度诱导HSV-2在SH-SY5Y细胞中激发的最适条件;加入25、50、75、100、125μmol/L福斯高林(Forskolin)诱导病毒在细胞中激活,探讨诱导的最佳浓度;对HSV-2在SH-SY5Y细胞中的潜伏及激发进行验证并测序;运用相差显微镜观察病毒激活后细胞形态的变化。【结果】60μmol/LACV的存在最适合HSV-2在SH-SY5Y细胞中建立潜伏状态;1-10MOI的感染量均能取得较好的病毒潜伏及激发效果;通过观察,病毒在SH-SY5Y细胞中最长可潜伏14d;43℃,1.5h及75μmol/LForskolin均为诱导病毒潜伏激发的最佳条件;相差显微镜观察病毒激发后细胞病变,从24h到72h,细胞变性、坏死的程度、数量随感染时间延长而增加;HSV-2LAT、gG基因PCR扩增及电泳结果,证实病毒在细胞中的潜伏及激活。【结论】初步在人神经母细胞瘤细胞株SH-SY5Y上建立了HSV-2潜伏感染及激活的细胞模型,为下一步研究HSV-2的潜伏与激发机理,了解HSV-2的致病机制打下基础。     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0mg/L, induced a 1.9-3.9-fold increase in MN frequency and a 1.5-1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P<0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15-80% decrease in mitotic indices (MI), but at the lowest concentration (0.005mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.