基于杂交链式(HCR)反应的甲型流感病毒检测技术研究

甲型流感病毒的现场快速检测对于流感的及时有效防控具有重要意义．本研究基于杂交链式(HCR)反应,利用GO对荧光基团的猝灭作用及共同实现了对甲型流感病毒的快速检测．当目标序列存在时,可引发HCR反应,使短链DNA形成长链,保护FAM荧光基团不被猝灭,从而实现目标物的检测．实验结果表明,该方法在10～40 nmol/L范围内荧光强度与目标检测物浓度表现出了良好的线性关系,检测范围为5～100 nmol/L．这种HCR等温扩增检测技术具有较好的样本检测能力,具有等温、无酶、反应体系简单、操作步骤简便等优点,表现出良好的现场检测应用前景．     

Long non‐coding RNA highly up‐regulated in liver cancer promotes epithelial‐to‐mesenchymal transition process in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is an oral and maxillofacial malignancy that exhibits high incidence worldwide. In diverse human cancers, the long non‐coding RNA (lncRNA) highly up‐regulated in liver cancer (HULC) is aberrantly expressed, but how HULC affects OSCC development and progression has remained mostly unknown. We report that HULC was abnormally up‐regulated in oral cancer tissues and OSCC cell lines, and that suppression of HULC expression in OSCC cells not only inhibited the proliferation, drug tolerance, migration and invasion of the cancer cells, but also increased their apoptosis rate. Notably, in a mouse xenograft model, HULC depletion reduced tumorigenicity and inhibited the epithelial‐to‐mesenchymal transition process. Collectively, our findings reveal a crucial role of the lncRNA HULC in regulating oral cancer carcinogenesis and tumour progression, and thus suggest that HULC could serve as a novel therapeutic target for OSCC.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导

在黄花蒿(Artemisia annua L.)发根液体培养中,黄花蒿内生炭疽菌(Colletotrichum sp. B501)细胞壁寡糖提取物可促进发根青蒿素的合成.经寡糖诱导子(20 mg/L)处理4 d后,发根青蒿素含量达1.15 mg/g, 比对照高出64.29%.诱导作用与诱导子浓度、作用时间相关.诱导处理1 d后,X射线能谱分析表明黄花蒿发根细胞中Ca2+积累量显著增高,电镜观察发现液泡内出现高电子致密物,具活性氧清除作用的过氧化物酶表现出高活性(6.5 unit*min-1*g-1 FW).诱导处理第三天,细胞核DNA呈梯度条带降解,部分细胞出现程序化死亡.内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成.     

汉滩病毒M和S基因不同拼接方式嵌合基因免疫效果的研究

本文在前期工作的基础上,构建了汉滩病毒76－118株M基因G2片段与S基因5'端0.7Kb片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7及pcDNA3.1-S0．7G2;用该质粒免疫BALB/c小鼠,结果表明两种质粒免疫小鼠可同时诱导产生抗滩滩病核蛋白（NP)及糖蛋白（GP）特异性的抗体,且前者刺激产生的抗体效价明显高于后者。淋巴细胞增殖实验表明,pcDNA3.1-G2S0.7组免疫小鼠脾细胞时NP及GP的增殖指数均明显高于空载体对照组,而pcDNA3.1-S0．7G2组未检测到其淋巴细胞有明显的增殖。这说明汉滩病毒M基因G2片段及S基因0.7Kb片段的嵌合基因既可刺激机体产生特异的抗汉滩病毒体液免疫应答,也可刺激机体产生特异的细胞免疫应答。不同拼接方式对嵌合基因免疫效果有很大影响,嵌合基因G2S0．7这种拼接方式明显优于S0．7G2。     

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphorylates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phosphorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphorylation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.     

小鼠beta-防御素2真核表达载体的构建及表达

构建小鼠heta-防御素2(murie beta-defensin 2,mBD2)的真核表达载体pcDNA-mBD2,并观察其在真核细胞中的表达.从小鼠肝组织中提取mRNA通过逆转录聚合酶链反应扩增得到mBD2基因,定向克隆至真核表达栽体pcDNA3.1(+)获得pcDNA-mBD2;经酶切和测序鉴定构建正确,应用脂质体法转染siha细胞,并通过蛋白免疫印迹法检测细胞内mBD2的表达.结果显示,构建了小鼠mBD2的真核表达载体,转染siha细胞培养并采用G418稳定筛选后,获得稳定转染细胞,收集并裂解转染细胞,蛋白免疫印迹法检测到转染细胞内mBD2的表达.成功构建了真核表达裁体pcDNA-mBD2,为研究mBD2在宫颈癌发生发展过程中的作用及作用机制奠定基础.     

乙型肝炎病毒核心抗原基因序列的启动子活性

将核心抗原基因起始码下游的序列,构建两个ＣＡＴ报告基因表达质粒,即ｐＣＡＴＮ Ｉ和ｐＣＡＴＮⅡ,转染ＨｅｐＧ２与ＣＶ－１细胞后,均可使ＣＡＴ报告基因表达,表现出启动子活性。