EOLA1基因突变体的克隆和鉴定

目的 对ECV304细胞株EOLAI基因进行克隆测序以及mRNA水平检测。方法 采用RT-PCR扩增EOLAI基因片断,连接到T载体进行测序。结果 测序发现一种目的基因mRNA剪接突变体,其第3外显子起始部分比Genebank数据库相应序列多出19个碱基。RT-PCR显示剪接突变体与野生型EOLAI基因具有不同的表达水平和LPS反应性。结论 发现一个EOLA1基因剪接突变体,其生物学功能尚不清楚。     

The expression of P63 protein in some keratinocyte original tissues and cells

Objective： To examine the expression patterns of p63 in tissues of particular keratinocyte original hyperproliferate diseases and variety cell types for determining if P63 is the marker of proliferative potential keratinocytes. Methods： P63 protein was detected and analyzed by immunoreactivity method and Western blot in biopsy specimens of keratinocyte original disorders including squamous cell carcinomas SCC, basal cell carcinomas BCC, Bowen＇ s disease and other tissues or cells, such as psoriasis vulgaris, normal skin tissues, primary cultured keratinocytes, immortal HaCaT cells, and epidermoid carcinoma cells A431. Results： P63 protein was expressed in the nuclei of basal and suprabasal layer of the epidermis, germinative cells of sebaceous glands in normal epidermal. P63 was strongly and diffusely detected in the majority of tumor cells in BCC and poorly-differentiated SCC. In Bowen＇ s disease, p63 expresses are remarkable in all cell layers. In the psoriasis plaque epidermal, p63 expressed mainly in basal cells and part of spinous cells. P63 expressed more strongly in primary cultured keratinocytes than in A431 cells or HaCaT cells. Conclusion： P63 is a nuclei marker of undifferentiated keratinocytes with the proliferative potential and may disrupt the terminal differentiation. The overexpression of p63 reflects immaturity of the tumor cells. The immunohistochemical staining of p63 may be useful for investigating the origin and differentiation of tumor cells.[第一段]     

SHIP1 基因过表达非小细胞肺癌细胞系的建立及其对细胞增殖能力的影响

目的：观察SHIP1 对NSCLC细胞增殖的影响。方法：由NCBI Gene 数据库查询获得人SHIP1 基因CDS区,插至载体pTSB-CMV-MCS-SBP-3Flag-EGFP,构建SHIP1 真核过表达质粒;进一步利用其构建SHIP1 过表达慢病毒。用该慢病毒感染A549、SPCA-1 和PC-9 细胞株,获得SHIP1 稳定过表达NSCLC细胞系。Western blotting 和qRT-PCR分别从蛋白水平和mRNA水平检测SHIP1 的表达变化。采用MTT法和克隆形成实验检测过表达SHIP1 的PC-9 细胞增殖活力和克隆形成能力。Western blotting检测AP-1 蛋白复合体各组分的表达。结果：SHIP1 真核过表达质粒经测序证实构建成功。稳定过表达SHIP1 的A549、SPCA-1 和PC-9 细胞,在荧光显微镜下可见均一表达绿色荧光。与阴性对照组比较,细胞中SHIP1 在mRNA（P<0.01）及蛋白水平均明显升高。在PC-9 细胞中,过表达SHIP1 使细胞增殖、克隆形成能力降低（均P<0.01）,p-c-Jun、FosB 等表达降低。结论：成功构建了稳定过表达SHIP1 的A549、SPCA-1和PC-9 细胞模型;过表达SHIP1可通过抑制AP-1 家族蛋白抑制NSCLC细胞增殖能力。     

基因组信息指导下海参共附生Penicillium sclerotiorum SD-36嗜氮酮类化合物的挖掘

目的 结合生物信息学与分子遗传操作技术深入挖掘海参共附生菌核青霉(Penicillium sclerotiorum)SD-36的活性次级代谢产物,并探析其生物合成基因簇中转录调控因子的作用。方法 基于本课题组前期P. sclerotiorum SD-36的基因组信息,利用antiSMASH预测了一条具有合成嗜氮酮类化合物潜力的双PKS基因簇。针对该簇中一个锌指转录因子PsAza1构建了含潮霉素抗性基因和强启动子pgpdA的转录因子基因过表达盒,转化P. sclerotiorum SD-36原生质体后,经过抗性筛选及PCR验证获得阳性转化子OE::PsAza1。发酵培养后HPLC检测次级代谢产物变化并通过qRT-PCR验证基因簇核心基因的转录水平。结果 OE::PsAza1的多种次级代谢产物产量增加,其中两个化合物通过质谱、核磁数据鉴定为活性嗜氮酮类isochromophilone VI和sclerotiorin C,其产量较野生型分别增加了约6倍和4.5倍。qRT-PCR检测该簇中两个聚酮合酶基因及Psaza1基因的转录水平,显示上调60-80倍。结论 首次明确P. sclerotiorum SD-36的双PKS基因簇编码活性嗜氮酮类化合物,且转录因子PsAza1正向调控该簇核心基因的表达水平及化合物产量。本研究结果为P. sclerotiorum SD-36中化合物isochromophilone VI和sclerotiorin C的生物制备及调控研究奠定理论基础。     

阳春砂HMGR和DXR在烟草中单独及共同过量表达调控萜类化合物的生物合成

目的：HMGR 和 DXR 分别是萜类生物合成途径———MVA 和 MEP 途径的关键酶,本研究探讨阳春砂AvHMGR 和AvDXR 在转基因烟草中的过量表达对不同萜类化合物生物合成的影响。方法：采用实时荧光定量PCR（RT-qPCR）技术分析AvHMGR 和AvDXR 的表达水平,应用专一底物法测定HMGR 和DXR 的酶活性,利用气相色谱-质谱（GC-MS）联用技术检测不同萜类化合物的变化。结果：AvHMGR 或AvDXR 单独过量表达抑制了HMGR 和DXR 的活性,提高了五针松烯、新植二烯、薄荷烯和甾醇的含量;而AvHMGR 和AvDXR 共同过量表达对不同植株中酶活性的影响有差异,提高了甾醇和植醇的含量,但抑制了新植二烯的积累。结论：AvHMGR 和AvDXR 单独或共同过量表达对烟草中不同萜类化合物的合成调控存在差异,同时也证明了MVA 和MEP 途径并不完全独立,而是有着相互交流,本研究为利用阳春砂AvHMGR 和AvDXR 进行萜类化合物的代谢调控提供了依据。     

ASRGL1 downregulation suppresses hepatocellular carcinoma tumorigenesis in a CDK1-dependent manner

The asparaginase-like protein 1 (ASRGL1) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Emerging evidences have shown a strong correlation between ASRGL1 expression and tumorigenesis. However, the expression and biological function of ASRGL1 in hepatocellular carcinoma (HCC) are still unclear. Here, we explored anti-tumor activity and fundamental mechanisms of ASRGL1 blockade in the HCC progression. Expression levels of ASRGL1 in patients with HCC were higher than those in the adjacent normal tissue. In addition, increased expression of ASRGL1 in HCC patients was correlated with poor overall survival. Knockdown of ASRGL1 gene in HepG2 and Li-7 cell lines inhibited cell proliferation, migration and invasion, but promoted apoptosis in vitro. ASRGL1 knockdown suppressed tumor growth in vivo. Conversely, ASRGL1 overexpression promoted cell proliferation, migration and invasion in HepG2 cells. Through bioinformatics analysis, we found that ASRGL1 might participate in the regulation of the cell cycle. Flow cytometry analysis conformed that ASRGL1 knockdown captured the cell cycle during the G2/M phase. ASRGL1 blockade promoted P53 protein expression and reduced expression of cyclin B and CDK1 proteins, as well as failed to binding. Moreover, CDK1 overexpression was able to reverse the decreased proliferation, migration and invasion of HepG2 cells induced by ASRGL1 knockdown. Collectively, our studies indicate that ASRGL1 blockade functions to inhibit cyclin B/CDK1-dependent cell cycle, leading to G2-to-M phase transition failure and tumor suppression in HCC.