表观遗传学与人类表观基因组计划

表观遗传学已被用来描述许多生物学过程,成为生物学与医学领域中热点的学科之一.本文简要介绍表观遗传学与表观遗传基因组学的概念、人类表观基因组计划研究的目标与意义,并阐述DNA甲基化、组蛋白修饰、染色质重塑和非编码微小RNA等表观遗传学调控基因表达的机制.我们已经认识到人类疾病基因缺损可能部分或完全与表观遗传有关.所以,研究疾病状态下非突变的、可逆的表观遗传调节,以及治疗的可能性具有重要实际意义.     

大肠杆菌中表达关键基因产异丁醇的研究

目的:改造大肠杆菌的代谢途径,使非生产菌株大肠杆菌具备产异丁醇的能力.方法:将乳脂乳球菌NIZO B1157的2-酮酸脱羧酶基因kdcA克隆到大肠杆菌中,使大肠杆菌产异丁醇;另外,采取两种方法过量表达alsS、ilvC、ilvD基因,增加前体物质酮酸的供应,以提高异丁醇的产量:一是与kdcA串联表达;二是在另一个相容质粒中表达.结果:大肠杆菌工程菌具备产异丁醇的能力,其中相关基因在一个质粒中串联表达的产量比其在两个相容质粒中共表达的产量高30倍,达到3g/L.结论:导入的酮酸合成途径与醇类生产途径结合,能使非生产菌株大肠杆菌生产异丁醇,并且单质粒表达代谢途径相关基因的效果优于双质粒表达.     

Inhibition of Filamin-A Reduces Cancer Metastatic Potential

Filamin-A cross-links actin filaments into dynamic orthogonal networks, and interacts with an array of proteins of diverse cellular functions. Because several filamin-A interaction partners are implicated in signaling of cell mobility regulation, we tested the hypothesis that filamin-A plays a role in cancer metastasis. Using four pairs of filamin-A proficient and deficient isogenic cell lines, we found that filamin-A deficiency in cancer cells significantly reduces their migration and invasion. Using a xenograft tumor model with subcutaneous and intracardiac injections of tumor cells, we found that the filamin-A deficiency causes significant reduction of lung, splenic and systemic metastasis in nude mice. We evaluated the expression of filamin-A in breast cancer tissues by immunohistochemical staining, and found that low levels of filamin-A expression in cancer cells of the tumor tissues are associated with a better distant metastasis-free survival than those with normal levels of filamin-A. These data not only validate filamin-A as a prognostic marker for cancer metastasis, but also suggest that inhibition of filamin-A in cancer cells may reduce metastasis and that filamin-A can be used as a therapeutic target for filamin-A positive cancer.     

Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.     

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.     

HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a．a-750a．a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB／c鼠。定期取兔血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1：3200。六周后,取鼠脾脏制各淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E：T＝200：1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCVE2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。     

PNAS-4 expression and its relationship to p53 in colorectal cancer

PNAS-4 is a novel pro-apoptotic protein activated during the early response to DNA damage; however, the molecular mechanisms and pathways regulating PNAS-4 expression in tumors are not well understood. We hypothesized that PNAS-4 is a p53 down-stream target gene and designed this study. We searched online for putative p53-binding sites in the entire PNAS-4 gene and did not find any corresponding information. In HCT116 colon cancer cells, after being transfected with small interfering RNA to silence p53, the expressions of PNAS-4 and other known p53 target gene (Apaf1, Bax, Fas and Dr5) were determined by real-time PCR. We found that PNAS-4 was up-regulated while Apaf1, Bax, Fas and Dr5 were down-regulated. We then examined the expression of PNAS-4 and p53 mutation in colorectal cancer patients. PNAS-4 expressed both in colorectal cancers and normal tissues, but compared with paired control, PNAS-4 was up-regulated in cancers (P = 0.018). PNAS-4 overexpression ratios were correlated to the p53 mutant status (P = 0.001). The mean PNAS-4 expression levels of p53 mutant homozygote group and heterozygote group were higher than that of p53 wild type group (P = 0.013). The expression ratios of PNAS-4 (every sample in relative to its paired normal mucosa) were different between negative lymph node metastasis (66% up-regulated, 34% down-regulated) and positive metastasis (42% up-regulated, 58% down-regulated). Taken together, these findings suggested that PNAS-4 was not a p53 target, but overexpression of PNAS-4 was correlated to p53 inactivity in colorectal cancer.     

滇金丝猴雌性个体间的理毛行为

相互理毛行为广泛存在于社会性群居灵长类动物中,通常具有清洁卫生和社会交往功能。2012 年10 月至2013 年6 月,我们在云南白马雪山国家级自然保护区对一人工辅助投食滇金丝猴群,采用全事件取样法和焦点动物取样法收集了雌性个体间相互理毛的行为数据,包括理毛的部位、理毛的姿势、理毛的时间和回合数。研究结果表明:滇金丝猴雌性个体之间每次相互理毛的平均时间为5. 7 min。相互理毛部位较多的发生在自我理毛不能进行(达到)的部位(61.1% );在不能自我理毛部位的相互理毛行为持续时间长,平均9.7 min;在个体能够进行自我理毛部位的相互理毛持续时间短,平均为3. 2 min。相互理毛的姿势以对坐为主(48. 4% ),不同理毛姿势的理毛时间差异显著。新迁入家庭单元的雌性个体为理毛的首先发起者,但其获得被理毛的时间却并不多。滇金丝猴雌性个体相互理毛部位、理毛姿势和理毛时间的差异表明,它们之间的相互理毛行为符合卫生功能假说和社会功能假说。     

醋酸纤维素膜为基础的葡萄糖生物传感器的研制

用共价法将酶固定在醋酸纤维素膜上,方法简便易行,制造的酶膜稳定,比活力高。同时采用该方法制备了葡萄糖氧化酶酶膜,与氧电极组装成测定葡萄糖的生物传感器,线性范围为50~800mg／dl,仪器工作的最适pH为6．0,最适温度为40℃。将该膜与过氧化氢电极组装得到的传感器具有以下特性：线性范围为10~200mg／dl,最适pH为6．0,测定结果与酶试制盒有良好相关性。