人工锌指核酸酶突变EGFP基因的功能分析

锌指核酸酶技术在基因定点修饰中具有效率高和特异性好等特点,并成功应用于数十种生物。目前,该技术是否能应用羊上尚未报道。为了敲除转基因山羊标记基因 (EGFP),构建了一对针对EGFP外显子上的锌指核酸酶表达载体,将其电转染至转EGFP基因胎儿成纤维细胞中,研究了锌指核酸酶突变EGFP基因的效率和方式,利用基因显微注射单细胞获得获得的转基因 (EGFP) 细胞系作为锌指核酸酶的靶细胞。结果显示,通过锌指核酸酶的突变作用,转染后的细胞发绿色荧光比例下降,测序结果显示在EGFP外显子中插入1个碱基G,导致编码EGFP基因的阅读框改变,从而起到基因突变的作用。结果表明,文中构建的锌指核酸酶对EGFP基因有突变作用,可以为以后获得无标记基因供核细胞进行体细胞核移植生产克隆羊奠定基础。     

Caspase-8及Caspase-9在肝细胞癌组织中的表达及意义

目的:观察凋亡相关因子半胱氨酸天冬氨酸蛋白酶8(caspase-8)和半胱氨酸天冬氨酸蛋白酶9(caspase-9)在肝细胞癌中的表达及临床病理意义。方法:选择我院自2012年7月~2013年4月的53例肝细胞肝癌患者作为观察组,选择同一时期在医院体检的50例正常肝组织患者作为对照组,采用原位分子杂交方法对两组患者的Caspase-8和Caspase-9mRNA进行检测。结果:观察组与对照组中caspase-8、caspase-9的阳性表达率分别为84.91%(45/53)、88.68%(47/53)和60.00%(30/50)、82.00%(41/50),观察组与对照组比较均有升高趋势,比值有显著性差异(P0.05)。结论:肝癌细胞中caspase-8、caspase-9的较高表达显示在肝癌的发生发展过程中起到重要作用,在肝癌组织细胞增殖凋亡中具有一定作用,肝癌中caspase-8和caspase-9蛋白的表达均对于判断肝癌预后具有一定的临床价值。     

Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.     

Inhibition of TREM-1 and Dectin-1 Alleviates the Severity of Fungal Keratitis by Modulating Innate Immune Responses

Purpose

To explore the possibility that inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) and Dendritic cell-associated C-type lectin-1(Dectin-1) could modulate the innate immune response and alleviate the severity of corneal fungal keratitis.

Method

TREM-1 and Dectin-1 expression was detected in fungus-infected human corneal specimens by real-time PCR. C57BL/6 (B6) mice were injected with Aspergillus fumigatus and divided into 4 groups that received subconjunctival injections of PBS and IgG as a control (group I), mTREM-1/IgG fusion protein (group II), the soluble β-glucan antagonist laminarin (group III), or mTREM-1/Fc and laminarin (group IV). Corneal virulence was evaluated based on clinical scores. TREM-1 and Dectin-1 mRNA levels were assayed using real-time PCR. The distribution patterns of TREM-1, Dectin-1 and cellular infiltrates in fungus-infected corneas were examined by immunohistochemistry. Moreover, changes in T Helper Type1 (Th1)-/ T Helper Type1 (Th2)- type cytokines and proinflammatory cytokines were measured.

Results

The expression of TREM-1 and Dectin-1 increased significantly and correlated positively with the progression of fungal keratitis. Most infiltrated cells were neutrophils and secondarily macrophages in infected cornea. The clinical scores decreased after interfering with TREM-1 and Dectin-1 expression in infected mouse corneas. Levels of Th1-type cytokines including interleukin-12 (IL-12), IL-18 and interferon-γ (IFN-γ) were decreased in the cornea, while the levels of Th2-type cytokines, including IL-4, IL-5 and IL-10, showed obvious increases.

Conclusion

TREM-1 and Dectin-1 function concurrently in the corneal innate immune response by regulating inflammatory cytokine expression in fungal keratitis. Inhibition of TREM-1 and Dectin-1 can alleviate the severity of corneal damage by downregulating the excessive inflammatory response.     

Role of Bmi-1 in Regulation of Ionizing Irradiation-Induced Epithelial-Mesenchymal Transition and Migration of Breast Cancer Cells

Radiotherapy is a widely used treatment for cancer. However, recent studies suggest that ionizing radiation (IR) can promote tumor invasion and metastasis. Bmi-1, a member of the polycomb group protein family, has been observed as a regulator of oxidative stress and promotes metastasis in some tumors. But, its potential role in the metastasis induced by IR of breast cancer has not been explored. In our study, we found that increased levels of Bmi-1 were correlated to EMT of breast cancer cells. Through analyzing the EMT state and metastasis of breast cancer induced by IR, we found the metastatic potential of breast cancer cells can either be inhibited or accelerated by IR following a time-dependent pattern. Silencing Bmi-1 completely abolished the ability of the IR to alter, reduce or increase, the migration of breast cancer cells. Also, when Bmi-1 was knocked down, the effect of inhibition of PI3K/AKT signaling on EMT affected by IR was blocked. These results suggest that Bmi-1 is a key gene in regulation of EMT and migration of breast cancer cells induced by IR through activation of PI3K/AKT signaling; therefore, Bmi-1 could be a new target for inhibiting metastasis caused by IR.     

N and P resorption as functions of the needle age class in two conifer trees

Aims Given the importance of resorption in nutrient conservations, nutrient resorption should change with leaf age if resorption depends on nutrient content, and if nutrient content changes with leaf age. However, no study has addressed this issue.     

Design,synthesis, and biological evaluation of novel substituted thiourea derivatives as potential anticancer agents for NSCLC by blocking K-Ras protein-effectors interactions

Abstract

Mutation of the proto-oncogene K-Ras is one of the most common molecular mechanisms in non-small cell lung cancer. Many drugs for treating lung cancer have been developed, however, due to clinical observed K-Ras mutations, corresponding chemotherapy and targeted therapy for such mutation are not efficient enough. In this study, on the basis of the crystal structure of K-Ras, 21 analogues (TKR01–TKR21) containing urea or thiourea were rationally designed, which can effectively inhibit the lung cancer cell A549 growth. The designing of these compounds was based on the structure of K-Ras protein, and the related groups were replaced by bioisosteres to improve the affinity and selectivity. Biological testing revealed that compound TKR15 could significantly inhibit the proliferation of A549 cell with IC₅₀ of 0.21?µM. Docking analysis showed that the TKR15 can effectively bind to the hydrophobic cavity and form a hydrogen bond with the Glu37. In addition, through flow apoptosis assay and immunofluorescence staining assay, it confirmed that this compound can inhibit A549 cell proliferation with the mechanism of blocking K-Ras^G12V protein and effector proteins interactions through the apoptotic pathway. In conclusion, our studies in finding novel potent compound (TKR15) with confirmed mechanism showed great potential for further optimisation and other medicinal chemistry relevant studies.     

霉酚酸酯对人肝癌细胞HepG-2抑制作用的研究

目的:研究霉酚酸酯体外对细胞生长抑制率、细胞凋亡以及对细胞黏附率的影响.方法:以霉酚酸酯在0.1μg/ml-100μg/ml,24-72h内作用于肝癌细胞,MTT法检测肿瘤细胞的生长抑制率,流式细胞仪检测细胞周期,Hoeehst33258荧光染色观察细胞凋亡的形态变化,细胞黏附实验检测细胞黏附率的影响.结果:霉酚酸酯显著的抑制了肿瘤细胞的增长,并显著的抑制其黏附率,在浓度为100μg/ml作用72小时时生长抑制率达78.8%,黏附率降低至42.1%,Hoechst33258染色实验发现随浓度增大细胞凋亡的发生增多,核固缩、核碎裂的现象发生越明显.流式细胞仪检测,细胞周期阻滞于GO/G1期,减少增殖细胞在S期的分布.结论:霉酚酸酯对肝癌细胞HepG-2的增长具有明显的抑制作用.