柴胡皂苷d对肝星状细胞内雌激素受体转录激活的影响

【目的】观察柴胡皂苷d对大鼠肝星状细胞内的雌激素受体转录激活的调节作用,探讨柴胡皂苷d的药理机制。【方法】体外培养大鼠肝星状细胞系HSC-T6,将雌激素受体的特异性报告基因ERE-tk-Luc用人工细胞膜介导细胞瞬时转染法转入HSC-T6细胞中,在不同药物浓度、不同时间作用点及加用雌激素受体抑制剂ICI182.780等条件下,采用双荧光素酶报告基因检测系统观察柴胡皂苷d及雌二醇对转染细胞荧光素酶(Luc)表达的影响。【结果】柴胡皂苷d浓度为0.01~5μmol/L、雌二醇浓度为0.01~1μmol/L时,荧光素酶活性呈剂量依赖性递增;柴胡皂苷d为5μmol/L,而雌二醇为1μmol/L时,荧光素酶活性最高;当雌二醇浓度到达5μmol/L时,效应减弱。且以5μmol/L柴胡皂苷D、1μmol/L雌二醇分别诱导细胞24 h时,荧光素酶表达活性最高。ICI182.780可明显抑制柴胡皂苷d、雌二醇对荧光素酶活性的诱导作用。【结论】柴胡皂苷d可促进HSC-T6细胞内雌激素受体转录激活。     

稳定表达人α-分泌酶adam10基因启动子荧光素酶报告基因细胞系的构建研究

目的 构建携带adam10基因启动子的荧光素酶报告载体,筛选稳定表达细胞系并分析其活性.方法 提取人神经母细胞瘤细胞(SH-SY5Y细胞)基因组DNA,以其为模板,PCR扩增adam10基因启动子并克隆至荧光素酶报告载体pGL4.17中,构建adam10基因启动子荧光素酶报告载体pGL4.17-adam10,将其转染SH-SY5Y细胞(无启动子的pGL4.17载体作阴性对照,带有CMV启动子的pGL4.51载体作阳性对照),经G418进行稳定表达株的筛选,用1μmol/L维甲酸处理细胞4d后检测其荧光活性.结果 成功扩增到438 bp的adam10基因启动子,pGL4.17-adam10经PCR和双酶切鉴定均正确.SH-SY5Y细胞被该载体转染后经G418筛选得到稳定表达adam10基因启动子的细胞株,经检测具有较强的转录活性;1μmol/L雏甲酸能诱导adam10基因启动子高效表达.结论 成功构建了人adam10基因启动子荧光素酶报告载体,adam10基因启动子在SH-SY5Y细胞中能稳定表达,为深入研究adam10基因的表达调控、多态性分析及其高通量药物筛选提供基础.     

Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation

Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.Optogenetics, which offers precise temporal control of neuronal activity, has been used widely in experimental neuroscience. Although optogenetic probes are indispensable tools, conventionally their application in vivo requires invasive optical fiber implants and thus imposes significant limitations for clinical applications and for applications involving multiple brain regions (1). On the other hand, chemogenetics can modulate neuronal activity throughout the brain using a genetically targeted actuator when combined with a systemically administered small molecule. Although systemic injection of a small molecule is far less invasive than implantation of fiber optics, chemogenetics has its own limitations, such as slow response kinetics and dependence on G protein signaling, which potentially elicits unwanted secondary effects in target neurons (2).Combining the distinct advantages of opto- and chemogenetic approaches would create unprecedented opportunities for interrogation of neural circuits at a wide range of spatial scales. To allow manipulation of activity of dispersed neuronal populations using optogenetic probes without fiber-optic implants, we proposed a different approach where bioluminescence—biological light produced by enzymatic reaction between a protein, luciferase, and its diffusible substrate, luciferin—activates an opsin, which is tethered to the luciferase (3). After injection to the peripheral bloodstream, luciferin reaches a target in the brain because it crosses the blood–brain barrier (4). Light is generated by the luciferase and then activates the opsin, resulting in activation (in case of channelrhodopsins) or inhibition (in case of proton or chloride pumps) of the target neurons. Capitalizing on the major advantage of opsins as powerful generators of electrical current, our approach integrates opto- and chemogenetic methods by preserving conventional photoactivation of opsins where desired, while at the same time providing chemogenetic access to the same molecules, thus allowing manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal.Initial proof-of-concept studies showed that Gaussia luciferase (GLuc)-emitted light is able to activate opsins when the two molecules are fused together (luminescent opsin or luminopsin, LMO) (3). Here we report a set of new LMOs, incorporating brighter versions of GLuc, with significantly improved performance. We found that the improved LMOs could modulate neuronal activity via bioluminescence in vitro, ex vivo, and in vivo and could elicit behaviors in freely moving mice.     

T7 RNA聚合酶体外转录合成siRNA的方法

目的：建立一种应用T7 RNA聚合酶体外转录合成大量siRNA的方法。方法：以萤火虫荧光素酶为靶标，应用T7 RNA聚合酶体外转录合成siRNA，脂质体转染法将其导入HepG2细胞中，通过测定荧光素酶的量，评价siRNA对萤火虫荧光素酶基因表达的抑制作用。结果：合成了以萤火虫荧光素酶为靶标的siRNA，合成的siRNA能特异性地抑制荧光素酶基因的表达，抑制率为80％。结论：建立了一种经济简便的体外合成siRNA的方法。     

Silencing of the SNARE protein NAPA sensitizes cancer cells to cisplatin by inducing ERK1/2 signaling, synoviolin ubiquitination and p53 accumulation

We found earlier that NAPA represents an anti-apoptotic protein that promotes resistance to cisplatin in cancer cells by inducing the degradation of the tumor suppressor p53. In the present study, we investigated the cellular mechanism underlying the degradation of p53 by NAPA. Knockdown of NAPA using short-hairpin RNA was shown to induce p53 accumulation and to sensitize HEK293 cells to cisplatin. On the other hand, this sensitization effect was not found in H1299 lung carcinoma cells which lack p53. Expression of exogenous p53 in H1299 cells was increased following knockdown of NAPA and these cells showed increased sensitivity to cisplatin-induced apoptosis. Notably, knockdown of NAPA induced the ubiquitination and degradation of the E3 ubiquitin ligase synoviolin and the accumulation of p53 in unstressed HEK293 cells. Conversely, NAPA overexpression decreased the ubiquitination and degradation of synoviolin, and reduced p53 protein level. Knockdown of NAPA disrupted the interaction between synoviolin and proteins that form the endoplasmic reticulum-associated degradation (ERAD) complex and in turn decreased the ability of this complex to ubiquitinate p53. In addition, knockdown of NAPA induced the activation of the MAPK kinases ERK, JNK and p38, but only inhibition of ERK reduced synoviolin ubiquitination and p53 accumulation. These results indicate that NAPA promotes resistance to cisplatin through synoviolin and the ERAD complex which together induce the degradation of p53 and thus prevent apoptosis. Based on these findings, we propose that the combination of cisplatin and knockdown of NAPA represents a novel and attractive strategy to eradicate p53-sensitive cancer cells.     

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties

In the mammalian circadian system, cell‐autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 ^Luc knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 ^Luc SCN cells and in serum‐shocked or SCN2.2‐co‐cultured mPer2 ^Luc fibroblasts. SCN mPer2 ^Luc cells generated self‐sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co‐cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 ^Luc fibroblasts decayed after 3–4 cycles in serum‐shocked cultures but robustly persisted for 6–7 cycles in the presence of SCN2.2 cells. In the co‐culture model, the circadian behavior of mPer2 ^Luc fibroblasts was dependent on the integrity of the molecular clockworks in co‐cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1^ldc/Per2 ^ldc). Because immortalized mPer2 ^Luc SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real‐time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.     

Development and characterization of a stable luciferase dengue virus for high-throughput screening

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10⁶ PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.     

Novel chimeric thyroid-stimulating hormone-receptor bioassay for thyroid-stimulating immunoglobulins

Thyroid-stimulating immunoglobulins (TSI) are a functional biomarker of Graves' disease (GD). To develop a novel TSI bioassay, a cell line (MC4-CHO-Luc) was bio-engineered to constitutively express a chimeric TSH receptor (TSHR) and constructed with a cyclic adenosine monophosphate (cAMP)-dependent luciferase reporter gene that enables TSI quantification. Data presented as percentage of specimen-to-reference ratio (SRR%) were obtained from 271 patients with various autoimmune and thyroid diseases and 180 controls. Sensitivity of 96% and specificity of 99% for untreated GD were attained by receiver operating characteristic analysis, area under the curve 0·989, 95% confidence interval 0·969-0·999, P = 0·0001. Precision testing of manufactured reagents of high, medium, low and negative SRR% gave a percentage of coefficient-of-variation of 11·5%, 12·8%, 14·5% and 15·7%, respectively. There was no observed interference by haemoglobin, lipids and bilirubin and no non-specific stimulation by various hormones at and above physiological concentrations. TSI levels from GD patients without (SRR% 406 ± 134, mean ± standard deviation) or under anti-thyroid treatment (173 ± 147) were higher (P < 0·0001) compared with TSI levels of patients with Hashimoto's thyroiditis (51 ± 37), autoimmune diseases without GD (24 ± 10), thyroid nodules (30 ± 26) and controls (35 ± 18). The bioassay showed greater sensitivity when compared with anti-TSHR binding assays. In conclusion, the TSI-Mc4 bioassay measures the functional biomarker accurately in GD with a standardized protocol and could improve substantially the diagnosis of autoimmune diseases involving TSHR autoantibodies.