雌激素受体亚型介导的荧光素酶报告基因试验方法的建立及应用

目的 建立人源性雌激素受体（hERα/β）介导的荧光素酶报告基因试验方法,比较不同雌激素受体亚型介导的报告基因试验的反应性和灵敏度,为检测内分泌干扰物拟雌激素活性提供研究工具。方法 以恒河猴肾细胞（LLC-MK2）为转染细胞,以萤火虫荧光素酶基因（Luc）为报告基因,利用瞬时转染的方法,将表达hERα/hERβ的质粒与重组Luc报告基因质粒pERE-minP-Luc2P、内对照质粒pGL4.74共转染LLC-MK2细胞,建立由hERα/β介导的Luc报告基因试验方法。以雌二醇(E 2)、己烯雌酚(DES)作为阳性受试物,以10-5 mol/L 雌激素拮抗剂（ICI 182,780）和不同浓度的E 2共同染毒,观察Luc表达的变化。用双酚A（BPA）和染料木黄酮（GS）对方法的有效性进行检验并比较雌激素受体两亚型反应性、灵敏度和特异性。结果 hERα报告基因系统对E 2的最低检测限为1.9×10-11 mol/L,在10-8 mol/L处获得最高诱导倍数,是对照组的30.7倍。hERβ报告基因系统对E 2的最低检测限为2.2×10-11 mol/L,在10-8 mol/L处获得最高诱导倍数,是对照组的14.4倍。ICI 182,780在10-5 mol/L浓度下能显著抑制两个报告基因系统E 2的雌激素活性。未转染受体表达载体hER-pcDNA3.1的LLC-MK2细胞用不同浓度E 2诱导,两个报告基因系统均与E 2无明显反应。BPA和GS在以上两个不同的报告基因系统中均能诱导Luc表达,但同一受试物在不同雌激素亚型介导的报告基因系统中表达倍数不同。结论 本研究建立ER不同亚型的报告基因试验有较高的灵敏度和重复性。hERα介导的报告基因试验灵敏度高于hERβ,在hERα系统中BPA雌激素活性强于GS,在hERβ系统中GS雌激素活性强于BPA。     

Insights from the ganglionic acetylcholine receptor autoantibodies in patients with Sjögren’s syndrome

Objective: It is not known whether autonomic neuropathy is a feature of Sjögren’s syndrome (SS) or whether it is related to circulating antiganglionic acetylcholine receptor (gAChR) antibodies. The goal of the present study was to investigate the autonomic dysfunction in patients with SS and the associations between autonomic dysfunction, anti-gAChR antibodies, and clinical features of SS.

Methods: (1) The first observational study tested for the presence of gAChR antibodies in the serum samples from 39 patients with SS (absent information regarding autonomic symptoms) and healthy volunteers. (2) In the second study, serological and clinical data from 10 Japanese patients diagnosed with SS were reviewed. These patients showed autonomic dysfunction, and luciferase immunoprecipitation systems (LIPS) test was conducted to detect anti-α3 and anti-β4 gAChR antibodies. (3) In the final analysis, we combined the data of seropositive SS patients with autonomic symptom from the first study with all of the patients from the second study, and analyzed the clinical features.

Results: (1) The LIPS assay revealed that anti-gAChRα3 and anti-gAChRβ4 antibodies were detected in the sera from patients with SS (23.1%, 9/39). Five of nine SS patients had autonomic symptoms. (2) Anti-α3 and anti-β4 gAChR antibodies were also detected in 80.0% (8/10) of patients with SS with autonomic symptoms. Six of the ten patients were diagnosed as having SS after neurological symptoms developed. These seropositive patients had predominant and severe autonomic symptoms and were diagnosed with autonomic neuropathy. (3) Thirteen of fifteen SS patients with autonomic symptoms (86.7%) were seropositive for anti-gAChR antibodies, and we confirmed sicca complex, orthostatic hypotension, upper and lower gastrointestinal (GI) symptoms, and bladder dysfunction at high rates.

Conclusion: The present results suggest the possibility of anti-gAChR antibodies aiding the diagnostics of SS with autonomic dysfunction.     

小鼠谷胱甘肽S转移酶K1启动子荧光素酶报告基因的构建及功能鉴定

目的:构建小鼠谷胱甘肽S转移酶K1(mGSTK1)启动子荧光素酶报告基因质粒,并分析其活性。方法:根据GeneBank中mGSTK1基因序列设计引物,应用PCR技术扩增mGSTK1启动子片段,插入pGL3-basic载体,获得mGSTK1启动子报告基因质粒pGL3-mGSTK1-2046。再以pGL3-mGSTK1-2046为模板,进一步构建了2个5’端部分缺失的报告基因质粒:pGL3-mGSTK1-936和pGL3-mGSTK1-76。将构建的报告基因质粒瞬时转染不同的细胞株3T3-L1和人胚肾(HEK)293,48 h后检测荧光素酶的活性。结果:所构建的质粒经酶切、测序鉴定正确。pGL3-mGSTK1-2046在3T3-L1和HEK293中均有不同程度的转录活性。将pGL3-mGSTK1-2046、pGL3-mGSTK1-936和pGL3-mGSTK1-76转染HEK293细胞,结果显示pGL3-mGSTK1-936转录活性最高,而pGL3-mGSTK1-76转录活性显著降低。结论:成功构建mGSTK1启动子荧光素酶报告基因质粒;翻译起始点上游的-971～-111 bp片段是mGSTK1启动子的重要区域。为深入了解mGSTK1启动子的调控机制以及今后对代谢性疾病的治疗提供一定的实验基础。     

Tumor tissue characterization evaluating the luciferase activity under the control of a hsp70 promoter and MR imaging in three tumor cell lines

We investigated the luciferase activity under the control of a hsp70 promoter and MR imaging for three tumor cell lines. Three tumor cell lines, SCCVII, NIH3T3 and M21 were transfected with a plasmid containing the hsp70 promoter fragment and the luciferase reporter gene and grown in mice. Bioluminescence imaging of the tumors was performed every other day. MR imaging, pre- and post-contrast T1-wt SE, T2-wt FSE, Diffusion-wt STEAM-sequence, T2-time determination were obtained on a 1.5-T GE MRI scanner at a tumor size of 600–800 mm³ and 1400–1600 mm³. Comparing the different tumor sizes the luciferase activity of the M21 tumors increased about 149.3%, for the NIH3T3 tumors about 47.4% and for the SCCVII tumors about 155.8%. Luciferase activity of the M21 tumors (r = 0.82, p < 0.01) and the SCCVII tumors (r = 0.62, p = 0.03) correlated significant with the diffusion coefficient. In the NIH3T3 tumors the best correlation between the luciferase activity and the MRI parameter was seen for the SNR (T2) values (r = 0.78, p < 0.01). The luciferase activity per mm³ tumor tissue correlated moderate with the contrast medium uptake (r = 0.55, p = 0.01) in the M21 tumors. In the NIH3T3 and SCCVII tumors a negative correlation (r = −0.78, p < 0.01, respectively, r = −0.49, p = 0.02) was found with the T2 time. Different tissue types have different luciferase activity under the control of the same hsp70 promoter. The combination of MR imaging with bioluminescence imaging improves the characterization of tumor tissue giving better information of this tissue on the molecular level.     

TMEM33 mRNA 3′-UTR报告基因载体的构建及功能验证

目的:根据microRNA-146a(miR-146a)预测靶点构建荧光素酶报告基因重组质粒并进行功能验证,为研究吸入性损伤中miR-146a通过靶向跨膜蛋白33(TMEM33)对Toll样受体/核因子-κB(TLRs/NF-κB)信号通路的调控作用提供前期的工作基础。方法:通过生物学软件预测TMEM33mRNA 3′端非翻译区(TMEM33mRNA 3′-UTR)可能是miR-146a的作用靶点,将设计合成的TMEM33及其突变序列TMEM33-mu序列分别克隆至荧光素酶报告质粒,采用脂质体法将这两种重组荧光素酶报告质粒pMIR-TMEM33和pMIR-TMEM33-mu分别与miR146amimics共转染HEK-293T细胞,以pMIR-TMEM33+miR-control转染HEK-293细胞作对照,双荧光素酶报告基因系统检测荧光素酶活性。结果:TargetScan和miRanda软件预测显示,miR-146a与TMEM33mRNA 3′-UTR存在互补结合位点,构建的荧光素酶报告质粒经酶切及测序鉴定正确。双荧光素酶报告基因检测系统显示,miR-146a对TMEM33mRNA 3′-UTR具有靶向作用,pMIR-TMEM33+miR146amimics组较pMIR-TMEM33+miR-control组荧光素酶活性降低62%左右,差异具有统计学意义(P<0.01);而miR-146a对TMEM33-mu 3′-UTR无靶向作用,pMIR-TMEM33-mu+miR-146a组与pMIR-TMEM33+miR-control组之间差异无统计学意义(P>0.05)。结论:成功构建TMEM33mRNA 3′-UTR荧光素酶报告载体,证实miR-146a对TMEM33mRNA 3′-UTR具有靶向调控作用,为从基因水平深入揭示吸入性肺损伤发病机制提供实验室数据和方法。     

牛β-乳球蛋白基因的克隆及其调控外源基因表达的研究

目的制备乳腺生物反应器所必需的乳腺特异性表达的调控序列,并验证其指导外源基因表达的能力.方法用PCR法从奶牛染色体上分5段扩增出了全长8.2Kb的牛β-乳球蛋白基因,包括1.8Kb的5′侧翼区、1.7Kb的3′侧翼区及4.7Kb的gDNA区.扩增出的各片段克隆到T-载体上,酶切鉴定及序列分析均证实了所扩增片段的正确性.将五个片段与荧光素酶cDNA拼接成荧光素酶瞬时表达载体并在小鼠乳腺中瞬时表达.结果注射荧光素酶瞬时表达载体的小鼠乳汁中明显测出了荧光素酶活性.结论所克隆的牛β-乳球蛋白基因表达调控序列能够指导外源基因在小鼠乳腺中表达.     

HCV5‘NCR调控抑制活性的反义寡核苷酸的筛选

目的 寻找高效特异的新型抗ＨＣＶ药物,探讨针对ＨＣＶ基因的硫代修饰反义寡核苷酸（Ｓ－ＡＳＯＤＮｓ）最佳作用靶序列。方法 参考ＨＣＶ５’ＮＣＲ计算机预测的二级结构图,设计合成了１５条Ｓ－ＡＳＯＤＮ、３条正义寡核苷酸及１条随机序列,采用ＨＣＶ５’ＮＣＲ调控荧光素酶基因的瞬时表达系统,将Ｓ－ＡＳＯＤＮ与ｐＨＣＶ－ｎｅｏ４共转染,通过荧光素激活性表达高低,反映Ｓ－ＡＳＯＤＮ对ＨＣＶ调控基因的抑制活性。结果     

Valproic acid and its derivatives enhanced estrogenic activity but not androgenic activity in a structure dependent manner

Steroid hormones affect metabolic pathways and cellular functions. Valproic acid (VPA), used as antiepileptic drug, inhibits histone deacetylases and interacts with intracellular receptors. We analyzed the impact of VPA and VPA derivatives on activation of estrogen and androgen receptors (ER and AR) using reporter gene assays. VPA and its long-chain derivatives 2-(2-propynyl)-hexanoic acid [butyl-4-yn-VPA], 2-(2-propynyl)-heptanoic acid [S-pentyl-4-yn-VPA] and 2-(2-propynyl)-nonanoic acid [heptyl-4-yn-VPA] enhanced 17β-estradiol-induced ERα and ERβ activation partly synergistically with a structure–activity correlation. The extent of this effect regarding to ERα activation increased with prolongation of the aliphatic side chain. Regarding AR activation, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA slightly induced AR activity when tested alone. In combination with the AR agonist 5α-dihydrotestosterone, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA showed anti-androgenic effects without an apparent structural relation. Our results indicate that VPA and its derivatives affect estrogen signaling with a structural specificity, while the (anti-)androgenic effects of these compounds are not structurally correlated.     

三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移的比较研究

目的采用活体成像技术比较三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移情况,为研究肿瘤转移提供理想的动物模型以及活体分析方法。方法以荧光素酶(luciferase,Luc)作为报告基因导入小鼠乳腺癌细胞4T1、66c14和4TO7中,经G418筛选获得稳定表达荧光素酶的细胞克隆并扩大培养。标记细胞稀释成1×107cells/mL,取0.1 mL进行乳腺原位及尾静脉接种BALB/c小鼠,制作小鼠乳腺原位和尾静脉移植瘤模型,比较三株细胞在小鼠体内生长及转移情况。结果获得稳定表达荧光素酶基因的细胞克隆,将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠乳腺原位接种后7 d,均有肿瘤生长,接种后28 d,4T1细胞乳腺原位移植瘤最大,66c14细胞瘤体次之,4TO7细胞瘤体最小;接种后35 d,三株细胞乳腺原位移植瘤大小较一致,但4T1和66c14原位移植瘤均发生转移,其中4T1细胞较66c14细胞转移严重,而4TO7细胞未见转移;接种后42 d,三株细胞乳腺原位移植瘤大小无明显差别,而4T1和66c14细胞随天数的增加,移植瘤转移程度逐渐严重,4T1较66c14细胞转移更严重,呈广泛性转移,4TO7细胞仍未见转移。将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠尾静脉接种后7 d,小动物活体成像发现小鼠肺部均能检测到荧光,其中4T1细胞接种的小鼠肺部荧光信号最强,且小鼠陆续死亡;4TO7细胞接种小鼠肺部荧光信号次之;66c14细胞接种小鼠肺部荧光信号最弱。尾静脉接种后14 d,4TO7和66c14细胞随着观察天数的增加,转移程度逐渐严重,4TO7细胞接种小鼠肺部荧光信号较66c14细胞强且小鼠陆续死亡。结论乳腺原位自发转移模型较尾静脉转移模型更真实反应了肿瘤细胞在体的转移特性,且能完整地呈现肿瘤转移的全过程,可作为研究肿瘤转移的最理想模型。