Mouse melanoma cell exosomes promote expression of Rac1 protein in fibroblasts

AIM To investigate the effect of exosomes secreted by mouse melanoma cells on the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1) protein in fibroblasts. METHODS Ultracentrifugation was adopted to separete exosomes secreted by mouse melanoma B16-F10 cells. The morphological structure of exosomes was observed by negative-staining electron microscopy. The size distribution of exosomes was determined by nanoparticle tracking analysis (NTA). The exosomal markers, tumor susceptibility gene 101 (Tsg101) and tyrosinase-related protein 2 (Tyrp2), were identified by Western blot. Laser confocal microscopy was used to observe the process that mouse embryonic fibroblasts (MEF) took in exosomes during co-culture. Immunocytochemical staining and Western blot were used to detect the expression of Rac1 protein in MEF. RESULTS B16-F10 cell exosomes showed a typical tea tray-like structure, with a size range of 141~255 nm, and expressed protein markers Tsg101 and Tyrp2. The results of laser confocal microscopy showed that compared with co-culture at 0 h, a small number of exosomes appeared in the MEF at 12 h, and a large number of exosomes accumulated in the MEF after co-cultured for 24 and 36 h. Western blot analysis showed that compared with co-culture at 0 h, the expression of Rac1 protein in the MEF was significantly increased at 24 h and 36 h of co-culture (P<0.01). The results of immunocytochemical staining showed that compared with co-culture at 0 h, the positive expression level of Rac1 in the MEF cells was significantly increased at 12 h, 24 h and 36 h of co-culture (P<0.05 or P<0.01). CONCLUSION Intake of exosomes secreted by mouse melanoma cells promotes the expression of Rac1 protein in fibroblasts.     

Involvement of histone H2B monoubiquitination in the regulation of mouse preimplantation development

Histone H2B monoubiquitination (H2Bub1) plays an important role in developmental regulation in various vertebrate species. However, the role of H2Bub1 in mammalian preimplantation development remains unclear. In the present study, we examined the role of H2Bub1 in the regulation of mouse preimplantation development. Based on immunocytochemical analysis using an anti-H2Bub1 antibody, no H2Bub1 signal was detected in the metaphase chromosomes of unfertilized oocytes or the pronuclei of early 1-cell stage embryos, but a weak signal was observed in late 1-cell stage embryos. The signal increased after cleavage into the 2-cell stage, and thereafter a strong signal was observed until the blastocyst stage. To assess the significance of H2Bub1 in the regulation of preimplantation development, RNF20 (an H2B-specific ubiquitin E3 ligase) was knocked down using small interfering RNA (siRNAs). In embryos treated with siRNA, the levels of Rnf20 mRNA and H2Bub1 decreased at the 4-cell and morula stages. Although these embryos developed normally until the morula stage, only one-third developed into the blastocyst stage. These results suggested that H2Bub1 is involved in the regulation of preimplantation development.     

No effect of exogenous melatonin on development of cryopreserved metaphase II oocytes in mouse

Background

This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation.

Results

First, mouse metaphase II (MII) oocytes were vitrified in the open-pulled straws (OPS). After warming, they were cultured for 1 h in M₂ medium containing melatonin at different concentrations (0, 10⁻⁹, 10⁻⁷, 10⁻⁵, 10⁻³ mol/L). Then the oocytes were used to detect reactive oxygen species (ROS) and glutathione (GSH) levels (fluorescence microscopy), and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10⁻³ mol/L melatonin group was significantly lower than that in melatonin-free group (control). The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp90aa1, Hsf1, Hspa1b, Nrf2 and Bcl-x1 with qRT-PCR in oocytes treated with 10⁻⁷, or 10⁻³ mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90aa1 expression in 10⁻⁷ mol/L melatonin-treated group than in the control (P < 0.05); the Hsf1, Hsp90aa1 and Bcl-x1 expression were significantly decreased in 10⁻³ mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10⁻⁷ or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them.

Conclusions

Our results indicate that the supplementation of melatonin (10⁻⁹ to 10⁻³ mol/L) in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.     

白术提取液急性毒性试验

测定白术提取液对小鼠腹腔注射和肌内注射的急性毒性,评价其安全性,为临床安全用药提供依据.采用最大给药量法,将60只小鼠随机分为3组,每组20只,分为白术提取液100g/kg腹腔注射组、20 g/kg肌内注射组和生理盐水100mL/kg腹腔注射组,注射给药后连续观察7 d,测定其1日最大给药量,确定白术提取液的急性毒性.结果表明,小鼠腹腔注射和肌内注射的1日最大给药量依次为100和20 g/kg,分别相当于临床犬日用量的400和80倍.说明白术提取液较为安全,可在临床研究试用.     

脱毒菜籽多肽对小鼠免疫功能的影响

[目的]研究脱毒菜籽多肽对小鼠免疫功能的影响。[方法]试验组以低、中、高剂量组0.334、0.667和2.000 g/(kg·d)的脱毒菜籽多肽灌胃小鼠,对照组以等体积纯净水灌胃,30 d后进行二硝基氟苯诱导小鼠迟发性变态反应试验、Con A诱导小鼠脾淋巴细胞转化试验、抗体生成细胞检测和血清溶血素测定试验、小鼠碳廓清试验、小鼠腹腔巨噬细胞吞噬鸡红细胞试验、NK细胞活性测定试验、试验前后小鼠体重以及脏器/体重比值试验。[结果]脱毒菜籽多肽对小鼠的耳廓肿胀、脾淋巴细胞转化、吞噬指数a和NK细胞活性均有显著影响,但对小鼠体重增长、胸腺/体重比值、脾脏/体重比值、脾抗体生成细胞量、血清溶血素水平和腹腔巨噬细胞吞噬能力均无影响。[结论]脱毒菜籽多肽具有增强免疫力的功能。     

牛MEN1基因真核表达载体的构建及其表达分析

多发性内分泌腺瘤致病因子1 (multiple endocrine neoplasia Ⅰ,MEN1)参与乳腺发育与泌乳行为的调控.本研究克隆了牛(Bos taurus) MEN1基因(bMEN1)的全长cDNA,并在不同细胞中检测bMEN1mRNA及其编码蛋白menin的表达情况.根据GenBank中bMEN1基因序列,设计特异性引物,用qRT-PCR方法得到附带EcoR Ⅰ和HindⅢ酶切位点的bMEN1片段,并将其克隆到真核表达载体pcDNA3.1-myc-his(A)中.体外转染物种同源性细胞牛乳腺上皮细胞(bovine mammary gland epithelial cell,MAC-T)和异源性细胞中国仓鼠(Cricetulus barabensis)卵巢细胞(Chinese hamster ovary cells,CHO)、小鼠(Mus musculus)成肌细胞(mouse myoblast cells,C2C12),利用qRT-PCR和Westem blot技术检测转染前后bMEN1 mRNA和蛋白menin的表达.双酶切和基因测序结果表明,成功构建了bMEN1基因的真核表达载体pcDNA3.1-myc-his-bMEN1.所建立的转染体系可以使目的基因bMEN1在3种不同细胞中成功表达mRNA和目的蛋白,转染后24 h都达到最高表达量,并达到极显著水平(P＜0.01),之后逐渐降低.其中,CHO细胞中的转染24 h后bMEN1 mRNA和menin蛋白的表达量分别是对照的28 415倍和5.65倍.本研究建立了bMEN1基因的真核表达载体及其转染体系,能够在不同细胞中成功表达,为体外研究MEN1基因对于乳腺的调节功能及其对于机体代谢的调节机制提供了一种工具和技术体系.     

小鼠Klf4基因的原核表达研究

[目的]对小鼠Klf4基因重组蛋白进行原核表达分析。[方法]利用双酶切从重组质粒p MXs-Klf4上回收小鼠Klf4基因片段,克隆入p ET-41a(+)载体后转化BL21大肠杆菌,用IPTG诱导表达,探讨诱导表达最佳浓度和时间,最后采用SDS-PAGE对重组蛋白进行鉴定及分析。[结果]重组质粒Klf4-p ET-41a(+)在IPTG诱导下可表达与预期相符的约为51 ku的KLF4蛋白;经IPTG刺激后重组蛋白表达增多,以0.8 mmol/L IPTG诱导6 h为佳;重组蛋白以包涵体形式存在于大肠杆菌BL21中。[结论]小鼠Klf4基因可在原核细胞中表达。     

小鼠催乳素释放肽的初步克隆

为克隆并分析小鼠催乳素释放肽基因,从小鼠下丘脑提取总RNA,进行RT-PCR,PCR产物与pMD-19T连接后转化E.coli DH5α,检测阳性克隆并测序。测序结果表明,所得序列与大鼠催乳素释放肽编码区序列的相似指数为86.3%。克隆所得序列为小鼠催乳素释放肽的基因片段。     

小鼠基因内含子中CpG岛和几个转录调控元件分析

内含子在基因转录调控中的作用已多次被实验报道,然而对其参与调控的普遍性还缺乏足够的理论支持。本研究利用计算分析方法,对小鼠基因内含子中的CpG岛（CpGisland）、TATA框（TATAbox）、CAAT框（CAATbox）以及GC框（GCbox）等元件的出现频率进行分析。结果发现,分别有56.01%、57.16%、65.88%和41.86%的第一内含子具有CpG岛、TATA框、CAAT框以及GC框,而它们在其它内含子中的平均出现频率则分别为14.07%、45.24%、49.91%和13.19%。即使考虑到不同位置的内含子,这些元件在第一内含子中的出现频率也显著高于它们在其它任何位置内含子中的出现频率。由于CpG岛、TATA框、CAAT框以及GC框均与基因的转录调控有关,据此推测小鼠第一内含子在基因转录调控过程中具有潜在的重要性。本研究结果为内含子参与转录调控提供了更多的理论依据。