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.     

温度对四川华鳊胚胎发育的影响

为探索温度对四川华鳊胚胎发育的影响,笔者观察16、19、22、25、28、31℃6个温度条件下四川华鳊的胚胎发育过程,描述总结(25±0.5)℃下胚胎发育各个阶段的形态特征。研究结果显示,四川华鳊胚胎发育过程可划分为受精卵、胚盘形成、卵裂、囊胚、原肠胚、神经胚、器官分化和出膜等8个连续发育阶段;在(25±0.5)℃下孵化总历时44.83 h;在温度为16℃时,胚胎发育至原肠胚晚期全部死亡;19℃时孵化率为30.00%,其中畸形致死占87.19%,显著高于其余4个温度组(P<0.05);温度由19℃升至31℃时,胚胎发育所需时间变短,各温度间差异显著(P<0.05),其中器官分化阶段均用时最长,占整个胚胎孵化历时的72.53%~77.07%;胚胎畸形率随着温度升高而上升,31℃的胚胎畸形率约为28℃的2倍;在22~28℃时,胚胎的受精率和孵化率、成活率最高,畸形率最低。研究结果表明,水温在(19±1)℃,卵径(y)与胚胎孵化时间(x)的关系为y=49.56-44.36x+47.92x^2(r^2=0.996);水温22~28℃为适宜孵化温度,最佳水温约为25℃;胚胎发育的生物学零度为12.69℃,有效积温为522.35~595.11℃·h。     

疣吻沙蚕胚胎发育观察及盐度对其孵化的影响

采用显微镜观察了疣吻沙蚕(Tylorrhynchus heterochaeta)胚胎发育不同时期特征,并依据疣吻沙蚕人工孵化经验设置6个盐度梯度组(A:0,B:5,C:10,D:13,E:15,F:20),每组3个平行,研究了盐度对疣吻沙蚕受精率和孵化率的影响。结果表明,疣吻沙蚕胚胎发育可分为受精卵、卵裂期、囊胚期、原肠期、前担轮幼虫期、后担轮幼虫期、膜内三刚节疣足幼虫期和三刚节疣足幼虫期8个时期;盐度对疣吻沙蚕孵化影响显著(P0.05),C组受精率和孵化率最高,与其他组存在显著差异(P0.05)。适宜受精的盐度范围为10~13,最适孵化的盐度为10。     

Knockdown of HMGA2 gene inhibits TGF-β1-induced human embryonic lung fibroblast growth and collagen synthesis

AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT signaling pathway.     

Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro

The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen‐day‐old, 16‐day‐old and 18‐day‐old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin‐ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14‐day‐old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14‐day‐old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.     

柳树愈伤组织形态和细胞学观察

以柳树叶片为材料,接种在诱导培养基上诱导愈伤组织发生,采用石蜡切片法对愈伤组织的内部细胞结构进行观察。结果表明,叶片组织脱分化的起始部位在微管束鞘;胚性细胞多发生在所形成的愈伤组织的表层或近表层,和非胚性细胞在形态和细胞学上有很大差别,胚性细胞核大、质浓,与周围细胞存在生理隔离;多细胞原胚以平周分裂为主,并由此表现出有序生长;胚性细胞内含有丰富的淀粉粒,随细胞的分裂和分化逐渐减少;愈伤组织有根产生。     

三种有机磷农药胚胎期暴露对新生鼠脑组织结构的影响

探讨了3种常用的有机磷农药胚胎期暴露对新生鼠脑组织结构的影响。在母鼠妊娠期7.5～11.5 d时,连续5 d每天分别经皮下注射2 mg/kg bw的二嗪磷(diazinon)、2 mg/kg bw的毒死蜱(chlorpyrifos)及50 mg/kg bw乙酰甲胺磷(acephate),显微镜下观察并计数大脑皮层S1区胶质细胞和神经元数量及比率,以及海马CA1、CA3区锥体细胞的数量及体积密度。结果表明,二嗪磷与毒死蜱处理分别使得大脑皮层S1区胶质细胞数减少了14.29%和21.43%;毒死蜱处理导致胶质细胞与神经元比率下降了24.19%;二嗪磷与毒死蜱处理后海马CA1区锥体细胞数量分别下降了9.30％与20.93％,毒死蜱处理后锥体细胞体积密度下降了27.05％;二嗪磷与毒死蜱处理后海马CA3区锥体细胞数量分别下降了22.22％和19.44％,锥体细胞体积密度分别下降了23.54％和18.98％;而乙酰甲胺磷对新生鼠大脑皮层与海马细胞均无明显影响。     

Effect of temperature on energetic demands during the last stages of embryonic development and early life of Octopus vulgaris (Cuvier, 1797) paralarvae

This study describes the effect of seasonal average temperatures (14 and 18°C) in the Ría of Vigo, on the utilization of external yolk over the last five Naef stages of development (XV–XX) for Octopus vulgaris embryos. Also, the transference of the outer yolk to the inner yolk sac, and its use during embryonic development and early life by O. vulgaris paralarvae. Temperature had a marked effect on embryonic development, except during stages XV–XIX (until the second inversion) where development time was the same (14 days), regardless of temperature. There were no significant differences in outer yolk decrease between consecutive Naef stages at 14°C and 18°C. Contrary, significant differences at all Naef stages from XV to XIX (both, with or without outer yolk) were observed for inner yolk between temperatures. A higher accumulation of inner yolk in embryos at 14°C was observed, due to lower yolk consumption. Paralarvae incubated at both temperatures were maintained independently at starvation during 4 days. At 18°C, a reduced accumulation of inner yolk, especially during Naef stage XIX, was observed. In 24 h old paralarvae, there was already significant higher inner yolk content at 14°C than at 18°C. Unfed paralarvae at 18°C lost weight faster than those at 14°C, due to higher energetic requirements. Finally, from these results, we propose a paralarvae rearing protocol during the first days after hatching and during the last five Naef stage (XV–XX) at lower temperatures, since the energy requirements are lower during the initial maturation stage.