小鼠整胚石蜡切片制备方法的改进

Objective:The morphology analysis of whole-mount mouse embryos was observed using an improved paraffin section technique.Methods:Mouse embryos of varying embryonic ages were collected and whole-mount embryo paraffin sections were prepared using PFA-intravenously injected fixation,prolonged dehydration,and paraffin embedding.Hematoxylin and eosin(H&E)staining and immunohistochemical staining were employed to evaluate the quality of sections,with different tissues being observed labeled by CD34.Results:Following a series of tissue processing and staining procedures,the structure of the whole-mount mouse embryo was well-preserved,and the staining was clear and easily distinguishable.Embryos of different embryonic ages were treated differently,yet the quality of tissue processing remained highly consistent.Conclusion:Tissue processing and staining have been significantly improved,allowing for the easy acquisition of whole-mount mouse embryos of different ages through simplified methods of tissue fixation and dehydration duration.The staining results are clear and stable,providing technical support for the study of mouse embryo development.

Value of MRI diffusion weighted imaging in localization of prostate cancer with whole-mount step section pathology

Objective To evaluate the value of MRI diffusion weighted imaging in localization of prostate cancer with whole-mount step section pathology. Methods We treated 36 patients using laparoscopic radical prostatectomy from Oct. 2009 to Jun. 2010. Patients who did not have an MRL /DWI examination or a surgical history of pros-

载脂蛋白E敲除(ApoE^(-/-))小鼠整装(whole-mount)主动脉免疫荧光组织化学染色的血管外膜淋巴管成像

目的 探索载脂蛋白E敲除(ApoE^(-/-))小鼠整装(whole-mount)主动脉免疫荧光组织化学染色法显示血管外膜淋巴管的可行性。方法 制备ApoE^(-/-)小鼠整装主动脉,进行淋巴管内皮受体1(LYVE1)、 α平滑肌肌动蛋白(α-SMA)免疫荧光组织化学染色,染色后组织经5 g/L苏丹黑B处理30 min、组织透明液透明,移至自制的样本容器中,荧光显微镜下成像。结果经5 g/L苏丹黑B处理的整装主动脉可见血管自发荧光显著降低,外膜淋巴管的特异性荧光强度显著增强,更易观察且不会对其他通道荧光产生干扰。结论 建立的ApoE^(-/-)小鼠整装主动脉免疫荧光组织化学染色显示血管外膜淋巴管的方法操作简便,特异性好。

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue(EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley(SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12 wk and the diabetic groups for 4, 8, and 12 wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation.RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was(0.54±0.23)%,(0.65±0.11)%,and(0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%,(0.03±0.05)%, and(0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were(0.53±0.22)%,(0.69±0.16)%, and(0.52±0.11)%. The leakage areas of deep blood vessels were(0.54±0.50)%,(1.42±0.16)%, and(1.80±0.07)% at 4, 8, and 12 wk, respectively. There was a statistically difference of the leakage area between the 8 th week and the 4 th week of diabetes group(P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4 wk and 8 wk(P<0.001).CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8 wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes （Lrp5/6, Dvl, APC, Ckla, CklS, Gsk3β, and Gro） of the Wnt/β-catenin signaling pathway from the amphioxus （Branchiostoma floridae） genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

Expression of the transcription factor Xvent-2 in <i>Xenopus laevis</i>embryogenesis

Till now the transcription factor Xvent-2 has been studied in Xenopus embryos only by the mRNA testing. We use immunochemical methods for testing of the Xvent-2 protein and gradient-centrifugation methods for estimation of activity of its mRNA. Our results show that the Xvent-2 protein is present in eggs and early embryos. The Xvent-2 mRNA is absent at any of these developmental stages. The majority of mRNA synthesized on the zygotic genome was stored in informosomes, while only its small part could be revealed in polysomes. The spatial patterning of the Xvent-2 protein at different developmental stages did not entirely agree with that of its mRNA. These data indicate that the Xvent-2 protein functioning in Xenopu embryos is regulated not only at the transcription, but at translation and posttranslation as well. We propose that the activation of translation on the masked Xvent-2 mRNA may lead to blood differentiation and cell migration.

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice

Embryo sac abortion is one of the major reasons for sterility in indicaljaponica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.

Molecular characterization and developmental expression of the gene encoding the prothoracicotropic hormone in the beet armyworm, Spodoptera exigua

Prothoracicotropic hormone (PTTH), a neuropeptide hormone stimulating the prothoracic glands to synthesize ecdysone, plays an important role in regulating postembryonic development in insects. The cDNA encoding PTTH was isolated and sequenced from the beet armyworm, Spodoptera exigua (Spe). The deduced a?mino acid sequence is composed of a signal peptide, a peptide (65 amino acids) of un-known function, and a mature PTTH molecule (111 amino acids). The Spe-PTTH shows similarities (45.5%―70.3%) to other known PTTHs reported in Lepidoptera species, but 7 cysteine r?esidues and the hydrophobic regions were conserved. Whole-mount immunocytochemistry by using an antiserum against recombinant Helicoverpa armigera PTTH showed that Spe-PTTH was synthesized in two pairs of neurosecretory cells in the S. exigua brain. Northern blot analysis demonstrates the presence of a 1.2-kb transcript in the brain. The Spe-PTTH mRNA is detectable at high levels at the wandering larval stage, early pupal stage, and pharate adult stage, suggesting that the Spe-PTTH gene might be corre-lated with molting, metamorphosis, and reproduction.