Differentiation of Embryonic Stem Cells into Neurons and Retina—like Structure in Nude Mice

Purpose: To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods: Murine embryonic stem cells (D3 cell line) were cultured and maintained in an undifferentiated state in vitro, then transplanted into the anterior chamber of nude mice. Mophological and immunohistochemical examinations were implemented. Results: Two to three days after transplantation, yellow-white floating granules, sheets and masses were seen inside the anterior chamber and vitreous cavity, and enlarged gradually. 14 - 20 days later, the mice were executed. Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity. The morphology of these differentiated cells were similar to that of the retina. The cells were highly positive in NSE staining. Conclusion: The tranplanted embryonic stem cells could grow in the eyes of nude mice with tendency to differentiate into neurons and r     

高效液相色谱—电喷雾质谱法同时测定人血浆中咖啡因、咪哒唑仑和代谢产物及其在药物代谢研究中的应用

目的:建立高效液相色谱-电喷雾质谱法同时测定人体血浆中咖啡因及代谢物1,7-二甲基黄嘌呤、咪哒唑仑及代谢产物1-羟咪哒唑仑。并将它应用于中药止咳橘红颗粒对CYP3A4和CYP1A2抑制作用的研究。方法:血浆样品先经液-液萃取纯化浓缩后,以ODS-C18为固定相,甲醇-0.01％甲酸不同比例为梯度洗脱流动相,内标物为阿普唑仑。经HPLC分离后的化合物在电喷雾离子源中被电离,电离后以选择离子方式检测各化合物的分子离子峰,以峰的保留时间及质荷比进行定性,以峰面积进行定量。结果:咖啡因、1,7-二甲基黄嘌呤、咪哒唑仑、1-羟咪哒唑仑分别在50～5000 ng·mL-1(r=0.9971),22.4～2240 ng·mL-1(r=0.9982),1.23～123 ng·mL-1(r=0.9997),0.84～84 ng·mL-1(r=0.9983)的范围内呈良好的线性关系。4种化合物的平均回收率均大于90％,日内及日间RSD均小于10％。结论:此方法简便,快速,灵敏,可同时测定人体血浆中咖啡因、咪哒唑仑及各自的主要代谢产物。     

中药活性成分生物转化的研究思路与方法

结合本课题组近几年来的研究结果,对中药活性成分的生物转化方法进行总结,并对今后生物转化的研究思路进行了展望。目前,生物转化在有机合成领域的应用比较广泛,而该技术用于中药活性成分的结构修饰还处于初级阶段,尤其缺乏对某类中药活性成分系统的生物转化研究,因此有很多方面的研究值得关注,尤其是生物催化酶的研究将是生物转化的重要发展方向。此外,生物转化在中药活性化合物合成中的应用以及利用“微生物模型”研究中药活性的体内代谢,也将成为今后生物转化研究中不可忽视的领域。     

Comparison of chemical compositions and osteoprotective effects of different sections of velvet antler

Ethnopharmacological relevance

Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA.

Materials and methods

Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats.

Results

Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells.

Conclusion

Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler.     

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

Self Renewal of Embryonic Stem Cells in the Absence of Feeder Cells and Exogenous Leukaemia Inhibitory Factor

Abstract

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the p-galactosidase transgenic Kli ES cells, revealed that LDF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.     

Parp‐1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment

Embryonic stem (ES) cells deficient in poly(ADP‐ribose) polymerase‐1 (Parp‐1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp‐1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild‐type (Parp‐1^+/+) and Parp‐1^?/? ES cells. The weights of the tumors derived from Parp‐1^?/? ES cells were lower than those of the tumors derived from Parp‐1^+/+ ES cells (P < 0.05). The Parp‐1^?/? tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp‐1^?/? tumors, but not for the Parp‐1^+/+ tumors (P < 0.05). Invasions were more frequently observed with the Parp‐1^?/? tumors compared with the Parp‐1^+/+ tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp‐1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.