Histological,Biochemical, and Ultrastructural Studies on Hyperpigmented Human Skin Xenografts

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post-grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4–6 weeks. The number of Dopa-positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre-grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (αTy-SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa-positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre- and post-grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw ～14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.     

OSBPL3在代谢相关脂肪性肝病中的作用及机制研究

摘要 目的：探究氧化固醇结合蛋白类似物3（Oxysterol Binding Protein-like 3,OSBPL3）在代谢相关脂肪性肝病中的作用及可能机制。方法：建立肝脏特异性沉默OSBPL3小鼠模型和空载体对照组,分别予以普食和高脂喂养12周。分为正常对照组、OSBPL3沉默组、肥胖对照组、肥胖OSBPL3沉默组。观察小鼠一般情况,Real-time PCR检测脂质合成基因及脂质分解基因mRNA水平,western blot检测Akt/mTOR通路关键蛋白的表达。人HepG2细胞株给予不同浓度油酸（oleic acid,OA）处理,观察油红O染色的变化,western blot检测OSBPL3表达水平。结果：正常对照组与OSBPL3沉默组小鼠各项指标相比无统计学差异（P＞0.05）;与对照组相比,肥胖对照组及肥胖OSBPL3沉默组体质量、内脏脂肪及内脏脂肪指数较高（P＜0.05）;与肥胖对照组相比,肥胖OSBPL3沉默组体质量、内脏脂肪及内脏脂肪指数较低（P＜0.05）。与对照组相比,肥胖对照组及肥胖OSBPL3沉默组总胆固醇(Total cholesterol,TC)、甘油三酯(triglycerides,TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)较高（P＜0.05）;与肥胖对照组相比,肥胖OSBPL3沉默组TC、TG、LDL-C及HDL-C较低（P＜0.05）。与正常对照组与OSBPL3沉默组小鼠SREBP-1C、FAS及PPARα表达水平相比无统计学差异（P＞0.05）;与对照组相比,肥胖对照组及肥胖OSBPL3沉默组SREBP-1C、FAS较高,PPARα表达水平较低（P＜0.05）;与肥胖对照组相比,肥胖OSBPL3沉默组SREBP-1C、FAS表达水平较低,PPARα表达水平较高（P＜0.05）。与对照组相比,肥胖对照组Akt及mTOR磷酸化表达水平较高（P＜0.05）;与肥胖对照组相比,肥胖OSBPL3沉默组Akt及mTOR磷酸化表达水平较低（P＜0.05）。随着OA作用浓度的升高,油红O染色逐渐加深。与0 μmol/L油酸相比,油酸以剂量依赖性方式增加HepG2细胞OSBPL3 mRNA水平（P<0.05）。结论：OSBPL3能够调控脂质代谢的表达,可能通过调控Akt/mTOR信号通路发挥生物学功能,有望为研究NAFLD疾病发生发展及治疗提供参考依据。     

Infectivity of preserved Cryptosporidium parvum oocysts for immunosuppressed adult mice

Abstract The present study was undertaken to determine the infectivity of Cryptosporidium parvum oocysts for immunosup-pressed adult C57BL/6N mice after the oocysts had been stored from 1–48 months at 4°C in 2.5% potassium dichromate. All mice inoculated with oocysts 1–18 months old developed patent infections, while mice inoculated with older oocysts remained uninfected. The prepatent period was extended from 2 to 6 or 7 days as the storage time for oocysts increased. The finding that C. parvum oocysts remain infective for mice for at least 18 months offers important economic and time-saving advantages for investigators who frequently require large numbers of oocysts that must be painstakingly purified from calf manure.     

A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

灵芝对小鼠空间分辨学习与记忆的影响

本文用Y-型迷宫法测试小鼠空间分辨行为。实验结果表明,每日ig灵芝2.58/kg共7d,有明显促进学习的作用。每日ig灵芝2.5g/kg共7d或ig灵芝5g/kg共7d都能显著地拮坑东莨菪碱所致学习障碍的作用。此外,学习训练后立即ig灵芝2.5g/kg或ig灵芝5g/kg也有明显地改善东莨菪碱损害记忆巩固的作用。     

复制型HBV转基因小鼠的遗传与繁育研究

本文采用类似系祖建系的方法,对复制型ＨＢＶ转基因小鼠模型的１７、２１、２５三个系列进行了繁育,通过ＰＣＲ和Ｓｏｕｔｈｅｒｎ分子杂交的方法,检测三个系列小鼠尾组织和血清的ＤＮＡ样品,以确定ＨＢＶ基因的整合和复制情况。结果表明,ＨＢＶ基因已稳定地遗传至第五代（Ｆ５）,且各世代小鼠血清中都存在ＨＢＶＤＮＡ,此外,整合阳性小鼠的血清中能测出ＨＢＶＤＮＡ的比率很高,Ｆ１代为９４．４４％（１０２／１０８）,Ｆ２、Ｆ３代为９５．３１％（６１／６４）,故在繁育中可以通过测血清中的ＨＢＶＤＮＡ来确定小鼠是否整合。     

远交系小鼠胚胎干细胞系的建立及嵌合鼠的获得

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｌｓ）是来源于小鼠早期胚胎的多潜能干细胞,它可以在体外大量培养。并以单细胞的形式注射到早期胚胎里,发育为嵌合体。到目前为止,通常使用的１２９小鼠品系是来源于近交系（ｉｎｂｒｅｄ）小鼠的胚胎．与之相比,远交系小鼠应当具有较强的生命力和抗病能力。曾有人报道过建成了远交系小鼠胚胎干细胞系,但是尚没有见到获得嵌合鼠的报道。有人甚至认为：由于不同品系小鼠所具有的遗传背景不同,有的小鼠不能建成ＥＳ细胞系。最近,本实验室在这方面做了有益的探索,成功地建成了远交系小鼠胚胎干细胞系,并在这里报导首例用远交系小鼠胚胎干细胞系培育成功嵌合体小鼠。采用源于Ｓｗｉｓｓ小鼠远交群的昆明（ＫＭ）品系小鼠囊胚建成了三个小鼠胚胎干细胞系（ＫＥ１．ＫＥ２．ＫＥ５）。核型正常率均达到７０％以上。自第八代起分批冻存,复苏后,培养至第１２代,消化成单细胞,通过囊胚显微注射,将其注射到６１５品系小鼠胚胎。在幸存的幼鼠中获得了一只来源于ＫＥ１细胞的嵌合鼠（Ｔａｂｌｅ１）．其毛色表现为受体鼠（６１５）的白色中嵌合有供体鼠（ＫＭ）的黑褐色（ＰｌａｔｅＩ－Ａ）．嵌合鼠与受体鼠的杂交后代鼠中仍然出现了受体鼠的毛色类型（     

氯喹导致大鼠肌组织载脂蛋白E大量表达

载脂蛋白E(ApoE)与迟发的家族性及孤发性阿尔茨海默(Alzheimer)病密切相关. 氯喹慢性中毒可诱发某些肌病理改变, 出现β淀粉样蛋白(βAP)与tau蛋白等的沉积, 与Alzheimer脑中见到的病理改变类似. 为分析这一改变的机制, 用逆转录结合多聚酶链反应技术(RT-PCR)对氯喹处理的大鼠肌肉中ApoE表达的改变进行了研究. 在PCR定量中采用了一种稳定表达的内源性甘油醛-3-磷酸脱氢酶mRNA作为内部参照. PCR扩增在很宽的循环数范围内成线性, 且靶mRNA与参照mRNA的扩增效率相当. 氯喹处理后大鼠肌肉中ApoE mRNA的表达从第6周开始增加, 第8周后超过对照组的20多倍. 结果提示, ApoE在氯喹慢性中毒所致的大鼠肌病理改变中发挥某些作用.     

Immunotherapy of human colon cancer by antibody-targeted superantigens

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.