动物体细胞克隆研究概况

介绍了动物体细胞克隆技术的方法、研究现状 ,指出体细胞克隆动物虽然存在成功率低、死亡率高、生长发育不正常等缺陷 ,但在快速扩繁优良种畜和转基因动物、保护动物遗传资源。

低温对动物体细胞的损伤影响

低温保存细胞是最常见的长期保存方法,但低温保存是有条件的,降温过快或过慢及其复温速率都直接影响细胞的存活。综述了降温与复温对动物体细胞的损伤。

“动物体细胞核移植”教学中的疑点分析

在初高中生物学教材中,对于动物体细胞核移植过程中怎样对核供体细胞进行处理有不同的表述。本文对该技术的发展历史、实验方法以及具体实验操作步骤进行梳理、分析和总结,为师生提供参考。

成年哺乳动物体细胞克隆的喜与忧

本文讨论了体细胞克隆和有性生殖的差异,分析了成年哺乳动物体细胞克隆的科学意义和技术上的成就,推测了这项技术可能的应用前景,着重讨论了克隆人的可能性,及其给社会可能带来的影响.由于哺乳动物细胞克隆的子代在发育环境和社会阅历等方面都与克隆细胞提供者不同,所以克隆不等于复制.克隆出来的人,未必就是原来的人.克隆人的出现将是生命科学发展的必然结果,在法律和伦理道德上给予正确的引导是必要.

表观重编程与体细胞克隆动物异常

从表观重编程的两大主要机制(DNA甲基化和组蛋白乙酰化)入手,对体细胞克隆动物的表观重编程异常展开了综述。

我国克隆技术步入世界前列

10月15日上午9:15,我国首例细胞克隆牛经剖腹产在深圳绿鹏公司繁育基地顺利出生。“体重33.5公斤,体温38.5℃,呼吸每分钟60次…….”,出生的小牛各项体征指标正常。

Emerging role of microRNAs in liver diseases

MicroRNAs are a class of small non-coding RNAs that are found in plants, animals, and some viruses. They modulate the gene function at the post-transcriptional level and act as a fine tuner of various processes, such as development, proliferation, cell signaling, and apopto-sis. They are associated with different types and stages of cancer. Recent studies have shown the involvement of microRNAs in liver diseases caused by various factors, such as Hepatitis C, Hepatitis B, metabolic disorders, and by drug abuse. This review highlights the role of microRNAs in liver diseases and their potential use as therapeutic molecules.

Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

Background： Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD （oxygen-glucose deprivation）-reperfusion. Methods： Viability of PC 12 cells which were injured at different time of OGD injury, was quantified by measuring MTT （2-（4,5-dimethylthia-zol-2-yl）-2,5-diphenyltetrazolium bromide） staining. In addition, PC 12 cells＇ viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by （OGD）. Furthermore, apoptotic population of PC 12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis, electron microscope and Hoechst/Pl staining. Finally, change of Bcl-2/Bax protein expression was detected by Western blot. Results： （1） The viability of PC12 cells decreased with time （1-12 h） after OGD. We regarded the model of OGD 2 h, then replacing DMEM （Dulbecco＇s Modified Eagle＇s Medium） for another 24 h as an OGD-reperfusion in this research. Furthermore, most PC 12 cells were in the state of apoptosis after OGD-reperfusion. （2） The viability of PC 12 cells preincubated with edaravone at high concentrations （1, 0.1, 0.01 μmol/L） increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. （3） Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion： Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Differential expression of cholangiocyte and ileal bile acid transporters following bile acid supplementation and depletion

AIM: We have previously demonstrated that cholangiocytes, the epithelial cells lining intrahepatic bile ducts,encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile acid vectorial transport. Cholangiocytes possess ASBT,an apical sodium-dependent bile acid transporter to take up bile acids,and t-ASBT,a basolateral alternatively spliced and truncated form of ASBT to efflux bile acids.Though hepatocyte and ileal bile acid transporters are in part regulated by the flux of bile acids, the effect of alterations in bile acid flux on the expression of t-ASBT in terminal ileocytes remains undear.Thus,we tested the hypothesis that expression of ASBT and t-ASBT in cholangiocytes and ileocytes was regulated by bile acid flux. METHODS: Expression of ASBT and t-ASBT message and protein in cholangiocytes and ileocytes isolated from pair- fed rats given control (C) and 1% taurocholate (TCA) or 5% cholestyramine (CY) enriched diets,were assessed by both quantitative RNase protection assays and quantitative immunoblotting.The data obtained from each of the control groups were pooled to reflect the changes observed following TCA and CY treatments with respect to the control diets. Cholangiocyte taurocholate uptake was determined using a novel microperfusion technique on intrahepatic bile duct units (IBDUs) derived from C,TCA and CY fed rats. RESULTS: In cholangiocytes,both ASBT and t-ASBT message RNA and protein were significantly decreased in response to TCA feeding compared to C diet.In contrast, message and protein of both bile acid transporters significantly increased following CY feeding compared to C diet.In the ileum,TCA feeding significantly up-regulated both ASBT and t-ASBT message and protein compared to C diet,while CY feeding significantly down-regulated message and protein of both bile acid transporters compared to C diet.As anticipated from alterations in cholangiocyte ASBT expression,the uptake of taurocholate in microperfused IBDUs derived from rats on TCA diet decreased 2.7-fold,whereas it increased 1.7-fold in those on CY diet compared to C diet fed groups. CONCLUSION: These data demonstrate that expression of ASBT and t-ASBT in cholangiocytes is regulated by a negative feedback loop while the expression of these transporters in terminal ileum is modified via positive feedback.Thus, while transcriptional regulatory mechanisms in response to alterations in bile acid pool size are operative in both cholangiocytes and ileocytes,each cell type responds differently to bile acid supplementation and depletion.

Future of liver transplantation: Non-human primates for patient-specific organs from induced pluripotent stem cells

Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.

Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis

The impact of starvation and anhydrobiosis on the number and size of the storage cells in the tardigrade species Milnesium tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens was investigated to gain more insight on the energetic side of anhydrobiosis. Storage cells are free floating cells within the body cavity of tardigrades and are presumed to store and release energy in form of glycogen, protein and fat to maintain a constant nutrient regime for the other tissues. The body size of the animals was not correlated with the size of the storage cells, however, M. tardigradum the largest species analysed also had the largest storage cells. A reduction in the size of the storage cells is apparent in all three species after seven days of starvation. A seven-day period of anhydrobiosis leads to a decrease in cell size in M. tardigradum but not in P. tonollii and M. sapiens. Although M. sapiens was raised on green algae, and M. tardigradum and P. tonollii were fed with rotifers and nematodes this difference in nourishment was not reflected in the response of the storage cells to anhydrobiosis

Effects of NHE-1 ribozyme gene transfection on apoptosis of rat pulmonary artery smooth muscle cells in vitro

Objective : To investigate the effects of the transfection of NHE-1 ribozyme gene on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in vitro. Methods: After NHE-1 ribozyme gene was designed, synthesized and then cloned into plasmid pLXSN, the recombined plasmid was tansfected into cultured rat PASMC. Expression of NHE-1 mRNA was detected with semi-quantitative RT-PCR. Intracellular pH (pHi) was measured by using fluorescence dye BCECF-AM. Cell cycle was measured with aid of flow cy-tometric DNA analysis. Cell apoptosis was observed with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNED respectively. Results: The NHE-1 mRNA expression level and pHi value were significantly lower in PASMCs transfected with NHE-1 ribozyme gene than those transfected with pLXSN or without transfection. Meanwhile, the apoptosis rate of cells transfected with NHE-1 ribozyme gene was increased significantly. Morphology of cell apoptosis was observed in the cells transfected with NHE-1 ribozyme gene under an electron microscope. Conclusion: The transfection of NHE-1 ribozyme gene induces the apoptosis of PASMCs by inhibiting NHE-1 expression and intracellular acidification.

STUDY ON THE MECHANISM AND EFFECTS OF GLY－TYR－NH_2 AND GLY－TYR－LYS ON RAT LUTEAL CELLS IN VITRO

The action of Gly-Tyr-NH_2, (GY-NH_2) and Gly-Tyr-LYS(GYK) on ￣(125)I-LH binding, cAMP accumulation and progesterone production was investigated. Incubation of rat luteal cells for 2.5 h with GY-NH_2 and GYK at dosage of 0. 2 mmol/L caused a significant inhidition of basal and gonadotropin-stimulated steroidogenesis. GY-NH_2 and GYK were also found to reduce cAMP formation in response to hCG. The activity of adenylate cycles of luteal cells was inhibitd by 0. 2 mmol/LGY-NH_2 and GYK. GY-NH_2 and GYK at a concentration of 0. 2mmol/L were not found to have an inhibitory effect on 8Br-cAMP-stimulated progesterone preduction. GY-NH_2 and GYK did not affect ￣(125)I-LH binding to LH receptors on the luteal cell surface. These results suggest that GY-NH_2 and GYK inhibit steroidogenesis at the step of gonadotropin-stimulated cAMP formation in luteal cells. Adenylate cyclase in luteal cells was also inhibited.

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS： Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS： Of 29 live-born recipients, 19 had the presence of human CD45^＋ cells in peripheral blood （PB） detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues （i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin） examined at the time of tissue collection, as confirmed by detecting human 132- microglobulin expression using immunohistochemistry. Tn this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CEHS-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION： Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.

Assembly and disassembly of mammalian chromosome pellicle

By means of indirect double immunofluorescent staining, the coordination of PI antigen and perichromonucleolin (PCN), the constituent of nuclear periphery and nucleolus respectively, in the assembly and disassembly of chromosome pellicle during mitosis was studied. It was found that in 3T3 cells, during mitosis PI antigen began to coat the condensing chromosome .surface earlier.than PCN did. However, both of them completed their coating on chromosome at approximately the same stage of mitosis, prometaphase metaphase. The dissociation of PI antigen from chromosome pellicle to participate the formation of nuclear periphery took, place also ahead of that of PCN. At early telophase PI antigen had been extensively involved in the formation of nuclear periphery, while PCN remained in association with the surface of decondensing chromosomes. At late telophase, when PI antigen was localized in an fairly well formed nuclear periphery, PCN was in a stage of forming prenucleolar bodies.

Human chromosome pellicle antibody recognizing centromere protein-C(CENP-C),the main component of the kinetochore

Recently the antichromosome antisera from several scleroderma patients have been found to recognize the pellicle of metaphase and anaphase chromosomes. In order to identify the pellicle components, we used these antichromosome antisera to screen a human embryonic cDNA library. The sequences of the positive clones are identical to the cDNA gene sequence of CENP-C (centromere protein C), a human centromere autoantigen. This result suggusts that CENP-C is a component of the pellicle of human metaphase and anaphase chromosomes.

ANTITUMOR ACTIVITY OF IMMUNOCONJUGATES COMPOSED OF BOANMYCIN AND MONOCLONAL ANTIBODY

Boanmycin (bleomycin A6 . BM) . an antitumor antibiotic, was conjugated to monoclonal antibodies including R19, H 111 and CCT2. The immunoconjugates exhibited selective cytotoxicity to related target cells including cecum cancer Hce-8693 cells, liver cancer BEL-7402 cells and leukemia CEM cells. They were highly effective against related human tumor xenografts in nude mice, and the inhibition rates by the conjugates were much higher than those by free BM. The inhibition rate by R19-BM conjugate against human cecum cancer xenografts reached 90%. BY immunoelectron microscopy, CCT2-BM conjugate showed specific binding and internalization in leukemia CEM cells. The results indicate that boanmycin-monoclonal antibody immunoconjugates are highly active both in vitro and in vivo.

The adenoviral E1A protein relieves gene repression by receptors in v/vo displaces corepressors and unliganded thyroid hormone

The human adenovirus type 5 early region 1A （E1A） is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone （T3） receptors （TRs）. However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.