Immortalized hippocampal astrocytes from 3xTg-AD mice,a new model to study disease-related astrocytic dysfunction:a comparative review

Alzheimer's disease(AD)is characterized by complex etiology,long-lasting pathogenesis,and celltype-specific alterations.Currently,there is no cure for AD,emphasizing the urgent need for a comprehensive understanding of cell-specific pathology.Astrocytes,principal homeostatic cells of the central nervous system,are key players in the pathogenesis of neurodegenerative diseases,including AD.Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways.Tumor-derived and immortalized astrocytic cell lines,alongside the emerging technology of adult induced pluripotent stem cells,are widely used to study cellular dysfunction in AD.Surprisingly,no stable cell lines were available from genetic mouse AD models.Recently,we established immortalized hippocampal astroglial cell lines from amyloid-βprecursor protein/presenilin-1/Tau triple-transgenic(3xTg)-AD mice(denominated as wild type(WT)-and 3Tg-iAstro cells)using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection,thereby maintaining natural heterogeneity of primary cultures.Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling,mitochondrial dysfunctions,disproteostasis,altered homeostatic and signaling support to neurons,and blood-brain barrier models.Here we provide a comparative overview of the most used models to study astrocytes in vitro,such as primary culture,tumor-derived cell lines,immortalized astroglial cell lines,and induced pluripotent stem cell-derived astrocytes.We conclude that immortalized WT-and 3Tg-iAstro cells provide a noncompetitive but complementary,low-cost,easy-to-handle,and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery.

The Immortal Taibai Mountain

The majestic Taibai Mountain boasts nature’s magic and wisdom, and has been known as the“Immortal Mountain”since ancient times.THE Taibai Mountain in Baoji,Shaanxi Province, is a range of major mountain peaks in the famous Qinling Mountains in China, having the highest peak east of the QinghaiTibet Plateau. Rising from the plains.

电影《Immortal Beloved》中交响曲音乐赏析

电影《Immortal Beloved》是1994年拍摄的一部关于贝多芬的传记电影。影片自始至终贯串了贝多芬作品的旋律,如按图索骥,便能深入贝多芬令人动容的音乐世界,同时也能领略到贝多芬每部作品如何成功地扮演推展剧情的说书人的功能。透过交响曲严肃而深奥的外表,发现其平易近人的本质与深刻的内涵。

Ricci流在任意度量时刻的Immortal解

通过解Poincaré-Lelong方程,完备非紧的n维的有着非负有界全纯双截曲率的Khler流形上的Ricci流方程被研究,如果它满足如下的条件:∫0skt(x,s)ds≤qC log(2+r).那么Ricci流在任意度量时刻t存在Immortal解的充分必要条件被得到,它是对文献[1]在度量t=0时刻得到Ricci流存在Immortal解条件的推广。

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

Detection of CYP2E1,a Genetic Biomarker of Susceptibility to Benzene Metabolism Toxicity in Immortal Human Lymphocytes Derived from the Han Chinese Population

Objective Cytochrome P450 2E1 （CYP2E1） is an important metabolizing enzyme involved in oxidative stress responses to benzene, a chemical associated with bone marrow toxicity and leukemia, We aimed to identify the CYP2E1 genetic biomarkers of susceptibility to benzene toxicity in support of environmental and occupational exposure prevention, and to test whether a model using immortal human lymphocytes might be an efficient tool for detecting genetic biomarkers. Methods Immortalized human lymphocyte cell lines with independent genotypes on four CYP2E1 SNP sites were induced with 0.01% phenol, a metabolite of benzene. CYP2E1 gene function was evaluated by mRNA expression and enzyme activity. DNA damage was measured by Single-Cell Gel Electrophoresis （SCGE）. Results Among the four SNPs, cells with rs2070673TT and rs2030920CC showed higher levels of ~YP2E1 transcription and enzymatic activity than the other genotypes in the same SNP site. Cells with higher gene expression genotypes also showed higher comet rates compared with lower gene expression genotypes. Conclusion These results suggest that CYP2E1 rs2070673 and rs2030920 might be the genetic biomarkers of susceptibility to benzene toxicity and that the immortalized human lymphocytes model might be an efficient tool for the detection of genetic biomarkers of susceptibility to chemicals.

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Conophylline Promotes the Proliferation of Immortalized Mesenchymal Stem Cells Derived from Fetal Porcine Pancreas (iPMSCs)

Conophylline, is a bis （indole） alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce β-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured rat pancreatic tissue. However, the precise role of conophylline in the growth and survival of immortalized pancreatic mesenchymal stem cells （iPMSCs） derived from fetal porcine pancreas were not understood at present. To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effects of conophylline on iPMSCs. We found that conophylline can robustly stimulate iPMSCs proliferation, even promote their potential differentiation into islet-like clusters analyzed by cell counting, morphology, RT-PCR and real-time PCR, Western blotting, glucose-stimulated insulin release and insulin content analysis. The effects of conophylline were inhibited by LY294002, which is the inhibitor of the PI3K pathway. These results suggest that conophylline plays a key role in the regulation of cell mass proliferation, maintenance of the undifferentiated state of iPMSCs and also promotes iPMSCs differentiated into insulin-producing cells.

Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag

Immortalized human precartilaginous stem cells （1PSCs） were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene （SV40Tag） was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southem blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells （IPSCs）, which were confirmed as fibroblast growth factor receptor-3 （FGFR-3） positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

Magnetic resonance image （MRI） systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields （SMF）. With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid （AL） cells, Chinese hamster ovary （CHO） cells, DNA double-strand break repair deficient mutant （XRS-5） cells, and human primary skin fibroblasts （AG1522） cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster ceils after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Establishment and characterization of immortalized bovine male germline stem cell line

Male germline stem cells （mGSCs） are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis. However, further studies are needed to establish a long-term cultural system of mGSCs in vitro, especially for large animals such as bovine mGSCs. In this study, we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40 （SV40） large T antigen. The proliferation of these cells was improved significantly. These cells could express spermatogonial stem cell （SSC）-specific markers, such as PLZF, PGP9.5, VASA, LIN28A, and CD49F, both in the mRNA and protein levels. Additionally, these cells could be differentiated into three germ layer cells to enter meiosis, form colonies, and proliferate in the seminiferous tubules of busulfan-induced infertile mice. The immortalized bovine mGSCs maintain the criteria of mGSCs.

Spontaneous immortalization of human dermal microvascular endothelial cells

AIM:To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line,iHDME1.METHODS:We developed a spontaneous immortalization method.This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens.Using this approach,we have successfully established a microvascular endothelial cell line,iHDME1,from primary human dermal microvascular endothelial cells.iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo.Using a GFP expressing retrovirus,we generated a GFP-stable cell line(iHDME1-GFP).RESULTS:iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin(α-SM-actin) and cytokeratin 18,markers for smooth muscle cells and epithelial cells respectively.These cells retain endothelial properties,migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel,similar to the parental cells.There is no signif icant difference in cell cycle prof ile between the parental cells and iHDME1 cells.Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells.iHDME1 cells display elevated expression of CD133 and hTERT.CONCLUSION:iHDME1 cells will be a valuable source for studying angiogenesis.

Establishment of Immortalized Cow Mammary Epithelial Cells Expressing Both h TERT and SV40 T

Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immortalized mammary epithelial cells in vitro. Both PT-PCR and immunohistochemical as- says of cells were carried out. Results showed that the expression of hTERT and SV40 T could effectively prolong the culture time in vitro of mammary epithelial cells, and enhance the cell passage number. The obtained cell line could be expressed normally, indicating that the in vitro cultured mammary epithelial cells expressing both hTERT and SV40 T could effectively prolong cell llfe without affecting the characteristics of mammary cells.

基于Android平台的IMMORTAL游戏开发

本作品基于Android平台开发一款IMMORTAL游戏,在MIT App Inventor编程环境下,采用积木式编程方法,即把流程、过程库等都变成“积木”,采用模块化代码和图形编程界面的方式,只需要拖动编程模块到代码区域就可以完成一个功能,不需要进行任何输入操作,类似于堆积木,这种方法完全可视化,生动有趣,以此编程出来的游戏,相比object C及JAVA等编程语言而言,互动性更强,更简单易于实现。同时,操作者可应用户要求轻松改动脚本,以适应需要。

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Emily Dickinson:An Interrogator of Death and Immortality

Emily Dickinson is widely regarded as one of American's premier poets of the nineteenth century.In both style and content,her verse was,"revolutionary" in her day.Owing to the deep exploration of death and immortality,Emily Dickinson has become a puzzle in literary.As show in her poems,Dickinson's spiritual journey led her from na?ve nature-mysticism through disappointment,to a sacramental approach to immortality afterlife and further discouragement,culminating in a mature attitude of faithful unknowing and even more interrogating the immortality.This paper will base on the theory of the death and immortality through common people's fear to death,the essence of death,the significance of death to show the poetess's whole life-childhood,love,life condition,spiritual contradiction,and mainly her skeptical concept of death and immortality.

Aesthetic Immortality in Speak Memory

In an effort to find out how Nabokov manages to achieve aesthetic immortality in Speak Memory,a detailed text analysis is conducted on the techniques he has utilized.Afterwards,it draws the conclusion that Nabokov,firmly grounded on his own con⁃sciousness,manages to command memory with the help of imagination,thus,transforming this recreation of memory into a work of aesthetic eternity.

EXPERIMENTAL STUDY OF THE DIFFERENTIATION HPV16 SUBGENES-IMMORTALIZED HUMAN ENDOCERVICAL CELLS INDUCED BY ALL-TRANS-RETINOIC ACID

Objective： To investigate the differentiation-inducing effects of all-trans-retinoic （ATRA） to HPV16 subgenesimmortalized human l：ndocervical cells （H8 cell） in vitro. Methods： HPV16 subgenes-immortalized human endocervical cells （H8 cells） were cultured in vitro. After treated with ATRA, the proliferation of immortalized human endocervical cells was measured by MTT assay; morphological changes were observed using M and TEM; cell cycle was analyzed by FCM; expression of Ki67 was tested using immunocytochemistry and the activity of telomerase was tested using PCR-ELISA. Results： ATRA could inhibit proliferation of H8 cells significantly and induce their morphodifferentiation. According to FCM, H8 cells accumulated in G1 phase and expression of Ki67 and activity of telomerase reduced significantly after treatment with ATRA. Conclusion： ATRA could induce the differentiation of H8 cell line obviously, which might be achieved by inhibiting proliferation, blocking cell cycle, and reducing activity of telomerase.

Inhibitory Effect of Nordy on HPV16 E6 Gene in Human Immortalized Endocervical Cells

Objective： To study the inhibitory effect of Nordy on HPV16 E6 gene in human immortalized endocervical cells （H8）. Methods： The expressions of E6 and p53 proteins of H8 cells were detected by immunocytochemistry, and the expression of HPV16E6 mRNA of H8 cells was detected by RT-PCR. Results： According to immunocytochemistry, expressions of E6 and p53 proteins of H8 cells after treatment with 100 μtmol/L Nordy for 72 h were weaker than control group obviously. After treatment with 50, 100, 200 μmol/L Nordy for 72 h. HPV16E6 mRNA expressions of H8 cells were lower than the control group significantly （P〈0.05）. Conclusion： Nordy could inhibit the expression of HPV16 E6 mRNA in H8 cells, influence transcription of E6 gene and result in decreased expressions of E6 and p53 proteins.