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.

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.

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.

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.

Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possible utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through mor-phology observation, immunnocytochemical staining and RT-PCR analysis. The Hath1 gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1(a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differentiate into hair cell-like cells when forced to express Hath1. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

Photobiomodulation:a novel approach to promote trans-differentiation of adipose-derived stem cells into neuronal-like cells

Photobiomodulation,originally used red and near-infrared lasers,can alter cellular metabolism.It has been demonstrated that the visible spectrum at 451-540 nm does not necessarily increase cell proliferation,near-infrared light promotes adipose stem cell proliferation and affects adipose stem cell migration,which is necessary for the cells homing to the site of injury.In this in vitro study,we explored the potential of adipose-derived stem cells to differentiate into neurons for future translational regenerative treatments in neurodegenerative disorders and brain injuries.We investigated the effects of various biological and chemical inducers on trans-differentiation and evaluated the impact of photobiomodulation using 825 nm near-infrared and 525 nm green laser light at 5 J/cm2.As adipose-derived stem cells can be used in autologous grafting and photobiomodulation has been shown to have biostimulatory effects.Our findings reveal that adipose-derived stem cells can indeed trans-differentiate into neuronal cells when exposed to inducers,with pre-induced cells exhibiting higher rates of proliferation and trans-differentiation compared with the control group.Interestingly,green laser light stimulation led to notable morphological changes indicative of enhanced trans-differentiation,while near-infrared photobiomodulation notably increased the expression of neuronal markers.Through biochemical analysis and enzyme-linked immunosorbent assays,we observed marked improvements in viability,proliferation,membrane permeability,and mitochondrial membrane potential,as well as increased protein levels of neuron-specific enolase and ciliary neurotrophic factor.Overall,our results demonstrate the efficacy of photobiomodulation in enhancing the trans-differentiation ability of adipose-derived stem cells,offering promising prospects for their use in regenerative medicine for neurodegenerative disorders and brain injuries.

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.

Advances in cell sources of hepatocytes for bioartificial liver

BACKGROUND: Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells. DATA SOURCES: A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the Key words such as liver failure bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed. RESULTS: Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines the incorporation of BAL with GS-HepG2 cells or alginate encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved. CONCLUSIONS: Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functionalhepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

Establishment of cell lines with porcine spermatogonial stem cell properties

Background:Spermatogonial stem cells(SSCs)are capable of both self-renewal and differentiation to mature functional spermatozoa,being the only adult stem cells in the males that can transmit genetic information to the next generation.Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine.However,studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.Results:In the present study,by lentiviral transduction of plasmids expressing the simian virus 40(SV40)large T antigen into porcine primary SSCs,we developed two immortalized cell lines with porcine SSC attributes.The established cell lines,with the expression of porcine SSC and germ cell markers UCHL1,PLZF,THY1,VASA and DAZL,could respond to retinoic acid(RA),and could colonize the recipient mouse testis without tumor formation after transplantation.The cell lines displayed infinite proliferation potential,and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.Conclusions:We have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation,thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.

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.

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.

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。

神仙文化在旅游规划中的运用研究——以仙市镇旅游开发为例(英文)

Through analyzing the relationship between immortal cultures and tourist activities, the authors proposed that birth of immortal thought was closely related to early tourist activities. The core idea of immortal cultures was in conformity with modern leisure cultures. Tourist resources in Ancient Xianshi Township were re-explored, and application of immortal cultures in tourism development of the town was studied. The authors proposed that both "immortal" and "salt" should be valued in the tourism development of ancient Xianshi Township, interaction and integration of immortal stories, immortal traces and scenic areas should be stressed in the application of immortal cultures, so as to incorporate "immortal bath" and modern salt bath, and to combine creation of fairyland with modern sightseeing and leisure agriculture.

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。

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.

Generation of functionally mature neurons from a telomerase-immortalized human glial progenitor cell line

BACKGROUND： It has long been thought that neurons and glial cells are produced from distinct progenitor pools, but recent studies suggest that the glial progenitor cell in the subventricular zone can generate neurons in the adult rodent brain. OBJECTIVE： To investigate the ability of a telomerase-immortalized human glial progenitor cell line to differentiate into functionally mature neurons. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Cell Biology Laboratory in the School of Basic Medical Sciences, Peking University Health Science Center, between July 2007 and May 2008. MATERIALS： A telomerase reverse transcriptase immortalized human glial progenitor cell line, was established in our laboratory. Dibutyryl cyclic AMP was purchased from Sigma （USA）. Specific antibodies against glial fibrillary acidic protein, ~ -tubulin-Ⅲand A2B5 were purchased from Chemicon, USA. Polyclonal antibodies against nestin and MAP2ab were obtained from Neomarker, USA. METHODS： The telomerase immortalized human glial progenitor cell line was passaged and maintained in growth medium consisting of DMEM/F12 （1：1） with N2 supplement （1%, v/v）, L-Glutamine （2 mmol/L）, epidermal growth factor （20 ng/mL）, basic fibroblast growth factor （20 ng/mL） and 3% fetal bovine serum. Neuronal differentiation was induced by the addition of 1 mmol/L dibutyryl cyclic AMP and 10% fetal bovine serum. MAIN OUTCOME MEASURES： Neuronal differentiation was evaluated by RT-PCR, quantitative PCR, immunofluorescence staining, Western blot analysis and electrophysiology. RESULTS： After dibutyryl cyclic AMP induction in the telomerase immortalized human glial progenitor cells, the expression of neuronal-specific marker mRNAs and proteins increased significantly. Concurrently, an apparent fast inward Na^＋ current was evoked in the cells after induction. CONCLUSION： This study suggests that some human glial progenitor ceils are indeed capable of generating functionally mature neurons, and such cells may be useful for treating human neurological disorders.

莎士比亚第12首十四行诗赏析(英文)

Shakespeare’s Sonnet 12 is about the destructive effect of time on the beauty of the nature as well as human beings and how one can be immortal. This paper analyzes the theme of this sonnet and the way the poet presents this theme.