Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Effect of Nrf2 overexpression on anti-hypoxia and anti-apoptotic ability of mesenchymal stem cells / 器官移植

Objective To investigate the effect of nuclear factor erythroid-2-related factor 2(Nrf2) on the anti-hypoxia and anti-apoptotic ability of mesenchymal stem cells(MSCs). Methods Human embryonic kidney cells(293FT) were transfected with recombinant plasmid which overexpressed Nrf2 and helper plasmid. High-titer lentivirus which overexpressed Nrf2 were obtained. MSCs were transfected with lentivirus with Nrf2 overexpression and empty lentiviral vector to establish Nrf2-MSCs which stably overexpressed Nrf2 (Nrf2 overexpression group) and green fluorescent protein (GFP)-MSCs(control group). The expression of green fluorescent in 2 groups was observed by fluorescence microscope. The expression level of Nrf2 protein in 2 groups was measured by Western Blot. The anti-hypoxia ability of 2 groups was observed by light microscope. The anti-apoptotic ability of 2 groups was measured by flow cytometry. Results Nrf2-MSCs which stably overexpressed Nrf2 were successfully established. Western Blot analysis revealed that the expression level of Nrf2 protein in the Nrf2 overexpression group was significantly higher than that in the control group(P<0.01). After 15 h hypoxia treatment, the cell activity in the Nrf2 overexpression group was significantly higher than that in the control group. Flow cytometry showed that the apoptosis rate in the Nrf2 overexpression group was (30.9±1.4)%, significantly lower than (61.3±1.3)% in the control group(P<0.05). Conclusions Nrf2-MSCs which can stably overexpress Nrf2 possess certain anti-hypoxia and anti-apoptotic ability in hypoxia environment.

Establishment and application of cell models with stable expression of hOCTN1/2 / 药学学报

Human carnitine/organic cation transporter 1 and 2(hOCTN1 and hOCTN2) mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1(+) plasmid vector containing hOCTN1 or hOCTN2(pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, Km and Vmax of ergothioneine, mediated by MDCK-hOCTN1, were 8.19±0.61 μmol·L-1 and 1427±49 pmol·mg-1(protein)·min-1; while Km and Vmax of mildronate by MDCKhOCTN2 were 52.3±4.3 μmol·L-1 and 2454±64 pmol·mg-1(protein)·min-1. Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCKhOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

Piloleiomiomas múltiples / Multiple piloleyomiomas

Los leiomiomas son tumores infrecuentes, los piloleiomiomas son el tipo clínico más común y puede ser solitario o múltiple; este último puede ser esporádico o familiar. Presentamos un caso clínico de piloleiomiomas múltiples en dorso, en un paciente joven, a quien se le realizaron estudios para descartar una patología asociada.

Leiomyomas are rare tumors, piloleiomyomas are the most common clinical type and can be solitary or multiple which can be sporadic or familial. We report a case of multiple piloleiomyomas in the back of a young patient, in which studies were performed to rule out pathology associated.

Establishment of canine kidney cell line for canine distemper virus replication / 동물의과학연구지

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

Effect of human tumor necrosis factor alpha-sercreting on proliferation of LOVO colon cancer cells / 局解手术学杂志

objective To observe the effects of co-culture of hTNFα-sercreting and human colon cancer cells LOVO on the proliferation of cancer cells. Methods The stable transfected hTNF-α/293 , mRNA of Hek-293 cell and protein expression were detected by RT-PCR and ELISA. The positive group was added hTNF-αfactor,and MTT assay was applied under the optical density 490 nm. Through human tumor cell proliferation inhibition experiment,the inhibitory effects on colon cancer cells ( LOVO) proliferation were observed. Results hTNF-α/293 cells and hTNF-α-positive group showed a significant lower A,which suggested that hTNF-α/293 cells and hTNF-α-positive group had significant inhibition on the proliferation of colon cancer cells. Conclusion The inhibition of hTNF-αsecreted by hTNF-α/293 cells on co-lon cancer cell proliferation shows significant dose-effect dependency,and hTNF-αexpresses a considerable inhibition on the colon cancer cell proliferation as positive drug.

Human growth hormone gene expression in vitro following rAAV2/1-mediated gene transfer.

Background: Viral vectors can produce longer-lasting effects than a recombinant protein. Objective: Develop a system of sustained GH1 (a human growth hormone gene) transfer and expression in baby hamster kidney cell (BHK-21) line using recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1 (rAAV2/1). Methods: The expression of GH1 in vitro was examined by reverse transcription polymerase chain reaction and Western blot analysis. Effects of GH1 on cell proliferation were measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) experiments. Mice serum IGF-1 and blood glucose, after injection of virus infected BHK-21 cells, were measured by enzyme-linked immunosorbent assays. Results: rAAV2/1-mediated GH1 gene transfer occurred effectively in vitro after 48 hours of transduction at 1 105 vg/mL (multiplicities of infection). MTT experiments indicated notable effects of the GH1 gene on cell proliferation in vitro. Subsequent animal experiment suggested that the injection of virus infected BHK-21 cells might induce increase of serum IGF-1. Conclusion: Our study shows the feasibility of rAAV2/1-mediated GH gene delivery in vitro, applicable for future experimental and clinical investigations.

Relationship between DNA of Rat Kidney and Post by Comet Assay / 中山大学学报(医学科学版)

[Objective] To study the correlation between kidney cell DNA degradation and postmortem interval within the span of 6-48 hour after the subject rats′ death.[Methods] To select 18 healthy mature female SD rats and equally divide them into 6 groups.To execute the rats with cervical spine articulation and put the rats under the incubator temperature of 25.1℃ (the average temperature of the 5 previous Decembers in Guangzhou prefecture).Sample kidney tissue from the rat separately 0 hour,6 hours,12 hours,24 hours,36 hours,48 hours,and 60 hours after the rats′ execution to prepare monoplast suspension,which is committed to comet assay.The comet images were captured by fluorescence CCD.Kinetic Comet 4.0 software was used to analyze images.Relevant data were collected by kinetic Comet 4.0 software and were subjected to Kruskal-Wallis test.[Results] Within the postmortem interval of 6-48 h,the number of SD rat kidney cell DNA fragments increased as the postmortem interval lengthens.So did the comet tail length.The Oliver tail moment and tail DNA of comet also showed sign of increase in positive proportion to the postmortem interval (their values corresponding to 60-hour-postmortem-interval were not obtainable.Kruskal-Wallis test indicated:the discrepancies of TL among the 6 groups were all significant (P ＜ 0.01).The difference of TM between 6 h group and 12 h group was not significant (P ＞ 0.05).The difference of TM between 24 h and 36 h was significant (P ＜ 0.05).The difference of TDNA among 6 h,12 h,and 36 h groups were not significant (P ＞ 0.05).The difference of TDNA between 36 h and 48 h was significant (P ＜ 0.05).[Conclusion] Degradation of nuclear DNA of the rat kidney cells increases as the postmortem interval lengthens and comet assay may provide important empirical evidence for determining the postmortem interval.

Meta-analysis on Serological Effect of the Primary Vaccination for Japanese Encephalitis of the Literatures / 中国疫苗和免疫

Objectives To summary and compare the different seroconversion rates after the primary vaccination for the Japanese encephalitis (JE),and to evaluate the serological effect of 3 kinds of JE vaccines.Method Searching "CHKD","Wanfang" database and "EMCC" databases,the studies of the immunogenicity after the primary JEV vaccination,all randomized controlled trials or non-randomized controlled trials were included,and statistical analysis were made by RevMan 4.2.10 software.Results A total of 12 literatures were included,7 studies had control groups.The seroconversion rates after the primary vaccination,JEV-L,JEV-I (Vero) and JEV-I(PHK),were 86% (95% CI:80% ～ 91%),83% (95% CI:72% ～ 94%) and 64% (95% CI:58% ～ 69%) respectively.Comparing the seroconversion rates of the 3 kinds of vaccines after primary immunization,the rate of JEV-I (Vero) was significantly higher than the rate of JEVI(PHK),other comparisons were no significant difference.Conclusion The serological effects of JEV-L and JEV-I (Vero) after the primary vaccination were higher than that of JEV-I (PHK).

Immunoelectron microscopy and ultrastructural studies of rotaviruses in Vero cell and primary monkey kidney cells

Background: The method of immunoelectron microscopy has been found more than 20 years. It is widely applied to detect and identify some types of virus in medical waste samples.\r\n', u'Objectives: To identify antigen location of Rota virus in organelle of the Vero cell and primary monkey kidney cells after infecting and to study the interaction between the virus and host cells.\r\n', u'Subjects and methods: The study was conducted on Rota virus G1P8 (KH0118) isolated from patients with symptoms of acute diarrhea, primary monkey kidney cells collected from Macaca mulatta monkey and the Vero cell of WHO. \r\n', u'Results: Gold particles (10nm) coated protein A and polyclonal antibodies were used to interact directly with Rotavirus proteins \r\n', u'These gold particles with high electron density revealed the antigen location of the Rota virus in the lysosome, pouch and other compartments of the cytoplasm.\r\n', u'Newly assembled viral particles could be identified only after 18-20hours post-infection. It is also noteworthy that viral particles and empty capsides (virus like particles) were comprised into cytoplasmic vesicles associated with the endoplasmic reticulum (ER)-Golgi system.\r\n', u'Conclusion: In order to better understand the interaction mechanism of virus and host cells, the use of this method together with specific monoclonal antibodies for each protein component of viruses and cells is essential.\r\n', u'\r\n', u'\r\n', u'

Rota virus G1P8 isolated from acute diarrhea children with rhesus kidney cells

Twenty Rota virus strains were isolated from 20 stool specimens derived from diarrhea children in Khanh Hoa province. After 3 times of passaged culturations on MAI04 cells, there were only 2 human Rota virus strains growing well in the medium of trypsin concentration at 5 mg/ml and 4 human Rotavirus strains multiflying well in the medium of trypsin concentration at 10 mg/ml.

Study on the technology to produce inactivated rabies vaccine on the primary hamster kidney cell at laboratory scale

Vnukovo-32 a cell culture rabies is used to study the vaccine strain technology of inactivated culture rabies vaccine production in primary hamster kidney cells. The master and working seeds meet the criteria of rabies vaccine strain titer (5.5 log LD 50 - 7.1 log LD 50/ml) according to WHO standard. During the study, we have used the procedure for inactivated, non concentrated rabies vaccine production of Russia and other countries procedures as references. The result was we constructed the procedure for inactivated, concentrated cell culture rabies vaccine production at laboratory scale.

Effects of Ulinastatin on Renal Apoptosis and Expression of bcl-2 in Rats with Severe Acute Pancreatitis / 中国普外基础与临床杂志

Objective To explore the effects of ulinastatin(UTI) on renal apoptosis and expression of bcl-2 in rats with severe acute pancreatitis(SAP).Methods Sixty rats weighing 250-300 g were randomized divided into 3 groups:pseudo-operation group(SO group,n=20),SAP group(n=20) and UTI treated group(UTI group,n=20).The model of SAP was established by retrograde injection of 5% sodium taurocholate solution into the biliopancreatic duct in the rats.Serum Cr and BUN were determined.The left kidneys were resected for light and electronic microscopic study.Renal cell apoptosis was determined by TUNEL.Expression of bcl-2 was detected by immunohistochemical staining of SABC.Results Serum Cr,BUN,renal cell apoptotic index and bcl-2 expression were markedly increased in SAP group compared with SO group(P

Trypsinization of fetal monkey kidney cells

Trypsine and dispase were used to produce fetal monkey kidney cells. The average field from one pair of kidney was 1.46 – 2.64 x 108 cells, enough to gain 15 Roux bottles of culture

The culture of rotavirus causing acute diarrhoea in under five children on African rhesus kidney cell

10 strains of rotavirus C-1P6 causing acute diarrhoea in under 5- year - old - children were cultured on African Rhesus monkey kidney cells. Only 4 strains were adapted in the mixture of monkey MA104 kid cells. Results showed that in the most favorable medium, not all strains of virus could be adapted and grown properly. Rotaviruses of A group is the main agents causing acute diarrhoea in children in winter

Protection in vitro of aFGF on human liver cell L-02 and human kidney cell 293 caused by nitidine chloride / 中成药

AIM: To detect the inhibition effect of nitidine chloride on proliferation of human liver cell L-02 and human kidney cell 293 and the protective effect of acidic fibroblast growth factor(aFGF) on human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride in vitro. METHODS: The MTT assay was used to assess the proliferation of human liver cell L-02 and human kidney cell 293 treated with nitidine chloride.The contents of SOD and MDA and LDH in cultural supernate were measured by ultraviolet spectrophotometry. RESULTS: Nitidine chloride inhibited the proliferation of human liver cell L-02 and human kidney cell 293 in a dose-dependent manner. CONCLUSION: Nitidine chloride has certain toxicity on human liver cell L-02 and human kidney cell 293.aFGF could protect human liver cell L-02 and human kidney cell 293 damaged by nitidine chloride.

INFLUENCE OF SELENIUM ON THE SYNTHESIS OF NUCLEIC ACIDS IN HUMAN AMNION CELLS AND MONKEY KIDNEY CELLS / 营养学报

The effect of selenium on the synthesis of nucleic acids in human am-nion cells and monkey kidey cells was studied by the techniques of cell culture and radioisotope assay. The cells were cultured in a medium containing varied dosages of sodium selenite for 24 hours at 37℃ in a CO2 incubator (5% CO2), then 3H-labeled precursors of DNA and RNA were added. The synthetic activity of DNA and RNA was determined by assaying the incorporation amount of radioisotope. The result suggested that selenium can promote intracellular DNA and RNA synthesis and may be essential to cell growth.