Microencapsulation of immunoglobulin Y: optimization with response surface morphology and controlled release during simulated gastrointestinal digestion / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Immunoglobulin Y (IgY) is an effective orally administered antibody used to protect against various intestinal pathogens, but which cannot tolerate the acidic gastric environment. In this study, IgY was microencapsulated by alginate (ALG) and coated with chitooligosaccharide (COS). A response surface methodology was used to optimize the formulation, and a simulated gastrointestinal (GI) digestion (SGID) system to evaluate the controlled release of microencapsulated IgY. The microcapsule formulation was optimized as an ALG concentration of 1.56% (15.6 g/L), COS level of 0.61% (6.1 g/L), and IgY/ALG ratio of 62.44% (mass ratio). The microcapsules prepared following this formulation had an encapsulation efficiency of 65.19%, a loading capacity of 33.75%, and an average particle size of 588.75 μm. Under this optimum formulation, the coating of COS provided a less porous and more continuous microstructure by filling the cracks on the surface, and thus the GI release rate of encapsulated IgY was significantly reduced. The release of encapsulated IgY during simulated gastric and intestinal digestion well fitted the zero-order and first-order kinetics functions, respectively. The microcapsule also allowed the IgY to retain 84.37% immune-activity after 4 h simulated GI digestion, significantly higher than that for unprotected IgY (5.33%). This approach could provide an efficient way to preserve IgY and improve its performance in the GI tract.

Microencapsulation of immunoglobulin Y: optimization with response surface morphology and controlled release during simulated gastrointestinal digestion / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Immunoglobulin Y (IgY) is an effective orally administered antibody used to protect against various intestinal pathogens, but which cannot tolerate the acidic gastric environment. In this study, IgY was microencapsulated by alginate (ALG) and coated with chitooligosaccharide (COS). A response surface methodology was used to optimize the formulation, and a simulated gastrointestinal (GI) digestion (SGID) system to evaluate the controlled release of microencapsulated IgY. The microcapsule formulation was optimized as an ALG concentration of 1.56% (15.6 g/L), COS level of 0.61% (6.1 g/L), and IgY/ALG ratio of 62.44% (mass ratio). The microcapsules prepared following this formulation had an encapsulation efficiency of 65.19%, a loading capacity of 33.75%, and an average particle size of 588.75 µm. Under this optimum formulation, the coating of COS provided a less porous and more continuous microstructure by filling the cracks on the surface, and thus the GI release rate of encapsulated IgY was significantly reduced. The release of encapsulated IgY during simulated gastric and intestinal digestion well fitted the zero-order and first-order kinetics functions, respectively. The microcapsule also allowed the IgY to retain 84.37% immune-activity after 4 h simulated GI digestion, significantly higher than that for unprotected IgY (5.33%). This approach could provide an efficient way to preserve IgY and improve its performance in the GI tract.

Granulocyte colony stimulating factor in COS-IUI cycles

Background: An unresolved assisted reproductive technique problem is the unresponsive, thin endometrium. Approximately 0.6%-0.8% of patients do not reach the minimum thickness. Using endometrial co culture, G-CSF>130pg/mL was associated with significantly improved pregnancy rate in ART cycles. This is a retrospective study that included all unexplained infertility cycles with controlled ovulation stimulation –IUI protocols. Aim was to note the effects of G-CSF on thin endometrium and pregnancy rate in G-CSF administered COS-IUI cycles.Methods: This study was done in the IVF department of Dr D Y Patil University, Navi Mumbai, India. Thin endometrium was defined as ET<7mm on transvaginal ultrasound. Clomiphene citrate was used for ovulation induction in strengths of 100mg or 50mg on day 2 of their cycle based on the antral follicle count. Trigger used was injection 10,000µg urinary hCG. On the same day when the trigger injection was given, 300 units G-CSF was instilled into the uterus. Post 36 hours IUI was done under aseptic precautions .After 16 days β-hCG levels were done to determine whether there is a pregnancy.Results: In present study,200 COS-IUI cycles were analysed.50 cycles showed a thin endometrium and in them G-CSF was used. The chemical pregnancy rates was 32%, the intrauterine pregnancy rate was 28%, ectopic pregnancy rate was 4%.Conclusions: Present study concluded that G-CSF increases ET significantly in COS-IUI cycles in the event of thin endometrium. In view of small cohort size further larger randomized controlled trials may be required  to substantiate the above conclusions.

Implicancia pronóstica de características basales en trombocitopenia esencial / Prognostic implication of basal characteristics in essential thrombocythemia

OBJETIVO: evaluar asociación entre características basales de Trombocitemia Esencial (TE) y eventos. Materiales-métodos: estudio observacional retrospectivo, datos capturados prospectivamente. Población: adultos con TE confirmada seguidos en CH del IIHEMA. Resultados: 67 pacientes evaluables. Md 68 años. Femenino 44, Masculino 23. Seguimiento Md 6 años. Score riesgo Alto 40, Intermedio 15, Bajo 12. Hematocrito Md 41%. Hemoglobina Md 13.4 g/dL. Leucocitos Md 9.7x109/L. Plaquetas Md 852x109/L. Esplenomegalia 12. Cariotipo normal 40, alterado 5. JAK2 V617F positivo 29/41. BCR/ABL1 negativo 45/45. FRV 40. Eventos: 67. Sangrado, 21 pacientes, presentó asociación con leucocitosis: Md 12.2x109/L en el grupo con sangrado y 8.8x109/L en el grupo sin evento (p=0.003). El 76% del grupo con sangrado y el 36,96% del grupo sin evento tenían leucocitos >10.0x109/L (p= 0.003). Trombocitosis Md 1.204x109/L en el grupo con sangrados y 814.5x109/L en el grupo sin evento (p=0.0098), no alcanzó significación estadística al comparar proporción de pacientes con recuento normal (p=0.46). Tromboembolia, 16 pacientes, se asoció con leucocitosis (Md 11.9x109/L en el grupo con tromboembolia y 9.2x109/L en el grupo sin evento (p=0.02). 75% del grupo con eventos y 41% del grupo sin eventos tenían leucocitos >10.0x109/L (p=0.018)) y con trombocitosis (Md 1.182x109/L en el grupo con tromboembolia y 809x109/L en el grupo sin evento (p=0.04), pero no alcanzó significación estadística al comparar proporción de pacientes con recuento normal (p=0.5)). CONCLUSIÓN: la asociación entre trombocitosis extrema/leucocitosis y sangrado coincidió con la literatura; leucocitosis se asoció a tromboembolia. El JAK2 V617F no mostró asociación: analizaremos prospectivamente aumentando la población para esclarecer asociación y posible rol de terapias dirigidas en esta entidad. (AU)

OBJETIVE: asses association basal characteristics of Essential Trombocitemia (ET) and events. MATERIALS-METHODS: retrospective observational study, data captured prospectively. Population: adults with confirmed ET treated in CH of IHEMA. RESULTS: 67 evaluable patients. Md 68-years-old. Female 44, Male 23. Follow up Md 6 years. Risk Score High 40, Intermeddle 15, Low 12. Hematocrit Md 41%. Hemoglobin Md 13.4 g/dL. Leukocytes Md 9.7x109/L. Platelets Md 852x109/L. Splenomegaly 12. Normal karyotype 40, abnormal 5. JAK2V617F positive 29/41. BCR-ABL1 negative 45/45. Vascular risk factors 40. EVENTS: 67. Bleeding, 21 patients, showed association with leukocytosis: Md 12.2x109/L in the group with bleeding and 8.8x109/L in the group without event (p=0.003). Leukocytes >10.0x109/L was seen in 76% of the group with bleeding versus 36.96% of the group without event (p= 0.003). Thrombocytosis Md 1.204x109/L in the group with bleeding and 814.5x109/L in the group without event (p=0.0098), but did not reach statistical significance when comparing with the proportion of patients with normal counts (p=0.46). Thromboembolism, 16 patients, showed association with both leukocytosis (Md 11.9x109/L in the group with thromboembolism and 9.2x109/L in the group without event (p=0.02). Leukocytosis >10.0x109/L was seen in 75% of the group with events and 41% of the group without event (p=0.018)) and with thrombocytosis (Md 1.182x109/L in the group with thromboembolism and 809x109/L in the group without event (p=0.04), but did not reach statistical significance when comparing with the proportion of patients with normal counts (p=0.5)). CONCLUSION: the association between extreme thrombocytosis/leukocytosis and bleeding was consistent with literature; leukocytosis was associated also with thromboembolism. JAK2 V617F mutated did not show association, we will prospectively analyze increasing the population to clarify its association and possible role of target therapies in this disease. (AU)

Application of a single-chain fragment variable (scFv) antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

Background: Hydatid disease is a serious parasitic disease threatening public health. Because of its rarity in non-endemic coastal areas, determining the nature and origin of a chronic, enlarged liver cystic mass is challenging in these regions. Under these circumstances, physicians need a confirmatory diagnostic tool beyond immunological and radiological examinations. This study investigated a novel human single-chain fragment variable (scFv) antibody for the confirmative diagnosis of 18 atypical hydatid disease cases in non-endemic coastal areas. Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P b 0.05). The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

Explosion on the core outcome set (COS) in the clinical evaluation in the TCM clinical studies / 国际中医中药杂志

Recently, more systemic reviews and randomized controlled trials were published to prove the effectiveness of TCM,and some of those were included by Cochrane Library. But due to the potential selective reporting bias and publication bias, few trials were included in the meta analysis, which failed to prove the evidence for the TCM treatment. The difference of reported outcomes comes to the big problem of the comparison between interventions. Such problem of difference was gradually brought to the attention. Therefore, Core Outcome Sets(COS), which stands for that the minimum standardized outcome set that must be repoted, may be the solusion to that problem. In this research we've introduced the formation and development of core outcome sets in TCM.

Propofol protects against oxidative-stress-induced COS-7 cell apoptosis by inducing autophagy

BACKGROUND: In oxidative stress, reactive oxygen species (ROS) production contributes to cellular dysfunction and initiates the apoptotic cascade. Autophagy is considered the mechanism that decreases ROS concentration and oxidative damage. Propofol shows antioxidant properties, but the mechanisms underlying the effect of propofol preconditioning (PPC) on oxidative injury remain unclear. Therefore, we investigated whether PPC protects against cell damage from hydrogen peroxide (H₂O₂)-induced oxidative stress and influences cellular autophagy. METHOD: COS-7 cells were randomly divided into the following groups: control, cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂) for 24 h without propofol; H₂O₂, cells were exposed to H₂O₂ (400 µM) for 2 h; PPC + H₂O₂, cells pretreated with propofol were exposed to H₂O₂; and 3-methyladenine (3-MA) + PPC + H₂O₂, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to H₂O₂. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) reduction. Apoptosis was determined using Hoechst 33342 staining and fluorescence microscopy. The relationship between PPC and autophagy was detected using western blot analysis. RESULTS: Cell viability decreased more significantly in the H₂O₂ group than in the control group, but it was improved by PPC (100 µM). Pretreatment with propofol effectively decreased H₂O₂-induced COS-7 cell apoptosis. However, pretreatment with 3-MA inhibited the protective effect of propofol during apoptosis. Western blot analysis showed that the level of autophagy-related proteins was higher in the PPC + H₂O₂ group than that in the H2O2 group. CONCLUSION: PPC has a protective effect on H₂O₂-induced COS-7 cell apoptosis, which is mediated by autophagy activation.

Anti-inflammatory effect of Chikusetsu oleanane saponin on RAW264. 7 cell through regulating SIRT1 activity / 中国药理学通报

Aim To investigate SIRT1 activity and an-ti-inflammatory effects of Chikusetsu oleanane saponin ( COS) on the RAW264. 7 macrophage cells induced by lipopolysaccharide ( LPS ) . Methods The nitric oxide ( NO) release was detected with Griess method. Western blot was used to detect the expression of tumor necrosis factor-α( TNF-α) , interleukin 1β( IL-1β) . Nuclear factor-κB ( NF-κB) and silent information ad-justment factor 1 ( SIRT1 ) were tested by immunofluo-rescence. Results 1 mg · L-1 LPS co-cultured with COS at 25~300 mg·L-1 had no significant effect on the growth of RAW264. 7 cells. Compared with the LPS group, COS effectively inhibited the NO release and suppressed the expression of TNF-α and IL-1β, and also inhibited the translocation of NF-κB and up-regulation of SIRT1 . Conclusion COS has protective effects on RAW264. 7 cells stimulated by LPS, which may be related to up-regulating the expression of SIRT1 and promoting the deacetylation of NF-κB, thereby in-hibiting the translocation of NF-κB and reducing the expression of TNF-α and IL-1β.

Remifentanil induces autophagy and prevents hydrogen peroxide-induced apoptosis in Cos-7 cells

BACKGROUND: This study investigated the effect of remifentanil pretreatment on Cos-7 cells exposed to oxidative stress, and the influence of remifentanil on intracellular autophagy and apoptotic cell death. METHODS: Cells were divided into 4 groups: (1) Control: non-pretreated cells were incubated in normoxia (5% CO₂, 21% O₂, and 74% N₂). (2) H₂O₂: non-pretreated cells were exposed to H₂O₂ for 24 h. (3) RPC+H₂O₂: cells pretreated with remifentanil were exposed to H₂O₂ for 24 h. (4) 3-MA+RPC+H₂O₂: cells pretreated with 3-Methyladenine (3-MA) and remifentanil were exposed to H₂O₂ for 24 h. We determined the cell viability of each group using an MTT assay. Hoechst staining and FACS analysis of Cos-7 cells were performed to observe the effect of remifentanil on apoptosis. Autophagy activation was determined by fluorescence microscopy, MDC staining, and AO staining. The expression of autophagy-related proteins was observed using western blotting. RESULTS: Remifentanil pretreatment increased the viability of Cos-7 cells exposed to oxidative stress. Hoechst staining and FACS analysis revealed that oxidative stress-dependent apoptosis was suppressed by the pretreatment. Additionally, fluorescence microscopy showed that remifentanil pretreatment led to autophagy-induction in Cos-7 cells, and the expression of autophagy-related proteins was increased in the RPC+H₂O₂ group. CONCLUSIONS: The study showed that remifentanil pretreatment stimulated autophagy and increased viability in an oxidative stress model of Cos-7 cells. Therefore, we suggest that apoptosis was activated upon oxidative stress, and remifentanil preconditioning increased the survival rate of the cells by activating autophagy.

Influence of cigarette smoking extract on fluorescence expression of thrombomodulin on surface of COS-7 cells / 心血管康复医学杂志

Objective:To explore whether cigarette smoking extract (CSE) has influence on fluorescence protein ex-pression of thrombomodulin (TM) on surface of African green monkey kidney fibroblast cells (COS-7) or not . Methods:When TM-green fluorescent protein (GFP) plasmid was successfully constructed ,COS-7 cells were trans-fected by it ,then incubated by prepared 5% CSE (5% CSE group) ,PBS of certain volume was added to serum-free minimal essential medium (MEM ,simple medium) ,which was cultured at the same time and treated as control group .Flow cytometry counting method was used to detect change of TM-GFP expression amount on COS-7 surface at different time point .Results:Compare with control group ,there were no significant difference in the expression of TM-GFP on COS-7 surface at 1h and 6h in 5% CSE group [1h :(134.99 ± 18.41) vs .(146.61 ± 12.06) ,6h :(116.89 ± 27.28) vs .(123.89 ± 39.24) ,P>0.05 both] .Conclusion:The 5% cigarette smoking extract has no influ-ence on fluorescence expression of thrombomodulin on surface of African green monkey kidney fibroblast cells .

Constructing an expression vector for human lncRNA H19 and the effect of its overexpression on MCF-7 cell proliferation / 中国药理学通报

Aims To construct an expression vector of human lncRNA H 1 9 ,and to determine the effect of H1 9 overexpression on MCF-7 cell proliferation. Methods Total RNA was extracted from MCF-7 cells,and the full-length of H1 9 lncRNA was amplified by RT-PCR and subcloned into pcDNA3.1 (-)ex-pression vector.The constructed H1 9 expression vector was transfected into HEK-293T and COS-7 cells and the H1 9 lncRNA expression was evaluated by real-time PCR.Following the transfection of H1 9 expression vec-tor into MCF-7 cells for 0,24h and 48h and H1 9 siR-NA interference fragment into MCF-7 cells for 24h, MCF-7 cell proliferation was determined by MTS as-say.Results A hH1 9-pcDNA3.1 (-)expression vector was successfully constructed. At Forty-eight hours after the transfection with H1 9 expression vector in to MCF-7 cells,cell proliferation was significantly increased in the transfected group compared to those without transfection and to those transfected with a neg-ative control vector,while twenty-four hours after the transfection with H1 9 siRNA interference fragment into MCF-7 cells,cell proliferation was significantly de-creased in the transfected group compared to those transfected with a negative control vector.Conclusion Ectopic overexpression of H1 9 lncRNA can promote breast cancer MCF-7 cell proliferation.

Construction and expression of a pEGFP-C2-HDAC2 recombinant plamid / 中国药理学通报

Aims HDAC2 gene was cloned into pEGFP-C2 vector to explore the efficiency of the plasmid trans-fection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to ob-serve the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I, then inserted into the eukaryotic expression vector pEGFP-C2 with T4 en-zyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the ex-pression of pEGFP-C2-HDAC2 was monitored by fluo- rescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double diges-tion, and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detec-ted by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed, the fusion expres-sion of HDAC2 and GFP protein can be detected in COS-7 cells.

Regionalização sob a ótica dos gestores: uma abordagem dialética / Regionalization as seen by managers: a dialectic approach

Este estudo é parte da pesquisa interdisciplinar Estudo da Regionalização e Organização da Assistência de Média e Alta Complexidade na Macrorregião Sudeste de Minas Gerais realizada por pesquisadores da Universidade Federal de Juiz de Fora (UFJF) e da Prefeitura de Juiz de Fora e alunos de iniciação científica da UFJF, concluída em 2008. Serão apresentados os dados e as reflexões relacionadas à organização da assistência à saúde do Sistema Único de Saúde na Macrorregião Sudeste de Minas Gerais que foram obtidos junto aos gestores municipais e ao Hospital Universitário da UFJF. Neste artigo, analisamos a regulação do acesso dos usuários à rede de saúde do município de Juiz de Fora. A preocupação principal é identificar como se estabelecem as relações entre os gestores dos municípios limítrofes com o município de Juiz de Fora, polo macrorregional de atenção à saúde e o Hospital Universitário da UFJF. A abordagem dialética formou a base metodológica deste estudo, considerando as categorias: contradição, totalidade e historicidade.

This study is part of the interdisciplinary study, Regionalization and Organization of Medium and High--Complexity Care in the Southeast Macro-region of Minas Gerais, undertaken by researchers and students from the Federal University of Juiz de Fora and researchers from the City Council, and concluded in 2008. We present data and reflections related to health care organization in the Brazilian Unified Health System (SUS) of the Southeast macro-region of Minas Gerais, which were obtained from the municipal managers and the Federal University of Juiz de Fora Hospital (HU-UFJF). In this article, we analyze the regulation of users` access to the health care network of the Juiz de Fora municipality. The main aim was to identify how the relationships between managers from neighboring municipalities and the HU-UFJF are built. The dialectic approach was the method used in this study, with the following categories being considered: contradiction, totality and historicity.

Acesso e regionalização em saúde: análise de um serviço ambulatorial de média complexidade / Access and regionalization in health: analysis of a medium complexity walk-in care service

Este artigo avalia o Serviço de Doenças Intestinais Inflamatórias do Hospital Universitário da Universidade Federal de Juiz de Fora, responsável pelo tratamento dos pacientes portadores de doença de Crohn ou de retocolite ulcerativa, em relação às normas que determinam a regionalização da assistência à saúde para identificar os limites e possibilidades do acesso e a caracterização dos usuários que, em face às peculiaridades da doença, são orientados para o atendimento fora dos padrões regulamentados no Plano Diretor de Regionalização do Estado de Minas Gerais. A situação diferenciada é o foco deste estudo, considerando-se o processo de contratualização entre a Secretaria Municipal de Saúde e o Hospital Universitário.

This article assesses the Inflammatory Intestinal Diseases Unit of the Federal University of Juiz de Fora Hospital, which is responsible for the care of patients with Crohn`s disease or ulcerative rectocolitis, as concerns adherence to the norms regulating the regionalization of health care, in order to identify access limitations and possibilities, and to characterize users who, owing to the peculiarities of the aforementioned diseases, are advised to seek care beyond the patterns standardized by the Regionalization Management Plan of the Minas Gerais state. This particular situation, taking into account the agreement process between the Municipal Health Authority and the University Hospital, is the focus of the study.

The adhesion of Neisseria gonorrhoeae to COS-1 cells mediated by human CEACAM1 / 中华微生物学和免疫学杂志

Objective To study the role of human carcinoembryonic antigen-related cellular adhesion molecule 1 (hCEACAM1)in mediating the specific adhesion of N. gonorrhoeae to its human host cells.Methods A recombinant eukaryotic expression vector pCDPGICEA1 was constructed by putting hCEACAM1 cDNA behind both hCD46 promoter and rabbit β-globulin intron 2,and with which,the COS-1 cells were transfected. Following G418 selection, the COS-1 cells expressing hCEACAM1 were sorted out with flow cytometry. The adhesion of N. gonorrhoeae to the gene transfected COS-1 cells was analyzed with bacterial binding assay. Results hCEACAM1 cDNA could be expressed effectively under the direction of hCD46 promoter and rabbit β-globulin intron 2,and N. gonorrhoeae could adhere to COS-1 cells expressing hCEACAM1. Conclusion hCEACAM1 can mediate the adhesion of N. gonorrhoeae to animal originated COS-1 cells. thus its transgenic mice may be used as a novel animal model for studying N. gonorrhoeae infection.

Cloning,weukaryotic expremion of the gene encoding glyceraidehydes-3-phosphate dehydrogenase fromperiodic Brugia malayi / 中国地方病学杂志

Objective To clone and express the encoding sequence of glyceraldehydes-3-phosphate dehydrogenase(GAPDH)from periodic Brugia molayi(Bm).Methods Total RNA was extraeted from periodic Brugic malayi.The BmGAPDH gene was amplified by RT-PCR.The PCR product was cloned and then subeloned into pcDNA3.1(+)vector.The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification,and were transformed into COS-7 cell subsequently.The expressed protein was identified by SDS-PAGE.Results BmGAPDH mRNA was highiy expressed in transfected COS-7 cell.The deduced amino acid sequence was identical with that of BmGAPDH.The recombinant pnotein wag about Nr 43 000.Conclusion The recombinant plasmid peDNA3.1(+)-BmGAPDH has been constructed and the protein has been expressed correctly.

Effects of Tetrahydroxy-stilbene Glycoside on Over-expression of α-synuclein Protein in Transgenic COS-7 Cells / 中国康复理论与实践

@#Objective To explore the effects of 2,3,5,4'-tetrahydroxy stilbene-2-β-D-glycoside(TSG)on the over-expression and aggregation of α-synuclein in vitro.Methods TSG in different concentrations was incubated with α-synuclein transgenic COS-7 cells for 24 h.The cell viability was measured by MTT method.The expression of α-synuclein protein was determined by immunocytochemistry and Western blotting method.Results Incubation of TSG at the range of 12.5～200.0 μmol/L with α-synuclein transgenic COS-7 cells for 24 h did not influence cell viability,but a dose-dependently inhibition for the over-expression of α-synuclein protein could be observed in the tests of immunocytochemistry and Western blotting.Conclusion TSG can inhibit the over-expression of α-synuclein protein in COS-7 cells in vitro.

Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells / 华中科技大学学报（医学）（英德文版）

CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.

Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro / 中南大学学报(医学版)

Objective To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK and were inserted into an eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and was observed through fluorescence microscope. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. Results A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells. Conclusion The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.

Construction and Expression of Recombinant Wild Lipoprotein Lipase Gene Plasmid / 上海第二医科大学学报

Objective To study the construction of recombinant wild lipoprotein lipase(LPL) gene plasmid and its expression in COS-1 cells. Methods The LPL cDNA was isolated from the human epiploon adipose tissue by means of RT-PCR.The LPL cDNA was ligated into the pcDNA3.1Zeo(+).The recombinant pcDNA3.1Zeo(+)-LPL cDNA was identified by endonucleases,PCR and DNA sequencing.COS-1 cells were transfected with the recombinant LPL gene plasmid using Lipofectamine 2000~(TM).The LPL mass in cells and the culture medium were determined by a Markit-M LPL Kit.Spectrophotometry was used to measure the LPL activity. Results The LPL gene was ligated into the pcDNA3.1Zeo(+) plasmid identified by endonucleases and PCR.The sequence of the LPL gene was the same as the sequence of the Gene Bank identified by DNA sequencing.Wild pcDNA3.1Zeo(+)-LPL(cDNA) plasmids was transformed into the COS-1 cells. Conclusion The recombinant plasmid pcDNA3.1Zeo(+)-LPL cDNA could be constructed and successfully transformed into the COS-1 cells.