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find Keyword "敲除" 9 results
  • Research Progress of Xenotransplantation

    Objective To summarize the research progress of xenotransplantation.Methods Domestic and international publications about xenotransplantation were summarized and reviewed. Results Hyperacute xenograft rejection was a huge problem for xenotransplantation, but it could be alleviated if the organs or tissues of donor were genetically modified. So far the graft survival time differed greatly due to characteristics of different organ. Conclusions By reviewing the studies of relevant papers about xenotransplantation, a comprehensive understanding of research background and a suitable research direction of xenotransplantation can be supplied. The graft organs or tissues from genetically modified donors are expected to avoid or alleviate hyperacute xenograft rejection.

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  • 敲除与Th2 细胞活化相关的电压依赖钙通道可防止实验性哮喘发生(Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma)

    敲除与Th2 细胞活化相关的电压依赖钙通道可防止实验性哮喘发生(Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma) 【摘要翻译】 研究理由: Th2 型细胞参与过敏性哮喘,这些细胞产生的细胞因子( IL-4、IL-5 及IL-13) 在过敏状态时分泌增加。因此, 研究Th2 型细胞表达的对其功能具有重要影响的关键信号分子至关重要。我们既往的研究显示二氢吡¤特异性调控Th2 细胞的功能。目的: 由于二氢吡¤可特异性与活化细胞的电压依赖钙通道( Cav1) 结合并调节其功能, 我们的主要目的是证实Th2 细胞特异性表达功能性的Cav1 相关通道, 抑制其功能可能抑制哮喘。方法: 我们通过定量PCR 和Western blot 检测Th2 和Th1 细胞Cav1 通道表达。我们将Th2 细胞表达的Cav1 的异构体进行测序, 并研究Cav1 反义寡核苷酸( Cav1AS) 是否影响Ca2 + 信号及细胞因子的产生。最后, 我们通过给OVA 鼻腔激发的BALB/c小鼠注射Cav1AS 转染的OVA 特异性Th2 细胞研究Cav1AS在被动免疫哮喘动物模型中的作用, 并通过鼻腔给予此前进行过OVA 及氢氧化铝免疫的BABL/ c 小鼠Cav1AS 和OVA溶液以研究Cav1AS 对主动免疫哮喘模型的影响。检测和主要结果: 我们发现小鼠Th2 细胞而非Th1 细胞表达Cav1. 2和Cav1. 3 通道。转染Cav1AS 抑制了钙通路和细胞因子产生, 并导致Th2 细胞丧失过继Th2 细胞诱导气道炎症功能。鼻腔内给予Cav1AS 可抑制主动免疫导致的哮喘气道炎症和气道高反应性。结论: 这些结果提示Th2 细胞特异性表达Cav1. 2 and Cav1. 3 通道, 以此作为治疗靶点可有效抑制动物模型的哮喘反应。 【述评】 哮喘是一种Th2 型慢性气道炎症反应疾病,目前机制未明。Th2 细胞在此过程中发挥关键作用, 但Th2细胞活化机制不清楚。本文的研究发现Th2 细胞特异性表达Cav1. 2 和Cav1. 3 通道, 以Cav1AS抑制Cav1 通道的表达可抑制Th2 细胞功能进而抑制哮喘炎症反应。该研究揭示了哮喘气道炎症反应的新机制, 为哮喘治疗提供了新靶点。但是, Cav1 通道如何影响Th2 细胞的功能尚需进一步研究。其次, 有些基因在人类和小鼠表达并不一致, 特别是一些异构体的表达水平不同, 甚至在功能上存在很大差异, 因此, 在哮喘患者中Cav1 通道的表达尚待研究。最后, Th2 功能失调在一些自身免疫性疾病中发挥重要作用, 因此, 如证实Cav1 通道可用于人类哮喘治疗, 则该方法可能对其他一些自身免疫性疾病有治疗作用。

    Release date:2016-08-30 11:54 Export PDF Favorites Scan
  • Generation of Mouse Embryonic Stem Cell Clones for Ghrelin Receptor Gene Knock Out

    Objective To establish the heterologous recombinant embryonic stem cell (ES cell) with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene. Methods PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing. Results The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination. Conclusion This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.

    Release date:2016-09-08 11:07 Export PDF Favorites Scan
  • Study of Influence of IGF-1 on Angiogenesis by Using IGF-1 Deficient Mice Breast Cancer Models

    Objective To determine the effect of insulin-like growth factor-1 (IGF-1) on angiogenesis in mouse breast cancer model of lower and normal serum IGF-1 levels after using angiogenesis inhibitor ginsenoside Rg3 (GS Rg3). Methods The breast cancer models were established in control mice and liver specific IGF-1 deficient (LID) mice by feeding DMBA and were treated with GS Rg3. Vascular endothelial growth factor (VEGF) and F8-RAg were detected by immunohistochemical method in breast cancer tissues. IGF-1 gene and angiogenesis relating genes were detected by gene chip in breast cancer and normal breast tissue. Results The incidence rate of breast cancer in LID mice was lower than that in control mice (P<0.05). VEGF expression and microvessel density of LID mice were lower than those in control mice (P<0.05). Compared to the control mice, IGF-1, FGF-1, TGF-β1 and HGF genes were increased, and FGFR-2, PDGF-A and PDGF-B genes were decreased in breast cancer of LID mice. After GS Rg3 treatment, VEGFa, EGF, EGFR, PDGF-A and FGFR-2 genes were increased, IGF-1 and TGF-β1 genes were decreased in breast cancer of LID mice compared with the control mice. Conclusion IGF-1 may be involved in mouse breast cancer progression and associated with the growth of blood vessels. Angiogenesis inhibitor may play an antitumor role by IGF-1 and TGF-β1.

    Release date:2016-09-08 11:07 Export PDF Favorites Scan
  • Establishment of a mice model with liver-specific AMP-activated protein kinase gene knockout

    AMP-activated protein kinase (AMPK) is involved in the development and progression of tumors including hepatocellular carcinoma (HCC). However, studies on AMPK and tumorigenesis were largely based on experiments in vitro or tumor xenografts model. Here, we introduce a liver-specific AMPKα1 knockout mice model, which is achieved by Alb-Cre recombinase system. The expression of AMPKα1 in the liver of AMPKα1-/--Alb-Cre mice is absent. AMPKα1 knockout in the liver does not affect the growth and histological structure of mouse liver. This model provides a favorable tool to the study of the roles of AMPKα1 in liver metabolism or tumorigenesis.

    Release date:2017-06-19 03:24 Export PDF Favorites Scan
  • The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy

    ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.

    Release date:2017-09-19 03:09 Export PDF Favorites Scan
  • Construction and characterization of a mutant outer membrane protein-A gene-deleted Escherichia coli strain

    Objective To construct an Escherichia coli outer membrane protein-A (OmpA) gene-deleted strain by Red homologous recombination, and laid the foundation for subsequent research. Methods Polymerase chain reaction (PCR) primers were designed according to the known OmpA gene sequence, and plasmid pKD3 for PCR amplification and integration; the fragment was transformed into Escherichia coli by λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation OmpA protein knocked out was observed by Western-blotting. Results The knock out gene product was correspond to a expected molecular weight. The western-blotting show that OmpA protein was knocked out. The difference in growth curve between the wild type and Escherichia coli △ OmpA gene-deleted strain was not significant. Conclusion OmpA gene deletion had no significant effect on the growth of Escherichia coli, which provides a foundation for further research on live vector vaccine.

    Release date:2017-12-25 06:02 Export PDF Favorites Scan
  • Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal

    The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.

    Release date:2021-04-21 04:23 Export PDF Favorites Scan
  • Report of 3 cases of transplantation of GGTA1 gene knockout porcine islet cells into type Ⅰ diabetic macaques

    ObjectiveTo explore the effect of transplanting neonatal porcine islet cells of pig via hepatic portal vein in type Ⅰ diabetic monkeys.MethodIn this study, three pig-monkey islet xenotransplantation experiments were carried out by using α-1, 3-galactosyltransferase (GGTA1) gene knockout neonatal pig islet cells.ResultsThree macaques were successfully transplanted with islet cells. After the operation, their vital signs were stable and no symptoms of venous embolism occurred. After transplantation, the blood glucose and the dosage of exogenous insulin were significantly reduced, and the specific porcine C-peptide could be detected. Three macaques developed symptoms of ketoacidosis, and one macaque developed wound infection. After symptomatic treatment, all of them survived for 16 weeks.ConclusionGGTA1 knockout neonatal porcine islet cells transplanted through hepatic portal vein is effective for the treatment of type Ⅰ diabetes.

    Release date:2021-05-14 09:39 Export PDF Favorites Scan
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