12 results about "Slow Virus Diseases" patented technology

The invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

The invention discloses a construction and verification method of an HEK293T cell line capable of stably overexpressing human ANT2, which comprises the following steps: cloning a human ANT2 gene, constructing the human ANT2 gene into a lentivirus expression plasmid pLVX-TRE3G, co-transfecting the lentivirus expression plasmid pLVX-TRE3G and a helper plasmid pLVX-Tet3G into an HEK293T-ACE2 cell, and carrying out puromycin and G418 pressurized screening and Western blot identification to obtain the HEK293T cell line capable of stably overexpressing human ANT2. A result shows that the expression quantity of the ANT2 is the highest when the HEK293T-ACE2-ANT2 cell is induced for 12 hours at the Doxycycline of 6mg / L; when the ANT2 is induced to express, the virus nucleic acid load and the N protein expression level after SARS-CoV-2 infection are obviously reduced, which indicates that the ANT2 has an inhibition effect on SARS-CoV-2 infection replication, and a cell model and an experimental basis are provided for exploring the effect of the ANT2 in virus infection resistance.

The application discloses a kind of immortalization human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line construction method and its application in liver differentiation, it is related to immortalized cell technical field.The application first connects target gene to vector, and the slow virus plasmid containing target gene is constructed, the slow virus plasmid containing target gene is introduced into 293T cell, and high titer slow virus containing target gene is generated;Immortalized human umbilical cord mesenchymal stem cell is infected with slow virus, and mixed clone cell strain is screened, and the immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line is obtained.The application proves that the differentiation efficiency of immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression can effectively improve liver-like cell to glycogen staining and Western blotting experiment.This research result is expected to provide new technical thought for the next step development to improve the yield of MSCs liver differentiation.

The invention belongs to the technical field of cell culture, and particularly relates to a construction method of an immortalized goat umbilical cord mesenchymal stem cell line. The method comprises the following steps: by taking newborn goat lamb umbilical cord tissues as a research object, cleaning goat lamb umbilical cords, stripping blood vessels, separating Wharton's jelly, cutting the Wharton's jelly into 1mm < 3 > tissues, culturing by using a DMEM / F12 complete culture medium containing fetal calf serum, carrying out passage, cryopreservation, repeated resuspension and centrifugation, and inoculating into a culture dish for culture; lentiviruses containing SV40 large T antigens are packaged in vitro and are transduced into goat umbilical cord mesenchymal stem cells, and immortalization is realized after long-term subculture. The result of the embodiment shows that the construction method of the immortalized goat umbilical cord mesenchymal stem cell line provided by the invention is strong in repeatability, simple and efficient in operation steps, good in stem cell growth state and stable in physiological state.

The invention discloses a tongue squamous cell carcinoma in-situ PDX model construction method based on SERINC3 high expression and application of SERINC3, and belongs to the technical field of tumor biology and animal model construction. The method comprises the following steps: segmenting small tissues from cancer tissues of a patient suffering from tongue squamous cell carcinoma, implanting the small tissues into the armpit of an NCG mouse to culture tumor tissues, and then subculturing the tumor tissues; the subcultured tumor tissue is taken and implanted into the lingual margin of an NCG mouse, and after the tumor is cultured, lentivirus carrying sh-NC / sh1-SERINC3 / sh2-SERINC3 plasmids is injected into the tumor at multiple points to obtain the tongue squamous cell carcinoma in-situ PDX model. The model constructed by the invention can better simulate the natural occurrence and development process of TSCC, reconstructs a tumor microenvironment closer to clinical practice, and particularly has remarkable advantages in the aspect of simulating head and neck lymph node metastasis of tongue squamous cell carcinoma.

The utility model relates to the technical field of separation and purification devices, and discloses a separation and purification device for extracting lentivirus, which comprises a filter cartridge, a placement rack detachably mounted in the filter cartridge, a clamping cover mounted at the end part of the filter cartridge, a rotating button mounted in the clamping cover, and a rotating component arranged at the bottom of the rotating button, a clamping block is arranged on the side portion of the rotating assembly, a clamping groove is formed in the side portion of the filter cartridge, the clamping block and the clamping groove are clamped in a sliding mode, the clamping cover and the filter cartridge are clamped through the arrangement of the clamping cover, the rotating button is pressed, the rotating button drives the rotating rod, and the rotating rod rotates and shrinks; meanwhile, a sliding block and a connecting rod at the side part of the sliding block are driven, a sliding rod slides in a fixed cylinder, so that the sliding rod drives a clamping block to be clamped with a clamping groove in a sliding manner, and the clamping block made of a rubber material has good elasticity and compressibility and can be tightly attached to a contact surface between the filter cylinder and a clamping cover, so that a gap is reduced; and leakage of air or pollutants is avoided.

The application belongs to the technical field of biological medicine, and discloses a spinal cord injury treatment drug based on a LINC00311 / miR-4318 / MYLK regulation axis and an application thereof, and aims to solve the technical problems of poor targeting and large side effects of the existing anti-inflammatory treatment of spinal cord injury. The effective component of the drug is an inhibitor of LINC00311, the inhibitor is selected from one or more of shRNA, siRNA, antisense oligonucleotide and CRISPRi system, can target the specific nucleotide sequence of LINC00311, and can be used in combination with miR-4318 mimic, and is administered by intrathecal injection, slow virus carrier delivery and the like. The in-vitro experiment proves that the inhibitor can significantly inhibit the release of TNF-alpha, IL-1beta, IL-6 and other pro-inflammatory factors by microglia cells; the in-vivo experiment shows that it can significantly improve the BBB motor function score of spinal cord injury rats, and reduce spinal cord tissue edema and necrosis. The application first discloses the mechanism of the LINC00311 / miR-4318 / MYLK ceRNA regulation axis in spinal cord injury, has the advantages of novel target, significant effect and high transformation potential, and provides a new scheme for the clinical treatment of spinal cord injury.

The invention provides a culture method for reversing replicative aging root tip tooth papilla stem cells and application of the culture method, and belongs to the technical field of biological medicine. The method comprises the following steps: overexpressing an OCT4 gene in the replicative senescence tooth apex papilla stem cells, and culturing the cells by adopting a culture medium containing methyl cellulose to obtain the reversed replicative senescence tooth apex papilla stem cells. The method can be used for dental differentiation and angiogenic differentiation of stem cells. Through the synergistic effect of stable overexpression of the slow virus-mediated OCT4 gene and a methylcellulose 3D balling culture system, reversal of replicative senescence of the stem cells of the apical papilla is realized, the proliferation efficiency and differentiation potential of the stem cells are remarkably improved, the source of the stem cells is further expanded, and the yield of the stem cells is greatly increased.

The invention discloses a cell model for detecting monocyte and macrophage IL-1beta response level by using secretory luciferase, and belongs to the technical field of cell models. The technical problem to be solved is that in the prior art, a method capable of tracking response levels of mononuclear and macrophage factors IL-1beta in a real-time and non-invasive manner under different stimuli is lacked. According to the technical scheme, the cell model for detecting the IL-1beta response level of the mononuclear and macrophages is provided, and a preparation method of the cell model comprises the steps that an IL-1beta promoter is placed on the upstream of a secretory luciferase gene, lentivirus plasmids are introduced, and recombinant lentivirus plasmids are obtained; co-transfecting a host cell with the recombinant lentivirus plasmid and a packaging plasmid to prepare a recombinant lentivirus; and introducing the recombinant lentivirus into the THP-1 cell to obtain the THP-1 cell for expressing luciferase.

The application relates to the technical field of the cross of microfluidic technology, chemical synthesis and biomedicine, and discloses a method for preparing EGFR CAR-NK cells based on polypeptide gel droplet confined transfection technology and application, which comprises the following steps: (1) preparing collagen-mimic-peptide-modified alginate; (2) constructing a confined transfection system: adopting a droplet microfluidic chip, wrapping NK cells and slow virus carrying an EGFR CAR gene in the collagen-mimic-peptide-modified alginate droplet prepared in the step (1), and obtaining a gel droplet after gelation of the droplet; and (3) confined transfection and cell culture. The method can significantly reduce the dosage of slow virus and cell toxicity, greatly improve the transfection success rate of the CAR gene, the survival rate and functional activity of the NK cells, and finally obtain EGFR CAR-NK cell products with higher purity and stronger target killing capacity.

The invention provides a preparation method and application of an in-vivo CAR-T cell for treating interstitial lung disease, and provides a T cell of a chimeric antigen receptor which is modified by genetic engineering and is used for expressing targeted fibroblast activating protein. A targeted fibroblast activation protein (FAP) CAR-T cell therapy model is constructed by targeting CD5 entrapped mRNA nano-liposome (LNP) transfection and lentiviral vector, and the method comprises the following steps: firstly, verifying the difference of the killing ability of FAP CAR-T constructed by CD5 LNP-mRNA and lentivirus in a 293T cell which stably co-expresses FAP, Luciferase and mCherry, and then verifying the difference of the killing ability of FAP CAR-T constructed by LNP-mRNA and lentivirus in the 293T cell which stably co-expresses FAP, Luciferase and mCherry; further verifying the effectiveness of the CAR-T cells constructed by transfecting the CD5 LNP-mRNA on cell lines of human fibroblasts (CDD19Lu, LL29 and LL97A) and primary fibroblasts of human and mice in vitro, and further verifying the effectiveness and safety of the CD5 LNP-FAP CAR-T in treatment of pulmonary fibrosis through in-vivo experiments in animals. A novel and effective anti-fibrosis treatment thought is provided for patients with fibrosis interstitial lung diseases.

The invention discloses a construction method and application of a membrane-bound IL-21 K562 cell strain, a human IL-21 mature sequence is fused with an IgG4 hinge region, a human immune globulin gamma-4 chain CH2 / CH3 region and a CD4 transmembrane structural domain through genetic engineering to construct an mbIL-21 fusion gene, and the mbIL-21 fusion gene is cloned to a PT-mnud carrier by using BamH I / Xho I double enzyme cutting sites to obtain a recombinant plasmid PCDH-mbIL-21. HEK 293T cells are transfected through a lentivirus packaging system, high-titer lentivirus particles are prepared, and the cell strain stably expressing mbIL-21 is obtained through puromycin screening after K562 cells are infected. The mbIL-21 K562 cell strain constructed by the invention has the characteristics of long acting, targeting and stable expression, and provides a powerful tool for basic research and clinical transformation of immunotherapy.