Application of long-chain non-coding RNA in preparation of tumor angiogenesis inhibitor
A long-chain non-coding, tumor vascular technology, applied in the field of genetic medicine, can solve the problems of unclear long-chain non-coding RNA related mechanisms, cell cycle arrest, etc.
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Embodiment 1
[0025] Example 1 Angiogenesis Experiment
[0026] The cloned long-chain non-coding RNA (sequence is SEQ ID NO: 1, prepared according to the existing method) was loaded on a conventional lentiviral vector, and directly infected K562 cells to construct a long-chain non-coding RNA high-expression stable cell line (LLEST). Virus-infected cells were used as controls (non-highly expressed leukemia K562 stable cell lines); the expression results of the two cell lines are shown in the appendix figure 1 . The specific methods of cloning, loading and infection are conventional techniques.
[0027] Angiogenesis Experimental Procedures
[0028] (1) Pre-cool the 96-well plate, spare cell supernatant, 200ul tip, and pipette in a 4°C refrigerator or on ice for 30 minutes, take out the Matrigel Matrigel from the -20°C refrigerator, and place it at 4°C Refrigerator until it thaws;
[0029] (2) Laying gel: Take 50 μl / well Matrigel matrigel and add it to a 96-well plate, and do it on ice. Av...
Embodiment 2
[0034] Example 2 Detection of PDGFA gene level
[0035] According to the method of Example 1, leukemia K562 stable cell lines with high expression and non-high expression of long-chain non-coding RNA were constructed, the cells were collected, RNA was extracted by Trizol one-step method, and the PDGFA gene level was detected by qRT-PCR method. Compared with the blank group, the PDGFA gene level decreased in the long non-coding RNA high expression group, see appendix image 3 . The existing commercially available Oblimersen drug (which is a single-stranded DNA molecule consisting of 18 bases and complementary to Bcl-2 mRNA, which can inhibit the expression of Bcl-2 family proteins by inhibiting Bcl-2 mRNA) was used for comparative experiments. According to the above method, it was found that compared with the blank group, the PDGFA gene expression level was similar (the difference was less than 3%), and it had almost no inhibitory effect on tumor angiogenesis. At the same tim...
Embodiment 3
[0036] Example 3 Detection of PDGFA protein level
[0037] According to the method of Example 1, leukemia K562 stable cell lines with high expression and non-high expression of long-chain non-coding RNA were constructed, and the cell supernatant was collected after routine culture, and the PDGFA protein level was detected by ELISA method. In the leukemia cell line K562, compared with the control group, the expression level of PDGFA decreased in the long-chain non-coding RNA high expression group, see appendix Figure 4 .
[0038] The invention packs long-chain non-coding RNA into a lentiviral vector to construct an angiogenesis inhibitor, detect angiogenesis, and detect tumor cell PDGFA gene and protein levels. By inhibiting its expression with gene medicine, it can inhibit the formation of tumor blood vessels, reverse the source of oxygen and nutrients for tumor cells, thereby inhibiting the growth of tumors and improving the anti-tumor efficacy.
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