Osteosarcoma stem cell marker and application thereof, and kit
A technology for osteosarcoma and kit, applied in the field of biomedicine, can solve the problem of no LIF patent application
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Embodiment 1
[0043] Example 1: Determination of enhancers common to osteosarcoma cell lines and osteosarcoma tissues by chromatin immunoprecipitation.
[0044] Osteosarcoma cell lines (143B, SJSA1, ZOS, ZOSM) and osteosarcoma tissues obtained through surgical resection and confirmed by pathological diagnosis were taken, fixed in formaldehyde, and lysed, and the lysed products were broken into 100-300 cells by ultrasound DNA fragments in the base pair range. Corrected and purified chromatin was incubated overnight with H3K27ac antibody. The antibody protein complex is immunoprecipitated with A / G magnetic beads, washed, eluted, and reverse cross-linked to obtain the product. The product was sequenced, and the sequencing results were consistent with the UCSC hg19 human genome reference, and visualized to obtain figure 1 a.
[0045] Result: see figure 1 A analysis found that in osteosarcoma cell lines and osteosarcoma tissues, LIF was present as an enhancer in each group of samples, and th...
Embodiment 2
[0046] Embodiment 2: Through the data analysis of GEO database, mining the expression difference of LIF gene in normal bone tissue and osteosarcoma tissue.
[0047] The GEO standard method was used to download the RNA-seq sequencing data of the two groups of osteosarcoma tissue samples, and statistical software was used to make the difference in the expression of LIF between the osteosarcoma tissue and the adjacent tissue.
[0048] Result: see figure 1 B analysis found that compared with normal osteoblasts, the expression level of LIF in osteosarcoma was significantly increased, which can be considered as an early diagnostic index of osteosarcoma.
Embodiment 3
[0049] Example 3: RT-qPCR verification of the expression difference of LIF gene in osteosarcoma tissue and normal bone tissue.
[0050] By measuring the LIF gene expression status in 12 pairs of osteosarcoma tissues and normal bone tissues, the osteosarcoma tissues were obtained by surgical resection and confirmed by pathological diagnosis.
[0051] First, total cellular RNA was extracted. Keep all operations and related reagents on ice. Use Trizol reagent to extract total RNA from cells, and the specific steps are as follows: Prepare to extract total RNA. Cells were directly digested and lysed with Trizol, and Trizol was added. After the cells were added with Trizol, they were left at room temperature for 5 min to fully lyse. Use a high-speed refrigerated centrifuge to centrifuge at 12,000 rpm at 4°C for 5 minutes, and discard the precipitate. Add chloroform at 200ul chloroform / ml Trizol, shake and mix for 15min, and place at room temperature for 15min. Centrifuge at 12,...
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