Applications of PBK/TOPK in diagnosis and treatment of intimal slim diseases
A thin, diagnostic reagent technology, applied in the direction of disease diagnosis, biochemical equipment and methods, microbial determination/inspection, etc.
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Embodiment 1
[0015] Example 1 Analysis of RNA-seq differentially expressed genes using bioinformatics methods
[0016] 1. Materials, reagents, equipment
[0017] 1.1 Source of human endometrial tissue
[0018] The endometrial tissues of 7 cases of control normal endometrium and 7 cases of patients with thin endometrium were collected. When obtaining endometrial tissues, according to the hormone level, it was ensured that all the stages of endometrial tissue acquisition were unified into the late stage of proliferation. All participants signed informed consent, which was approved by the Ethics Committee of Nanjing Drum Tower Hospital.
[0019] 1.2 Main reagents
[0020] RNasy Plus Micro Kit (Qiagen, Germany), absolute ethanol, DEPC water, enzyme-free water, reverse transcription reagent (Yase), SYBR Green (Yase).
[0021] 1.3 Main instruments
[0022] Qubit 3.0 Fluorometer, Agilent 2100 Bioanalyzer, Illumina Hiseq X10 Platform (Illumina, California, USA), Nano Drop.
[0023] 1.4 Main m...
Embodiment 2
[0030] Example 2 Expression of PBK / TOPK in Endometrial Tissue of Patients with Normal and Thin Endometrium
[0031] 1. Materials, reagents, equipment
[0032] 1.1 Source of human endometrial tissue
[0033] Collect 8 cases of normal endometrial tissue and 10 cases of endometrial tissue from patients with thin endometrium. When obtaining endometrial tissue, according to the hormone level, ensure that the endometrial tissue acquisition stage is unified into the late proliferation stage. All participants signed informed consent, which was approved by the Ethics Committee of Nanjing Drum Tower Hospital.
[0034] 1.2 Main reagents
[0035] TRNzol (Tiangen), chloroform, isopropanol, absolute ethanol, DEPC water, enzyme-free water, reverse transcription reagent (Yase), SYBR Green (Yase), xylene, 95%, 80% and 75% Ethanol, 3% hydrogen peroxide, citric acid repair solution, PBK / TOPK antibody (abcam), DAB chromogenic solution, hematoxylin, ammonia water, neutral resin.
[0036] PBK / T...
Embodiment 3
[0048] Example 3 Effect of PBK / TOPK on proliferation of human endometrial stromal cells
[0049] 1. Materials, reagents, equipment
[0050] 1.1 Main reagents
[0051]CCK8 kit (Vazyme), hyaluronidase (Sigma), DNAase (Roche), PBS, DMEM / F12 medium (Gibco), FBS serum (Gibco), antibiotics, trypsin 1.2 Main instruments
[0052] Filter, centrifuge, cell incubator, cell counter, microplate reader
[0053] 1.3 Main method
[0054] 1.3.1 Isolation and culture of human endometrial stromal cells
[0055] The human endometrial tissue scraped under the hysteroscope was taken, rinsed with normal saline, and cut into pieces. Add 0.1% type Ⅰ collagenase digestion solution, and digest at a constant temperature of 37°C for 1 hour. Pass through a 100-mesh sieve to remove tissue residues, centrifuge the filtrate at 230×g for 5 minutes, and discard the supernatant. Resuspend the pellet in serum-free DMEM / F12, pass through a 400-mesh sieve, centrifuge at 230×g for 5 minutes, and discard the su...
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