Monoclonal nonspecific inhibitory factor-beta as index for clinical evaluation of recurrent spontaneous abortion
A technology of recurrent spontaneous abortion and factors, applied in the fields of biotechnology and medicine, can solve the problems of RSM identification and poor knowledge of prediction methods
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Embodiment 1
[0077] Example 1. Detection of MNSFβ protein expression in human decidual tissue during early pregnancy
[0078] In this example, the inventors detected the expression of MNSFβ protein in human decidual tissue during early pregnancy.
[0079] Specifically, the inventors performed tissue immunofluorescence detection as follows:
[0080] The freshly obtained tissue was placed in 4% paraformaldehyde (PFA) as soon as possible and fixed overnight at 4°C; after successively dehydrated in a gradient of 10% and 25% sucrose solution, frozen tissue was embedded with embedding medium O.C.T, -80 Store at ℃ until use. After cutting 8 μm frozen sections with a cryostat, they were directly placed in pre-cooled 4% PFA for 10 min; washed with PBS for 3×5 min, punched with 1% Triton-X100 / PBS solution for 10 min; added 3% BSA or corresponding serum and IgG , block at room temperature for 1h; add primary antibody (purchased from Abcam), overnight at 4°C (concentration is adjusted according to the...
Embodiment 2
[0082] Example 2. Detection of dMφ cell purity and cell morphological characteristics
[0083] In this example, the inventors examined the dMφ cell purity and cell morphological characteristics.
[0084] Specifically, it is detected by the following methods:
[0085] (1) Wash the collected fresh decidua in the first trimester with sterile sugar-free PBS solution for 2 to 3 times until there is no obvious blood clot in the decidua.
[0086] (2) Take 5 ml of decidual tissue and put it into a C-type tube, crush the tissue with a Gentle MACS Dissociator, the mode is M-heart-01.01 (16s), and pour the crushed decidual tissue into a digestion bottle, In a 1:1 ratio, add serum-free 1640 medium (5ml) and repeat this step until the volume of the solution in the digestion bottle reaches 20ml.
[0087] (3) After the digestion, the decidua tissue mixture in the digestion bottle was introduced into a 50ml centrifuge tube, supplemented with serum-free 1640 medium to 45ml, 400rpm, centrifug...
Embodiment 3
[0100] Example 3. Detection of mRNA and protein expression levels of MNSFβ in human dMφ cells
[0101] In this example, the inventors detected the expression of MNSFβ at the mRNA and protein levels in human dMφ cells.
[0102] Specifically, it is detected by the following methods:
[0103] 1. Extraction of total cellular RNA and total protein:
[0104] (1) Cell lysis: isolated dMφ cells, each 5×10 5 Add 500 μl of Trizol to each cell, pipet slowly with a pipette tip until no obvious flocs appear, and store in a -80°C ultra-low temperature freezer for later use.
[0105] (2) Layering: Take out the Trizol lysate from the -80°C refrigerator, dissolve it slowly on ice, add chloroform (200 μl / 1ml Trizol), mix up and down, stand still on ice for 2-3 minutes, 4°C, 12000g, and centrifuge for 15 minutes ; After centrifugation, take out the EP tube, there will be layers as shown in the figure below, in which RNA is fused to the upper layer, DNA is fused to the middle and lower layers,...
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