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Method for detecting lung cell NLK (Nemo Like Kinase) expression, application of method, and application of NLK

A cell, expression technology, applied in the field of cell biology

Inactive Publication Date: 2013-10-02
万春华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of NLK in cancer tissues and its regulatory effect on carcinogenesis are currently less studied. Studies have shown that it may be highly expressed in some cancer tissues, or it may be low expressed in some cancer tissues

Method used

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  • Method for detecting lung cell NLK (Nemo Like Kinase) expression, application of method, and application of NLK
  • Method for detecting lung cell NLK (Nemo Like Kinase) expression, application of method, and application of NLK
  • Method for detecting lung cell NLK (Nemo Like Kinase) expression, application of method, and application of NLK

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Western blot detection

[0036] Western blot was used to detect the expression of NLK in lung cancer tissues (Tumor tissue, T) and adjacent normal tissues (Non-tumor tissue, N) of 8 pairs of clinical non-small cell lung cancer patients.

[0037] Western blot identification method

[0038] (1) The cells or tissue samples were taken out from -80°C and thawed on ice for 5 minutes;

[0039] (2) Add appropriate cell lysis solution, and for tissue samples, use a manual grinder to grind for ten minutes; for cell samples, resuspend cells, lyse on ice for 30 minutes, and then centrifuge at 13,000 rpm and 4°C for 15 minutes;

[0040] (3) take the supernatant, measure the protein concentration after cleavage by BCA method;

[0041] (4) After determining the protein concentration, adjust the protein concentration with 5× loading buffer (500mM Tris-Cl, pH=6.8, 10% Glycerol, 10% SDS, 0.1% BPB, 0.2M DTT) and deionized water, and the sample Boil for 5min;

[0042] (5) The...

Embodiment 2

[0047] Example 2 NLK immunohistochemical detection

[0048] Adenocarcinoma and squamous cell carcinoma were taken for immunohistochemical detection of NLK;

[0049] The detection method is as follows:

[0050] (1) Slices and baked slices: 5mm serial sections of the tissue to be inspected that are embedded in paraffin, affixed to polylysine-treated glass slides, and baked in an electric oven at 60°C for more than 8 hours;

[0051] (2) Dewaxing and hydration steps of tissue sections: xylene 15min×2 times (placed in a 60°C oven)→anhydrous ethanol 10min (2 times)→95% ethanol 5min (2 times)→80% ethanol 5min→ 70% ethanol for 5 min → rinse with running water → dd H 2 Soak in O for 5min, rinse with PBS for 3min×3 times;

[0052] (3) with H 2 O 2 Inhibits endogenous peroxidase activity: 3% H 2 O 2(37°C in a constant temperature incubator, the same below) incubate for 40min, rinse with PBS for 3min×3 times;

[0053] (4) Blocking: block with blocking solution (1% BSA, 10% normal ...

Embodiment 3

[0062] Example 3 NLK expression and cancer patient survival rate

[0063] 100 cancer patients were selected for NLK expression identification and follow-up visits. The statistical results are as follows image 3 . The survival rate of patients with low NLK expression was significantly lower than that of patients with high NLK expression, indicating that high NLK expression can predict better patient prognosis.

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Abstract

The invention discloses a method for detecting lung cell NLK (Nemo Like Kinase) expression. The method adopts immunoblotting detection or immunohistochemical detection. The immunoblotting detection utilizes GAPDH (Reduced Glyceraldehyde-Phosphate Dehydrogenase) as a reference gene and PCNA (Proliferating Cell Nuclear Antigen) as a cell proliferation marker. The method disclosed by the invention is used for detecting NLK expression amounts of lung cells to be detected and normal lung cells; when the NLK expression amount of the lung cells to be detected and the NLK expression amount of the normal lung cells are the same, the cells are normal; when the NLK expression amount of the lung cells to be detected is lower than the NLK expression amount of the normal lung cells, the lung cells to be detected are non-small lung cancer cells; the NLK expression amount of the non-small lung cancer cells and the non-small lung cancer cells are in malignant negative correlation. The method disclosed by the invention can be used for identifying the expression of the NLK of the lung cells.

Description

technical field [0001] The cell biology of the present invention particularly relates to the pulmonary cell protein kinase Nemo-like kinase (NLK). Background technique [0002] At present, a variety of molecular targets for the diagnosis and treatment of lung cancer have been discovered, including epidermal growth factor receptor (EGFR), HER2, Akt, KRAS and ERCC1, etc. The discovery of related molecular markers and the research on targeted therapy are important for lung cancer. It is of great significance to improve the clinical treatment and survival rate of patients. However, there are still many deficiencies in the diagnosis and treatment of lung cancer. Due to the existence of various pathological types and molecular marker changes in lung cancer, there are still no recognized molecular markers that can be used as diagnostic indicators in lung cancer, and the targeted therapy for lung cancer is still limited. Protein kinase NLK is an important class of cancer regulator...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/574G01N33/569
Inventor 万春华胡宝英吕丽婷
Owner 万春华
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