| Objective: Immunohistochemical method was used to analyze the difference in ILK protein expression in primary cancer foci of patients with BRAF mutations and unmutated PTC,to explore the pathological parameters related to ILK and malignant biological behaviors,and to assist in clinical early screening of high metastatic risk and poor prognosis.High-risk PTC disease;cultured in vitro BRAF mutant papillary thyroid cancer cell B-CPAP,using a variety of molecular techniques to detect changes in the malignant biological behavior indicators of Prunella vulgaris on B-CPAP cells,in order to explore Prunella vulgaris The specific mechanism of action on thyroid cancer is expected to open up a new path for the prevention and treatment of thyroid cancer by traditional Chinese medicine.Methods: 1.The first part of histopathology experiment: 1.1 Collect 46 patients who underwent thyroidectomy at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from December 2018 to May 2019,and were diagnosed with papillary thyroid carcinoma by the pathology department of our hospital Basic information of patients,statistics of BRAF gene mutation and gender,age,diameter of primary foci,distribution of cancer foci,color Doppler ultrasound TI-RADS grade,cancer TNM and pathological staging,whether primary foci invaded the thyroid The degree of difference between pathological parameters such as membrane and lymph nodes,whether or not accompanied by autoimmune thyroiditis.1.2 Immunohistochemical staining was performed on the primary cancer foci of all enrolled patients to evaluate the strength of ILK protein.1.3 According to the results of the previous step,record the grade of the ILK protein in 46 cases,and analyze the correlation between ILK and various clinical pathological parameters of PTC.1.4 Statistic and analyze the relationship between the degree of ILK expression and the PTC patients with BRAF V600 E gene mutation.2.The second part of the cell experiment: 2.1 Culture the isolated BRAF mutant papillary thyroid carcinoma cell B-CPAP under suitable conditions.The CCK8 experiment screened out the optimal concentration of Prunella vulgaris to inhibit cell proliferation.2.2 Taking the concentration of 0.5-1.5 mg/m L of Prunella vulgaris as the scope of the subsequent experiment,observe the transformation process of BCPAP cell morphology after the action of the drug under the microscope.2.3 Flow cytometry was used to detect the degree of annexin staining on the surface of B-CPAP cells after the action of Prunella vulgaris,and analyze the ratio of cells in the apoptotic phase.2.4 Draw several parallel “-” traces on the bottom of the six-well plate inoculated with cancer cells,and measure the difference in drug concentration and action time respectively.2.5 Inoculate B-CPAP cells in the upper layer of the Transwell cell,add Prunella vulgaris solution to the lower layer to induce the cells to do chemotaxis,observe and count the number of cells permeated by the bottom membrane of the cell.2.6 Western Blot experiment to verify the inhibitory effect of the appropriate concentration of Prunella vulgaris on B-CPAP cell apoptosis proteins BAX,BCL-2,Caspase3 and invasion protein ILK from the protein level,and the regulation of the phosphorylation level of MAPK pathway protein ERK / p-ERK effect.Results: 1.BRAF V600 E gene mutation is positively correlated with multiple PTC cancer lesions,higher stage and grade,lymph node and extracapsular infiltration(P <0.05),and no significant statistical relationship with other factors,such as gender,age,TIRADS grade,diameter of primary focus,whether it is associated with autoimmune thyroiditis,etc.(P <0.05).2.ILK protein showed positive expression in epithelial papillary tissues with different strengths.The strong positive signal was closely related to malignant biological behaviors such as larger tumor diameter,multifocal infiltration,lymph node metastasis and capsular invasion(P< 0.05).3.The positive rate of ILK protein detected in the primary cancer lesions of BRAF mutant PTC patients is much higher than that of the non-mutated patient group(P<0.05).4.CCK8 experiment results show that high concentration of Prunella can significantly inhibit the activity of B-CPAP cells,the drug IC50 is about 0.94 mg / m L.5.Under the inverted phase contrast microscope,the growth of B-CPAP cells was restricted after 24 hours,the integrity of the cell membrane was destroyed,and the number of rounded and brightened cells increased.6.The results analyzed by flow cytometry are consistent with the performance under a phase-contrast microscope.The higher the dose of Prunella vulgaris solution,the higher the percentage of cells in the apoptotic area.7.The results of the scratch experiment showed that the scratched area had been regrown with cells after 24 h in the drug-free group,while the cell-free zone was obvious in the Prunella vulgaris group,and the cell chemotactic growth was limited.The amount and duration of action increase.8.Transwell experiment results show that the higher the drug concentration,the fewer the number of stained cells that migrate through the membrane,and the high concentration of Prunella can completely inhibit the cell process.9.Western Blot protein electrophoresis found that compared with the blank group,Prunella vulgaris can down-regulate the levels of anti-apoptotic protein Bcl-2 and invasion protein ILK in B-CPAP cells,which in turn makes the proapoptotic proteins BAX and Caspase-3 The level is up-regulated,while reducing the phosphorylation degree of the MAPK pathway representative protein p-ERK / ERK.Conclusion: 1.BRAF mutant papillary thyroid carcinoma has the characteristics of strong invasiveness and high grade.ILK presents strong positive expression in this type of cancer,which is associated with malignant tumors of larger diameter,multifocal infiltration,lymph node metastasis,and capsule invasion.Related to biological behavior.2.The antagonistic effect of Prunella vulgaris on thyroid tumors is mainly triggered by the mitochondrial pathway,and the mechanism of weakening cancer invasion may be that the BRAF gene mutation induces the activation of the MAPK pathway,which cross-links with ILK protein and interferes with downstream signal transmission. |
PDF Full Text Request