Effects Of High Energy X-rays On Expression Of SPC-A-1 BM ERCC1 In Bone Metastatic Cells Of Lung Cancer

Objective:To investigate the effect of high-energy radiation on the expression of ERCC1 protein in SPC-A-1BM cell line with metastasis to lung cancer,Prompted the cell line SPC-A-1BM for platinum drug resistance point of time and the establishment of animal models of ERCC1 protein expression.Methods : Human lung cancer bone metastasis cell line SPC-A-1BM culture,Cells from the exponential growth phase were divided into experimental group and reference group.The experimental group with a medical linear accelerator 6MV X radiation,each 2Gy,1 time /day,a total of 10 Gy.After irradiation,every other week,exponentially growing part of the cells were frozen,the reference group also took part of the cells frozen,and finally got 5 experimental specimens.Fixed with 4%paraformaldehyde 2h,glycerin storage,minus 20℃ save.The expression of ERCC1 was detected by immunohistochemical SABC method,Mouse anti-human ERCC1 m Ab kit According to the reagent instructions: 3% H₂O₂ inactivation of peroxidase;Microwave antigen repair Add serum blocking solution;Add mouse anti-human ERCC1 monoclonal antibody reagent;Biotin secondary antibody was added dropwise;Add reagent SABC;Color with DAB;Infection with hematoxylin;Sample dehydration;Transparent with xylene;Seal with neutral resin。ERCC1 positive for the nucleus showed brown or brown-red particles precipitation。The number of positive cells in 100 cells was counted using a high power microscope Repeat each film 5times in different positions.The animal model was established after the reference group and the high energy ray irradiation 10 Gy group cell line,Exponentially growing cells were made into suspension。For tail vein inoculation of 10 experimental mice,After the vaccination,keep for 4 weeks in the SPF environment,Accept dissection to observe the tumorigenesis of the mice,Tumor tissue made from tissue pathology paraffin block。After pathological section,through dewaxing,hydration。Peroxidase is inactivated with hydrogen peroxide,Repeatedly washed with distilled water。Repair with microwave antigen,Wash 3 times with PBS。Add serum blocking solution。Incubate for 30 minutes at room temperature。Remove excess liquid。Mouse anti-human ERCC1 monoclonal antibody was added 。37℃ C lasted 1h,4 degrees within the refrigerator 12h。Wash repeatedly with PBS solution and the like。Add biotin secondary antibody,37℃,for 1h。Wash repeatedly with PBS three times。Add reagent SABC,37℃ dropwise for 1 h 。 Again,PBS was washed three times repeatedly。Color with DAB。Infection with hematoxylin。Sample dehydration。Transparent with xylene 。 Seal with neutral resin 。 The same high magnification microscopy visual field counts the number of positive cells in 100 cells。Repeat each film 5 times in different positions 。 Measurement data using mean ± standard deviation,Using SPSS19.0 software to process the data,X₂ test was used to analyze the data。P <0.05 for the difference was statistically significant。Results:The expressions of ERCC1 protein in the reference group and the 10 Gy group irradiated with high energy radiation at different time points after 1 4 weeks of irradiation were（22.07 ± 6.05%）,（47.13 ± 7.32%）,（69.51 ± 8.17%）and（51.37 ±9.10）,（31.57±5.89%）ERCC1 protein expression was significantly different from the reference group 1 to 3 weeks after irradiation(P<0.05.There was no significant difference between the 4-week group and the non-irradiated group（P> 0.05）.The nude mouse in the reference group developed 6 tumors,and the irradiated tissues formed 3 tumors,The cells that are irradiated by the rays cause the tumor to weaken during the inoculation process.There was no significant difference in the expression of ERCC1 in the pathological specimens。Conclusion:The human lung cancer bone metastasis cell line SPC-A-1BM in high-energy radiation within 1 to 3 weeks after the emergence of ERCC1 high expression,suggesting resistance to platinum drugs.The cells that are irradiated by the rays cause the tumor to weaken during the inoculation of nude mouse.