| BackgroundHeat shock protein 70 (HSP70) is a class protein with the role of molecular chaperone and immune regulatory. Many studies have shown that HSP70 protein expresses highly in many tumors and involves in tumor occurrence, development, treatment and prognosis. Researches about HSP70 in tumor tissues and cells became the hotspot in recently years. So it is necessary to obtain highly purified HSP70. But the methods of purifing HSP70 from tumor tissues are imperfect.ObjectiveIn this study, we plan to separate and purify tumor-derived HSP70 peptide complex (HSP70-PC) from human lung cancer by fast protein liquid chromatography (FPLC), then study on the human lung adenocarcinoma cell line A549 co-cultured with HSP70-PC, to research the effect of HSP70-PC on the proliferation of A549.Materials and methods1. The Preparation of mixed protein:The tissue sample of lung cancer was collected by pneumonectomy and confirmed by histopathology. The protein was derived from lung cancer tissues by means of splitting, ultrasonication and centrifuge. Then, we collected the clear supernatant liquid.2. The Separation and Purification of HSP70-peptide complex:The mixed protein was separated by affinity chromatography on ConA-Sepharose and DEAE ion-exchange chromatography3. The qualitative and quantitative analysis of HSP70-peptide complex:The molecular weight and identity of the HSP70 was confirmed by SDS-PAGE and Western blot. The concentration was measured by the means of Bradford.4. Cell Test:We used MTT cell proliferation assay to measure cell proliferation rate and investigated whether HSP70-peptide complexes purified from human lung cancer is able to influence A549 cell proliferation. 5. Statistical analysisDatabases were created by Excel 2003, which was used to establish databases. Further analysis was carried out by using the statistical analysis software SPSS 12.0 package (SPSS Inc, Chicago, Illinois, USA). The results were analyzed by two different mapping softwares. The data with normal distribution were measured by x±s. On the data with Equal Variance, ANOVA was used to compare multiple mean and the pairwise comparison. On the data with Unequal Variances, the chi-square test (χ2) was used to study the statistical discrepancy.Results1. Base on the above-mentioned research, we can purify HSP70 from human lung cancer by using ConA-Sepharose and DEAE ion-exchange chromatography, which was confirmed by SDS-PAGE and Western blot. The 110μg quantity of HSP70 measured by Bradford assay could be obtained from 1g lung cancer tissue.2. Although the value of OD in HSP70 was higher than control group, we found some groups were not significant. It is worthy of being noticed we found that there was not significantly positive correlation between concentration of HSP70 and cell multiplication. After 24 hours in culture, the proliferation of four experimental groups (OD was 0.899±0.0515,1.004±0.0406,1.114±0.0195,1.214±0.0285 and 1.323±0.206) were significantly higher than the control group (OD=0.678±0.0586)(P <0.05), except for 0.1μg/ml group (OD=0.678±0.0586). After 48 hours in culture, the cell proliferation rate of 1.0μg/ml (OD=1.72±0.0245) and 2.5μg/ml (OD=1.796±0.0245) experimental groups was significantly higher than the control group (OD=1.357±0.0477) (P<0.05), but this condition was not found in the other three experimental groups. After 72 hours in culture, cell proliferation rate of 0.5μg/ml (OD=2.49±0.0336), 1.0μg/ml (OD=2.604±0.010) and 2.5μg/ml (OD=2.462±0.0326) three experimental groups was significantly higher compared to the control group (OD=2.005±0.0187) (P<0.05), but 0.1μg/ml and 5μg/ml two experimental groups did not find this condition.Conclusions1. HSP70-peptide complexes could be purified by ConA-Sepharose column and DEAE ion-exchange chromatography from lung adenocarcinoma. 2. At the certain range of concentration, HSP70-peptide complexes can promote A549 cells growth. |
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