| Objective:To observe the effect of Qing You II Lotion on cell proliferation and HPV E7 and PCNA protein expression of SiHa cell, illumated the effective therapy mechanism of traditional Chinese medicine in condyloma acuminatum. Methods:1. SiHa cells were cultured in vitro in the condition of 37℃ 5% co2 and 10% rpmi 1640 culture medium. After cells pasted with the wall of cultrue bottle entirely, through the convert microscope observed the morphological change of SiHa cells co-cultrued with different concentration Qing You II Lotion within 48h.2. Divided groups: blank control group(SiHa cell group), Qing You II Lotion groups (the end concentration were 10-3g/ml, 10-4g/ml, 10-5g/ml, 10-6g/ml) and negative control group. Logarithm growth period cells were digested to unicellular suspension by 0.25% pancreas enzyme, then inoculated to 96 hole board. After adding different concentration medicine above mention, the cell proliferation level was detected by the 3-(4, 5-Dimethylthiazol-2-yl) 2, 5-diphenyltetrazo-liumbromid (MTT) at 48h.3. Divided groups: blank control group (SiHa cell group), Qing You II Lotion groups (the end concentration were 10-3g/ml, 10-4 g/ml, 10-5 g/ml, 10-6g/ml) and negative control group. Inoculated unicellular suspension to the cover glass slices, added different concentration of Qing You II Lotion, after co-cultrued 48h, checked the protein expression of HPV E7 and PCNA by indrect immunofluorescene technology, calculated the grey level of average fluorescence and positive unit.4. All experiment datas were showed by mean ± std.deviation, and were analysed by One Way ANOV, t test, Correction analysis and Regression analysis with SPSS11.5 for windows software.Results:1. We could see that SiHa cells grow well after secondary cultrue, cell bodies were satiety, sizes were even, cells present shuttle, spindle or triangle, sticked to wall of cultrue botton tightly, the ovoid core in the center, and the purity was extremely high. After adding different concentrate Qing You II Lotion, SiHa cells still grew well, there was no obvious variety in the shape of SiHa cells.2. In the experiment of MTT, the OD490 value of blank control group was 0. 8450±0. 0271, the OD490 values from 10-6g/ml group to 10-3g/ml group sequentially were 0.7351±0.0428, 0.5963 ± 0.0514, 0.5103 ± 0.0455, 0.3810±0.1704; And following the rising of medicine concentration gradient, the cell proliferation activation gradually reduced, the cell proliferation levels in Qing You II Lotion groups were obvious lower than that in SiHa cell group (P<0. 01) ,among Qing You II Lotion groups the cell proliferation levels olso had significance difference (P <0.01). In company with the rising of medicine concentration gradient, the inhabition rate gradually increased, the values from blank control group to10-3g/ml group sequentially were (12.9860±4. 7639)%,(29.4780 ± 4.8062) %, (39.6520 ± 4.1867) % and (54.9140±2.4769) %; the cell proliferation inhabition rates of Qing You II Lotion groups compared with the blank group had significance difference (P<0.01), the proliferation inhabition rates between QingYou II Lotion groups also had significance difference (P<0.01); by Correlation analysis, it was high relation between the proliferation inhabition rates and medicine concentration gradient, r=0.994, P<0.01.It shew that Qing You II Lotion groups could evidently suppress the SiHa cell proliferation.3. From the result of indrect immunofluorescene technology, we could see that the positive cell counts which strongly expressed bright green immunofluorescenes of HPV E7 and PCNA gradually decreased, and the negative cell counts which faintly expressed green immunofluorescenes of HPV E7 and PCNA gradually increased. The average grey level of PCNA immunofluorescence expressed by 10-6g/ml group had no significance difference compared with blank control group (P>0.05), Positive units of PCNA immunofluorescence expressed by 10-6g/ml group had significance difference compared with blank control group (P<... |
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