| Object: The purpose of this study was to compare chemoprevention activity between Sal B-PLC-NPs(Nano)and Sal B-Free(Free)to Oral Mucosa potentially malignant disorders(OPMDs).Method: The topic was designed about cells experiments in vitro and animal model experiments in vivo.1.Cells experiments in vitro.Cells qualitative uptake differences between Nano and Free by HN13,HN30 and Leuk1 respective for 2h were observed with laser confocal microscope.Cells quantitative uptake differences between Nano and Free at 25,50,100 and200?g/ml concentration-gradient action to HN13,HN30 and Leuk1 for 2h and200?g/ml for 5,10,20,30,60 and 120 min time-gradient were analyzed by fluorescence spectrophotometer.MTS method used to compare Nano with Free about inhibition cell proliferation about HN13,HN30 and Leuk1.The study was set negative control group,Nano and Free drug treatment groups with 20,50,100 and 200?g/ml concentration-gradient,observed 24,48,72 and 96 h time-gradient.Differences about cell cycle retardation were to analyze and compare with flow cytometry on condition that Nano and Free effect on HN13(200?g/ml-24h),HN30(100?g/ml-48h)and Leuk1(200?g/ml-48h).Differences about induction cell apoptosis at 24 h and 48 h were to analyze and compare with flow cytometry on condition that Nano and Free effect on HN13(200?g/ml),HN30(100?g/ml)and Leuk1(200?g/ml).2.Animal model experiments in vivo.The research used the classic animal model about 4NQO inducing C57BL/6mice tongue mucosa cancerization.C57BL/6 mice were intervened by intragastric administration Nano and Free for 18 weeks,stopped drugs and observed another 4weeks,total 22 weeks.The chemoprevention activity difference between Nano and Free to C57BL/6 mice tongue mucosa epithelial canceration were compared based on gross inspection,anatomical records and pathological diagnosis,tongue mucosa epithelial cell proliferation inhibition and cell cycle arrest with immunohistochemical method.Result:1.Cells experiments in vitro.The cells treated with Sal B-PLC-NPs intuitively showed stronger fluorescence than the cells treated with Free qualitatively observed with Laser confocal microscopy,which present Nano is easier to be absorbed by cells than Free.Nano was more uptaken by HN13 at 25,50,100 and 200?g/ml,HN30 at200?g/ml,Leuk1 at 25 and 200?g/ml than Free all at 2h,which suggest that it is different about the drug concentration and time-point uptaken by different cancern cells and OPMDs cells.Compared with negative control group,Nano and Free inhibition cell proliferation about HN13,HN30 and Leuk1 all had statistically significant difference.Nano inhibition cell proliferation about HN13(200?g/ml-48h)and HN30(100?g/ml-96h)was more significant than Free.Nano inhibition cell proliferation about Leuk1(200?g/ml-96h)was stronger than Free.Which indicated that Sal B has activity of inhibition cell proliferation to oral squamous cancer cells and OPMDs cells,and nanofomulation of Sal B has stronger inhibitory activity to different cell lines with a specific time and concentration conditions.Nano arrest cell cycle of HN13(200?g/ml-24h)and HN30(100?g/ml-48h)were more obvious trend than Free.Free arrest cell cycle to Leuk1(200?g/ml-48h)was more significant than Nano.200?g/ml Free inducing HN13 apoptosis for 24 h were more obvious than Nano.100?g/ml Free inducting HN30 early apoptosis for 48 h was more obvious than Nano.200?g/ml Free inducing Leuk1 apoptosis at 24 h and 48 h were more obvious with statistically significant than Nano.The last two results were consistent with the curve of cell growth inhibition,which prompt that anti-cancer activity about nanoformulation of Sal B under the condition of certain concentration and action time is better than non-nanoformulation through cell cycle and cell apoptosis with difference perspective.2.Animal model experiments in vivo.Growth and development of mice were in good condition from 8 to 30 week-age during the whole procedure of experiment.Mice drinking water first decreased then increased,and last decreased;weights of mice increased at first then decreased.Mice had no accidental death and other diseases.According to mice drinking 4NQO aqueous solution ad arbitrium,average water intake had no statistical differences between groups at the same time-point,which suggested that each group mice had been exposured to 4NQO equivalently;According to curves of body weight about mice,weights of mice grew parallelly and slowly at early stage and had no statistically difference between groups,which illustrated that various drugs had no obvious toxicity to mice.Gross examination and dissection,pathological diagnosis of 4NQO positive control group at 4th,8th,14 th,18th,22 nd weekend showed that the lingual mucosa epithelium of mice had undergone normal mucosa epithelium,benign hyperplasia,mild dysplasia,moderate dysplasia,severe dysplasia,mucosa epithelial canceration and cervical lymph node metastasis,the illness of mice aggravated gradually along with the progress of the experiment,which proved that this experiment successfully set up animal model about 4NQO inducing C57BL/6 mice tongue mucosa canceration.Scoring and statistical analysis basing on gross inspection,anatomical records and pathology diagnosis of mice about drug intervention groups at the 8th,14 th,18th and 22 nd weekend,we got that mice of Nano Group had relatively the lightest illness conditions,followed by mice of Free Group,the worse conditions with mice of 4NQO positive control Group which had no drug chemoprevention.Mice of three negative control groups were completely normal at the end of experiment,which declared that Nano and Free had no carcinogenicity,influencing mice growth and development and other side effects.Activity of Nano is stronger than Free to chemoprevent 4NQO induced C57 BL / 6 mice lingual mucosa canceration.The results about expression of Ki-67 and PCNA showed that Nano had more activity about inhibition cell proliferation of C57BL/6 mice tongue mucosa epithelial cells than Free.The results about expression of cyclin D1 and p16 showed that Nano had more activity about arrest cell cycle of C57BL/6 mice tongue mucosa epithelial cell than Free.Conclusion: The topic from cells experiments in vitro and in vivo animal model experiments confirmed that :Nanoformulation and non-nanoformulation Sal B all have inhibition or retardation activity to oral squamous cell cancer lines(HN13 and HN30)and OPMDs cell line(Leuk1),as well as the animal model about 4NQO induced C57BL/6 mice oral mucosa canceration,which shows that has no obvious toxic effect.The results of cell experiments in vitro and animal experiments in vivo manifested that Nanoformulation Sal B has stronger anti-cancer activity than non-nanoformulation Sal B under condition of certain concentration of drug and time-point.Uptaken of nanoformulation Sal B is more than non-nanoformulation's,as well as activity of nanoformulation Sal B is better than non-nanoformulation's about inhibition cell proliferation,block cell cycle and induction cell apoptosis,which deserves further research whether these phenomena could be used to account for cancer chemoprevention activity of nanoformulation Sal B than non-nanoformulation. |
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