| Objective Cervical cancer is the most common gynecological malignant tumor,the second largest cancer leading to death in women.There is no fully effective treatment to cervical cancer at present.The traditional radiotherapy and chemotherapy cannot reduce mortality,and the inevitable side effects greatly reduces the patients' quality of life.Up to now we treat human papilloma virus(HPV)persistent infection as the major pathogen of cervical cancer.In addition,considering the complexity of antigen,virus subtype diversification,the vaccine researches have become increasingly difficult as well.Therefore,we have been trying to find a new treatment to prolong patients life,and to improve the quality of life.Lactic acid bacteria is a group normal advantage bacterium in the woman's vagina,play a vital role in maintaining vaginal normal ecological balance.Several studies have shown that cervical cancer is closely associated with vaginal microecological balance disorders.The lactobacillus decrease,leading to vaginal Gardner or mixed anaerobic bacteria produce harmful substances,with other carcinogenic factors(such as the human papilloma virus)joint action in the cervix,accelerated the development of cervical cancer.The present study suggests that epithelial/interstitial transformation(EMT)is not only a basis of embryonic development process in multi-cellular organism and organs,but also plays an important role in tumorigenesis,invasion and metastasis,and the occurrence of a variety of chronic diseases development.Epithelial calcium mucins-cadherin(E)is one of the center of the most important factor in the EMT,can promote the connections between cells,maintain the stability of the stent.Its down-regulated expression is often an important factor of cervical cancer metastasis.This study aims to explore whether lactobacillus intervention affect the cervical cancer cells growth and metastasis,to explore the corresponding mechanism,aiming to provide a new perspective for the prevention and treatment of cervical cancer.Method: 1 Experiment in vitro 1.1 Cultivate lactobacillus(DE's lactobacillus DM8909)1.2 Lactobacillus directly stimulate cervical cancer cell Hela and cervical cancer source of mice U14 cell(MOI: 10:1,100:1 or 1000:1)for 24 h,48h,72 h,CCK-8 cell proliferation kits to learn the influence of lactobacilli affect on cervical cancer cell proliferation 1.3 Through the scratch experiment test to learn the effects of lactobacillus on cervical cancer cell migration 1.4 Lactobacillus respectively direct stimulation of cervical cancer cells and through trans-well Chambers indirectly stimulate cervical cancer cells,collected cells at different time points,to extract proteins,Western blot to detect factor E-cadherin,vimentin,FOXO4,snail,Twist and beta-catenin expression which may affect cell growth and migration.1.5 Lactobacillus are inactivated by 65?,30 min,then affect on cervical cancer cells,collected cell at different time points,testing the above might involve factor expression by Western blot.2.In vivo testsTwenty-five 4~6 week old female BALB/C mice,will be randomly divided into 5 groups(control group,the therapeutic dose(2.25x106/0.2 ml)and the corresponding inactivated bacteria group,high dose group(5x106/0.2ml)and corresponding inactivated dose group),each group has 5 mice.The 2x106/0.2ml U14 cells were then subcutaneously injected into the BALB/C mice.Tumor-inhibiting experiment was conducted the day after tumor-burdened.Therapeutic dose groups and corresponding dosage inactivated way respectively around the tumor multi-point injection activated or inactivated live lactobacillus 2.25x106/0.2 ml(therapeutic dose).High dose group and corresponding inactivated dose group mice were injected with live or inactivated lactobacilli 5mlx106/0.2(high dose)in the same way,the control group injected the same amount of PBS,only 1 times a day,for 20 consecutive days.Test mice weight and the size of the tumor everyday.After a day's withdrawal,execute mice.Weighed tumor and use vernier calipers to measure tumors length diameter(L)and short diameter(W),and to calculate the tumors volume according to the formula V=L.W2/2.Cut the tumor,observe its internal color,texture,with dead zone or not.Divide tumor tissue into two parts,one part use 10% paraformaldehyde fixed liquid to fixation,to do immune-histo-chemical,the other part to lift protein to do Western Blot,detection the above factors.Result:1 Lactic acid bacteria had no effect on cervical cancer cell proliferation.2 After 24 hours Lactobacilli in cervical cancer cell,it showed its inhibition on cervical cancer cell migration3 Lactobacillus can promote cervical cancer cells E-cadherin expression.In human cervical cancer cells Hela,cut the expression of Snail,but had no effect in mice cervical cancer U14.There has no effect on vimentin,FOXO4,snail,Twist and beta-catenin expression.4 Lactobacillus indirectly stimulus Hela and U14 cervical cancer cells by transwell,the expression which can promote E-cadherin increased significantly.5 Inactivated lactobacilli in cervical cancer cell,E-cadherin expression has no significant differences compared with control group.6 A tumor-burdened mice weight after lactobacillus intervente 3 days begin to have differences,treatment dose group were alleviated than the other four groups,with statistically differences.The weight of high dose group and the corresponding inactivated dose group were lighter than a tumor-burdened group and inactivating treatment dose group,with statistically differences;But the control group and inactivating treatment dose group,high dose group and corresponding inactivated dose group there is no statistical differences.7 The net volume of treatment dose group and high dose group were less than the other three groups,with statistically differences;but between treatment dose group and high dose group;a tumor-burdened group,the inactivated treatment dose group and inactivated high group there is no statistical differences.8 Treatment dose and high dose group of tumors in the E-cadherin expression significantly higher than the other three groups,but between the treatment dose group and high dose group;tumor-burdened group,the inactivated treatment dose group and inactivated high dose group there is no statistical difference.Vimentin,FOXO4,snail,Twist and beta-catenin expression shows no statistical differences between 5 groups.9 Immunohistochemical HE staining confirmed a tumor-burdened mouse model for squamous cell carcinomas.Between Treatment dose group and high dose group,the proportion of E-cadherin is more than corresponding inactivated dose group and PBS group with statistically significant.Conclusion:1 Lactic acid bacteria had no effect on cervical cancer cell proliferation.2 Lactic acid bacteria inhibitate cervical cancer cells migration3 The migration effect is achieved by raising E-cadherin expression4 In human cervical cancer,it is possibly not affected by cutting the expression of Snail to promoted the expression of E-cadherin.5 Lactobacillus increase E-calcium sticky protein expression is not affected by Twist and beta-catenin.6 Lactobacillus increase E-calcium sticky protein expression by its secretion to extracellular component implementation. |
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