Lactobacillus' Role Approach In Cervix Cancer And Mechanism Study About Enhanceing Dendritic Cell(DC) Effect In Resisting Tumor

Cervix cancer is one of frequent femme malignant tumor,Incidence rate of cervix cancer steps up year by year, And age of onset is gradually younger. Cervix cancer is a persistent infection result of Human papilloma virus( HPV). It possibly has a intimate relation to vagina dysbacteria with Lactobacillus decreasing. Present abroad study consider that Lactobacillus has a fine role in resisting cervix cancer and cervical intraepithelial neoplasia (CIN). There is less domestic research about it . In the first study , we detect and analyze vagina lactobacillus and the other common pathogen (infusorian, mycetes, bacterial vaginosis, mycoplasma, chlamydia, gonococcus, HPV),And preferably understand Lactobacillus′role in cervix cancer.Functional defect of dendritic cell(DC) is primary cause resulting in cervix cancer. Dendritic cell(DC)vaccine tumor immunity treatment about cervix cancer is an Important and hot spot at present.In the second study,We approached lactobacillus′role and mechanism in cervix cancer invasion. We study lactobacillus′effect in assisting DC vaccine treatment to cervix cancer,And approach lactobacillus′clinical application prospect of DC vaccine treatment about cervix cancer at the third study.PartⅠDistribution feature and analysis of Vagina microorganism in cervix cancer patientsobjective: Approach distribution feature of Vagina lactobacillus and common pathogen, Study their relation to cervix cancermethod: Detected vagina mucosa lactobacillus planting density and common pathogen (mycetes, bacterial vaginosis, mycoplasma, Chlamydia, infusorian, gonococcus, HPV) of 387 women(Normal cervix 65, Chronic cervicitis 70,cervical intraepithelial neoplasia 110,cervix cancer 142),Observe their distribution feature and roles in cervix cancer invasion.Results:1 Vagina mucosa lactobacillus planting densitys of Normal cervix(294/HP), Chronic cervicitis (282/HP),CIN(221/HP)and cervix cancer(125/HP)are gradually decreaseing (X2=47.07 P<0.01),But detection rates of mycetes, infusorian, mycoplasma, Chlamydia, bacterial vaginosis, HPV are gradually increasing(P<0.01). Detection rates of mycetes, infusorian, mycoplasma, Chlamydia, bacterial vaginosis, HPV in Un-Lactobacillus group are higher than those in Lactobacillus group about Normal cervix, Chronic cervicitis and cervical intraepithelial neoplasia (CIN ) gruops (P<0.01).But in cervix cancer,there is no statistics difference (P>0.05). Detection rates of gonococcus is very low in every group, there is no statistics difference among four groups(P>0.05).2 With Lactobacillus decreaseing in CIN and cervix cancer groups, Infection rate,mixed infection rate,poly-mixed infection rate of vagina pathogen were gradually increasing (P<0.01);Infection rate,mixed infection rate,poly-mixed infection rate of vagina pathogen in Un-Lactobacillus group are higher than those in Lactobacillus group about Normal cervix, Chronic cervicitis and cervical intraepithelial neoplasia (CIN ) gruops (P<0.01), But ,there is no statistics difference in cervix cancer (P>0.05).3 Vagina mucosa lactobacillus planting densitys in infection are less than those in no-infection about Normal cervix, Chronic cervicitis and cervical intraepithelial neoplasia (CIN ) gruops (P<0.01), But ,there is no statistics difference in cervix cancer (P>0.05).Compared with Normal cervix and Chronic cervicitis, Vagina mucosa lactobacillus planting densitys of infection are gradually decreaseing in CIN and cervix cancer groups(P<0.01).4 Lactobacillus depletion rates of HPV infections in cervix cancer(66.9%)and CIN(60.9%)groups are higher than those in Normal cervix (37.5%)and Chronic cervicitis(35.7%)groups(P<0.01). There is no statistics difference about Lactobacillus depletion rates of HPV infections between CIN and cervix cancer groups, between normal cervix and Chronic cervicitis groups(P>0.05).Vagina mucosa lactobacillus planting densitys of lactobacillus group with HPV infections in cervix cancer(114/HP)and CIN (129/HP)groups are obviously less than those in Normal cervix (249/HP)and Chronic cervicitis(251/HP)groups(P<0.01), but there is no statistics difference about lactobacillus planting densitys between CIN and cervix cancer groups, between normal cervix and Chronic cervicitis groups(P>0.05). 5 Infection rate of HPV16/18,BV are obviously increased in CIN and cervix cancer groups first, Second Infection rate of mycoplasma, Chlamydia, mycetes, infusorian are also increased . Detection rates of gonococcus is very low in every group.Conclusion: Lactobacillus can restrain multiply and infection of Vagina common pathogen, It can protect cervix cellula epithelialis, Restrain its paraplasm and cancerization. Vagina normal flora Disproportion and lactobacillus decreaseing are relation to cervix cancerPartⅡEx vivo study of anti-cervix cancer cytotoxic T cell effect by Dendritic cell vaccine enhanced with lactobacillusObjective:Approach effect and significance of lactobacillus in anti- cervix cancer tumor immunity killing by dendritic cell vaccine.Methods:1 Freeze thaw uterine cervix cancer antigen repeatedly and form sensitized antigen, Then load dendritic cell: A Dendritic cell (DC): loaded by antigen lysate with lactobacillus stimulating, B Dendritic cell (DC): loaded by pure antigen lysate; C Dendritic cell (DC): loaded by lactobacillus;DNo-loaded DC. 2 Determine lactobacillus′influence to CD1a,C D83,CD80,CD86,HLA-DR and mature condition of dendritic cell through flow cytometry(FCM) in four groups. 3 Function of DC loaded by freeze-thawing cervix cancer antigen stimulated with lactobacillus was evaluated by their ability to induce proliferation of allogeneic T cells by mixed lymphocyte reaction (MLR),IL-12 and IFN-γproduction in cell supernatant was assayed by using ELISA method. 4 The cytotoxicity to uterine cervix cancer HeLa cells of CTL activated by dendritic cells loaded with antigen and Lactobacillus was detected by MTT method.Results: 1 Lactobacillus can obviously promote mature of DC cells and express of cell phenotypic molecule CD1a,CD83,CD80,CD86,HLA-DR of DC cells(P<0.01).2 Compared with DC cells allergized by pure freeze thawing cervix cancer antigen or pure lactobacillus, DC cells loaded by freeze thawing uterine cervix cancer antigen stimulated with lactobacillus can obviously reinforce cell proliferation of T lympholeukocyte(P<0.01)and IL-12,IFN-γproduction in cell supernatant of T cell culture(P<0.01). It'kill power to cervix cancer Hela cell is higher than those of the other groups (P<0.01). 3 Proliferation of T lympholeukocyte(P<0.01); IL-12,IFN-γproduction in cell supernatant of T cell culture and immune kill power to cervix cancer Hela cell in loaded DC group is obviously higher than that of un-loaded DC group(P<0.01)Conclusion: Lactobacillus can reinforce angtigen presentation ability of dendritic cell and immunity lethal effect of DC vaccine to uterine cervix cancer.PartⅢIn Vivo study about anti-cervix cancer effect of dendritic cell vaccine enhanced by lactobacillusObjective:Approach Lactobacillus′role and mechanism in anti-cervix cancer transplantation tumor inducted by dendritic cell (DC) vaccineMethods:1 Lactobacillus′role in anti-cervix cancer transplantation tumor of DC vaccine : (1) Establish 30 cervix cancer Hela cell transplantation tumors with femina athymic mouse; They were randomly divided into three group when tumor volume is about 4cm3:In the first group, vaccinate DC vaccine loaded by freeze thawing cervix cancer antigen after Lactobacillus stimulating; In the second group, vaccinate DC vaccine loaded by freeze thawing cervix cancer antigen; In the third group, inject PBS buffer as control group. (2) Measure tumor bulk every 4 days and draw growth curve of transplantation tumor.(3)Make tumor unicell suspension,Detect tumour cell apoptosis with flow cytometry(FCM).(4)Make tumor unicell suspension,Detect Fas protein with flow cytometry(FCM X-mode).(5)the express of Survivin protein were observed by immunohistochemistry echnique(SP). 2 Lactobacillus′role in preventing cervix cancer transplantation tumor about DC vaccine (1)Choose 18 female athymic mice which were randomly divided into three group: A group:vaccinate DC vaccine loaded by freeze thawing cervix cancer antigen after Lactobacillus stimulating; B group: vaccinate DC vaccine loaded by freeze thawing cervix cancer antigen; C group: inject PBS buffer as control group. (2) In the third day after vaccination,Inject cervix cancer Hela cell, Establish transplantation tumors; Measure tumor bulk every 4 days and draw growth curve of transplantation tumor.Results:1 Lactobacillus can enhance DC vaccine′inhibitory role to cervix cancer transplantation tumor: In DC vaccine group assisted by Lactobacillus, 10 days after treatment, tumor(2.10±0.60cm3)was obviously smaller than that of before treatment(4.05±0.53cm3)(P<0.01); In DC vaccine group, tumor grow after treatment (6.19±1.36 cm3)was obviously restrained compared to that in control group (before treatment 4.15±0.84cm3)(P<0.01);But in control group, Tumor′growth is faster compared to treatment group (before treatment 3.92±0.78cm3; after treatment 14.56±1.67cm3 )(P<0.01).2 In DC vaccine group assisted by Lactobacillus, DC vaccine can obviously reinforce Apoptosis of tumor cell(Apoptosis rate 38.26±3.45%) compared to DC vaccine group(21.68±3.26%) and control group(3.77±0.99%). 3 The expression of Fas protein in DC vaccine group(570.42土66.72)are higher Than those of DC vaccine group(400.50土86.05) and control group(247.32土54.06). 4 Opposite to Fas protein, In DC vaccine group assisted by Lactobacillus ,survivin protein expression is Negative in most tumor cell. But in control group, the expression of survivin protein in most tumor cell was positive , there was weak survivin protein expression in DC vaccine group. 5 DC vaccine assisted by Lactobacillus can obviously prevent cervix cancer transplantation tumor with tumor form rate(16%) and small node (0.52cm3); But in DC vaccin with 50% and big tumor (2.34±0.38cm3);And in control group, tumor rate is 100% , node is the biggest(4.89±1.48cm3)(P<0.01).Conclusion: 1 The DC vaccine assisted by Lactobacillus can prevent growth of cervix cancer Hela cell transplantation tumor very well. Lactobacillus enhance Apoptosis role of cervix cancer tumour cell treated by DC vaccine. There are two Apoptosis mechanism: (1) Up-regulate Fas protein expression(considered Apoptosis protein), (2) Down-regulate the expression of Survivin protein((considered refraining Apoptosis protein). 2 As a good immunopotentiating agent to DC vaccine, Lactobacillus can provide a new think or method for exploitation and clinical application of cervix cancer treatment by DC vaccine.