【作者】 逯彩虹；

【导师】 程建新；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 博士

【摘要】 宫颈癌是目前常见的妇科恶性肿瘤之一,其发病率逐年升高,并且逐渐年轻化。宫颈癌是人乳头瘤状病毒(Human papilloma virus HPV)长期持续感染的结果,可能与阴道以乳酸杆菌减少为主的菌群失调有密切关系。目前国外研究多数认为乳酸杆菌有很好的抗宫颈癌及宫颈上皮内瘤变作用,国内报道很少。本研究第一部分首先对宫颈癌患者阴道乳酸杆菌及其它常见病原体滴虫、霉菌、细菌性阴道病、支原体、衣原体、淋菌、HPV进行检测与分析,进一步了解乳酸杆菌在宫颈癌发病中的作用。树突状细胞(Dendritic Cell DC)功能的缺陷是导致宫颈癌的主要原因,以DC疫苗为主进行宫颈癌肿瘤免疫治疗是目前宫颈癌研究的重点,本文第二部分内容探讨了乳酸杆菌在DC疫苗抗宫颈癌中的作用以及作用机制。第三部分利用动物实验进一步研究乳酸杆菌在DC疫苗治疗宫颈癌中的效果,探讨其临床应用前景。第一部分宫颈癌患者阴道微生物分布特点与分析目的:探讨乳酸杆菌与常见阴道病原体在阴道黏膜上的分布特点以及与宫颈癌的关系。方法:检测正常宫颈妇女65例、慢性宫颈炎70例、宫颈上皮内瘤变(cervical intraepithelial neoplasia CIN)110例、宫颈癌患者142例阴道黏膜分泌物中乳酸杆菌的定植密度(乳酸杆菌数目/高倍镜)、霉菌、细菌性阴道病(Bacterial vaginosis BV)、支原体、衣原体、滴虫、淋菌、人乳头瘤状病毒(Human papilloma virus HPV16/18)的感染情况,了解宫颈癌患者阴道黏膜乳酸杆菌与常见病原体的分布特点、以及在宫颈癌发病中作用。结果:1、正常宫颈组(294个/HP)、慢性宫颈炎(282个/HP)、CIN(221个/HP)、宫颈癌组(125个/HP)阴道黏膜乳酸杆菌的定植密度逐渐减少(X2=47.07 P<0.01);霉菌、滴虫、支原体、衣原体、BV、HPV的检出率逐渐增加(P<0.01)。在正常宫颈组、慢性宫颈炎、CIN组内乳酸杆菌阴性患者阴道霉菌、滴虫、支原体、衣原体、BV、HPV的检出率明显高于乳酸杆菌阳性组(P<0.01),但在宫颈癌组内比较无差异(P>0.05)。淋菌在各组中发病率低,各组之间统计比较无差异(P>0.05)。2、在CIN组、宫颈癌组中随着乳酸杆菌数量减少,阴道病原体总感染率、混合感染率以及多种混合感染率也明显增加(P<0.01);在正常宫颈组、慢性宫颈炎、CIN组内乳酸杆菌阴性组病原体总感染、混合感染、多种混合感染率均明显高于乳酸杆菌阳性组(P<0.01),但在宫颈癌组内比较无差异(P>0.05)。3、在正常宫颈组、慢性宫颈炎、CIN组内感染组乳酸杆菌定植密度明显少于非感染组(P<0.01);但在宫颈癌组内比较无差异(P>0.05)。与正常宫颈组、慢性宫颈炎组比较,宫颈上皮内瘤变组、宫颈癌组感染者乳酸杆菌的定植密度逐渐减少(P<0.01)。4、宫颈癌组(66.9%)、CIN组(60.9%)中HPV感染患者阴道乳酸杆菌缺失率高于慢性宫颈炎(35.7%)、正常宫颈组(37.5%)。宫颈癌与CIN组;慢性宫颈炎与正常宫颈组比较无差异(P>0.05)。宫颈癌组(114个/HP)、CIN组(129个/HP)中HPV感染患者阴道乳酸杆菌阳性组乳酸杆菌定植密度明显少于慢性宫颈炎(251个/HP)、正常宫颈组(249个/HP)(P<0.01)。宫颈癌与CIN组;慢性宫颈炎与正常宫颈组比较均无差异(P>0.05)。5、在CIN组、宫颈癌组中HPV16/18、BV的感染率增加最明显,其次是衣原体、支原体、霉菌、滴虫;淋菌的检出率在各组中均很低。结论:乳酸杆菌抑制阴道常见病原体的繁殖与感染,保护宫颈上皮细胞、抑制其异常增生与癌变。宫颈癌发生与阴道黏膜乳酸杆菌减少、阴道菌群失调相关。第二部分乳酸杆菌增强树突状细胞疫苗诱导抗宫颈癌细胞毒性反应的体外研究目的:探讨乳酸杆菌在增强树突状细胞(DC)疫苗抗宫颈癌肿瘤免疫杀伤中的作用与意义。方法:1、反复冻融宫颈癌Hela细胞形成致敏抗原,并致敏树突状细胞:A乳酸杆菌作用后宫颈癌细胞冻融抗原致敏树突状细胞组;B单纯Hela细胞冻融抗原致敏树突状细胞组;C单纯乳酸杆菌致敏树突状细胞组;D未致敏树突状细胞组。2、采用流式细胞技术(Flow Cytometry FCM)测定各组树突状细胞成熟以及树突状细胞特征性抗原CD1a、CD83、CD80、CD86、HLA-DR表达。3、采用混合淋巴细胞反应(mixed lymphocytereaction MLR)、ELISA法分别测定经乳酸杆菌作用后的冻融宫颈癌抗原负载树突状细胞(DC)体外刺激T细胞增殖的能力以及上清液中IL-12与IFN-γ的含量。4、MTT法测定乳酸杆菌冻融抗原致敏树突状细胞(DC)激活的CTL细胞对宫颈癌HeLa细胞的杀伤作用。结果:1、乳酸杆菌明显促进树突状细胞(DC)的发育成熟、提高树突状细胞特征性抗原CD1a、CD83、CD80、CD86、HLA-DR表达(P<0.01)。2、与单纯冻融抗原、单纯乳酸杆菌致敏DC比较,经乳酸杆菌作用后的冻融宫颈癌细胞抗原负载树突状细胞(DC)体外明显增强T细胞增殖的能力(P<0.01);增加T细胞培养上清液中IL-12与IFN-γ的含量(P<0.01);增强冻融抗原致敏DC激活的CTL细胞对宫颈癌HeLa细胞的杀伤力(P<0.01)。3、各致敏组的树突状细胞(DC)体外刺激T细胞增殖的能力、培养上清液中IL-12与IFN-γ的含量、激活的CTL细胞对宫颈癌HeLa细胞的杀伤力均明显高于未致敏组(P<0.01)。结论:乳酸杆菌增强树突状细胞抗原提呈能力,增强树突状细胞疫苗对宫颈癌肿瘤细胞的免疫杀伤作用。第三部分乳酸杆菌增强树突状细胞疫苗抗宫颈癌的的体内研究目的:探讨乳酸杆菌在树突状细胞(DC)疫苗抑制宫颈癌移植瘤中的作用。方法:1、乳酸杆菌在DC疫苗治疗宫颈癌移植瘤中作用:(1)宫颈癌Hela细胞建立30只雌性裸鼠移植瘤动物模型,在移植瘤大小为4cm3左右时随机化分为三组实验:第1组,接种乳酸杆菌作用后宫颈癌Hela细胞冻融抗原致敏的DC疫苗;第2组,接种单纯宫颈癌Hela细胞冻融抗原致敏的DC疫苗;第3组,注射PBS缓冲液作为对照组。(2)4天测量一次瘤体大小,绘制移植瘤生长曲线。(3)制备肿瘤单细胞悬液,采用FCM检测各实验组肿瘤细胞凋亡率。(4)制备肿瘤单细胞悬液,采用FCM检测各实验组肿瘤细胞Fas蛋白的含量。(5)采用免疫组化技术(SP法)观察各组移植瘤Survivin蛋白的表达。2、乳酸杆菌在DC疫苗预防宫颈癌移植瘤中作用:(1)选取18只雌性裸鼠:A组:接种乳酸杆菌辅佐冻融抗原致敏DC活化的疫苗;B组:接种冻融抗原致敏DC活化疫苗;C组:注射PBS缓冲液作为对照组。(2)3天后将对数生长期的宫颈癌Hela细胞系接种于裸鼠。4天观察一次裸鼠移植瘤的生长情况,绘制移植瘤生长曲线。结果:1、乳酸杆菌增强DC疫苗对宫颈癌移植瘤的抑制作用:乳酸杆菌DC疫苗组移植瘤在治疗后10天体积大小为2.10±0.60cm3,明显小于治疗前4.05±0.53cm3(P<0.01);单纯DC疫苗治疗组可明显抑制瘤体的生长,治疗前4.15±0.84cm3,治疗后6.19±1.36cm3(P<0.01);而对照组移植瘤呈快速生长状态,治疗前3.92±0.78cm3,治疗后14.56±1.67cm3(P<0.01)。2、乳酸杆菌DC疫苗组明显增强肿瘤细胞的凋亡,凋亡率为38.26±3.45%,高于单纯DC疫苗组21.68±3.26%与对照组3.77±0.99%)(P<0.01)。3、乳酸杆菌DC疫苗治疗组肿瘤细胞Fas蛋白的含量为570.42土66.72,高于单纯DC疫苗治疗组400.50土86.05与对照组247.32土54.06。4、与Fas蛋白表达相反,survivin蛋白在乳酸杆菌辅助DC疫苗治疗组肿瘤细胞中表达大部分为阴性;单纯DC疫苗治疗组肿瘤细胞大部分表达为弱阳性;而对照组大部分肿瘤细胞survivin蛋白表达为强阳性。5、乳酸杆菌DC疫苗预防组宫颈癌Hela移植瘤的成瘤率为16%,而且瘤体小0.52cm3;单纯DC疫苗预防组成瘤率为50%,瘤体较大2.34±0.38cm3;而对照组成瘤率为100%,瘤体最大4.89±1.48cm3(P<0.05)。结论:乳酸杆菌辅佐DC疫苗可以很好预防并明显抑制裸鼠宫颈癌Hela移植瘤的生长,增强DC疫苗促进肿瘤细胞凋亡。其凋亡的机制一方面上调Fas膜蛋白,另一方面下调抑制凋亡Survivin蛋白。乳酸杆菌可作为DC疫苗很好的免疫增强剂,为宫颈癌DC疫苗的进一步开发与临床应用提供一条新的思路。更多还原

【Abstract】 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.更多还原