【作者】 王浩；

【导师】 王志明； 唐利立；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 第一章铜绿假单胞菌注射液治疗乳腺癌癌性溃疡的临床研究目的研究铜绿假单胞菌注射液治疗乳腺癌癌性溃疡的有效性和不良反应。方法对乳腺癌癌性溃疡患者随机分组,分别采种不同的治疗方法：单纯化疗(对照组)、铜绿假单胞菌注射液联合化疗(治疗组)。分别测量治疗前后溃疡大小以及肿块大小,观察铜绿假单胞菌注射液在乳腺癌癌性溃疡治疗中的疗效以及可能出现的不良反应。结果同对照组相比,铜绿假单胞菌注射液联合化疗促进患者乳腺癌癌性溃疡面积缩小(治疗组有效率85.7%,对照组有效率33.3%,P<0.05),促进乳腺癌性肿块缩小,但无统计学差异(治疗组有效率57.1%,对照组有效率33.3%,P>0.05)；在铜绿假单胞菌注射液治疗过程中除发热外无明显不良反应发生。结论铜绿假单胞菌注射液对于乳腺癌癌性溃疡面愈合有促进作用,且无明显不良反应。第二章铜绿假单胞菌注射液对于乳腺癌治疗作用的体外杀伤机制研究目的铜绿假单胞菌注射液在体外实验中对人乳腺癌细胞(MCF-7)凋亡和增殖的影响。方法使用体外培养至对数生长期的MCF-7细胞设立实验组和对照组,实验组给予不同浓度的铜绿假单胞菌注射液,对照组给予等量RPMI 1640培养基处理后,MTT比色法检测铜绿假单胞菌注射液对体外培养的MCF-7细胞增殖的抑制作用；流式细胞术检测其对MCF-7细胞凋亡及细胞周期的影响；荧光染色法观察其诱导体外培养的MCF-7细胞凋亡的形态学改变。结果与对照组相比,多个浓度的铜绿假单胞菌注射液均可抑制MCF-7细胞增殖(P<0.05)；促进MCF-7细胞的凋亡(PA-MSHA组凋亡率2.14±0.2457%,对照组凋亡率1.19±0.1778%,P<0.01),升高MCF-7细胞中G0／G1期细胞比例(PA-MSHA组73.00±7.10%,对照组为49.83±9.83%,P<0.05)、降低S期细胞所占百分比(P<0.05),荧光显微镜观测示MCF-7细胞呈现明显的凋亡图像。结论铜绿假单胞菌注射液可能通过阻滞乳腺癌MCF-7细胞周期中G0／G1期,诱导细胞凋亡,从而抑制体外培养的乳腺癌细胞的增殖。第三章铜绿假单胞菌注射液对于乳腺癌治疗作用的体内治疗作用及机制研究目的：1、研究铜绿假单胞菌注射液(PA-MSHA)在高自发转移乳腺癌小鼠模型中对乳腺癌的治疗作用,并探讨其可能的作用机理。2、研究铜绿假单胞菌注射液对于晚期乳腺癌患者细胞免疫功能的影响。方法：1、使用4T1细胞株通过细胞悬液注射法建立高自发转移乳腺癌小鼠模型,将荷瘤小鼠随机分成4组,即阴性对照组(A组),CTX治疗组(B组),PA-MSHA组(C组),联合治疗组(D组),给予不同处理,同时培养正常小鼠设为空白对照组(E组)。比较治疗后各组间荷瘤小鼠的肿块大小及肿块重量、生存期、肺转移率、脾指数；并采用流式细胞检测法分析比较各组小鼠外周血T淋巴细胞亚群,采用液相多重蛋白定量技术(CBA)检测小鼠外周血细胞因子TNF-αIFN-γ、IL-5、IL-4、IL-2含量。Pearson相关性分析法分析实验小鼠外周血T细胞亚群、TNF-α、IFN-γ、IL-5、IL-4、IL-2水平与小鼠生存期之间的关系。2、选择临床上有转移的晚期乳腺癌患者14例,随机分为化疗组和化疗联合PA-MSHA组,两组同期进行化疗,实验组加用铜绿假单胞菌注射液,隔日一次进行治疗。于三周期化疗后,分别抽取外周血,采用流式细胞术(FCM)检测CD3+总T淋巴细胞、CD4+辅助T细胞、CD8+杀伤／抑制T细胞,计算CD4+／CD8+比值。结果：1、肿块大小及瘤重比较中,各治疗组均小于阴性对照组(P<0.05),CTX治疗组同PA-MSHA组间无统计学差异(P=0.160),均大于联合治疗组(P<0.05)。肺转移率比较中,阴性对照组转移情况明显高于联合治疗组(P=0.029),其余各组之间的转移情况差异无统计学意义(P>0.05)。生存期比较中,PA-MSHA组同CTX治疗组间差别无统计学意义(P=0.056),均高于阴性对照组(P<0.05),低于联合治疗组(P<0.05)。脾指数比较中,空白对照组小于其他四组,联合治疗组高于其他四组(P均小于0.05)；CTX治疗组与PA-MSHA组之间差异无统计学意义(P=0.352)。同阴性对照组相比,铜绿假单胞菌注射液可提高外周血T淋巴细胞中CD4+T细胞所占比例,降低外周血T淋巴细胞中CD8+T细胞所占比例,提高CD4+／CD8+比值(P<0.05)。升高外周血细胞因子IL-2水平.(P<0.05)；与CTX共同使用可提高TNF-αIFN-γ水平(P<0.05). Pearson相关性分析显示,CD4+T细胞比例与处理组小鼠生存期之间存在强正相关性(r=0.714,P<0.01)；CD8+T细胞比例与生存期之间存在强负相关性(r=0.790,P<0.01)；CD4+／CD8+与生存期之间存在强正相关性(r=0.781,P<0.01)；IL-2与生存期之间存在一般正相关性(r=0.552,P<0.05)；其余细胞因子与生存期之间无明显相关性(P>0.05)。2、铜绿假单胞菌注射液治疗组同对照组患者外周血中T淋巴细胞占总淋巴细胞比例无差别(P>0.05),T细胞中,实验组CD4+T细胞比例高于对照组、CD8+T细胞比例低于对照组、CD4+／CD8+比值高于对照组(P<0.05)。结论：1、在使用4T1小鼠乳腺癌细胞株建立的高自发转移乳腺癌小鼠动物模型体内,存在着免疫失衡的现象。2、PA-MSHA能够调节乳腺癌动物模型中的T细胞亚群和多个细胞因子水平(IL-2、TNF-a水平,IFN-γ),改变其免疫失衡状态,从而发挥抑制肿瘤生长、减少转移、延长生存期作用；并可与化疗发挥协同作用。3、PA-MSHA对于临床Ⅳ乳腺患者有明确的改善免疫功能的作用,可能有利于改善患者预后。更多还原

【Abstract】 Part I A Clinical Research on Pseudomonas Aeruginosa Injection for the Treatment of Malignant Ulcer of Breast CancerObjective:To evaluate the effectiveness and adverse reaction of pseudomonas aeruginosa injection (PA-MSHA) for the treatment of malignant ulcer of breast cancer.Methods:Patients with malignant ulcer of breast cancer were randomly divided into two groups such as the control group receiving chemotherapy alone and the treatment group receving chemotherapy combined with PA-MSHA. Tumor size and ulcer area were measured before and after treatment. The adverse reactions were also evaluated following with the process of the therapy.Results:Contrast with the control group(33.3%), the treatment group(85.7%) could promote malignant ulcer area growing downwards(P<0.05), but there was no statistical significance(P>0.05) about breast cancer tumor between the treatment group(57.1%) and the control group(33.3%).Conclusion:Administration of PA-MSHA is effective in the treatment of malignant ulcer in breast cancer without obvious adverse reactions. PartⅡKilling Mechanism Research on Pseudomonas Aeruginosa MSHA (PA-MSHA) Injection in Breast Cancer in VitroObjective:To investigate the effects of PA-MSHA on the apoptosis and proliferation of human breast cancer cells (MCF-7) cultured in vitro.Methods:With culture in vitro of MCF-7 cells being in logarithmic phase, then divided them into two groups such as the expertiment group and the control group. The experiment group were given PA-MSHA injection, and the contro group were mitte tales doses of RPMI 1640 culture medium. With MTT colorimetric method detecting the inhibition effect of PA-MSHA on MCF-7 cultured in vitro, and flow cytometry dectected the influence of PA-MSHA affecting MCF-7 apoptosis and cell cycle. Then used Hoechst fluorescent staining to observe it induce the morphology chage of MCF-7 cultured in vitro.Results:Contrast with the control group, different densities of PA-MSHA all could inhibit MCF-7 cell proliferation(P<0.05), promote MCF-7 cell apoptosis (PA-MSHA group apotosis rate:2.14±0.246, the control group rate:1.19±0.178, P＜0.01), raised G0/G1 stage cell ratio(PA-MSHA group apotosis ratio:73.00±7.10, the control group ratio:49.83±9.83) of MCF-7 cell and cut down the percentage of G2/M and S stages. Fluorescence microscope detecting showed the obvious apoptosis image of MCF-7 cell.Conclusion:PA-MSHA mighg through inducing MCF-7 apoptosis, played inhibition effect in the G0/G1 stage of cell cycle, thus inhibited MCF-7 cell proliferation. Part III Therapeutic Action and Mechanism Study on PA-MSHATreating Breast cancer in VivoObjective:1. To investigate the effects of PA-MSHA for the treatment of brease cancer in high spontaneous metastasis breast cancer of mouse model, and explore its mechanism of action.2. To study the effects of PA-MSHA on the immune function in patients with advanced stage breast cancer.Methods:Use of 4T1 cell suspension of cell strains building up high spontaneous metastasize breast cancer mice model, divided mice with tumor into four groups, such as negative control group(A group), CTX treatment group(B group), PA-MSHA group(C group) and therapeutic alliance group (D group). Compared tumor size and weight, life span, pulmonary metastasis rate among groups after treatments. And use of flow cytometry detection method compared peripheral blood lymphocyte subpopulation among different groups, applied with cytometric bead array(CBA) detecting TNF-a, IFN-y, IL-5, IL-4, IL-2 content of peripheral blood cytokines in mices. Pearsonian correlation analysis was used to investigate the relationship between peripheral blood lymphocyte subpopulation, TNF-a, IFN-y, IL-5, IL-4, IL-2 and survival time.Of 14 patients with advanced stage breast cancer were randomly divided into two groups as chemotherapy group and chemotherapy associated with PA-MSHA group. Both groups were treated with chemotherapy and the PA-MSHA group were injected with PA-MSHA by oneself, while the two groups were treated with every other day. The contents of T cells(CD3+, CD4+, CD8+) phenotype in blood was detected by flow cytometry (FCM), and computed the ratio of CD4+/CD8+.Results:Compared with the negative group, the treatment groups had smaller size of tumor and lighter weight of tumor, but there were no statistical significance between the CTX group and PA-MSHA group (P=0.160), both larger than therapeutic alliance group (P<0.05). Compared with Pulmonary metastasis rate, the negative group was much higher than the therapeutic alliance group (P<0.05), but there were no statistical significance among other groups (P>0.05). Contrast with life span, there was no significance between the PA-MSHA group and the CTX group (P=0.056), both higher than the negative group (P<0.05),both lower than the therapeutic alliance group(P<0.05). Compared with spleen index, the control group was smaller than the other four groups (P<0.05), the therapeutic alliance group was higher than the other four groups(P<0.05), but there were no statistical significance between the CTX group and the PA-MSHA group (P=0.352). Contrast with the negative group, PA-MSHA could elevate the ratio of CD4 cells of peripheral blood T lymphocytes, cut down the ratio of CD8 cells of peripheral blood T lymphocytes, and raise the ratio of CD4/CD8 (P<0.05). The level of IL2 was higher in PA-MSHA group (P<0.05), and the levels of TNF-a and IFN-y were higher in PA-MSHA& CTX group (P< 0.05). There was good positive correlation between CD4+and survival time (r=0.714, P＜0.01), but there was negative correlation between CD8+and survival time r=0.790, P＜0.01). There was good positive correlation between CD4+/CD8+ and survival time (r=0.781, P<0.01), and there was positive correlation between IL-2 and survival time (r=0.552,P<0.05). Other indexes didn’t exist correlation with survival time(P>0.05).In the clinical research, there was no statistical significance between the PA-MSHA group and the control group(P>0.05). The CD4+T cells and CD4+/CD8+ ratios of the experiment group were higher than the control group(P<0.05), but the CD8+T cells ratio of the experitment group was lower than the control group (P< 0.05).Conclusions:1.There are immune imbalance in the high spontaneous metastasis breast cancer mouse model that set up with 4T1 cell.2. The PA-MSHA could change the immune state in the tumor-bearing mices so as to treat breast cancer, and prolong the lifetime. And it was more effective together with CTX.3. PA-MSHA could enhance cellular immune function in advanced stage of breast cancer patients.更多还原