Development of DC--Ctl and Study the Therapeutic Effects of Different Routes in Ov Arian Cancer Mice

摘要

[背景和目的]
　　卵巢癌为女性生殖系统三大恶性肿瘤之一，死亡率高居妇科肿瘤之首[1].卵巢癌的发展非常隐匿，超过70％的患者在发现时已属晚期，有全身或腹腔等多处转移，其五年生存率低于40％[1]，且预后不良。现在卵巢癌主要的临床治疗方案主要是手术和化疗，但具有较高的复发率和耐药率。寻找卵巢癌新的治疗方法已迫在眉睫。免疫治疗作为癌症治疗的热点，已经用于治疗各种恶性肿瘤的临床试验。大量的研究表明CTL对卵巢癌细胞的细胞毒作用，但研究没有探索注射途径对治疗效果的影响。
　　本文将重点讨论树突状细胞(DC)免疫治疗的发展，调查DC-CTL治疗对卵巢癌小鼠和不同途径的治疗效果以及不同输液方式的分析。
　　[方法]
　　从正常人外周血制备DC-CTL细胞，负载抗原的DC组（A1组）和无抗原负载组（A2组）。通过酶联免疫吸附试验检测A1和A2对卵巢癌细胞的杀伤作用。为了在清洁的工作台中获得60只6周龄的雌性SPF小鼠，将SKOV3细胞皮下接种到裸鼠中以建立背部异种移植模型。模型建立约15天。选择移植瘤模型建立成功的小鼠48只，随机分为四组，分别为皮下注射组及其对照组，尾静脉注射组及其对照组。两组实验组第21天分别采用皮下注射和尾静脉注射法注入5*103的DC-CTL细胞，对照组注射相同剂量的生理盐水作为安慰剂。第35天处死小鼠，分离瘤组织称重并统计数据。
　　[结果]
　　抗原负载的DC-CTL细胞对卵巢癌细胞具有更好的杀伤作用。SKOV3细胞可以通过注射在小鼠背部和颈部皮肤下使谣言约15天。实验组肿瘤重量显着低于对照组(P＜0.05)，尾静脉注射组的肿瘤重量显着低于小鼠皮下注射组(P＜0.05)。
　　[结论]
　　DC-CTL细胞能有效的抑制小鼠卵巢癌的发展。将DC-CTL细胞注射到患有卵巢癌的小鼠的尾静脉中可以比小鼠皮下注射具有显着更好的治疗效果。本结果为DC-CTL细胞在卵巢癌治疗选择输注中的临床应用提供了一定的依据。

目录

Abstract

摘要

APPENDIX

1．GENERAL INTRoDUCTION

1．1 Ovarian Cancer Introduction

1．1．2 Types of ovarian cancer

1．1．3 RISK FACTORS OF OVAIUAN CANCER

1．1．4 PATHOGENESIS

1．1．5 SYMPTOMS

1．1．6 FIGO CLASSIFICATION

1．1．7 EARLY DETECTION

1．2 DISCOVERY oF OVARIAN CANCER BIo MARKERS VIA PROTEOMICS

1．2．2 THE IDEAL TUMOR MARKER

1．2．3 MECHANISM OF BIOMARKER ELEVATION

2．1 MANAGEMENT OF OVARIAN CANCER

2．1．1 SURGERY FOR OVARIAN CANCER

2．1．4 SIDE EFFECTS OF CHEMOTHERAPY

2．1．6 TARGETED THERAPY OF OVARIAN CANCER

2．1．9 IMMUNOTHERAPY FOR OVARIAN CANCER

3．1 IMMUNE THERAPY VIA DENDRITIC CELLS

3．1．2 DENDRITIC CELLS

3．1．3 DENDRITIC CELLS SOURCE

3．1．4 DENDRITIC CELLS MATURATION

3．1．5 Cancer immunoediting：the natural process

3．1．6 Dendritic cell immunotherapy as an answer to escape

3．1．7 FUNCTI0NS oF DENDRITIC CELLS

3．1．8 HOW D0 THEY KILL CANCER CELLS

3．1．9 CLINICAL USE OF DENDRITIC CELLS FOR LMMUNOTHERAPY

3．1．10 FUTURE DIRECTION OF DC VACCINES

4．1 COMBINATION OF DENRITIC CELLS-CYTOTOXIC TLYMPHOCYTES DC-CTL

4．1．1 ANTIGEN LOADED DENDRITIC CELLS

4．1．2 GENERATION OF DENDRITIC CELLS

4．2 ISOLATION AND CRYOPRESERVATION of DCs FROM PBMCs：(day 0)

4．2．1 INDUCTION OF IL-4 AND rhGM-CSF(DAY 0)

4．2．2 MEDIUM CHANGE ANfD FEEDING OF DC BY rhGM—CSF,IL-4(DAY 3)

4．2．3 MATURATION AND ANTIGEN LOADING OF DENDRITIC CELLS(DAY 6)

4．2．5 DIVIDING MATURE DC INTO GROUPS

4．2．6 FLOW CYTOMETRY

4．3．1 Background Information on SK-OV-3 cell line

4．3．2 STORAGE

4．3．3 REAGENTS

4．3．4 ESTABLISHMENT OF SKOV3 FROM FROZEN CELLS BYTHAWING AND PROPOGATING

4．3．5 CRYOPRESERVATION OF CELLS

4．3．7 ISOLATION AND PUIUFICATION OF CD34+CELLS

4．3．8 DC induction and sensitization

4．3．9 Morphological observation

4．3．10 Cell phenotype identification

4．3．11 Allogeneic Mixed Lymphocyte Reaction

4．4 MTT assay

4．4．1 PRINCIPLES oF ASSAY

4．4．2 MATERIALS

4．4．3 METHODS

4．4．4 RESULTS

4．5 BENEFITS OF DC-CTL VACCINE

5．0 ANIMAL EXPERIMENT

5．1．2 ESTABLISHMENT OF SUBCUTANEOUS XENOGRAFTMODELIN MICE

5．2 ANIMAL VACCINATIoN oF DC-CTL

5．2．1 MATERIALS FOR TAIL VEIN INJECTION

5．3 PRINCIPLES OF DISSECTION

5．4 STATISTICAL ANALYSIS

5．4．1 RESULTS

5．5 CONCLUSION

6．1 ROUTES OF INJECTING DC VACCINES OVARIANCANCER

6．2 CLlNICAL TRIALS OF DC VACCINES IN OVARIANCANCER

7．0 DISCUSSION

REVIEW

References

ACKNOWLEDGEMENTS

Major academic papers and participating activities in tlle Period of Studying

声明