基于三维微流控和FRET技术的阿霉素诱导卵泡毒性体外机理研究

发布时间：2021-11-08 13:53

　　阿霉素常作为有效的抗肿瘤药物使用。然而,由于阿霉素对卵巢具有不可逆转的毒副作用,常常导致严重的妇科并发症,因此在临床应用方面受到一定限制。卵泡作为卵巢的功能单位,一旦受损则无法再生。前人尝试了多种技术来研究阿霉素对卵泡的毒性作用,但分子层面的毒性机制尚不清楚。卵泡的细胞功能主要依赖于某些重要的信号蛋白,在其不同生长阶段,这些信号蛋白的活性处于动态变化中。在神经和体液因素的干扰下,在体内监控其变化过程非常困难。因此,为深入探究阿霉素诱导的毒性的分子机制,需要解决两个关键问题。第一,研制载体工具,体外模拟卵泡的生理微环境,并控制卵泡动态生长;第二,构建基因工程分子探针,监测卵泡生长过程中重要信号蛋白的活性动态变化。因此,本文开发了一种新的微流控芯片进行体外卵泡三维培养,通过检测卵泡直径、激素分泌和凋亡相关基因的mRNA水平研究了不同信号分子在阿霉素诱导卵泡毒性变化中的作用。此外,利用荧光共振能量转移（fluorescenceresonanceenergy transfer,FRET）技术揭示了阿霉素诱导卵泡细胞内钙离子浓度（[Ca2+]i）增加的钙源,以及通过Src/Ca2+/PIM信号通... 

【文章来源】：大连理工大学辽宁省 211工程院校 985工程院校 教育部直属院校

【文章页数】：137 页

【学位级别】：博士

【文章目录】：
Abstract
摘要
1 Introduction
    1.1 Research Background and Significance
    1.2 Progress in Related Research
        1.2.1 DOX Mechanism
        1.2.2 Challenges Accompanying the DOX Therapy
        1.2.3 DOX Induces Ovarian Toxicity
        1.2.4 Microfluidics and Organ Simulation
        1.2.5 FRET-based Biosensors
        1.2.6 Src Kinases
        1.2.7 Calcium Signaling
        1.2.8 Src Kinase as Upstream of Calcium Signaling
        1.2.9 PIM Kinases
    1.3 Purpose and Structure of the Thesis
        1.3.1 Purpose of the Thesis
        1.3.2 Structure of the Thesis
2 Microfluidics Chip for Culturing a Single Ovarian Follicle
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 List of Equipment and Reagents
        2.2.2 Isolation of OFs
        2.2.3 Encapsulation of the OFs
        2.2.4 Culture of OF in Dish
        2.2.5 Fabrication of the Microfluidic Model
        2.2.6 Assembly of Microfluidic Chip System
        2.2.7 An OF Culture on the Chip
        2.2.8 Diameter Measurement
        2.2.9 Hormone Tests
        2.2.10 Statistical Analysis
    2.3 Results
        2.3.1 The Diameter of the OF
        2.3.2 Hormone Tests for the OF
    2.4 Discussion
    2.5 Conclusion
3 Doxorubicin-induced Toxicity to 3D-Cultured OFs on a Microfluidic Chip
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 List of Equipment and Reagents
        3.2.2 OFs Isolation
        3.2.3 An OF Cultured on the Microfluidic Device
        3.2.4 Diameter Measurements
        3.2.5 Hormone Tests
        3.2.6 RNA Extraction and RT-qPCR
        3.2.7 TUNEL Assay
        3.2.8 Statistical Analysis
    3.3 Results
        3.3.1 DOX Reduces the OFs Diameter and E2 Secretion
        3.3.2 Src Inhibitor (PP1)Increases the DOX Toxicity to OFs
        3.3.3 ER-Ca~(2+) Inhibitor (2APB) Decreases DOX Toxicity to OFs
        3.3.4 PIM kinases Inhibitor (AZD1208) Enhances the DOX Toxicity to OFs
    3.4 Discussion
    3.5 Conclusion
4 Doxorubicin Induces ER-Ca~(2+) Release in OFs
    4.1 Introduction
    4.2 Materials and Methods
        4.2.1 List of Equipment and Reagents
        4.2.2 Isolation and Culturing of OFs
        4.2.3 Transfection of FRET Biosensors
        4.2.4 Microscopy and Image Analysis
        4.2.5 Statistical Analysis
    4.3 Results
        4.3.1 DOX Application Increases the [Ca~(2+)]i Level in the OF
        4.3.2 DOX Affects Early and Late Phases of [Ca~(2+)]i Independent of ExtracellularCalcium
        4.3.3 [Ca~(2+)]i Increase Depends upon ER-Ca~(2+) Release on DOX Application
        4.3.4 DOX can Increase Src Kinase
        4.3.5 DOX Causes [Ca~(2+)]i Increase via Src Kinase
    4.4 Discussion
    4.5 Conclusion
5 Doxorubicin Enhances the PIM Kinase Activity
    5.1 Introduction
    5.2 Material and Methods
        5.2.1 List of Equipment and Reagents
        5.2.2 Design and Establishment of PIM FRET Biosensor
        5.2.3 Design of Primers
        5.2.4 PCR
        5.2.5 Agarose Gel Preparation and Electrophoresis
        5.2.6 Gel Purification
        5.2.7 Double Enzyme System
        5.2.8 DNA Ligation
        5.2.9 Plasmid Transformation
        5.2.10 Plasmid Amplification
        5.2.11 Plasmid Extraction
        5.2.12 Detection of the Concentration
        5.2.13 Culturing of HeLa Cells
        5.2.14 Cell Transfection
        5.2.15 Isolation,Culture, and Transfection of OFs
        5.2.16 Microscopy and Image Analysis
        5.2.17 Statistical Analysis
    5.3 Results
        5.3.1 The Preparation of FRET-based Biosensors
        5.3.2 FRET-based Biosensor (EPHY)Reflects PIM1 Kinase Activity
        5.3.3 DOX Enhances PIM Kinase Activity
        5.3.4 DOX Enhances PIM Kinase Activity via Src
        5.3.5 DOX Enhances PIM Kinase Activity via ER-Ca~(2+) Release
    5.4 Discussion
    5.5 Conclusion
6 Conclusion and Future Horizons
    6.1 Conclusion
    6.2 Future Horizons
7 Abstract of Innovation Points
创新点摘要
References
Appendix A
Appendix B
Appendix C
Research Projects and Publications during Ph.D.Period
ACKNOWLEDGEMENT
About the Author


【参考文献】：
期刊论文
[1]Di-2-Ethylhexyl Phthalate Induces Ovarian Toxicity and Alters Protein Expression of Hormone-regulated Receptors in Rats[J]. QIN Xiao Yun,MA Quan,XU Xin Yun,TAN Qin,ZHENG Kai,WANG Bing Yu.  Biomedical and Environmental Sciences. 2019(07)
[2]The PI3K/Akt/m TOR pathway in ovarian cancer: therapeutic opportunities and challenges[J]. Bianca Cheaib,Aurélie Auguste,Alexandra Leary.  Chinese Journal of Cancer. 2015(01)
[3]SGI-1776钝化Pim-1对卵巢癌细胞增殖和侵袭的抑制作用及其机制（英文）[J]. 谢晶,白军.  中南大学学报(医学版). 2014(07)



本文编号：3483829