發(fā)布時(shí)間：2021-11-08 13:53

　　阿霉素常作為有效的抗腫瘤藥物使用。然而,由于阿霉素對(duì)卵巢具有不可逆轉(zhuǎn)的毒副作用,常常導(dǎo)致嚴(yán)重的婦科并發(fā)癥,因此在臨床應(yīng)用方面受到一定限制。卵泡作為卵巢的功能單位,一旦受損則無(wú)法再生。前人嘗試了多種技術(shù)來(lái)研究阿霉素對(duì)卵泡的毒性作用,但分子層面的毒性機(jī)制尚不清楚。卵泡的細(xì)胞功能主要依賴于某些重要的信號(hào)蛋白,在其不同生長(zhǎng)階段,這些信號(hào)蛋白的活性處于動(dòng)態(tài)變化中。在神經(jīng)和體液因素的干擾下,在體內(nèi)監(jiān)控其變化過程非常困難。因此,為深入探究阿霉素誘導(dǎo)的毒性的分子機(jī)制,需要解決兩個(gè)關(guān)鍵問題。第一,研制載體工具,體外模擬卵泡的生理微環(huán)境,并控制卵泡動(dòng)態(tài)生長(zhǎng);第二,構(gòu)建基因工程分子探針,監(jiān)測(cè)卵泡生長(zhǎng)過程中重要信號(hào)蛋白的活性動(dòng)態(tài)變化。因此,本文開發(fā)了一種新的微流控芯片進(jìn)行體外卵泡三維培養(yǎng),通過檢測(cè)卵泡直徑、激素分泌和凋亡相關(guān)基因的mRNA水平研究了不同信號(hào)分子在阿霉素誘導(dǎo)卵泡毒性變化中的作用。此外,利用熒光共振能量轉(zhuǎn)移（fluorescenceresonanceenergy transfer,FRET）技術(shù)揭示了阿霉素誘導(dǎo)卵泡細(xì)胞內(nèi)鈣離子濃度（[Ca2+]i）增加的鈣源,以及通過Src/Ca2+/PIM信號(hào)通... 

【文章來(lái)源】：大連理工大學(xué)遼寧省 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：137 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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
創(chuàng)新點(diǎn)摘要
References
Appendix A
Appendix B
Appendix C
Research Projects and Publications during Ph.D.Period
ACKNOWLEDGEMENT
About the Author


【參考文獻(xiàn)】：
期刊論文
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