發(fā)布時(shí)間：2018-08-20 10:53

【摘要】：第一部分無血清培養(yǎng)、化療及放療富集前列腺癌干細(xì)胞的研究 目的：腫瘤干細(xì)胞(Cancer stem cells, CSCs)的發(fā)現(xiàn)為腫瘤研究及治療帶來了新的希望,但由于其在腫瘤細(xì)胞中所占比例極低,較難獲得數(shù)量充足的CSCs以對其進(jìn)行深入的研究。因此,我們采用三系前列腺癌細(xì)胞通過無血清懸球培養(yǎng)法、化療藥物法及放射治療法三種方法分別富集前列腺癌干細(xì)胞,為前列腺癌干細(xì)胞領(lǐng)域的研究奠定基礎(chǔ)。 方法：分別用含血清的培養(yǎng)基及添加了人表皮生長因子(EGF)、人堿性成纖維細(xì)胞生長因子(bFGF)和人白血病抑制因子(LIF)的無血清培養(yǎng)基培養(yǎng)DU145、PC-3、LNCap前列腺癌細(xì)胞,流式細(xì)胞儀檢測兩種不同培養(yǎng)條件下三系細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞的含量。采用無血清懸球培養(yǎng)法、化療藥物法(多西紫杉醇0.1μM)及放射治療法(4Gy/次,2次／周,連續(xù)照射兩周)三種方法分別富集DU145細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞,并使用流式細(xì)胞儀檢測三種方法富集后DU145細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞的含量。 結(jié)果：在含血清的常規(guī)貼壁培養(yǎng)條件下,三系細(xì)胞中僅有DU145細(xì)胞能檢測到CD133+/CD44+細(xì)胞,且含量極低(0.1%±0.01%)。少量DU145細(xì)胞和PC-3細(xì)胞能夠在無血清培養(yǎng)基中存活并形成懸浮細(xì)胞球,且無血清懸球培養(yǎng)后兩種細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞比例顯著增高(DU145:10.3%; PC-3:3.0%)。使用化療藥物多西紫杉醇富集后DU145細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞比例增高至9.8%,放療富集后DU145細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞比例增高至3.5%。 結(jié)論：常規(guī)培養(yǎng)條件下,前列腺癌三系細(xì)胞僅DU145細(xì)胞系中可檢測到極微量的CD133+/CD44+前列腺癌干細(xì)胞。經(jīng)無血清懸球培養(yǎng)富集后,在DU145及PC-3細(xì)胞系中可檢測到CD133+/CD44+前列腺癌干細(xì)胞,而LNCap細(xì)胞中未能檢測到。通過化療及放療富集后,DU145細(xì)胞中CD133+/CD44+前列腺癌干細(xì)胞比例明顯上升。無血清培養(yǎng)、化療及放療三種方法均能有效富集CD133+/CD44+前列腺癌干細(xì)胞,為后期前列腺癌干細(xì)胞特性研究奠定基礎(chǔ)。 第二部分CD133+/CD44+前列腺癌干細(xì)胞特性研究 目的：腫瘤干細(xì)胞(Cancer stem cells, CSCs)具有無限增殖、自我更新及多向分化潛能,是“腫瘤的種子”,是驅(qū)動腫瘤形成和生長、保持腫瘤異質(zhì)性并促進(jìn)腫瘤無限增殖、復(fù)發(fā)和轉(zhuǎn)移的根源,也是化療和放療抵抗的重要原因之一。我們將富集、分選出的CD133+/CD44+前列腺癌干細(xì)胞通過體內(nèi)、體外實(shí)驗(yàn)進(jìn)一步證實(shí)其CSCs特性。 方法：DU145細(xì)胞經(jīng)無血清培養(yǎng)富集后,利用流式細(xì)胞儀分選出CD133+/CD44+前列腺癌干細(xì)胞。平板克隆形成實(shí)驗(yàn)及Transwell細(xì)胞侵襲實(shí)驗(yàn)比較CD133+/CD44+DU145細(xì)胞和未分選親代DU145細(xì)胞的增殖能力及侵襲性的差異。20只雄性BALB/c裸鼠,隨機(jī)分為實(shí)驗(yàn)組(10只)及對照組(10只),分別皮下注射CD133+/CD44+DU145細(xì)胞(1×104)及未分選親代DU145細(xì)胞(1×106),比較二者體內(nèi)致瘤能力的差異。 結(jié)果：研究結(jié)果顯示,在平板克隆形成實(shí)驗(yàn)中CD133+/CD44+DU145細(xì)胞的克隆形成率(colony-formation efficiency, CFE)為68.5±4.7%,而未分選親代DU145細(xì)胞的CFE為19.7±3.4%,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。在Transwell細(xì)胞侵襲實(shí)驗(yàn)中,顯微鏡下計(jì)數(shù)穿透小室膜的CD133+/CD44+DU145細(xì)胞數(shù)與未分選親代DU145細(xì)胞數(shù)分別為416±47與109±24,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。在裸鼠移植瘤實(shí)驗(yàn)中,實(shí)驗(yàn)組中10只裸鼠全部荷瘤成功,而對照組中僅有5只裸鼠荷瘤成功,同時(shí)實(shí)驗(yàn)組的出瘤時(shí)間早于對照組,且出瘤后生長速度顯著快于對照組(P0.001)。 結(jié)論：體外和體內(nèi)實(shí)驗(yàn)證實(shí)CD133+/CD44+DU145細(xì)胞與未分選親代DU145細(xì)胞相比具有更高的增殖、侵襲能力及更強(qiáng)的致瘤能力,證實(shí)其具有干細(xì)胞特性,為前列腺癌干細(xì)胞。 第三部分Notch-1在前列腺癌干細(xì)胞化療耐藥中的作用研究 目的：目前針對腫瘤干細(xì)胞(Cancer stem cells.CSCs)耐藥機(jī)制的研究,成為干細(xì)胞研究領(lǐng)域的熱點(diǎn)也是難點(diǎn)。腫瘤干細(xì)胞產(chǎn)生耐藥的機(jī)制相當(dāng)復(fù)雜,目前尚不完全清楚。Notch信號通路在進(jìn)化上高度保守,調(diào)控著細(xì)胞的增殖、分化、生存和凋亡,在細(xì)胞命運(yùn)決定中起關(guān)鍵作用。最近的研究顯示Notch信號通路與腫瘤耐藥有關(guān),NOtch通路能調(diào)控CSCs的形成以及上皮間質(zhì)化(Epithelial-mesenchymal transition, EMT),這都與腫瘤的化療耐藥密切相關(guān).以Notch信號通路為靶標(biāo)的基因療法及新藥開發(fā)將為腫瘤耐藥,特別是CSCs耐藥研究開辟新的領(lǐng)域。我們將進(jìn)一步探討Notch-1在CD133+/CD44+前列腺癌干細(xì)胞化療耐藥中的作用,以期為前列腺癌的治療提供新的靶點(diǎn)。 方法：MTT實(shí)驗(yàn)分別檢測CD133+/CD44+DU145細(xì)胞及未分選親代DU145細(xì)胞對化療藥物多西紫杉醇的敏感性。Real-time PCR分別檢測兩者中干細(xì)胞特有基因(Oct-4、Nanog)和Notch-1mRNA表達(dá)量的差異。6-8周的雄性BALB/c裸鼠16只,DU145細(xì)胞裸鼠移植瘤造模成功后,隨機(jī)分為兩組,實(shí)驗(yàn)組(8只),給予多西紫杉醇化療,劑量為10mg/kg,每周一次,連續(xù)3周；對照組(8只)：未行化療。3周后,裸鼠被處以安樂死,分別剝?nèi)φ战M裸鼠皮下移植瘤及實(shí)驗(yàn)組裸鼠皮下殘存腫瘤行Notch-1、Jagged-1的檢測及流式細(xì)胞檢測CD133+/CD44+前列腺癌干細(xì)胞的含量。 結(jié)果：研究結(jié)果顯示,在MTT實(shí)驗(yàn)中給予不同濃度化療藥物多西紫杉醇48h后,CD133+/CD44+DU145細(xì)胞存活顯著多于未經(jīng)分選的親代DU145細(xì)胞(P0.05),與第一部分實(shí)驗(yàn)中化療富集CD133+/CD44+DU145前列腺癌干細(xì)胞結(jié)果一致。在相同多西紫杉醇濃度下,其對CD133+/CD44+DU145細(xì)胞的抑制率顯著低于親代DU145細(xì)胞,多西紫杉醇對CD133+/CD44+DU145細(xì)胞及親代DU145細(xì)胞的IC50分別為0.075μM和0.665μM,耐藥指數(shù)為8.87。Real-time PCR實(shí)驗(yàn)結(jié)果顯示CD133+/CD44+DU145細(xì)胞與未分選親代DU145細(xì)胞相比,高表達(dá)干細(xì)胞特定基因：Nanog、Oct-4,并且Notch-1表達(dá)水平顯著增高(P0.01)。裸鼠移植瘤實(shí)驗(yàn)中,實(shí)驗(yàn)組與對照組相比,Notch-1、Jagged-1表達(dá)明顯增高；流式細(xì)胞檢測實(shí)驗(yàn)組殘存腫瘤中CD133+/CD44+前列腺癌干細(xì)胞比例顯著高于對照組(實(shí)驗(yàn)組：2.6%；對照組：0.1%)。 結(jié)論：CD133+/CD44+DU145前列腺癌干細(xì)胞對多西紫杉醇具有化療耐藥性,且高表達(dá)Notch-1�；熀髿埓婺[瘤高表達(dá)Notch-1、Jagged-1,且CD133+/CD44+前列腺癌干細(xì)胞比例顯著升高,提示CD133+/CD44+前列腺癌干細(xì)胞對化療耐藥,Notch-1可能在前列腺癌干細(xì)胞化療耐藥中起一定作用。
[Abstract]:The first part is a study on the enrichment of prostate cancer stem cells by serum-free culture, chemotherapy and radiotherapy.
Objective: The discovery of cancer stem cells (CSCs) has brought new hope for tumor research and treatment, but because of its very low proportion in tumor cells, it is difficult to obtain enough CSCs for further study. Prostate cancer stem cells can be enriched by three methods: biological method and radiotherapy, which lay a foundation for the research of prostate cancer stem cells.
Methods: Du145, PC-3, LNCap prostate cancer cells were cultured in serum-containing medium and serum-free medium supplemented with human epidermal growth factor (EGF), human basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF). CD133 +/CD44 + prostate cancer cells were detected by flow cytometry under two different culture conditions. The concentration of CD133 + / CD44 + prostate cancer stem cells in DU145 cells was enriched by serum-free suspension culture, chemotherapy (docetaxel 0.1 mu M) and radiotherapy (4 G Y / time, 2 times / week, continuous irradiation for 2 weeks), respectively. CD133 + / CD44 + prostate cancer stem cells in DU145 cells were detected by flow cytometry. The content of prostate cancer stem cells.
Results: Only DU145 cells could detect CD133 + / CD44 + cells in the three cell lines under the normal adherent culture condition with serum, and the content was very low (0.1% + 0.01%). The proportion of CD133 + / CD44 + prostate cancer stem cells in DU145 cells was increased to 9.8% after enrichment with chemotherapy drug docetaxel, and the proportion of CD133 + / CD44 + prostate cancer stem cells in DU145 cells was increased to 3.5% after enrichment with radiotherapy.
Conclusion: CD133 + / CD44 + prostate cancer stem cells can be detected only in DU145 cell lines under conventional culture conditions. CD133 + / CD44 + prostate cancer stem cells can be detected in DU145 and PC-3 cell lines after enrichment by serum-free suspension culture, but not in LNCap cell lines. The proportion of CD133 +/CD44 + prostate cancer stem cells in DU145 cells increased significantly after collection. Serum-free culture, chemotherapy and radiotherapy could effectively enrich CD133 +/CD44 + prostate cancer stem cells, which laid a foundation for the study of the characteristics of prostate cancer stem cells in the later stage.
The second part of CD133+/CD44+ prostate cancer stem cell characteristics
Objective: Cancer stem cells (CSCs) have the potential of infinite proliferation, self-renewal and multidirectional differentiation. They are the seeds of tumor, which drive tumor formation and growth, maintain tumor heterogeneity and promote tumor infinite proliferation, recurrence and metastasis. They are also one of the important reasons for chemotherapy and radiotherapy resistance. The CD133+/CD44+ prostate cancer stem cells were further confirmed by CSCs in vivo and in vitro.
Methods: CD133 + / CD44 + prostate cancer stem cells were separated by flow cytometry after enrichment of DU145 cells in serum-free medium. The proliferation and invasiveness of CD133 + / CD44 + DU145 cells and their unselected parental DU145 cells were compared by plate cloning assay and Transwell cell invasion assay. Twenty male BALB / C nude mice were randomly divided into two groups. The experimental group (10 rats) and the control group (10 rats) were subcutaneously injected with CD133+/CD44+DU145 cells (1 X 104) and the unselected parental DU145 cells (1 X 106), respectively. The differences of tumorigenicity between the two groups were compared.
Results: The results showed that the colony-formation efficiency (CFE) of CD133+/CD44+DU145 cells was 68.5 (+ 4.7%) and that of unselected parent DU145 cells was 19.7 (+ 3.4%). There was a significant difference in the number of CD133+/CD44+DU145 cells penetrating the ventricular membranes in the Transwell cell invasion test (P 0.001). The number of CD133+/CD44+DU145 cells and the number of unselected parental DU145 cells were 416+47 and 109+24, respectively. The difference was statistically significant (P 0.001). In the nude mice transplantation experiment, all the 10 nude mice in the experimental group were successful in tumor-bearing, while only 5 nude mice in the control group were successful in tumor-bearing. The degree was significantly faster than that of the control group (P0.001).
CONCLUSION: CD133+/CD44+DU145 cells have higher proliferation, invasion and tumorigenicity than the unselected parental DU145 cells in vitro and in vivo. It is proved that CD133+/CD44+DU145 cells have the characteristics of stem cells and are prostate cancer stem cells.
The third part is the role of Notch-1 in chemoresistance of prostate cancer stem cells.
OBJECTIVE: At present, the mechanism of drug resistance of cancer stem cells (CSCs) has become a hot and difficult point in the field of stem cell research. Recent studies have shown that Notch signaling pathway is associated with tumor resistance, and NOtch signaling pathway can regulate the formation of CSCs and epithelial-mesenchymal transition (EMT), which are closely related to chemotherapeutic drug resistance. We will explore the role of Notch-1 in the chemoresistance of CD133+/CD44+ prostate cancer stem cells in order to provide a new target for the treatment of prostate cancer.
METHODS: MTT assay was used to detect the sensitivity of CD133 +/CD44 + DU145 cells to docetaxel. Real-time PCR was used to detect the differences of stem cell-specific gene (Oct-4, Nanog) and Notch-1 mRNA expression between CD133 +/CD44 + DU145 cells and non-selected parental DU145 cells. After 3 weeks of chemotherapy, the nude mice were euthanized. The nude mice in the control group and the nude mice in the experimental group underwent Notch-1, Jagged-1 detection and flow cytometry respectively. The content of CD133+/CD44+ prostate cancer stem cells was measured.
Results: The results showed that CD133 + / CD44 + DU145 cells survived significantly more than their unselected parental DU145 cells (P 0.05) 48 hours after treatment with different concentrations of docetaxel in MTT assay, consistent with the results of CD133 + / CD44 + DU145 prostate cancer stem cells enriched by chemotherapy in the first experiment. The inhibitory rate of docetaxel on CD133+/CD44+DU145 cells was significantly lower than that of parental DU145 cells. The IC50 of docetaxel on CD133+/CD44+DU145 cells and parental DU145 cells were 0.075 and 0.665 mu M, respectively. The resistance index was 8.87. Real-time PCR results showed that CD133+/CD44+DU145 cells expressed stem cell-specific characteristics higher than that of parental DU145 cells. The expression of Notch-1 and Jagged-1 was significantly higher in nude mice than in the control group. The proportion of CD133+/CD44+ prostate cancer stem cells in the experimental group was significantly higher than that in the control group (experimental group: 2.6%; control group: 0.1%).
Conclusion: CD133+/CD44+DU145 prostate cancer stem cells are resistant to docetaxel and highly express Notch-1. After chemotherapy, the remaining tumors overexpress Notch-1 and Jagged-1, and the proportion of CD133+/CD44+ prostate cancer stem cells is significantly increased, suggesting that CD133+/CD44+ prostate cancer stem cells are resistant to chemotherapy. Notch-1 may be dry and thin in prostate cancer. Cellular chemotherapy plays a role in drug resistance.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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