【目的】

探索超声介导的微泡空化效应对前列腺癌化疗的增敏作用及对肿瘤免疫微环境的影响。

【方法】

1. 在联合紫杉醇和微泡的条件下，分别以不同频率(3 MHz、4 MHz、5 MHz)和机械指数(0.2、0.4、0.6)的超声辐照RM-1细胞，检测治疗后细胞活力，确定最适频率及机械指数。使用该频率和机械指数的超声+微泡联合紫杉醇处理RM-1细胞，检测处理后细胞活力和凋亡率，明确其对化疗的增敏效果。进一步利用钙黄绿素检测超声+微泡辐照后即刻细胞通透性变化，探索其增敏化疗的可能机制。

2. 利用RM-1细胞构建小鼠前列腺癌原位肿瘤模型，应用超声+微泡联合白蛋白结合型紫杉醇治疗前列腺癌小鼠，观察肿瘤生长及小鼠生存情况，并设置单纯化疗组、超声+微泡组和空白对照组进行对比，在动物层面验证超声介导的微泡空化效应增敏化疗效果。进一步提取治疗后各组小鼠前列腺癌中肿瘤浸润淋巴细胞，流式检测CD8⁺ T细胞表面PD-1和CTLA-4的表达情况、CD4⁺ T细胞内Foxp3表达和其表面CTLA-4的表达情况，以探索空化效应对肿瘤免疫微环境的影响。

【结果】

1. 频率为3 MHz时细胞活力(37.97 ± 3.51%)显著低于4 MHz (P<0.001)和5 MHz (P<0.001)；机械指数为0.6时细胞活力(29.05 ± 5.33%)显著低于机械指数为0.2 (P<0.001)和0.4时(P=0.001)。使用频率为3 MHz、机械指数为0.6的超声+微泡辅助紫杉醇化疗，RM-1细胞的活力显著低于单纯化疗(33.35 ± 8.23% vs 70.42 ± 8.72%, P<0.001)，凋亡率则显著增多(14.46 ± 0.22% vs 6.63 ± 0.93%, P<0.001)。经过超声+微泡辐照后，细胞通透性增加，钙黄绿素荧光信号阳性细胞率显著高于对照组(15.79 ± 0.93% vs 2.37 ± 0.72%, P<0.001)。

2. 成功构建小鼠前列腺癌原位肿瘤模型，联合治疗组的肿瘤体积明显小于单纯化疗组(166 ± 34 mm³ vs 249 ± 18 mm³, P=0.005)，该组小鼠的平均存活时间长于单纯化疗组(16.20 ± 1.99 d vs 14.24 ± 2.77 d, P=0.031)；超声+微泡组与对照组的肿瘤体积 (481 ± 96 mm³ vs 444 ± 145 mm³, P=0.687)和小鼠的存活时间(11.54 ± 3.30 d vs 12.25 ± 2.93 d, P=0.507)均无明显差异。肿瘤淋巴细胞的流式检测结果显示，超声+微泡组的CD8⁺ T细胞中CTLA-4⁺细胞比例(P=0.017)和PD-1⁺/ CTLA-4⁺细胞(P=0.032)比例低于Control组。而在PTX+US+MB组中，这两个比例均略高于PTX组，但是缺乏明显的统计学意义。

【结论】

超声介导的微泡空化效应能增敏前列腺癌的化疗效果，其机制可能为空化效应增加肿瘤细胞的通透性，从而提高局部化疗药浓度。单纯的空化效应可下调肿瘤微环境中CD8⁺ T细胞表面PD-1及CTLA-4的表达，但是在化疗的同时联合空化效应呈现出上调CD8⁺ T细胞表面PD-1及CTLA-4表达的趋势。

[Objective]

To explore the effect of ultrasound (US)-mediated microbubble (MB) cavitation on enhancing the efficacy of chemotherapy for treatment of prostate cancer and its effect on the tumor immune microenvironment.

[Methods]

1. RM-1 cells were treated with US at different frequencies (3 MHz, 4 MHz and 5 MHz) and mechanical index (MI) (0.2, 0.4 and 0.6) in combination with paclitaxel (PTX) and MB. Cell viabilities were measured after treatment to determine the optimal frequency and MI. Then RM-1 cell suspensions were then treated with US at the optimal frequency and MI in combination with PTX and MB. And cell viabilities and cellular apoptosis were detected to demonstrate the role of US+MB in enhancing the efficacy of chemotherapy. Membrane permeability was evaluated by calcein after treated with US+MB to explore the mechanism of increasing the efficacy of chemotherapy.

2. The orthotopic mouse model of RM-1 prostate cancer was developed and these mice were divided into four groups: a US+MB+PTX group, a PTX group, a US+MB group and a control group. In vivo, the chemotherapeutic drug PTX was albumin-bound paclitaxel. Tumor growth and survival were recorded and compared in different groups to verify the effect of US-mediated MB cavitation on enhancing the efficacy of chemotherapy of prostate cancer in vivo. Tumor infiltrating lymphocytes were extracted from prostate cancer in each group. The expression of PD-1 and CTLA-4 on CD8⁺ T cells and the expression of Foxp3 in CD4⁺ T cells and CTLA-4 on CD4⁺ T cells were detected by flow cytometry to explore the influence of cavitation on tumor immune microenvironment.

[Results]

1. The cell viability in 3 MHz group (37.97 ± 3.51%) was significantly lower than that in 4 MHz group (P<0.001) and 5 MHz group (P<0.001) and the cell viability was significantly lower when MI was 0.6 (29.05 ± 5.33%) than MI was 0.2 (P<0.001) and 0.4 (P=0.001). When RM-1 cells were treated with US (3 MHz and MI of 0.6) +MB+PTX, the cell viability was lower than cells treated with PTX alone (33.35 ± 8.23% vs 70.42 ± 8.72%, P<0.001) and the cellular apoptosis rate was significantly increased (14.46 ± 0.22% vs 6.63 ± 0.93%, P<0.001). After treated with US+MB, the fluorescence signal positive cell rate was higher than control group (15.79 ± 0.93% vs 2.37 ± 0.72%, P<0.001), which means that US-mediated MB cavitation can increase the membrane permeability.

2. The orthotopic mouse model was established successfully. In US+MB+PTX group, the tumor volume was statistically smaller than that in PTX group (166 ± 34 mm³ vs 249 ± 18 mm³, P=0.005), and the mean survival was longer than that in PTX group (16.20 ± 1.99 d vs 14.24 ± 2.77 d, P=0.031). Both tumor size (481 ± 96 mm³ vs 444 ± 145 mm³, P=0.687) and mean survival (11.54 ± 3.30 d vs 12.25 ± 2.93 d, P=0.507) were similar between US+MB group and control group. The proportion of CTLA4⁺ cells and PD-1⁺/CTLA4⁺ in CD8⁺ T cells were both lower in the US+MB group than that in the control group (P=0.017 and P=0.032). However, in the PTX+US+MB group, PD-1 and CTLA4 expression on CD8⁺ T cells were slightly higher than that in the PTX group, although the difference was not statistically significant.

[Conclusion]

US-mediated MB cavitation can enhance the efficacy of chemotherapy for treatment of prostate cancer by increasing membrane permeability of prostate cancer cells so that increasing the local concentration of chemotherapeutic agents. Cavitation alone can downregulate the expression of PD-1 and CTLA-4 on the surface of CD8+ T cells in the tumor microenvironment, however, the cavitation has a tendency to upregulate the expression of PD-1 and CTLA4 on the surface of CD8⁺ T cells in the presence of chemotherapy.

第一章 文献综述 1
1.1前言 1
1.2前列腺癌临床研究进展 1
1.2.1 前列腺癌术后复发和化疗疗效的预测 1
1.2.2 前列腺癌化疗临床进展 3
1.3 前列腺癌化疗的毒性反应 5
1.4 血-前列腺屏障与前列腺癌化疗 6
1.5 前列腺癌化疗增敏的研究进展 8
1.5.1 纳米技术 8
1.5.2 化学-光热协同治疗 9
1.5.3 磁驱动药物输送装置 11
1.5.4 超声空化增敏前列腺癌化疗 12
1.6 前列腺癌免疫研究进展 13
1.7 总结与展望 15
第二章 超声介导的微泡空化效应对RM-1细胞的化疗增敏研究 17
2.1 引言 17
2.2 实验器材和试剂材料 18
2.2.1 主要仪器设备及耗材 18
2.2.2 主要试剂和药品 20
2.2.3 细胞系 21
2.2.4 主要溶液及配制 21
2.3 实验方法 21
2.3.1 细胞培养 21
2.3.2 CCK-8法细胞活力检测 23
2.3.3 Annexin V/7-AAD 细胞凋亡检测 23
2.3.4 PTX抑制RM-1细胞活力的浓度梯度实验 24
2.3.5超声介导的微泡空化效应对RM-1细胞化疗增敏的最优频率探索 24
2.3.6超声介导的微泡空化效应对RM-1细胞化疗增敏的最优机械指数探索 25
2.3.7 超声介导的微泡空化效应增敏PTX抑制RM-1细胞活力实验 26
2.3.8 超声介导的微泡空化效应对PTX诱导的RM-1细胞凋亡的增敏研究 26
2.3.9 超声介导的微泡空化效应对RM-1细胞通透性的影响 27
2.3.10 超声介导的微泡空化效应对RM-1细胞活力的影响 28
2.3.11 超声介导的微泡空化效应对RM-1细胞凋亡的影响 28
2.4 统计学方法 28
2.5 结果 29
2.5.1 不同浓度PTX对RM-1细胞活力的影响 29
2.5.2超声介导的微泡空化效应对RM-1细胞化疗增敏的最适频率及机械指数 30
2.5.3 超声介导的微泡空化效应对RM-1细胞的化疗增敏效果 31
2.5.4 超声介导的微泡空化效应对RM-1细胞的通透性的影响 33
2.5.5 超声介导的微泡空化效应对RM-1细胞的活力和凋亡的影响 35
2.6 讨论 37
2.7 小结 40
第三章 改良小鼠前列腺癌原位移植瘤模型构建及空化效应增敏小鼠前列腺癌化疗和免疫调节初步探索 41
3.1 引言 41
3.2 实验器材和试剂材料 42
3.2.1 主要仪器设备及耗材 42
3.2.2 主要试剂和药品 44
3.2.3 实验动物和细胞 45
3.2.4 主要溶液及配制 45
3.3 实验方法 46
3.3.1 小鼠前列腺癌原位荷瘤模型的构建 46
3.3.2 小鼠前列腺癌原位模型的验证 47
3.3.3 组织病理检测 47
3.3.4 超声介导的微泡空化效应对小鼠前列腺癌化疗增敏实验 49
3.3.5 超声介导的微泡空化效应对小鼠前列腺癌的肿瘤免疫调节研究 50
3.4 统计学方法 51
3.5 结果 52
3.5.1 改良小鼠前列腺癌原位移植瘤模型的构建 52
3.5.2 超声介导的微泡空化效应对小鼠前列腺癌的化疗增敏效果 57
3.5.3 超声介导的微泡空化效应对小鼠前列腺癌的肿瘤免疫调节 60
3.6 讨论 62
3.7 小结 68
第四章 结论与展望 69
4.1 研究结论 69
4.2 创新点 69
4.3 不足之处 70
4.4 展望 70
参考文献 71
致谢 83
北京大学学位论文原创性声明和使用授权说明 85
学位论文答辩委员会名单 87
个人简历、在学期间发表的学术论文与研究成果 88

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