【作者】 曹菲；

【导师】 田玉科；

【作者基本信息】 华中科技大学 ， 麻醉学， 2008， 博士

【摘要】 研究背景恶性肿瘤是危及人类生命的首要原因,临床上较为常见的恶性肿瘤,如乳腺癌、前列腺癌及肺癌等,易发生骨转移,加上原发的骨肉瘤,均会引起病理性的骨痛(简称骨癌痛),极大的干扰了患者的日常生活,并可导致患者死亡率的增高、体能状态的下降及焦虑或抑郁的发生,因此如何改善患者的生活质量成为当前亟待解决的问题。由于对骨癌痛相关机制认识的不足,目前的治疗方案难以达到令人满意的疗效,且常伴有严重的副作用,延误了疼痛的缓解,某种程度上甚至加速了癌症的恶化,因此极有必要寻求一种基于机制的、新的治疗方案。脊髓上高级中枢以极为精细的方式下行调控脊髓水平痛觉信息的传递和处理,延髓头端腹内侧区(rostral ventromedial medulla,RVM)作为该调控系统最主要的下行通路,直接影响了大脑和脊髓间痛觉信息的“中继”,大量行为学和电生理学的研究提示RVM区内表达μ阿片受体的下行易化神经元可能直接参与了病理性疼痛的形成,对脊髓水平“中枢敏化”的维持发挥了极为关键的作用,因此这些易化神经元可能成为治疗慢性顽固性疼痛的新靶点。基于此,通过μ阿片受体介导选择性阻断RVM区下行易化作用可能成为治疗骨癌痛的一种有效措施。本实验首先构建大鼠骨癌痛模型,通过应用靶向毒素——皮啡肽-皂角素耦联体,靶向毁损大鼠RVM区内μ阿片受体阳性的下行易化神经元,探讨此治疗方案应用于骨癌痛的有效性和安全性。另外鉴于皮啡肽-皂角素耦联体起效慢、制作工艺复杂、价格昂贵、难以形成产业化等缺点,本实验模拟皂角素在靶细胞内诱导凋亡的过程,进一步构建大鼠源性自发活化的caspase-3重组促凋亡基因,以期为骨癌痛的靶向基因治疗奠定实验基础。研究方法与结果1. Mu阿片受体介导靶向毁损骨癌痛大鼠下行易化系统镇痛效应的研究方法:成年雌性Wistar大鼠96只,随机分为6组:正常对照组(6只,不接受任何处理)、肿瘤细胞接种组(18只,仅构建骨癌痛模型,RVM区不接受任何微注射)、PBS(Phosphate buffered saline,磷酸盐缓冲液)组(18只)、皮啡肽组(18只)、皂角素组(18只)和皮啡肽-皂角素组(18只),其中后四组大鼠RVM区依次接受PBS、皮啡肽、皂角素及皮啡肽-皂角素耦联体单次微注射处理。微注射后第28d,除正常对照组外所有大鼠右侧胫骨接种Walker 256乳腺癌细胞构建大鼠骨癌痛模型,肿瘤细胞接种后第3d开始观察各组大鼠痛觉行为学改变,包括机械性痛觉超敏、机械性痛觉过敏、冷痛觉超敏、热痛觉过敏及移动诱发痛评分至细胞接种后第20d。肿瘤细胞接种后第7d、14d及20d,影像学观察肿瘤细胞接种侧胫骨骨质破坏的程度,免疫组化法检测各组脊髓背角FOS阳性神经元的数目和星形胶质细胞的活化,并采用ELISA法检测脊髓实质中前炎症因子IL-1β和TNF-α的改变。结果:1)疼痛行为学检测:肿瘤细胞接种组、PBS组、皮啡肽组及皂角素组大鼠与正常对照组大鼠相比均有痛觉超敏(nociceptive hypersensitivity)的发生(P<0.05),而RVM区微注射皮啡肽-皂角素耦联体可在痛觉超敏发生后的4~7d内明显降低痛觉超敏的程度(P<0.05,与肿瘤细胞接种组相比)。2)影像学检查:所有接种肿瘤细胞的大鼠接种侧胫骨骨质呈现进行性的破坏,各种RVM区微注射处理对局部骨质的破坏无明显影响;3)脊髓Fos阳性神经元的数目:肿瘤细胞接种组、PBS组、皮啡肽组及皂角素组大鼠脊髓双侧Fos阳性的神经元数目较正常对照组显著增加(P<0.05),而细胞接种后第14d和第20d,皮啡肽-皂角素组大鼠脊髓双侧Fos阳性神经元数目与肿瘤细胞接种组相比明显降低(P<0.05),且与正常对照组相比差异无统计学意义(P>0.05)。4)活化星形胶质细胞的数目及前炎症因子IL-1β和TNF-α的表达:肿瘤细胞接种组、PBS组、皮啡肽组及皂角素组大鼠脊髓双侧活化的星形胶质细胞数目和前炎症因子IL-1β和TNF-α的表达均较正常对照组大鼠显著增加(P<0.05),而RVM区微注射皮啡肽-皂角素耦联体可明显降低活化的星形胶质细胞的数目并抑制前炎症因子IL-1β和TNF-α的产生(P<0.05,与肿瘤细胞接种组大鼠相比),并在细胞接种后第20d回落至正常对照组水平(P>0.05)。2. Mu阿片受体介导靶向毁损大鼠痛觉下行易化系统安全性评估方法:成年雌性Wistar大鼠102只,随机分为五组:正常对照组(6只,不做任何处理)、PBS组(24只)、皮啡肽组(24只)、皂角素组(24只)及皮啡肽-皂角素组(24只),其中后四组大鼠RVM区依次接受PBS、皮啡肽、皂角素及皮啡肽-皂角素耦联体单次微注射处理。从微注射后第4d开始至第28d,每4d对大鼠生命体征进行检测。微注射后第4d、7d、14d及28d,通过超声心动图检测大鼠心功能的反应,免疫组化法检测RVM区内神经元数目以及局部星形胶质细胞和小胶质细胞的活化, Real-time PCR及ELISA法检测局部前炎症因子IL-1β和TNF-α的表达。结果:1)生命体征: RVM区内微注射皮啡肽-皂角素耦联体对大鼠基本生理功能无显著影响(P>0.05,与正常对照组相比),包括体重、肛温、呼吸频率、心率及收缩压。2)超声心动图检测心功能:微注射后第7d、14d及28d,皮啡肽-皂角素耦联体组大鼠左心室射血分数(left ejection fraction, EF)、左心室短轴分数(left fractional shortening, FS)、左心室收缩末期内径(left ventricular internal dimension systole at end-diastole, LVIDS)和左心室舒张末期内径(left ventricular internal dimension diastole at end-diastole, LVIDD)与正常对照组相比差异无统计学意义(P>0.05)。3)神经元数目:皮啡肽-皂角素组大鼠RVM区NeuN标记的神经元总数与正常对照组相比差异无统计学意义(P>0.05);4)胶质细胞的活化及前炎症因子IL-1β和TNF-α的表达:与正常对照组相比,皮啡肽-皂角素组大鼠RVM区内小胶质细胞和星形胶质细胞无显著活化,前炎症因子IL-1β和TNF-α持续维持在基线水平(与正常对照组相比,P>0.05)。3.大鼠源性自发活化caspase-3重组基因的构建及其促凋亡效应的研究方法:模拟皂角素在细胞内诱导凋亡的过程,通过重组PCR技术获得大、小亚基顺序颠倒的大鼠源性自发活化的重组caspase-3基因,经酶切电泳及测序分析后,克隆入真核表达载体pcDNA3.1(+)或EGFP-C1,转染大鼠永生化神经前体细胞(Immortalized neural progenitor cells,INPC)和人胚肾293T细胞,分别通过荧光倒置显微镜观察细胞形态学改变、Annexin V-PI双标后流式细胞仪检测转染细胞的早期凋亡率、MTT法检测转染后细胞的生长抑制率及Western blot检测转染后细胞内活化caspase-3蛋白质的表达。结果:1)转染24h后荧光倒置显微镜显示转染重组caspase-3基因的细胞呈现明显的细胞碎裂、浓缩及细胞突起回缩等典型的凋亡形态学改变。2)转染重组caspase-3基因的细胞Annexin V-PI双染后流式细胞仪检测显示细胞早期凋亡率分别为: 16.01%±1.03%(INPC)和30.67%±1.53%(293T),与正常对照组、空白载体组或野生型caspase-3组细胞相比显著增高(P<0.05);3)MTT结果提示转染重组caspase-3基因的细胞生长抑制率分别为: 44.61%±0.15%(INPC)和48.35±0.16%(293T),与正常对照组、空白载体组或野生型caspase-3组细胞相比生长抑制率明显增加(P<0.05);4)Western blot结果提示转染重组caspase-3基因的细胞中活化caspase-3蛋白的相对表达量分别为:2.44±0.01 (INPC)和3.42±0.21(293T),较正常对照组、空白载体组或野生型caspase-3组细胞显著增加(P<0.05)。4.统计学处理采用SigmaStat 3.0统计软件进行处理。计量资料以均数±标准差(±s)表示,实验第一部分组间、组内比较采用双因素方差(Two way ANOVA)分析后post-hoc检验(SNK法),余实验部分中组间、组内比较采用单因素方差(One way ANOVA)分析后SNK法。P<0.05设定为差异有统计学意义。研究总结1. RVM区微注射皮啡肽-皂角素耦联体选择性毁损μ阿片受体表达阳性的下行易化神经元,可降低脊髓中枢敏化的程度,抑制脊髓内星形胶质细胞的活化和前炎症因子的产生,显著抑制骨癌痛大鼠痛觉超敏的程度。2. RVM区微注射皮啡肽-皂角素耦联体选择性毁损μ阿片受体表达阳性的下行易化神经元对实验大鼠生理功能无明显影响,超声心动图结果亦提示对大鼠心功能影响较小。耦联体注射后也未引起局部神经元的显著丢失和明显的神经炎症或神经免疫反应。3.通过模拟皂角素在靶细胞内诱导凋亡的过程,成功构建了大鼠源性自发活化的caspase-3重组基因,在无前凋亡信号的作用下可自发诱导细胞凋亡。研究意义本研究从慢性病理性疼痛脊髓上内源性调控系统入手,根据RVM区在该系统下行投射至脊髓过程中的“中继”作用,应用皮啡肽-皂角素耦联体选择性毁损了RVM区内μ阿片受体表达阳性的下行易化神经元,有效降低了骨癌痛的程度,同时研究结果也证实了靶向毁损效应应用的安全性。本研究是对骨癌痛基于机制治疗的一种新探索,为将该靶向毁损的治疗方案应用于临床提供了实验依据。鉴于皂角素自身的诸多缺点,本研究模拟皂角素诱导细胞凋亡的过程,构建了大鼠源性自发活化的caspase-3重组促凋亡基因,为骨癌痛的靶向基因治疗提供了理想的杀伤基因。更多还原

【Abstract】 BackgroundAs advances in cancer detection and therapy extend the life expectancy of cancer patients, there is an increasing focus on improving patients’quality of life. Bone cancer pain is the most common pain in patients with advanced cancer as most common tumors including breast, prostate, and lung cancer have a remarkable affinity to metastasize to bone. Once tumors metastasize to bone, they are usually major causes of morbidity or mortality, and consequently compromise patients’survival and quality of life. Currently, factors that drive cancer pain are poorly understood; however, in the just past decade several animal models of cancer induced bone pain (CIBP) have been developed. In terms of tumor growth, bone remodeling, and bone pain, these models seem to mirror several aspects of human bone caner pain, providing insight into mechanisms that drive bone cancer pain. The researchers realize by degree that bone cancer pain is a unique persistent pain state, including elements of both neuropathy and inflammation.Currently, the treatment of pain from bone metastases, involving the use of multiple complementary approaches, is frequently relatively ineffective. And the neurobiological basis for the treatment is largely empirical or based on scientific studies arising from some aspects of cancer pain. In clinic, the severity of this pain is variable from patient to patient, tumor to tumor, and time to time, even from site to site plausibly because their underling mechanisms are inconsistent. In addition, the metastases are generally not limited to a single site and analgesics that are most commonly used to treat bone caner pain are limited by significant adverse side effects. Therefore, it is a big challenge for us to explore some novel mechanism-based approaches to relieve bone cancer pain for most patients.Complex networks of pathways project from various supraspinal structures to modulate spinal processing of sensory input in a top-down fashion. The rostral ventromedial medulla (RVM) in the brainstem is one major final common output of this endogenous modulatory system and is involved in the relay of sensory information between the spinal cord and brain. The net output of descending neurons that exert inhibitory or facilitatory effects will determine whether neuronal activity in the spinal cord increased or decreased. Extensive behavioral and electrophysiological studies suggest that descending facilitation from the RVM is critical for maintenance of central sensitization in neuropathic pain. Thus, targeting these descending facilitatory neurons may be a novel and as yet clinically unexploited method for treating chronic intractable pain. However, little is known that whether this modality is also workable in CIBP. Thus, through selective ablating ablationμreceptor positive facilitatory neurons with dermorphin-saporin, this research investigated analgesic effects through blocking descending facilitation from RVM in the rat model of CIBP and evaluated the safety of this targeted lesion. Furthermore, considering complex making and high price of dermorphin-saporin, we continued to clone a rat constitutively active recombinant caspase-3 gene in order to lay the foundation for future constructing dermorphin and constitutive active recombinant caspase-3 gene conjugate for gene therapy of CIBP. Methods and Results1. Inhibition of cancer induced bone pain through selective ablation ofμ-opioid receptor mediated descending facilitation from the rostral ventromedial medulla Methods: A total of 96 adult female Wistar rats were randomly divided into six groups: 1) na?ve group (n=6, without any intervention); 2) carcinoma cell group (n=18, without any intra-RVM microinjection); 3) PBS group (n=18, received a single intra-RVM microinjection with PBS alone); 4) dermorphin group (n=18, received a single intra-RVM microinjection with dermorphin); 5)saporin group (n=18, received a single intra-RVM microinjection with saporin); 6) dermorphin-saporin group (n=18, received a single intra-RVM microinjection with dermorphin-saporin). On day 28 post-microinjection, all rats except na?ve rats were inoculated with Walker 256 carcinoma cells into the right tibiae. Commencing from day 3 to day 20 post-inoculation, mechanical allodynia, mechanical hyperalgesia, cold allodynia, thermal hyperalgesia and the score of ambulatory pain were recorded to investigate changes of nociceptive behaviors. The development of the bone tumor and structural destruction to the bone was monitored by radiological analysis. After repetitive non-noxious tactile stimulation, the total number of FOS positive neurons in the spinal cord horn was regarded as a marker indicative of central sensitization. The GFAP (Glial fibrillary acidicprotein) expression in the spinal cord was detected through immunohistochemistry. And changes of proinflammatory cytokins, such as IL-1βand TNF-αwere observed by ELISA.Results: 1) Nociceptive behaviors: Rats in carcinoma cells group, PBS group, dermorphin group or saporin group demonstrated nociceptive hypersensitivity compared with na?ve rats (P<0.05), while rats in dermorphin-saporin group showed decreased behaviors within 4-7 days after the onset of nociception in comparison with carcinoma cells group (P<0.05). 2) Radiological analysis: All of rats inoculated with carcinoma cells showed ongoing damage to cortical bone and the trabeculae in the ipsilateral tibial bone from day 7 post-inoculation, and by day 20 the damage threatened the integrity of the tibial bone. All intra-RVM treatments failed to influence the bone destruction during the observation. 3) FOS positive neurons: In parallel with behavioral tests, the number of FOS positive neurons in the bilateral spinal cord significantly enhanced in all groups treated with carcinoma cells compared with na?ve rats on day 7 post-inoculation (P<0.05), whereas dermorphin-saporin declined significantly the amount of FOS labeled neurons on day 14 and day 20 post-inoculation compared with non-microinjection or other intra-RVM treatment (P<0.05). 4) The amount of activated spinal astrocytes and the protein levels of proinflammatory cytokines: The amount of activated astrocytes and the protein levels of IL-1βor TNF-αin the bilateral spinal cord from carcinoma cell group, PBS group, dermorphin group and saporin group increased significantly compared with that from na?ve rats (P<0.05). In contrast, intra-RVM microinjection with dermorphin-saporin evidently decreased the amount of activated astrocytes and down-regulated the production of proinflammatory cytokines relative to non-microinjection (P<0.05), and reversed them to the baseline levels on day 20 post-inoculation (P>0.05).2. Safety evaluation of microinjection of the targeted neuropepetide-toxin conjugate, dermophine-saporin, into the rat rostral ventromedial medullaMethods :A total of 102 adult Wistar rats were randomly divided into five groups: 1) na?ve group(n=6, without any intervention); 2) PBS group (n=24, received a single intra-RVM microinjection with PBS alone); 3) dermorphin group (n=24, received a single intra-RVM microinjection with dermorphin); 4)saporin group (n=24, received a single intra-RVM microinjection with saporin); 5) dermorphin-saporin group (n=24, received a single intra-RVM microinjection with dermorphin-saporin). From day 4 to day 28 after microinjection, physiological parameters, such as body weight, rectal temperature, respiratory rate, heart rate and systolic blood pressure, were observed every 4 days. On day 4, 7, 14 and 28 post-microinjection, cardiovascular functions were recorded through echocardiogram. Meanwhile, both the number of neurons and the activity of glia were determined via immunohistochemistry, and the mRNA and protein levels of proinflammatory cytokines, such as IL-1βand TNF-α, were analyzed by real-time PCR and ELISA, respectively.Results: 1) Intra-RVM with dermorphin-saporin had no significant influence on the physiological parameters (P>0.05), including body weight, rectal temperature, respiratory rate, heart rate and systolic blood pressure; 2) With echocardiogram there was no significant difference in EF (left ejection fraction), FS (left fractional shortening), LVIDS (left ventricular internal dimension systole at end-diastole), and LVIDD (left ventricular internal dimension diastole at end-diastole) between dermorphin-saporin group and na?ve group on day 7, day 14 and day 28 post-microinjection (P>0.05). However, EF and FS significantly increased in all of groups receiving intra-RVM microinjection compared with na?ve group on day 4 after microinjection (P<0.05); 3) The total number of NeuN labeled neurons in the RVM failed to exert significant differences between dermorphin-saporin group and niave group (P>0.05). 4) In the local micro-environment the resident microglia or astrocytes kept quiescent, and the mRNA or protein levels of IL-1βor TNF-αmaintained at the baseline during the observation (P>0.05).3. Construction of rat constitutively active recombinant caspase-3 and evaluation of its apoptotic effects in vitroMethods: The rat constitutively active recombinant caspase-3 gene was constructed through reversing two subunits of rat wild type caspase-3 gene by overlap-PCR, and cloned into expression vector EGFP-C1 or pcDNA3.1(+).Then, the recombinant caspase-3 was transfected into human 293T cells or rat immortalized neural progenitor cells. The target gene expression and the morphology of transfected cells were observed by fluorescence microscope, and apoptotic effects of the recombinant gene were analyzed by Annexin V-Fluorescein∕PI flow cytometry, MTT assay and Western blot. Results: The recombinant caspase-3 can be expressed efficiently in both 293T cells and INPC. The transfected cells with recombinant caspse-3 gene presented typical characteristics of apoptosis, including cellular fragmentation and cytoplasm concentration with fluorescence microscope. Results from Annexin V-Fluorescein∕PI flow cytometry suggested the gene resulted significantly in early apoptosis in both cells compared with controls (P<0.05). In MTT assay both cells displayed evident growth inhibition after transfection with the recombinant caspase-3 relative to controls (P<0.05). And the protein levels of active caspase-3 increased significantly in cells transfected with the recombinant capase-3 compared to cells with other treatment (P<0.05).4. Statistical analysisAll of analyses were performed by SigmaStat 3.0 software package. All data were expressed as the mean±standard deviation (SD). Significant differences within the first part were detected by two-factor ANOVA followed by post-hoc test (SNK), and one-way ANOVA was utilized to detect significant differcence in other parts followed by SNK. Significance was set at P<0.05.Conclusion1. In our rat model of CIBP, selective ablation ofμopioid receptor expressing descending facilitatory neurons in the RVM with dermorphin-saporin reduced a variety of nociceptive behaviors through inhibiting spinal sensitization, decreasing the amount of activated astrocytes and down-regulating the proinflammatory cytokines (IL-1βor TNF-α) production.2. Microinjection of dermorphin-saporin into the RVM had no significant impacts on the physiological functions and heart functions. And neither significant loss of NeuN labelsled neurons nor evident responses of the neuroinflammtory or neuroimmune were absent during the observation.3. The constitutively active rat recombinant caspase-3 induced apoptosis without pro-apoptotic signals stimulation, and imitated cell killing effects of saporin in the targeted cells.SignificanceAccumulating evidence suggests that inappropriate tonic-descending facilitation arising from the RVM in the brainstem has been established to underlie some chronic pathologic pain. Our research selectively ablatedμopioid receptor expressing neurons in the RVM, presumed to be a source of spinopetal facilitatory projection, by a single intra-RVM microinjection with dermorphin-saporin, and subsequently reversed the experimental CIBP effectively. On the other hand, results of this research established the targeted lesion is a safe and reliable approach to treat pathologic pain. Thus, this research is a novel exploration of mechanism-based therapy for CIBP, providing us some experimental evidence for future clinical application. Furthermore, considering the drawbacks of saporin, we mimicked cell killing effects of saporin in the targeted cells, and cloned the constitutively active recombinant caspase-3, laying the foundation for our future targeted gene therapies in CIBP.更多还原