【作者】 胡兴斌；

【导师】 韩骅；

【作者基本信息】 第四军医大学 ， 医学发育生物学， 2009， 博士

【摘要】 肿瘤是人类的重大健康威胁之一,给人类带来沉重的经济和社会负担。实体肿瘤发病率和死亡率很高,彻底根治肿瘤的治疗方案十分有限,深入研究肿瘤的发生发展机理,是探索肿瘤治疗新策略的前提。肿瘤血管和抗肿瘤免疫应答是影响实体肿瘤生长的关键因素,肿瘤体积小于2 -3mm3时,瘤细胞可通过周边弥散的方式获得营养支持而缓慢生长;瘤体体积继续增大则需要大量血管新生来支持瘤细胞生长。同时,肿瘤抗原能刺激机体产生不同类型的抗肿瘤免疫应答,进而产生抗肿瘤的细胞免疫和体液免疫反应,直接决定肿瘤患者的预后。由于血管在肿瘤生长中的重要作用,以血管生成调控相关分子为靶位的治疗手段是近年来抗肿瘤新策略的重要方向。该策略治疗可有效治疗某些肿瘤,然而以此为基础的抗肿瘤药物仍存在耐药、无效等诸多不足,因此,探寻肿瘤血管新生的调控机制,是研发肿瘤抗血管治疗药物的必然前提。Notch信号是经典而保守的发育调控通路,该信号通路由表达在相邻细胞上的4种配体和5种受体、细胞内转录因子、下游分子和调节相关分子组成。细胞间Notch受体和配体相互结合以后,受不同的蛋白酶切割,使Notch胞内段发生细胞内转位并入核,进入细胞核的胞内段与转录因子RBP-J相互作用,使转录共抑制物变为转录共激活物,从而启动含有RBP-J识别位点的启动子转录,进而调节相关的下游基因表达,参与诸多重要的个体发育事件。Notch信号在成年个体和胚胎血管发育中具有不可替代的作用,在动静脉选择、Tip /stalk细胞命运决定、血管内皮细胞增殖、迁移及伪足形成和成年个体血管稳定性维持等方面都发挥了调控作用。因此Notch信号途径成员成为近来肿瘤血管干预的候选靶位之一。以Dll4为靶标去抑制Notch信号,可导致小鼠体内肿瘤生长速度减缓,但瘤体内微血管密度却明显高于对照,这些新生的肿瘤微血管灌流异常,可能影响了实体肿瘤生长;然而矛盾的是活化Notch信号,同样能够抑制某些肿瘤的生长。与此同时,Notch信号还参与免疫细胞发育调控。包括T细胞、B细胞、DC和髓系细胞以及它们的亚群,发育过程都受到了Notch信号的影响。抗肿瘤治疗时,鉴于抗肿瘤免疫对肿瘤生长的重要作用,有必要分析以血管抑制为目的的Notch信号干预带来的免疫改变。Notch信号通路存在多种配体及受体,而不同配体或受体间可能存在功能互补性。RBP-J是Notch信号的关键分子,因此对RBP-J分子的抑制,比单一阻断受体或配体更能代表Notch信号的整体失活。故本课题采用了条件性RBP-J基因剔除小鼠模型,分析不同实体肿瘤在小鼠体内的生长情况,并对其免疫细胞命运和作用进行了探讨,为干预Notch信号治疗实体肿瘤提供了更完整的实验室数据。主要研究结果如下:1.获得了RBP-J条件性基因敲除小鼠,筛选了可在基因敲除小鼠直接接种的实体肿瘤细胞系,确定了肿瘤接种时间。通过繁育RBP-Jflox/wr和Mx-Cre小鼠,获得了Mx-Cre-RBP-Jflox/wr和RBP-Jflox/flox小鼠;通过ROSA和Mx-Cre小鼠交配,获得了Mx-Cre-ROSA小鼠。Mx-Cre-ROSA小鼠经500μg总剂量的poly:IC诱导(以腹腔注射的方式分5次间隔进行)后,耳部皮肤行X-gal染色见明显蓝染,表明小鼠血管开始高效表达Cre重组酶。对小鼠源性的4种肿瘤细胞系进行了Notch信号成员的RT-PCR定性分析,Notch4、Dll4在S180细胞和H22细胞中有转录mRNA,而Notch1在B16细胞中有转录,Notch2在LLC细胞中有表达。这些结果表明,用RBP-J条件性基因敲除小鼠模型,进行上述肿瘤细胞的接种分析是可行的。2.不同肿瘤在RBP-J基因敲除小鼠表现出差异性生长的特征,至少包括肝癌在内的某些肿瘤会因Notch信号的缺失而出现转移。将S180、H22、B16和LLC细胞接种到RBP-J基因敲除小鼠和对照鼠腹部皮下。和对照小鼠相比,基因敲除小鼠体内成骨肉瘤S180和肺癌LLC的生长减缓,瘤体指数缩小,而B16黑色素瘤生长加快,瘤体指数增加;H22肝癌细胞皮下接种后,有些基因敲除小鼠出现肝脏表面结节,肝组织切片H.E染色见到肿瘤细胞肝脏内部浸润;发生肿瘤转移的基因敲除小鼠生存时间缩短。这些数据表明,彻底抑制Notch信号治疗不同类型的肿瘤,可能会有不同的治疗结果。3. Notch信号缺失导致的肿瘤血管增生,不一定带来所有肿瘤生长减缓,缺氧和免疫细胞浸润改变不是RBP-J基因敲除小鼠肿瘤差异性生长的原因。对诱导小鼠的S180、H22、B16和LLC肿瘤瘤体进行包埋、切片和组织学分析,RBP-J基因敲除小鼠来源的4种肿瘤细胞的抗CD31染色信号增加,微血管密度计数显示其肿瘤新生血管增多;这些瘤组织的抗HIF-1α染色信号强度均增加。流式细胞术分析瘤体内部免疫细胞,4种RBP-J基因敲除小鼠来源的肿瘤瘤体内T细胞、B细胞和DC的浸润与对照相比,无明显改变,而髓系细胞Gr-1+/Mac-1+细胞均有显著增加。对H22细胞进行了背部气囊诱导的肿瘤血管生成分析,RBP-J基因敲除小鼠气囊内典型的迂曲、不规则肿瘤样血管显著增多。这些结果说明,Notch信号缺失导致的肿瘤血管增生,可能和肿瘤生长速度不一致,缺氧和免疫细胞浸润改变并不导致RBP-J基因敲除小鼠体内肿瘤差异性生长;H22肝癌转移的原因可能在于该细胞诱导RBP-J基因敲除小鼠迂曲、不规则肿瘤样血管的显著增多。4. Notch信号缺失后Gr-1+细胞向DC发育受阻;RBP-J基因敲除后DC多局限于血管内,可能妨碍其抗原呈递功能。体外培养诱导小鼠骨髓的Gr-1+细胞,使其向DC发育方向分化,RBP-J基因敲除小鼠的Gr-1+细胞发育明显受挫;Dio染料标记野生小鼠骨髓Gr-1+细胞后进行单系骨髓移植,也可见到RBP-J基因敲除小鼠的Gr-1+细胞向DC发育受阻。血管内皮细胞和Gr-1+细胞共培养显示,RBP-J缺陷血管内皮诱导Gr-1+细胞向DC方向发育的作用减弱。分选脾脏CD11c+细胞,用Dio染料标记后尾静脉注射后分析其在脾脏和肿瘤瘤体内与血管的共定位,RBP-J基因敲除小鼠的Dio和凝集素叠加信号明显增强。这些结果说明,RBP-J依赖的Notch信号参与维持Gr-1+细胞向DC分化方向的选择;血管内皮细胞和Gr-1+细胞的相互作用,可能是该发育过程中Notch信号传导的基础;RBP-J基因敲除后,DC多局限于血管内,可能妨碍其发挥抗原呈递功能,进而影响肿瘤生长。5.结论本课题研究证实,不同的肿瘤在RBP-J基因敲除小鼠表现出差异性生长的特征,至少包括肝癌在内的某些肿瘤会因Notch信号的缺失而出现转移。Notch信号缺失导致的肿瘤血管增生,不一定带来所有肿瘤生长减缓,而缺氧和免疫细胞浸润改变不是RBP-J基因敲除小鼠差异性生长的根本原因,但免疫细胞尤其是Notch信号缺失后DC和髓系-单核细胞的功能,是否会影响肿瘤生长,需要进一步研究。RBP-J依赖的Notch信号参与维持Gr-1+细胞向DC分化方向的选择;血管内皮细胞和Gr-1+细胞的相互作用,可能是该发育过程中Notch信号传导的基础;RBP-J基因敲除后,DC多局限于血管内,可能妨碍其发挥抗原呈递功能,进而影响肿瘤生长。血管提供营养支撑来促进肿瘤生长,DC细胞呈递肿瘤抗原抑制肿瘤生长,而Notch信号可同时调控血管因素和免疫因素,因此无论上调还是下调Notch信号,都有可能起到治疗作用;据此推测上调或下调Notch信号治疗实体肿瘤,关键在于平衡向哪边倾斜。因此以Notch信号作为实体肿瘤治疗靶点,需要重新慎重考虑,至少需要针对不同实体肿瘤类型来做出上调或下调Notch信号的选择。更多还原

【Abstract】 Tumor, a big threat to human life, brought about a big economic burden for sufferers and the whole society. Solid tumors cause numerous incidence and mortality, while no perfect therapy is available up to now. It is necessary to explore the tumor genesis and development mechanism for the sake of producing better tumor control strategies.Tumor angiogenesis and anti-tumor immune response are the most important determinants of tumor growth. Neoplasm, within 2 or 3 cubic micrometers, can slowly expand by diffusions of nutritients in cell neighborhood. But when the tumor size gets bigger and bigger, the growth requires tumor neovascularization to supply the nutrition. On the other hand, immune responses can be stimulated by tumor antigen, leading to cellular and humoral responses against tumor cells.The latest tumor control strategy emphasizes the angiogenesis inhibition because neovascularization was thought indispensable to tumor growth. Lab and clinic practice have proved that this strategy is effective in some solid tumors. But drug resistance and lower efficacy has been observed also. As a result, the understanding of tumor angiogenesis mechanism is the premise of vascular network formation inhibition.Notch signal, a classical and conserved pathway associated with development regulation, is consisted of ligands and receptors on cell membrane, transcription factors in cytoplasm, downstream targets and regulators. Notch intracellular domain can be cleaved by different proteases after ligand and receptor binding, then the cleaved domain translocates into nuclear and interact with the transcription factor, RBP-J. After that, the RBP-J-containing co-repressor is switched into co-activator and triggers target transcription factors to initiate downstream genes expression. These activated genes then participate in developmental events.Notch signal is an important player in vascular network formation in embryos and adults. Notch pathway was reported to be involved with the choice of artery or vein fate, commitment of tip cell or stalk cell, endothelial proliferation, filopodia formation, migration and stability maintenance. Thus Notch pathway has been hought as a potential target in tumor angiogenesis inhibition. When Notch signal is interrupted by Dll4 intervention, some reports have observed retarded tumor growth while tumor angiogenesis augmented. In those new formed vascular, poor perfusion of blood was recorded. But the paradox is that after Notch signal activation, some solid tumor growth is slowed down.Furthermore, Notch signal is related to immune cell development. Bulk of evidence has showed that T cell, B cell, dendritic cell and myloid cell commitments require Notch singal. Consquently, when tumor angiogenesis inhibition is central for therapy, the immunological changes should be evaluated since anti-tumor responses contribute to prognosis.Because of functional redundancy in Notch pathway result from varied receptors and ligands, the completely interruption of this signal can be fulfilled by RBP-J inhibition. Actually, RBP-J is the key player in Notch pathway. Thus in the current study conditional RBP-J gene knockout mice were employeed to analyze solide tumor growth. In the same mice, immune cells involved in tumor growth were evaluated also. These following results provided some important informations for Notch singal intervention in solid tumor therapy.1. Conditional RBP-J gene knockout mice were obtained and some solid tumor cell lines were selected for direct inoculation.Mating RBP-Jflox/wr and Mx-Cre mice got Mx-Cre-RBP-Jflox/wr and RBP-Jflox/flox mice. Mating ROSA and Mx-Cre got Mx-Cre-ROSA mice. Ear from Mx-Cre-ROSA mice were stained by X-gal after peritoneal injection with 500μg polyI:C. The obvious blue color indicated that Cre recombinants began is expressed in vascular cells. RT-PCR analysis showed that Notch4、Dll4 transcripts presented in S180 and H22 cell line, while Notch1 was expressed in B16 cell line and Notch2 presented in LLC cell line.2. In the RBP-J gene knockout mice, different tumor growth was characterized. Hepatocarcinoma could metastasize to the liver in the RBP-J knockout mice.Compared to the tumors in the control mice, the S180, H22 and LLC tumor grew slower in the RBP-J gene knockout mice. Melanoma grew faster in the RBP-J gene knockout mice. Furthermore, liver metastasis was observed in RBP-J gene knockout mice after H22 cell inoculation. H.E staining showed maglinant cell invasion in the metastasized livers. The control mice suvived the metastasized mice after H22 cell inoculation. These results implied that completely interruption of Notch signal mighg lead to different growth for different tumors.3. Tumor angiogenesis, resulted from completely Notch signal interruption, did not necessarily bring about retarded growth; Tumor hypoxia and immune cell infiltration were not the reasons of different tumor growth after RBP-J gene knockout.Tissue section from S180, H22, LLC and B16 tumor were stained with anti-CD31 antibody to show the micro-vessel density. All the micro-vessel density in the tissue from different tumors growing in RBP-J gene knockout mice increased. Anti-HIF-1αstaining indicated that all the hypoxia area in the tissue from different tumors growing in RBP-J gene knockout mice increased. FACS analysis showed that T cell, B cell and DC infiltration in the tissue from different tumors growing in RBP-J gene knockout mice did not changed, while Gr-1+/Mac-1+ cell infiltration increased in the tissue from different tumors growing in RBP-J gene knockout mice. Air sac analysis based on H22 cells showed more typical tumor-like vessels in RBP-J gene knockout mice. These data suggested that tumor angiogenesis result from Notch signal interruption dissociated with melanoma growth. Hypoxia and immune cell infiltration were not the cause of different tumor growth after RBP-J gene knockout. H22 hepatocarcinoma with higher metastasis in RBP-J gene knockout mice might be linkedwith more typical tumor-like vessels.4. The process of Gr-1+ cell developing into DC was retarded after Notch signal blocking; More dendritic cells located in the vascular network when RBP-J gene was knockout, which meant poor antigen process ability.Culture analysis in vitro showed that the Gr-1+ cell from bone marrow of RBP-J gene knockout mice slowed down the process of commitment into DC. Wild type Gr-1+ cells were marked by Dio and tranfered into RBP-J gene knockout mice proved that the commitment of DC was blocked. What’s more, after splenic CD11c+ cells were sorted and marked by Dio, co-location staining result implied that the merge signal based on CD11c+ and agglutinin staining increased in RBP-J gene knockout group. These results indicated that RBP-J dependent Notch pathway played a role in the commitment DC from Gr-1+ cells. Dendritic cells located more in the vascular network when RBP-J gene was knockout, which meant poor antigen process ability and possible retarded tumor growth.5. ConclusionThe current study showed that in the RBP-J gene knockout mice, different tumor growth was characterized. At least hepatocarcinoma can metastasize to the liver in the Notch signal interruption mice. Tumor angiogenesis, resulted from completely Notch signal interruption, did not necessarily bring about retarded growth; Hypoxia and immune cell infiltration were not the reason of different tumor growth after RBP-J gene knockout. But the function of DCs and myeloid cells after Notch sigal intervention need further analysis to ascertain the possible effects on tumor growth.Tumor angiogenesis supports tumor growth while DCs process tumor antigen to inhibit tumor growth. Notch signal is an important player in tumor angiogenesis and immune cell commitment. Thus intervention of Notch signal, whether up or down regulated, can retard tumor growth. The therapy result depends on the plolarization after Notch signal is changed. So it is important to take the different tumor types into account for the sake of therapy targeting on Notch singal.更多还原