基于复杂化三维共培养模型解析微环境对乳腺上皮细胞命运的调控及其分子基础

Breast cancer is the second leading course of death in the world. Unfortunately, much is yet known to uncover the mechanisms of the neoplastic transformation of human breast tissue due to its complexity and the limited culture model system in vitro. It has been demonstrated that the process of physiologically development of mammary gland shared some features with that of the neoplastic transformation, evidenced by their cellular proliferation, migration and morphogenesis. This is important because a number of investigations have found that tissue microenvironment in the mammary gland plays a critical role in determining the fate of mammary epithelial cells. Thus, to elucidate the effect of the microenvironment factors especially ECM and stromal cells, on the fate of mammary epithelial cells (MECs) would enlighten us to better understanding the carcinogenesis mechanism of breast tissue. In this proposal, the PI is proposing to bioengineering an improved 3D co-culture model of MECs by using tissue engineering techniques. It is hypothesized that a more physiologically relevant microenvironment of breast tissue could be reconstructed through co-culturing MECs and multiple types of mammary stromal cells embedded within silk nanofiber-collagen-matrigel hydrogel matrix with bioreactor culture system incorporation. It is expected that this bioengineered 3D co-culture compartment could replicate the tissue microenvironment more closely, allowing the mechanism exploration of the influence exerted by the microenvironment on the fate of MECs. Based on this engineered 3D co-culture model of human breast tissue, a systemic study on the mammary microenvironment effect on morphogenesis, development and even neoplastic transformation would be carried out and its related molecular mechanism will be explored. Also, a platform of characterizing the phenotype and functionality of the epithelial structure generated by the 3D co-cultures will be established, which is important for the future elucidation of the microenvironmental effect on the transformed 3D culture model of MECs. Taken together, the model system constructed through integrating approaches from the multi-disciplines of tissue engineering and mammary developmental biology would offer a robust tool to better understand the effect of the mammary microenvironment effect on the MECs and the related mechanism. This would also enlighten our future dissection the underlying mechanism of carcinogenesis in human breast tissue.

乳腺肿瘤是威胁世界女性生命健康的重要疾病，但有关乳腺组织发生癌性转化的机制却仍远未被揭示。研究证明：乳腺组织的生理发育与癌性转化可能是机制共享且相互转归的生理与病理过程。因此，深入解析“乳腺上皮组织表型及功能的调控因素并阐明其作用机制” 将是揭示癌性转化机制的科学关键。本项目基于“乳腺组织微环境诱导”理论，结合团队前期工作基础，拟采用组织工程技术优化构建复杂化人乳腺上皮细胞3D共培养模型，在理想模拟乳腺组织微环境的基础上，建立并完善相关的检测评价技术平台；并基于该平台技术，定性及定量考察微环境各因素尤其是ECM和间质细胞对乳腺上皮细胞基本生物学行为的影响、对上皮结构发生、发展及转化的调控并揭示其具体的分子基础。这种基于组织工程技术体外优化再造人乳腺组织3D培养模型以揭示微环境对乳腺上皮命运调控的研究策略对于乳腺发育生物学研究具有重要的指导意义，也将为乳腺癌的防治奠定夯实的理论基础。

乳腺肿瘤是威胁世界女性生命健康的重要疾病，但有关乳腺组织发生癌性转化的机制却远未被揭示。乳腺组织的生理发育与癌性转化是“机制共享”且相互转归的生理与病理过程。深入解析“乳腺上皮组织表型及功能的调控因素并阐明其作用机制” 将是揭示癌性转化机制的基础和关键。本项目基于“乳腺组织微环境诱导”理论，首先成功制备了兼具有生物相容性和基质刚度可调性的纳米丝蛋白-胶原复合水凝胶支架；再以人乳腺上皮细胞和间质细胞为模型，利用三维共培养平台技术优化构建了复杂化人乳腺上皮细胞3D共培养模型。我们发现：所构建的复合水凝胶支持乳腺上皮细胞的活性、增殖以及类乳腺上皮结构的发生。这些上皮结构（包括类腺泡和类导管结构）具有正确的细胞极性，特定的功能蛋白和基因的表达。基于所构建的模型体系，本研究还深入探讨了乳腺微环境因素对乳腺上皮细胞表型与功能的影响和作用规律。采用组织学、细胞生物学以及分子生物学技术，定性及定量考察了培养微环境中ECM组分、基质刚度以及间质细胞对乳腺上皮细胞生物学行为的影响，并初步阐释了作用规律与分子机制。结果发现：ECM成分、基质刚度以及间质细胞共培养均能影响乳腺上皮细胞分化形成乳腺上皮结构。其中,胶原组分更利于导管样结构的形成，而基质胶则促进腺泡结构的发生，且利于维持正常细胞极性；间质细胞的掺入促进导管样上皮结构的发生，细胞极性的建立和功能蛋白的表达，而这种上皮-间质的相互作用与细胞直接接触和细胞因子旁分泌密切相关。基质刚度是调控上皮细胞发生EMT的关键因素。高基质刚度抑制规则性上皮结构的形成，促进细胞骨架改变，极性丧失，功能性蛋白表达水平的降低，而与EMT相关的基因与蛋白的表达水平则显著增高。该研究结果充分证明了本课题所提出的科学假设，揭示了微环境与乳腺上皮细胞命运调控密切相关。这不但为乳腺发育生物学研究提供了理想的模型体系，也为乳腺癌的防治研究奠定了科学的理论基础。