基于Smad-7和VEGF基因修饰的脂肪干细胞复合生物补片构建功能性阴茎白膜的可行性研究

Some penis disorders, such us Peyronie’s disease, severe penis flexion deformity or congenital penis dysplasias often involve the reconstruction or increase the abnormal tunica albuginea. In practice, many materials, such as leather, fascia are used to repair or reconstruction the abnormal tunica albuginea. However, whether it is a synthetic material, allogeneic or autologous tissue material, the most major problem is the local fibrosis leading to postoperative recurrence of penile buckling and decreased sexual function. The emergence of tissue engineering techniques makes scientists get new ideas in the field of tunica albuginea defect therapy. In our previous study, adipose derived stem cells (ADSCs) combining with porcine small intestinal acellular matrix (SIS) were used to reconstruction rats tunica albuginea defect and we found the fibrosis and erectile function was improved in a certain extent which had not been reported previously. We first reported these exciting findings in the 2012 PNAS (proceedings of the national academy of sciences). It was considered as a revolutionary breakthrough in the functional reconstruction technique of tunica albuginea. Two factors, including inhibition of fibrosis, protection and promotion of micro vascular growth, are important to the penis morphology and good erection after the tunica albuginea reconstruction. In order to further solve the fibrosis and microvascular damage problem after the tunica albuginea reconstruction, With the help of genetic engineering technology, we try to research and development a new kind tissue-engineered biological patch which is reunited by SIS and ADSCs modified by samd7 and VEGF. The ultimate purpose of this research is to explore the feasibility of constructing gene enhanced tissue engineering patch to repair the tunica albuginea defect.

Peyronie’s病、重度阴茎屈曲畸形或先天性阴茎发育不良等阴茎病变常涉及重建或增加病变处白膜，实践中真皮、筋膜等很多材料被使用，但不论是合成材料、同种异体材料还是自体组织，术后最大问题就是局部纤维化导致阴茎屈曲畸形以及大量微血管毁损继发阴茎勃起功能障碍。组织工程技术的兴起使白膜缺损的修复获得了新思路，本研究小组用脂肪干细胞复合猪小肠脱细胞基质构建组织工程补片修复了大鼠阴茎白膜缺损，发现修补后阴茎纤维化和勃起功能均较单纯补片修补有改善，该研究结果在PNAS作了初步报道。以此为切入点，本研究拟将基因工程融合组织工程技术，构建表达smad-7和VEGF的慢病毒，以此为基因修饰的脂肪干细胞构建组织工程生物补片付诸于大面积阴茎白膜修复，探索构建基因强化组织工程补片来重建功能性阴茎白膜的可行性。

本项目1）成功构建了GV492-Smad7-P2A-VEGF双基因共表达慢病毒载体，能有效感染脂肪源性干细胞（ADSCs）使之稳定表达目标基因；2）结合基因工程技术，成功构建了稳定、有效共表达Smad7和VEGF的基因修饰ADSCs，为组织工程化阴茎白膜提供了种子细胞；3）结合体外器官3D培养的方法，进一步改良常规的种子细胞-支架复合技术，成功构建了Smad7/VEGF双基因修饰ADSCs-小肠黏膜下层（SIS）复合补片材料；4）利用单纯SIS补片对SD大鼠阴茎白膜移植和重建作为实验模型组（SIS组），观察了ADSCs-SIS复合补片材料和Smad7/VEGF双基因修饰ADSCs（mADSCs）-SIS复合补片材料对SD大鼠阴茎勃起组织结构和功能的影响，并进行比较。与正常对照组和假手术组相比，SIS组大鼠阴茎海绵体平滑肌含量明显减少且纤维化程度增高，阴茎海绵体内压（ICP）波形明显压低，ICPmax和ICPmax/平均动脉压（MAP）均明显下降（P均<0.01），海绵体组织中内皮源性一氧化氮合酶（eNOS）、神经源性一氧化氮合酶（nNOS）、Smad7及VEGF蛋白和mRNA相对表达水平均受到明显抑制（P<0.05或P均<0.01）；与SIS组相比，ADSCs-SIS组大鼠阴茎海绵体平滑肌含量升高且纤维化改善，ICP波形上抬，ICPmax和ICPmax/平均动脉压（MAP）均显著上升（P<0.05或P<0.01），海绵体组织中eNOS、nNOS及VEGF蛋白相对表达水平均明显提高（P均<0.05）；与SIS组相比，mADSCs-SIS组大鼠阴茎海绵体平滑肌含量升高且纤维化改善，ICP波形明显上抬，ICPmax和ICPmax/平均动脉压（MAP）均显著上升（P均<0.01），海绵体组织中eNOS、nNOS、Smad7及VEGF蛋白及mRNA相对表达水平均明显提高（P<0.05或P<0.01）。值得关注的是，与ADSCs-SIS组相比，mADSCs-SIS组大鼠ICPmax、海绵体组织中eNOS和Smad7蛋白相对表达水平及Smad7 mRNA相对表达水平明显提高（P<0.05或P<0.01），反映了mADSCs-SIS复合补片材料用作阴茎白膜移植与重建时，不仅可改善单纯SIS补片造成的阴茎勃起组织结构和功能损害，且较传统ADSCs-SIS复合补片材料更优。