Stem Cell Homing for Treatment of Female Pelvic Floor Disorders

描述(由申请人提供)：在美国，多达一半的女性经历女性盆底疾病(FPFD)：压力性尿失禁(SUI)、大便失禁和盆腔器官脱垂(POP)。这些情况都有共同的风险因素，包括阴道分娩，因此应该一起研究。FPFD的终生手术风险为11%，再手术率为29%。由于年龄、糖尿病和肥胖症是FPFD的风险因素，美国人口老龄化以及肥胖症和糖尿病发病率的增加预示着未来几十年美国将出现一场FPFD疫情。女性生殖器官和盆底含有丰富的弹性纤维，在怀孕和分娩期间经历了大规模的重塑。赖氨酰氧化酶样1(LOXL1)是一种对弹性蛋白重塑和动态平衡至关重要的蛋白质。与正常小鼠不同，LOXL1基因敲除(KO)小鼠的阴道分娩会导致POP和SUI，使其成为FPFD的理想动物模型。干细胞参与正常的修复过程，有可能被用作预防性治疗，以促进分娩后的修复。我们的初步数据表明，干细胞增加了下尿路组织中弹性蛋白的含量，这表明干细胞应用于促进产后恢复和预防FPFD是可行的。损伤组织产生的细胞因子梯度将循环干细胞吸引或聚集到损伤部位，在那里它们促进修复。由于引起FPFD的损伤太过弥漫，不适合直接干细胞注射治疗，干细胞归巢可以提供一种可行的治疗方法，以促进分娩后的修复和预防FPFD。最近的研究表明，干细胞可以通过分泌旁分泌作用蛋白(如生长因子)来促进损伤后的修复。识别这些因素可能提供一种可行的非细胞疗法，以促进产后修复和预防FPFD。我们将利用LOXL1 KO小鼠模型来研究是否可以利用基于细胞或非细胞干细胞的修复疗法来预防FPFD。这个项目要检验的假设是，间充质干细胞在分娩后回到盆腔器官中，促进恢复，并通过分泌旁分泌因子来预防由此产生的FPFD。这一假设将以以下具体目标进行检验：SA1。确定LOXL1 KO小鼠阴道分娩或剖腹产后，泌尿生殖器官上调参与干细胞归巢和组织修复的细胞因子的时间进程。我们将使用趋化因子/趋化因子受体阵列系统来筛选潜在的干细胞归巢细胞因子的上调。使用RT-PCR和免疫组织化学方法进行确认和定位。SA2.优化MSCs的剂量和时机，以最大限度地促进母体骨盆损伤的恢复，预防FPFD。荧光标记的MSCs将在雌性LOXL1 KO小鼠分娩后的几个时间点进行输注。结果将包括POP的分期、尿动力学、弹性蛋白数量的定量组织形态计量学评估以及泌尿生殖器官的聚集模式。SA3.确定受体介导的MSC归巢和加速阴道分娩后功能恢复的机制。雌性LOXL1 KO小鼠将接受阴道分娩，并将接受MSCs的输注，其中归巢细胞因子受体已通过小干扰RNA(SiRNA)转染而被击倒。经阴道分娩后输注MSC对SA2最有效的剂量和时间点将用于SA3的实验。SA3的结果将与SA2相同。SA4.确定阴道分娩加速功能恢复的非细胞机制。骨髓间充质干细胞的输注将被比作注射来自这些干细胞的浓缩条件培养液(CCM)。我们将同时利用来自野生型和LOXL1 KO小鼠的MSCs和CCM。我们还将使用siRNA来敲除MSCs中分泌的旁分泌因子，并利用这些细胞中的MSCs和CCM。经阴道分娩后输注MSC对SA2最有效的剂量和时间点将用于SA4的实验。SA4的结果将与SA2和SA3相同。意义重大。分娩的母体损伤是弥漫性的，涉及许多小结构的损伤，如盆底筋膜和盆腔器官，使得直接向这些部位注射干细胞治疗是不可能的。因此，基于干细胞归巢或干细胞的全身效应的治疗可能为促进受损组织的修复和预防FPFD提供一种可行的方法。此外，识别干细胞产生的旁分泌因子，并在分娩后引入这些因子，可能会提供一种可行的非细胞修复疗法来预防FPFD。因此，本文的研究具有很强的创新性，对浮式浮雕的研究具有重要意义。 公共卫生相关性：在美国，多达一半的女性经历过女性盆底疾病：压力性尿失禁、大便失禁和盆腔器官脱垂，但对它们的病理生理知之甚少，可用的非手术治疗也很少。在这个项目中，我们将使用动物模型来确定成体间充质干细胞的归巢是否可以提供一种可能的非手术治疗或预防选择。我们将探索使这种疗法成为非细胞疗法的可能性，以避免细胞疗法的并发症。本研究对改进女性盆底疾病的治疗方法具有重要意义。

DESCRIPTION (provided by applicant): Up to half the women in the U.S. experience female pelvic floor disorders (FPFD): stress urinary incontinence (SUI), fecal incontinence, and pelvic organ prolapse (POP). These conditions share common risk factors, including vaginal delivery, and therefore ought to be studied together. The lifetime risk of surgery for FPFD is 11%, with a re-operation rate of 29%. Since age, diabetes, and obesity are risk factors for FPFD, the aging US population and increasing prevalence of obesity and diabetes portend an FPFD epidemic in the U.S. in the coming decades. Female reproductive organs and the pelvic floor are rich in elastic fibers and undergo massive remodeling during pregnancy and childbirth. Lysyl oxidase-like 1 (LOXL1) is a protein essential for elastin remodeling and homeostasis. Unlike in normal mice, vaginal delivery in LOXL1 knockout (KO) mice results in POP and SUI, making it an ideal animal model of FPFD. Stem cells participate in normal repair processes and have the potential to be harnessed as a preventative therapy to facilitate repair after childbirth. We have preliminary data demonstrating that stem cells increase the elastin content of lower urinary tract tissues, suggesting the feasibility of stem cell administration to facilitate recovery after delivery and prevent FPFD. Cytokine gradients produced by injured tissues attract or home circulating stem cells to sites of injury, where they facilitate repair. Since the injuries that cause FPFD are too diffuse to be amenable to direct stem cell injection therapy, stem cell homing could be utilized to provide a viable therapy to facilitate repair after delivery and prevent FPFD. Recent research has demonstrated that stem cells can facilitate repair after injury by secreting paracrine-acting proteins such as growth factors. Identification of these factors may provide a viable noncellular therapy to facilitate repair after childbirth and prevent FPFD. We will utilize the LOXL1 KO mouse model to study if a cellular or noncellular stem cell-based reparative therapy could be utilized to prevent FPFD. The hypothesis to be tested in this project is that mesenchymal stem cells home to pelvic organs after delivery, facilitate recovery, and prevent resultant FPFD via secretion of paracrine factors. This hypothesis will be tested with the following Specific Aims: SA1. Determine the time course over which cytokines involved in stem cell homing and tissue repair are upregulated by the genitourinary organs after vaginal delivery or C-section delivery in LOXL1 KO mice. We will screen for upregulation of potential stem cell homing cytokines with a chemokine/chemokine receptor array system. Confirmation and localization will be made using RT-PCR and immunohistochemistry. SA2. Optimize the dose and timing of MSCs to best facilitate recovery from the maternal pelvic injuries of delivery and prevent FPFD. Fluorescently labeled MSCs will be infused at several time points after pup delivery in female LOXL1 KO mice. Outcomes will include staging of POP, urodynamics, quantitative histological morphometry assessment of elastin amount and pattern of clustering in genitourinary organs. SA3. Determine the receptor-mediated mechanisms of MSC homing & accelerated functional recovery from vaginal delivery. Female LOXL1 KO mice will undergo vaginal delivery and will receive an infusion of MSCs in which homing cytokine receptors have been knocked down via transfection with small interfering RNA (siRNA). The dose and time point of MSC infusion after vaginal delivery identified as most effective in SA2 will be used for the experiments of SA3. Outcomes of SA3 will be the same as in SA2. SA4. Determine the noncellular mechanisms of accelerated functional recovery from vaginal delivery. Infusion of MSCs will be compared to injection of concentrated conditioned media (CCM) from these stem cells. We will utilize both MSCs and CCM of MSCs generated from wild type and LOXL1 KO mice. We will also use siRNA to knock down secreted paracrine factors in MSCs and utilize both the MSCs and CCM from these cells. The dose and time point of MSC infusion after vaginal delivery identified as most effective in SA2 will be used for the experiments of SA4. Outcomes of SA4 will be the same as in SA2 & SA3. Significance. The maternal injuries of childbirth are diffuse and involve injury to many small structures, such as pelvic floor fascia as well as pelvic organs, making direct injection stem cell therapy to all these sites impossible. Therefore, therapies based on stem cell homing or on the systemic effects of stem cells may provide a viable method to facilitate repair of injured tissues and prevent FPFD. In addition, identification of the paracrine factors produced by stem cells and introduction of these factors after delivery may provide a viable noncellular reparative therapy to prevent FPFD. Thus, this research is highly innovative and is of high significance for FPFD. PUBLIC HEALTH RELEVANCE: Up to half the women in the U.S. experience female pelvic floor disorders: stress urinary incontinence, fecal incontinence, and pelvic organ prolapse, but little is known about their pathophysiology and few nonsurgical treatments are available. In this project, we will use an animal model to determine if homing of adult mesenchymal stem cells could provide a possible nonsurgical treatment or preventative option. We will explore the possibility of making this therapy noncellular to avoid complications of cell therapy. This research is of high significance for development of improved therapies for female pelvic floor disorders.