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过女性盆底疾病（FPFD）：压力性尿失禁（SUI），大便失禁和盆腔器官脱垂（POP）。这些情况有共同的危险因素，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、糖尿病和肥胖是FPFD的危险因素，美国人口老龄化以及肥胖和糖尿病患病率的增加预示着FPFD在未来几十年将在美国流行。女性生殖器官和骨盆底富含弹性纤维，在怀孕和分娩期间经历了大量的重塑。赖氨酸氧化酶样1 （LOXL1）是弹性蛋白重塑和体内平衡所必需的蛋白质。与正常小鼠不同，LOXL1敲除（KO）小鼠阴道分娩可导致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，并有潜力被利用为一种预防性治疗，以促进分娩后的修复。我们有初步的数据表明，干细胞增加了下尿路组织的弹性蛋白含量，这表明干细胞给药促进分娩后恢复和预防FPFD的可行性。损伤组织产生的细胞因子梯度吸引或回家循环干细胞到损伤部位，在那里它们促进修复。由于导致FPFD的损伤过于分散，无法直接进行干细胞注射治疗，因此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并预防FPFD。最近的研究表明，干细胞可以通过分泌生长因子等旁分泌作用蛋白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治疗，以促进产后修复和预防FPFD。我们将利用LOXL1小鼠模型来研究基于细胞或非细胞干细胞的修复疗法是否可以用于预防FPFD。本项目要验证的假设是，间充质干细胞在分娩后回到盆腔器官，促进恢复，并通过分泌旁分泌因子预防由此产生的FPFD。这一假设将通过以下具体目的进行检验：确定LOXL1 KO小鼠阴道分娩或剖腹产分娩后，生殖泌尿器官上调参与干细胞归巢和组织修复的细胞因子的时间过程。我们将使用趋化因子/趋化因子受体阵列系统筛选潜在干细胞归巢细胞因子的上调。使用RT-PCR和免疫组织化学进行确认和定位。SA2。优化MSCs的剂量和时间，以最好地促进产妇分娩后骨盆损伤的恢复和预防FPFD。荧光标记的MSCs将在雌性LOXL1 KO小鼠幼鼠分娩后的几个时间点注入。结果将包括POP的分期、尿动力学、定量组织形态测量法评估弹性蛋白的数量和泌尿生殖器官的聚集模式。SA3。确定受体介导的MSC归巢机制和加速阴道分娩后的功能恢复。雌性LOXL1 KO小鼠将通过阴道分娩，并接受输注MSCs，其中通过小干扰RNA （siRNA）转染敲除归巢细胞因子受体。在SA2中经阴道分娩后输注MSC最有效的剂量和时间点将用于SA3的实验。SA3的结果与SA2相同。SA4。确定加速阴道分娩功能恢复的非细胞机制。输注间充质干细胞将与注射这些干细胞的浓缩条件培养基（CCM）进行比较。我们将利用野生型和LOXL1小鼠产生的MSCs和CCM。我们还将使用siRNA敲除MSCs中分泌的旁分泌因子，并利用这些细胞中的MSCs和CCM。在SA2中阴道分娩后输注MSC最有效的剂量和时间点将用于SA4的实验。SA4的结果与SA2和SA3相同。的意义。分娩的产妇损伤是弥漫性的，涉及许多小结构的损伤，如盆底筋膜和盆腔器官，这使得直接注射干细胞治疗所有这些部位是不可能的。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损伤组织的修复和预防FPFD提供一种可行的方法。此外，鉴定由干细胞产生的旁分泌因子，并在分娩后引入这些因子，可能为预防FPFD提供一种可行的非细胞修复疗法。因此，本研究具有很强的创新性，对FPFD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.