人多能干细胞分化而来的平滑肌前体细胞治疗压力性尿失禁大鼠的研究

发布时间：2018-05-24 22:13

本文选题：多能 + 干细胞　；参考：《南方医科大学》2016年博士论文

【摘要】：背景：压力性尿失禁(Stress urinary incontinence, SUI)是指在咳嗽、喷嚏、大笑等腹压增高时出现的不自主地尿液渗漏。SUI是女性最为常见的尿失禁类型,尤其是老年妇女。SUI症状经常发生在日常活动中,因此严重影响患者的生活质量和社交活动,亦带来心理精神负担。SUI发病机制尚不明确,但尿道固有括约肌功能不全(Intrinsic sphincter deficiency, ISD)是导致SUI的重要因素。ISD主要由于尿道括约肌的平滑肌层或横纹肌层或其支配神经出现损伤引起。具有ISD的SUI患者通常有更严重的尿失禁症状,且对常规的治疗反应不佳。当保守治疗失败后,必须进行手术治疗。目前首选的手术治疗方式,如中段尿道悬吊术等,仅是增加对尿道的支持、固定作用,并不涉及对损伤或老化的尿道括约肌进行组织重建。而这类手术的长期成功率约为40-50%,有很大一部分患者会出现术后复发的情况。手术的局限性及逐渐进展的尿道括约肌平滑肌层老化衰退被认为是导致术后复发的原因。因此,有效恢复受损的尿道括约肌是需要积极探索的重要治疗方式。干细胞因其具备自我更新和多向分化的潜能,可应用于SUI患者的尿道括约肌再生,这引起了研究领域的极大兴趣。目前,已有成体干细胞(肌肉来源、泌尿系来源、脂肪来源、骨髓来源等)在SUI动物研究或临床研究中表现出在排尿功能的恢复、组织学的改善方面有较好的效果。但是,这些成体干细胞的临床应用仍存在众多问题,如提取的自体干细胞数量有限、细胞扩增准备时间过长、移植细胞为多种细胞类型混合、携带表观遗传改变等等。这些障碍不利于干细胞疗效的优化,也影响了我们对干细胞具体作用机制进行深入研究。因此,为避免上述问题,人胚胎干细胞或诱导多能干细胞或许可以成为新的干细胞来源。另外,现阶段的干细胞实验主要修复的是尿道外括约肌(横纹肌),尚未有仅针对尿道内括约肌(平滑肌)再生的研究报道。研究人员发现,在SUI研究中应用最广的成体间充质干细胞在体内仅有一小部分能够分化为平滑肌细胞,而由于成体间充质干细胞移植混杂有多种细胞类型,分化而来的平滑肌细胞是否对尿道括约肌功能重建有促进作用及有关机制都无法明确。目的：我们试图探索人胚胎干细胞和人诱导多能干细胞(Induced pluripotent stem cells, iPSCs)分化而来的单一平滑肌前体细胞(Progenitor smooth muscle cells, pSMCs)对重建SUI动物模型尿道功能的作用及机制。首先,通过在不同时间点对不同SUI大鼠建模方式的建模效果进行比较,以期找到更稳定更适宜干细胞移植研究的长期SUI模型。在此基础上,分化而来的pSMCs在移植入SUI大鼠尿道周围一段时间后,根据LPP和尿道外周括约肌肌电图(Electromyography, EMG)基线的测试,判断尿道功能的恢复情况。根据尿道括约肌组织学检查、细胞外基质(Extracellular matrix, ECM)主要组成弹性纤维和胶原纤维的蛋白比较、移植细胞体内融合实验等分析平滑肌前体细胞恢复尿道功能的作用机制。方法：1、根据终末观察时间的不同将150只产后SD大鼠随机分为三个独立实验：1周短期观察实验、4周和8周长期观察实验。每个实验中,大鼠随机分为对照组、尿道松解组、耻骨-尿道韧带损伤(Pubo-urethral ligament injury, PULI)组和多次阴道球囊扩张组(Multiple vaginal distention, MVD)。除对照组及1周短期实验中的尿道松解组和PULI组外,其余组均在手术的同时附加了双侧输卵管卵巢切除术(Bilateral salpingo-oophorectomy, BSO)。1周短期观察实验通过Crede法对三个时间点进行漏尿点压力(Leak point pressure, LPP)测试：术前、术后立即和术后一周。4周和8周长期观察实验通过Crede法和垂直-倾斜板(vertical tilt tabel, VT)法在术后4周和8周的终末时间点对LPP进行测试。术后8周LPP测试后收集大鼠的尿道进行组织学分析。2、108只成年未经产的Rowett裸鼠随机分为四组：对照组(无干预)、生理盐水组(手术+生理盐水注射)、膀胱平滑肌细胞(Bladder smooth muscle cells, BSMC)组(手术+人BSMC注射)、平滑肌前体细胞(pSMCs)处理组(手术+pSMCs注射,包括人胚胎干细胞H9、游离型载体介导重编程的iPSC (Episomal reprogrammed induced pluripotent stem cells, Epi-iPSC)、逆转录病毒介导重编程的iPSC分化而来的pSMCs)。根据第一部分实验得出的更好的SUI建模方式对Rowett nude (RNU)大鼠进行手术。手术后3周,在尿道周围注射分化好的平滑肌前体细胞(2×106个细胞/只)。注射5周后测试LPP和尿道外周括约肌EMG基线。3、对H9-pSMCs实验中的四组(正常对照组、生理盐水组、BSMC组、H9-pSMCs组)的尿道组织,进行平滑肌特异性蛋白、骨骼肌肌动蛋白双重免疫荧光染色,评价尿道括约肌肌层的变化；进行弹性纤维、胶原蛋白双重染色,评价尿道ECM结构的变化；RT-qPCR检测大鼠尿道组织中的人弹性蛋白、人ERV-3,判断H9-pSMC是否在大鼠体内大量增殖及分化；Western Blot比较四组大鼠尿道组织中的弹性蛋白、胶原蛋白Ⅲ含量,对组织学结果进行验证；在5只SCID小鼠单侧后肢长收肌注射荧光素标记的1×106H9-pSMCs,进行体内生物信号BLI追踪,并对注射部位的组织进行切片染色检测pSMCs移植后的融合情况。结果：1、在1周观察实验中,尿道松解组术后立即(33.69±2.27 cmH2O)和术后1周时的LPP (39.34±2.08cmH2O)与术前(50.40±1.99cmH2O) (p0.05)相比显著降低。MVD+BSO组的术后一周LPP (35.17±1.76 cmH2O)与术前(49.39±4.44 cmH2O) (p0.05)相比也有同样的结果;在4周观察实验中,各组与对照组相比均没有显著差异；而在术后8周时,仅有尿道松解组经VT法测得的的LPP与对照组相比显著降低(28.26±1.11 vs 38.89±3.91 cmH20) (P0.05),其余组LPP均无差异。组织学结果也证实术后8周时,尿道松解组尿道中的弹力纤维和胶原纤维破坏得最为严重。2、iPSC分化而来的平滑肌前体细胞处理组,不论是游离型载体方法重编程的iPSC(N=9, mean=19.4±1.33 cm H20),还是逆转录病毒方法重编程的iPSC(N=8, mean= 18.45± 1.41cm H20),与生理盐水组(N=14, mean=14.16±1.07 cm H2O)(p0.05)相比,LPP显著升高,与尿道功能恢复相一致。而H9分化的平滑肌细胞处理组的LPP(N=22, mean=16.66±0.74 cm H2O)与生理盐水组(N=10, mean=15.52±1.09 cm H2O)的差异并不显著,但仍然显示出了其LPP向正常对照组的LPP水平(N=13,mean=18.75±0.96 cm H2O)靠拢的发展趋势。3、尿道组织学显示H9-pSMCs处理的大鼠尿道与生理盐水组相比具有更丰富的弹性纤维和更厚的肌层。Western blot结果证实H9-pSMCs处理组的大鼠尿道和膀胱中的弹性蛋白、胶原蛋白含量显著增加。人弹性蛋白的基因表达在大鼠尿道组织中未检测到,说明细胞外基质的合成是来源于大鼠自身组织,而不是移植的人类细胞。严重联合免疫缺陷(Severe combined immunodeficiency, SCID)小鼠的体内pSMCs生物信号追踪和免疫荧光染色结果证明pSMCs可以长期存活,并融合入SCID小鼠组织且表达平滑肌细胞表型。结论：尿道松解术方法建立的SUI大鼠模型较为稳定,更适宜干细胞移植研究；人多能干细胞(hESC和iPSC)分化而来的平滑肌前体细胞可以恢复括约肌功能,这一发现为多能干细胞分化大量平滑肌细胞用于泌尿系统的功能恢复提供了可能性；H9-pSMC的尿道周移植能够促进大鼠受损尿道的结构重建,其作用机制包括通过旁分泌调节ECM代谢、刺激自身括约肌修复、pSMC可能增殖并整合入再生的括约肌肌层。意义：本研究首次揭示人多能干细胞(H9 hESC、iPSCs、Epi-iPSCs)来源的pSMCs能够恢复SUI大鼠的尿道功能。iPSCs来自自体细胞,能够减少宿主免疫排斥反应,同时也避免了hESCs应用的伦理问题。本研究使用的iPSCs有两种诱导方式,一种是通过逆转录病毒,这种方式可以使病毒颗粒整合到宿主基因组中,导致基因突变,因而不能应用于临床,另一种是通过非整合方式诱导完成的Epi-iPSCs,对人体相对无害。因此,Epi-iPSCs来源的pSMCs非常有希望在SUI治疗的实际临床中得到应用。另外,我们通过对H9-pSMC的作用机制研究发现,pSMC主要通过某种旁分泌作用调节大鼠自身尿道的ECM代谢,帮助受损尿道括约肌的修复,也不排除pSMC分化增殖并整合入大鼠的尿道括约肌层。
[Abstract]:Background: stress urinary incontinence (Stress urinary incontinence, SUI) refers to the involuntary urine leakage in coughing, sneezing, and laughing,.SUI is the most common type of urinary incontinence in women, especially in old women's.SUI symptoms often occurring in daily activities, which seriously affects the quality of life and social work of the patients. The pathogenesis of mental and mental burden.SUI is not clear, but the urethral intrinsic sphincter dysfunction (Intrinsic sphincter deficiency, ISD) is an important factor causing SUI,.ISD is mainly caused by the injury of the smooth muscle layer or the rhabdomyosus layer of the urethral sphincter or its dominant nerve. The SUI patients with ISD are usually more serious. The symptoms of urinary incontinence and poor response to conventional treatment. Surgical treatment must be performed when conservative treatment fails. The preferred surgical approach, such as the middle urethral suspension, is only an increase in support for urethra, fixed, and does not involve tissue reconstruction of the injured or aged urethral sphincter. The success rate is about 40-50%, and a large number of patients have recurrence. The limitations of the operation and the progressive deterioration of the smooth muscle layer of the urethral sphincter are considered to be the cause of postoperative recurrence. Therefore, the effective recovery of the damaged urethral sphincter is an important treatment for the need to explore. The potential of self renewal and multidirectional differentiation can be applied to the rebirth of the urethral sphincter in SUI patients. This has aroused great interest in the research field. At present, the existing adult stem cells (muscle sources, urinary sources, fat sources, bone marrow sources, etc.) have shown the recovery of urination function and histology in SUI animal research or bed study. However, there are many problems in the clinical application of the adult stem cells, such as the limited number of autologous stem cells, the long preparation time of the cells, the mixed cell types, the epigenetic changes and so on. These obstacles are not conducive to the optimization of the effect of stem cells, but also affect us. In order to avoid these problems, human embryonic stem cells or induced pluripotent stem cells may be a new source of stem cells in order to avoid these problems. In addition, the current stem cell experiments mainly repair the external sphincter of the urethra (rhabdomyus muscle), and have not yet been regenerated only for the inner sphincter of the urethra (smooth muscle). The researchers found that the most widely used adult mesenchymal stem cells used in the SUI study could differentiate into smooth muscle cells in a small fraction of the body, and whether the transplanted mesenchymal stem cells are mixed with a variety of cell types, and whether the differentiated smooth muscle cells can promote the reconstruction of the urethral sphincter function and have a good effect on the reconstruction of the urethral sphincter. Objective: we try to explore the effect and mechanism of the single smooth muscle precursor cells (Progenitor smooth muscle cells, pSMCs) on the reconstruction of the urethra function of the SUI animal model of human embryonic stem cells and human induced pluripotent stem cells (Induced pluripotent stem cells, iPSCs). The modeling effect of different SUI rat modeling methods was compared in order to find a more stable and more suitable long-term SUI model for stem cell transplantation research. On this basis, the differentiated pSMCs was tested by LPP and the baseline of the Electromyography (EMG) electromyography (Electromyography, EMG) after the transplantation of the SUI rat to the urethra for a period of time. To determine the recovery of urethral function. According to the histological examination of the urethral sphincter, Extracellular matrix (ECM) mainly consists of the protein comparison between the elastic fiber and the collagen fibers. The mechanism of the restoration of the urethra function by the smooth muscle precursor cells in the body of the transplanted cells is analyzed. Methods: 1, according to the end observation time 150 postpartum SD rats were randomly divided into three independent experiments: 1 weeks short term observation, 4 weeks and 8 weeks. The rats were randomly divided into control group, urethral release group, pubis urethral ligament injury (Pubo-urethral ligament injury, PULI) group and multiple vaginal balloon dilatation group (Multiple vaginal distenti). On, MVD). Except for the control group and the 1 week short term experimental urethral loosening group and the PULI group, the other groups were performed simultaneously with bilateral fallopian tube oophorectomy (Bilateral salpingo-oophorectomy, BSO) in the short term observation experiment of.1 weeks by Crede method at three time points (Leak point pressure, LPP) test: pre operation Crede and vertical tilt tabel (VT) methods were used to test LPP at the end time of 4 and 8 weeks after the operation at.4 weeks and 8 weeks after the operation. The urethra of rats was collected after 8 weeks of LPP test, and the histological analysis of the urethra of rats was collected, and the Rowett nude mice of.2108 adulthood were randomly divided into four groups Control group (no intervention), saline group (operation + physiological saline injection), Bladder smooth muscle cells (BSMC) group (operation + BSMC injection), smooth muscle precursor cell (pSMCs) treatment group (operation +pSMCs injection, including H9 in human embryonic stem cells, iPSC of free vector mediated reprogramming iPSC (Episomal reprogrammed) Ed pluripotent stem cells, Epi-iPSC), pSMCs from iPSC differentiated by retrovirus mediated reprogramming. A better SUI modeling method based on the first part of the first experiment was performed on Rowett nude (RNU) rats. 3 weeks after the operation, well differentiated smooth muscle precursor cells (2 x 106 cells / only) were injected around the urethra. After 5 weeks of injection, the test was measured. The urethral tissue of four groups (normal control group, saline group, BSMC group, H9-pSMCs group) was tested for LPP and EMG baseline of peripheral peripheral sphincter of urethra. Double immunofluorescence staining of smooth muscle and skeletal muscle actin was carried out in four groups of H9-pSMCs experiments (normal control group, saline group, BSMC group, H9-pSMCs group), and the changes of urethral sphincter muscle layer were evaluated; elastic fiber and collagen double staining were performed. To evaluate the changes in ECM structure of urethra; RT-qPCR detection of human elastin in urethral tissue of rats and human ERV-3 to determine whether H9-pSMC proliferates and differentiation in rats; Western Blot compares the elastin and collagen III content in the urethra tissue of four groups of rats to verify the results of the histology; in the unilateral hind limbs of the 5 SCID mice The long adductor muscle was injected with fluorescein 1 x 106H9-pSMCs, and the biological signal BLI was traced in vivo, and the tissue of the injection site was stained to detect the fusion of pSMCs after pSMCs transplantation. Results: 1, in the 1 week observation experiment, the urethral loosening group was immediately (33.69 + 2.27 cmH2O) and LPP (39.34 + 2.08cmH2O) and 1 weeks after the operation (50.40. LPP (35.17 + 1.76 cmH2O) compared with the preoperative (49.39 + 4.44 cmH2O) (49.39 + 4.44 cmH2O) (P0.05) in the group of.MVD+BSO group was also compared with that of the control group. In the 4 week observation experiment, there was no significant difference between the groups and the control group. At the 8 weeks after the operation, only the LPP and the control group of the urethral release group were measured by VT method. Compared with a significant reduction (28.26 + 1.11 vs 38.89 + 3.91 cmH20) (P0.05), no difference was found in the rest of the other groups. The histological results also confirmed that at 8 weeks after the operation, the elastic and collagen fibers in the urethra urethra group were most severely damaged by.2, iPSC differentiated smooth muscle precursor cells, whether the free vector method was reprogrammed iPSC. N=9, mean=19.4 + 1.33 cm H20), or retroviral reprogramming iPSC (N=8, mean= 18.45 + 1.41cm H20), compared with the physiological saline group (N=14, mean=14.16 + 1.07 cm), which was significantly higher and consistent with the recovery of urethra function. The difference in the saline group (N=10, mean=15.52 + 1.09 cm H2O) was not significant, but it still showed the trend towards the LPP level of the normal control group (N=13, mean=18.75 + 0.96 cm H2O), and the urethra histology showed that the urethra treated with the H9-pSMCs treatment had a richer elastic fiber and a thicker muscularis than the saline group. The results of tern blot confirmed that the elastin in the urethra and bladder of the H9-pSMCs treatment group increased significantly. The gene expression of human elastin was not detected in the urethral tissue of rats, indicating that the synthesis of extracellular matrix was derived from the rat's own tissues, not the transplanted human cells. Sev was a serious combined immunodeficiency (Sev). The results of pSMCs biological signal tracing and immunofluorescence staining in ere combined immunodeficiency, SCID mice showed that pSMCs could survive for a long time and fused into SCID mouse tissues and expressed the phenotype of smooth muscle cells. Conclusion: the SUI rat model established by urethral release method is more stable, more suitable for stem cell transplantation, and human multiple energy. The differentiation of stem cells (hESC and iPSC) to smooth muscle precursor cells can restore the function of the sphincter. This discovery provides the possibility for the pluripotent stem cells to differentiate a large number of smooth muscle cells for the functional recovery of the urinary system; H9-pSMC's pericurethral transplantation can promote the reconstruction of the damaged urinary tract in rats. The secretion of ECM metabolism and the stimulation of its own sphincter repair, pSMC may proliferate and integrate into the regenerated sphincter myometrium. Significance: This study revealed for the first time that pSMCs from human pluripotent stem cells (H9 hESC, iPSCs, Epi-iPSCs) can restore the urethral function of SUI rats from self body cells, which can reduce immune rejection in the host, and also to reduce the immune rejection of the host. The ethical problems of hESCs application are avoided. There are two ways to induce iPSCs in this study. One is through retrovirus, which can integrate virus particles into the host genome, causing gene mutation, which can not be applied to the clinic, the other is to induce the completed Epi-iPSCs by non integration, and is relatively harmless to the human body. Therefore, the pSMCs source of Epi-iPSCs is very promising to be used in the practical clinical treatment of SUI. In addition, through a study of the mechanism of action of H9-pSMC, we have found that pSMC regulates the ECM metabolism of the rat's own urethra by some paracrine effect, helps to repair the damaged urethral sphincter, and does not exclude the pSMC differentiation and proliferation and integration into the urethra. The urethral sphincter layer of the rat.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.59

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