發(fā)布時間：2018-05-24 22:13

  本文選題：多能 + 干細(xì)胞　； 參考：《南方醫(yī)科大學(xué)》2016年博士論文

【摘要】：背景：壓力性尿失禁(Stress urinary incontinence, SUI)是指在咳嗽、噴嚏、大笑等腹壓增高時出現(xiàn)的不自主地尿液滲漏。SUI是女性最為常見的尿失禁類型,尤其是老年婦女。SUI癥狀經(jīng)常發(fā)生在日常活動中,因此嚴(yán)重影響患者的生活質(zhì)量和社交活動,亦帶來心理精神負(fù)擔(dān)。SUI發(fā)病機制尚不明確,但尿道固有括約肌功能不全(Intrinsic sphincter deficiency, ISD)是導(dǎo)致SUI的重要因素。ISD主要由于尿道括約肌的平滑肌層或橫紋肌層或其支配神經(jīng)出現(xiàn)損傷引起。具有ISD的SUI患者通常有更嚴(yán)重的尿失禁癥狀,且對常規(guī)的治療反應(yīng)不佳。當(dāng)保守治療失敗后,必須進(jìn)行手術(shù)治療。目前首選的手術(shù)治療方式,如中段尿道懸吊術(shù)等,僅是增加對尿道的支持、固定作用,并不涉及對損傷或老化的尿道括約肌進(jìn)行組織重建。而這類手術(shù)的長期成功率約為40-50%,有很大一部分患者會出現(xiàn)術(shù)后復(fù)發(fā)的情況。手術(shù)的局限性及逐漸進(jìn)展的尿道括約肌平滑肌層老化衰退被認(rèn)為是導(dǎo)致術(shù)后復(fù)發(fā)的原因。因此,有效恢復(fù)受損的尿道括約肌是需要積極探索的重要治療方式。干細(xì)胞因其具備自我更新和多向分化的潛能,可應(yīng)用于SUI患者的尿道括約肌再生,這引起了研究領(lǐng)域的極大興趣。目前,已有成體干細(xì)胞(肌肉來源、泌尿系來源、脂肪來源、骨髓來源等)在SUI動物研究或臨床研究中表現(xiàn)出在排尿功能的恢復(fù)、組織學(xué)的改善方面有較好的效果。但是,這些成體干細(xì)胞的臨床應(yīng)用仍存在眾多問題,如提取的自體干細(xì)胞數(shù)量有限、細(xì)胞擴增準(zhǔn)備時間過長、移植細(xì)胞為多種細(xì)胞類型混合、攜帶表觀遺傳改變等等。這些障礙不利于干細(xì)胞療效的優(yōu)化,也影響了我們對干細(xì)胞具體作用機制進(jìn)行深入研究。因此,為避免上述問題,人胚胎干細(xì)胞或誘導(dǎo)多能干細(xì)胞或許可以成為新的干細(xì)胞來源。另外,現(xiàn)階段的干細(xì)胞實驗主要修復(fù)的是尿道外括約肌(橫紋肌),尚未有僅針對尿道內(nèi)括約肌(平滑肌)再生的研究報道。研究人員發(fā)現(xiàn),在SUI研究中應(yīng)用最廣的成體間充質(zhì)干細(xì)胞在體內(nèi)僅有一小部分能夠分化為平滑肌細(xì)胞,而由于成體間充質(zhì)干細(xì)胞移植混雜有多種細(xì)胞類型,分化而來的平滑肌細(xì)胞是否對尿道括約肌功能重建有促進(jìn)作用及有關(guān)機制都無法明確。目的：我們試圖探索人胚胎干細(xì)胞和人誘導(dǎo)多能干細(xì)胞(Induced pluripotent stem cells, iPSCs)分化而來的單一平滑肌前體細(xì)胞(Progenitor smooth muscle cells, pSMCs)對重建SUI動物模型尿道功能的作用及機制。首先,通過在不同時間點對不同SUI大鼠建模方式的建模效果進(jìn)行比較,以期找到更穩(wěn)定更適宜干細(xì)胞移植研究的長期SUI模型。在此基礎(chǔ)上,分化而來的pSMCs在移植入SUI大鼠尿道周圍一段時間后,根據(jù)LPP和尿道外周括約肌肌電圖(Electromyography, EMG)基線的測試,判斷尿道功能的恢復(fù)情況。根據(jù)尿道括約肌組織學(xué)檢查、細(xì)胞外基質(zhì)(Extracellular matrix, ECM)主要組成彈性纖維和膠原纖維的蛋白比較、移植細(xì)胞體內(nèi)融合實驗等分析平滑肌前體細(xì)胞恢復(fù)尿道功能的作用機制。方法：1、根據(jù)終末觀察時間的不同將150只產(chǎn)后SD大鼠隨機分為三個獨立實驗：1周短期觀察實驗、4周和8周長期觀察實驗。每個實驗中,大鼠隨機分為對照組、尿道松解組、恥骨-尿道韌帶損傷(Pubo-urethral ligament injury, PULI)組和多次陰道球囊擴張組(Multiple vaginal distention, MVD)。除對照組及1周短期實驗中的尿道松解組和PULI組外,其余組均在手術(shù)的同時附加了雙側(cè)輸卵管卵巢切除術(shù)(Bilateral salpingo-oophorectomy, BSO)。1周短期觀察實驗通過Crede法對三個時間點進(jìn)行漏尿點壓力(Leak point pressure, LPP)測試：術(shù)前、術(shù)后立即和術(shù)后一周。4周和8周長期觀察實驗通過Crede法和垂直-傾斜板(vertical tilt tabel, VT)法在術(shù)后4周和8周的終末時間點對LPP進(jìn)行測試。術(shù)后8周LPP測試后收集大鼠的尿道進(jìn)行組織學(xué)分析。2、108只成年未經(jīng)產(chǎn)的Rowett裸鼠隨機分為四組：對照組(無干預(yù))、生理鹽水組(手術(shù)+生理鹽水注射)、膀胱平滑肌細(xì)胞(Bladder smooth muscle cells, BSMC)組(手術(shù)+人BSMC注射)、平滑肌前體細(xì)胞(pSMCs)處理組(手術(shù)+pSMCs注射,包括人胚胎干細(xì)胞H9、游離型載體介導(dǎo)重編程的iPSC (Episomal reprogrammed induced pluripotent stem cells, Epi-iPSC)、逆轉(zhuǎn)錄病毒介導(dǎo)重編程的iPSC分化而來的pSMCs)。根據(jù)第一部分實驗得出的更好的SUI建模方式對Rowett nude (RNU)大鼠進(jìn)行手術(shù)。手術(shù)后3周,在尿道周圍注射分化好的平滑肌前體細(xì)胞(2×106個細(xì)胞/只)。注射5周后測試LPP和尿道外周括約肌EMG基線。3、對H9-pSMCs實驗中的四組(正常對照組、生理鹽水組、BSMC組、H9-pSMCs組)的尿道組織,進(jìn)行平滑肌特異性蛋白、骨骼肌肌動蛋白雙重免疫熒光染色,評價尿道括約肌肌層的變化；進(jìn)行彈性纖維、膠原蛋白雙重染色,評價尿道ECM結(jié)構(gòu)的變化；RT-qPCR檢測大鼠尿道組織中的人彈性蛋白、人ERV-3,判斷H9-pSMC是否在大鼠體內(nèi)大量增殖及分化；Western Blot比較四組大鼠尿道組織中的彈性蛋白、膠原蛋白Ⅲ含量,對組織學(xué)結(jié)果進(jìn)行驗證；在5只SCID小鼠單側(cè)后肢長收肌注射熒光素標(biāo)記的1×106H9-pSMCs,進(jìn)行體內(nèi)生物信號BLI追蹤,并對注射部位的組織進(jìn)行切片染色檢測pSMCs移植后的融合情況。結(jié)果：1、在1周觀察實驗中,尿道松解組術(shù)后立即(33.69±2.27 cmH2O)和術(shù)后1周時的LPP (39.34±2.08cmH2O)與術(shù)前(50.40±1.99cmH2O) (p0.05)相比顯著降低。MVD+BSO組的術(shù)后一周LPP (35.17±1.76 cmH2O)與術(shù)前(49.39±4.44 cmH2O) (p0.05)相比也有同樣的結(jié)果;在4周觀察實驗中,各組與對照組相比均沒有顯著差異；而在術(shù)后8周時,僅有尿道松解組經(jīng)VT法測得的的LPP與對照組相比顯著降低(28.26±1.11 vs 38.89±3.91 cmH20) (P0.05),其余組LPP均無差異。組織學(xué)結(jié)果也證實術(shù)后8周時,尿道松解組尿道中的彈力纖維和膠原纖維破壞得最為嚴(yán)重。2、iPSC分化而來的平滑肌前體細(xì)胞處理組,不論是游離型載體方法重編程的iPSC(N=9, mean=19.4±1.33 cm H20),還是逆轉(zhuǎn)錄病毒方法重編程的iPSC(N=8, mean= 18.45± 1.41cm H20),與生理鹽水組(N=14, mean=14.16±1.07 cm H2O)(p0.05)相比,LPP顯著升高,與尿道功能恢復(fù)相一致。而H9分化的平滑肌細(xì)胞處理組的LPP(N=22, mean=16.66±0.74 cm H2O)與生理鹽水組(N=10, mean=15.52±1.09 cm H2O)的差異并不顯著,但仍然顯示出了其LPP向正常對照組的LPP水平(N=13,mean=18.75±0.96 cm H2O)靠攏的發(fā)展趨勢。3、尿道組織學(xué)顯示H9-pSMCs處理的大鼠尿道與生理鹽水組相比具有更豐富的彈性纖維和更厚的肌層。Western blot結(jié)果證實H9-pSMCs處理組的大鼠尿道和膀胱中的彈性蛋白、膠原蛋白含量顯著增加。人彈性蛋白的基因表達(dá)在大鼠尿道組織中未檢測到,說明細(xì)胞外基質(zhì)的合成是來源于大鼠自身組織,而不是移植的人類細(xì)胞。嚴(yán)重聯(lián)合免疫缺陷(Severe combined immunodeficiency, SCID)小鼠的體內(nèi)pSMCs生物信號追蹤和免疫熒光染色結(jié)果證明pSMCs可以長期存活,并融合入SCID小鼠組織且表達(dá)平滑肌細(xì)胞表型。結(jié)論：尿道松解術(shù)方法建立的SUI大鼠模型較為穩(wěn)定,更適宜干細(xì)胞移植研究；人多能干細(xì)胞(hESC和iPSC)分化而來的平滑肌前體細(xì)胞可以恢復(fù)括約肌功能,這一發(fā)現(xiàn)為多能干細(xì)胞分化大量平滑肌細(xì)胞用于泌尿系統(tǒng)的功能恢復(fù)提供了可能性；H9-pSMC的尿道周移植能夠促進(jìn)大鼠受損尿道的結(jié)構(gòu)重建,其作用機制包括通過旁分泌調(diào)節(jié)ECM代謝、刺激自身括約肌修復(fù)、pSMC可能增殖并整合入再生的括約肌肌層。意義：本研究首次揭示人多能干細(xì)胞(H9 hESC、iPSCs、Epi-iPSCs)來源的pSMCs能夠恢復(fù)SUI大鼠的尿道功能。iPSCs來自自體細(xì)胞,能夠減少宿主免疫排斥反應(yīng),同時也避免了hESCs應(yīng)用的倫理問題。本研究使用的iPSCs有兩種誘導(dǎo)方式,一種是通過逆轉(zhuǎn)錄病毒,這種方式可以使病毒顆粒整合到宿主基因組中,導(dǎo)致基因突變,因而不能應(yīng)用于臨床,另一種是通過非整合方式誘導(dǎo)完成的Epi-iPSCs,對人體相對無害。因此,Epi-iPSCs來源的pSMCs非常有希望在SUI治療的實際臨床中得到應(yīng)用。另外,我們通過對H9-pSMC的作用機制研究發(fā)現(xiàn),pSMC主要通過某種旁分泌作用調(diào)節(jié)大鼠自身尿道的ECM代謝,幫助受損尿道括約肌的修復(fù),也不排除pSMC分化增殖并整合入大鼠的尿道括約肌層。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.59

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