發(fā)布時間：2018-01-12 14:21

  本文關鍵詞：人臍血間充質(zhì)干細胞成肌分化及對尿道括約肌功能影響的實驗研究　出處：《第三軍醫(yī)大學》2007年博士論文　論文類型：學位論文

  更多相關文章： 壓力性尿失禁 臍血 間充質(zhì)干細胞 Myocardin 誘導 分化 基因治療 平滑肌 尿動力學 細胞移植

【摘要】： 女性壓力性尿失禁(Stress Urinary Incontinence,SUI)是一種常見疾病,其發(fā)生率隨婦女年齡的增加而增加,影響婦女的生活質(zhì)量和身心健康。SUI發(fā)生的主要原因是尿道高移動性和尿道括約肌功能障礙。女性SUI的治療方法雖多,但均難從根本上糾正尿道括約肌功能障礙。本課題擬利用基因工程技術將生肌調(diào)節(jié)因子Myocardin基因轉(zhuǎn)染人臍血間充質(zhì)干細胞(UB-MSCs),并移植于SUI模型大鼠尿道括約肌周圍,研究基因調(diào)控的UB-MSCs是否具有向平滑肌細胞分化的能力以及其在尿道括約肌周圍存活、分布情況和對尿道括約肌功能的影響,為細胞移植治療SUI提供理論依據(jù)。 主要實驗結果和結論如下: 1.采用密度梯度離心法從臍血中分離培養(yǎng)出間充質(zhì)干細胞(UB-MSCs),并通過流式細胞儀對其表面抗原進行鑒定。UB-MSCs均穩(wěn)定地表達相關的抗原標記CD29、CD44、CD105,但不表達造血細胞系的表面標記CD34、CD45,這與源于骨髓MSCs的表面抗原標記相一致。 2.構建了真核表達質(zhì)粒載體pEGFP-Myocl。以pAdApt.Myocl為模板進行PCR和瓊脂凝膠電泳,切膠回收純化;反應產(chǎn)物經(jīng)KpnⅠ和AgeⅠ雙酶切后與線性化的載體用T4 DNA連接酶連接,形成pEGFP-Myocl,將之轉(zhuǎn)入工程菌JM109,鋪板,挑選陽性克隆,搖菌,提取質(zhì)粒。目的片段測序分析,與Gene Bank報道序列完全一致。 3.應用脂質(zhì)體轉(zhuǎn)染的方法,將pEGFP-Myocl轉(zhuǎn)入UB-MSCs中。轉(zhuǎn)染后的細胞經(jīng)G_(418)篩選,轉(zhuǎn)染效率為15.3%。轉(zhuǎn)染后的MSCs用RT-PCR可檢測出Myocardin的表達;熒光顯微鏡下觀察可見胞體內(nèi)有報告基因產(chǎn)物的綠色熒光;免疫組化檢測Myocardin以及其下游蛋白myosin表達為陽性,而對照組為陰性表達。這說明UB-MSCs在Myocardin的調(diào)控下可誘導為成平滑肌細胞。 4.通過模擬產(chǎn)傷和卵巢切除成功制作SUI大鼠動物模型。對模型大鼠進行尿動力學檢測,最大膀胱容量和漏點壓均明顯低于正常;HE染色可見近端尿道括約肌萎縮,肌纖維有斷裂。 5.將轉(zhuǎn)染Myocardin基因的UB-MSCs擴增后局部植入模型鼠尿道括約肌周圍,通過Brdu法觀察發(fā)現(xiàn)其可在注射點附近短時生長,并表達肌相關蛋白Desmin;通過尿動力學指標的統(tǒng)計分析,細胞移植后SUI模型鼠的控尿功能可以得到改善,但此效應是移植細胞對損傷的修復還是單純的填充效應,還需進一步研究。 總之,本實驗成功地構建了質(zhì)粒載體pEGFP-Myocl,并將其成功地轉(zhuǎn)染人UB-MSCs,使MSCs在Myocardin的調(diào)控下分化為成平滑肌細胞;并觀察了向平滑肌分化的MSCs對SUI模型大鼠的尿道括約肌周圍的分布、分化與生存情況以及對尿道括約肌功能的影響,初步探討了細胞移植治療SUI的可行性。本實驗為SUI治療的基礎研究,旨在為SUI治療提供新的思路。
[Abstract]:Female stress urinary incontinence (Stress Urinary, Incontinence, SUI) is a common disease, its incidence with age increasing, the main reason for women's health and life quality of.SUI is of high mobility and urethral sphincter dysfunction. Although there are many methods to treat the female SUI, but are difficult to fundamentally correct the urethral sphincter dysfunction. This project intends to use the technology of gene engineering myogenic regulatory factor Myocardin gene transfection of human umbilical cord blood mesenchymal stem cells (UB-MSCs), and transplanted into SUI rat model of urethral sphincter surrounding, the research of gene regulation whether UB-MSCs can differentiate into smooth muscle cells and its ability to survive in the urethral sphincter around the effect of the distribution, and urethral sphincter function, provide a theoretical basis for cell transplantation in the treatment of SUI.
The main experimental results and conclusions are as follows:
1. by density gradient centrifugation. Mesenchymal stem cells isolated from human umbilical cord blood (UB-MSCs), and were identified by flow cytometry for.UB-MSCs surface antigen for stable expression of antigen CD29, related CD44, CD105, surface marker CD34, but did not express hematopoietic cell line CD45, consistent surface this antigen from the bone marrow of MSCs.
2. to construct the eukaryotic expression plasmid vector pEGFP-Myocl. with pAdApt.Myocl as the template for PCR and agar gel electrophoresis, gel extraction purification; carrier reaction products by Kpn I and Age I digested and linearized by T4 DNA ligase, the formation of pEGFP-Myocl, will be transferred to the engineering bacteria JM109, plank, positive clones, shake bacteria, plasmid extraction. Analysis of DNA sequencing, and Gene Bank consistent with the sequence reported.
Methods 3. application of liposome transfection, pEGFP-Myocl was transformed into UB-MSCs. After transfection, the cells were treated with G_ (418) screening, the transfection efficiency was 15.3%. after transfection with MSCs RT-PCR can detect the expression of Myocardin; observation of green fluorescence reporter gene product in vivo were observed under fluorescence microscope; immunohistochemical detection of Myocardin and its the lower expression of myosin protein was positive, but negative in control group. This shows that UB-MSCs can be induced into smooth muscle cells under the control of Myocardin.
4., SUI rat model was successfully produced by simulating birth injury and ovariectomy. The maximal bladder volume and leak point pressure of model rats were significantly lower than those of normal rats. HE staining revealed proximal urethral sphincter atrophy and muscle fibers breaking.
Around 5. the transfected Myocardin gene was amplified by UB-MSCs after implantation of rat model of urethral sphincter, by the method of Brdu were observed in the growth in the short-term near injection points and the expression of muscle related protein Desmin; urodynamic indexes through statistical analysis, SUI model can be improved in the control of urinary function after transplantation, but this effect is repair of transplanted cells to injury or simply filling effect, further research is needed.
In conclusion, this study successfully constructed pEGFP-Myocl plasmid, and successfully transfected into UB-MSCs, MSCs under the regulation of Myocardin differentiation into smooth muscle cells; and to observe the differentiation of MSCs into smooth muscle around the urethral sphincter of SUI rat model of the distribution, differentiation and survival situation and effect on urethral sphincter the function, discussed the feasibility of cell transplantation for the treatment of SUI. Based on this experiment for the treatment of SUI, in order to provide new ideas for the treatment of SUI.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R329

【參考文獻】

相關期刊論文 前5條

1 鄧黎,梁志清;壓力性尿失禁動物模型的建立[J];重慶醫(yī)學;2004年05期

2 羅凱,單根法,鐘z，

本文編號：1414627