脂肪间充质干细胞治疗压力性尿失禁的实验研究
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摘要
目的：本研究在体外通过改良两步酶消化法结合差速贴壁技术分离ADSCs并标记,体外定向诱导ADSCs分化为成肌细胞。将ADSC移植至SUI大鼠的近端尿道,检测了ADSCs移植后体内形态和分布以及尿动力学变化,及尿道及其周围组织形态学改变,观察间充质干细胞移植治疗压力性尿失禁大鼠是否有效可行,为压力性尿失禁的组织工程学治疗奠定基础。
方法：1、采用改良两步酶消化法结合差速贴壁技术分离ADSCs,对连续传代细胞进行计数并描绘生长曲线；收集P3细胞,进行细胞表面抗原流式细胞术鉴定及成脂、成骨定向分化功能鉴定；2.以携带绿色荧光蛋白(GFP)的慢病毒载体系统作为基因转导的媒介对ADSCs进行转染。3、选用第2代ADSCs,经5-氮杂胞苷体外定向向成肌细胞诱导分化。在倒置显微镜、透射电镜下及激光共聚焦显微成像下观察细胞形态学和超微结构。采用免疫细胞化学对诱导前后细胞进行Desmin检测,采用RT-PCR和Western Blot方法对诱导后细胞sarcomeric、Desmin基因mRNA和蛋白表达的检测。4、以双侧阴部神经离断的方法,制备大鼠SUI动物模型。模拟人体尿动力学检查的方法,测定实验动物的最大膀胱容量(MBV)、腹压漏尿点压(ALPP)、最大尿道闭合压(MUCP)、能性尿道长度(FUL)各项指标,证实SUI动物模型制作成功。并通过HE染色、Masson特殊染色、Mallory特殊染色、电镜超微结构对大鼠尿道横纹肌性括约肌的显微解剖进行研究。5、将建模成功的33只大鼠分成ADSCs移植治疗组和培养基注射对照组,以5×106个细胞／只的细胞数移植注射到大鼠尿道中下段3、9、12点。移植2周后进行尿动力学检测,评价动物尿动力学变化情况。通过HE染色、Masson染色、Mallory染色及对尿道评价细胞移植后尿道周围组织病理变化。同时对横纹肌性括约肌的超微结构的形态学变化观察及采用肌肉分割法评估干细胞治疗后控尿系统的修复。激光共聚焦扫描显微镜观察移植细胞存活情况,以及采用二次谐波(SHG)成像技术探测尿道周围SHG峰值信号,定量评价胶原蛋白含量的变化。
结果：①体外培养的ADSCs主要呈梭形和多角形,细胞表达CD44、CD90,不表达CD34、CD45、CD106;细胞成脂、成骨定向分化诱导后油红-0染色、茜素红染色阳性。倒置相差荧光显微镜下,GFP转染大鼠ADSCs发出绿色荧光,荧光强度随MOI值的增加而增强,但以MOI为8的病毒感染大鼠ADSCs荧光强度最强,传至P3时GFP阳性细胞达82%-86%。②ADSCs经5-Aza等诱导分化后其形态发生改变,细胞大小不一致；在透射电镜、激光共聚焦显微成像系统下观察到,逐渐由长梭形变为多角形及星形,细胞表面有许多微绒毛；细胞器丰富,胞质内可见丰富的线粒体、粗面内质网,胞浆内可见肌丝样结构。诱导后细胞表达结蛋白Desmin。RT—PCR、Western Blot结果证实,诱导细胞结蛋白Desmin基因的表达明显高于对照组(P＜0.05)。③雌性SD大鼠的尿道为一长约2.0 cm的管状结构。其组织结构由外向内依次为环形横纹肌层、环形平滑肌层、纵行平滑肌层、致密结缔组织层和上皮层。双侧阴部神经离断的方法能成功制造大鼠SUI模型。尿动力学检测结果：模型建立后大鼠膀胱最大容量增加(0.89±0.04)ml(P＜0.01),腹压漏尿点压下降(15.46±5.49)cmH2O(P<0.01),最大尿道闭合压和功能性尿道长度亦下降(7.97±3.25)cmH2O、(0.69±0.32)cm(P＜0.01)。建模后尿道及其周围组织形态学观察：括约肌排列松散,部分肌肉有断裂现象,肌层变薄；结缔组织所占比重增加,胶原纤维稀疏,排列紊乱。④尿动力学检测ADSCs移植后尿控功能变化,ADSCs治疗后大鼠膀胱最大容量下降(0.78±0.35)ml,腹压漏尿点压增加(15.06±4.36)cmH2O,最大尿道闭合压和功能性尿道长度亦增加(9.69±2.57)cmH2O、(0.69±0.32)cm,与对照组比较各指标均有显著性意义(P＜0.01)。ADSCs移植治疗后可以使注射局部尿道肌层得到明显的加强和增厚,括约肌形态及功能得到明显的改善,周围结缔组织紧密,支撑作用得到加强。横纹肌肌肉分割分析显示干细胞治疗4周横纹肌肌肉面积及其长短轴比值均得到明显增加。移植治疗后尿道周围的胶原蛋白含量明显高于对照组(P<0.01)。
结论：
1、通过消化分离,percoll密度梯度离心和preplate差速贴壁技术,可以获得纯度较高的SD大鼠ADSCs。本实验使用携带GFP基因重组的慢病毒成功高效地转染了ADSCs,该方法为长期观测ADSCs的体内状况创造了条件。
2、本研究提示：在特定诱导条件下ADSCs能被定向诱导分化为成肌细胞；并为SUI的干细胞移植治疗提供了新的思路。
3、本部分研究通过双侧阴部神经离断的方法,成功建立了SUI的动物模型,为后续实验奠定了基础。尿道横纹括约肌呈封闭环形分布于雌性大鼠尿道的中外段,提示此处为控尿机制的主要部位。
4、ADSCs移植至压力性尿失禁模型大鼠后能存活、分化而整合至宿主尿道括约肌,改善压力性尿失禁大鼠的尿控功能,并且主要可能通过在体内定向分化为肌细胞,增强尿道括约肌系统,并改善尿道粘膜闭合机制及尿道周围组织中胶原蛋白的含量而实现。组织工程学技术的发展为压力性尿失禁的治疗提供了新的思路和手段。
Objective:ADSCs were purified through percoll density gradient centrifugation and preplate technique,and to differentiate rat ADSCs into myoblasts in vitro.Labeled ADSCs will be transplatated into the SUI rats around the posterior urethral.By urodynamics,morphological,transmission electron microscope and multiphoton microscopic imaging changes of urethra and its surrounding tissue.Our objective is to observe whether ADSCs transplant in the treatment of SUI rats is effective and feasible or not and its possible mechanism.Rat ADSCs transplantation can be provided a new way for cell transplantation therapy of stress urinary incontinence.
Methods:1. ADSCs were purified through percoll density gradient centrifugation and preplate technique. ADSCs were further identified by flow cytometry technology for CD44、CD90, CD34、CD45、CD106 antibodies and by adipogenesis,osteogenesis cellular differentiation.2. A lentiviral vector with green fluorescent protein(GFP) was be used to transfection ADSCs.3. The twice passage ADSCs were treated for 24 h with 5-azacytidine (5-Aza, 10μmol-L-1) to induce cellular differentiation. The morphology and ultrastructure of treated ADSCs were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging. The expression of desmin identified by immunohistochemistry, was used to identify myoblasts differentiation.The sarcomeric and Desmin expression were detected with RT-PCR and Western Blot.4. Bilateral pudendal nerve transaction was preparation of rat animal model of SUI. Simulated human body urodynamic method of the maximum bladder capacity of experimental animals (MBV), abdominal leak point pressure (ALPP), maximum urethral closure pressure (MUCP), and the functional length of the urethra (FUL);and HE staining, Masson special stain, Mallory staining and special nature of the urethral sphincter striated muscle ultrastructure in transmission electron microscopy studies confirmed the successful production of animal models of SUI.5. Rats were randomly assigned to transplant Group I (n=16) injected with ADSCs and control GroupⅡ(n=9) injected with culture medium without serum. Urodynamic testing, evaluation of dynamic changes in animal,morphology and ultrastructure of were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging to evaluation the effective and feasible of ADSCs transplantation.
Results:1. Method combining improved two-step enzymatic digestion with preplating technique is a kind of easy and practicable in some degree to obtain relative high purity of ADSCs.The purified ADSCs by limited-dilution appeared fibroblast-like shapes and proliferated rapidly in vitro. Results of flow cytometry showed that ADSCs were positive for CD44 and CD90, and negative for CD34, CD45 and CD106. Florescence intensity increased was companied with MOIs increased,but MOI of 8 fluorescence intensity was the strongest.82%-86%of successively passaged P3 ADSCs were GFP-positive.2. The morphology of ADSCs treated with 5-Aza under inverted microscope and multiphoton microscopic imaging changed from fusiform to polygon or astrocyte. With transmission electron microscope, the ultrastructure of the treated ADSCs was visible.The magnitude of cells was not identical, the surface of some cells had abundance of microvilli. Intracytoplasm had rich organelles, mitochondrium and rough endoplasmic reticulum.The induced ADSCs demonstrated positive staining for desmin by immunohistochemistry.The outcome of RT-PCR and Western Blot manifested that the gene expression of Desmin was up regulated in induced ADSCs.3.The urethral of female SD rat is a tubular structure about 2.0 cm. Its structure includes circular striated muscle layer,circular smooth muscle layer,longitudinal smooth muscle layer,dense connective tissue and epithelium from outside to inside. Bilateral pudendal nerve transection of the method can be successfully produced in rat model of SUI.Compared with those at baseline, the average quantity of maximum bladder volume (MBV) were increased by (0.89±0.04)ml (P<0.01); Abdomen pressure leak point pressure (ALPP) were significantly decreased by (15.46±5.49)cmH2O, respectively (P<0.01).Maximum urethra closure pressure (MUCP) and functional length of the urethra (FUL) were decreased by (7.97±3.25) cmH2O and (0.69±0.32)cm,respectively (P<0.01). Modeling of the posterior urethra and surrounding tissue morphology:sphincter loose arrangement of some muscles fracture phenomenon, muscle thinning; the proportion of connective tissue increase in sparse collagen fibers arranged in disorder.
4. Two weeks after transplantation, by urodynamic testing, compared with the control group all parameters of MBV, ALPP, MUCP, FUL were statistically significant (P <0.01). By ADSCs transplantation,the urethral circular striated muscle layer can be significantly strengthened and thickened, sphincter morphology and function was significantly improved, the surrounding connective tissue close, supportive role to be strengthened. Striated muscle segmentation analysis revealed that ADSCs therapy for 4 weeks the ratio of muscle area and the length of the shaft were to be increased significantly. Collagen contents around the urethra and its surrounding was significantly higher compared with control group(P<0.01).
Conclusion
1. This part study manifests that high purify ADSCs can be obtained through digestion,percoll density gradient centrifugation and preplate technique. Lentiviral vector with green fluorescent protein(GFP) was be successfully used to transfection ADSCs, which creates good condition for long time observation of ADSCs in vivo.
2. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.
3. In this part study,rats models of SUI were obtained by transaction of bilateral pudendal nerve,which established the foundation of extending experiment. The striated urethral sphincter of female rats was considered as a closed ring surrounding the urethral from the middle to the distal. It suggests that this may be the main site of urinary continence mechanism.
4. ADSCs transplanted into rats with stress urinary incontinence can survive, divide and integrated into the host urethral sphincter,and it can be improved the control of urinary function. The possible mechanism of ADSCs may be adopted to differentiate into myoblasts, increased urinary sphincter system, and to improve urethral closure mechanism mucosal achieved. With the development of tissue engineering technology, it provided a new concept and method to treat stress urinary incontinence.
方法：1、采用改良两步酶消化法结合差速贴壁技术分离ADSCs,对连续传代细胞进行计数并描绘生长曲线；收集P3细胞,进行细胞表面抗原流式细胞术鉴定及成脂、成骨定向分化功能鉴定；2.以携带绿色荧光蛋白(GFP)的慢病毒载体系统作为基因转导的媒介对ADSCs进行转染。3、选用第2代ADSCs,经5-氮杂胞苷体外定向向成肌细胞诱导分化。在倒置显微镜、透射电镜下及激光共聚焦显微成像下观察细胞形态学和超微结构。采用免疫细胞化学对诱导前后细胞进行Desmin检测,采用RT-PCR和Western Blot方法对诱导后细胞sarcomeric、Desmin基因mRNA和蛋白表达的检测。4、以双侧阴部神经离断的方法,制备大鼠SUI动物模型。模拟人体尿动力学检查的方法,测定实验动物的最大膀胱容量(MBV)、腹压漏尿点压(ALPP)、最大尿道闭合压(MUCP)、能性尿道长度(FUL)各项指标,证实SUI动物模型制作成功。并通过HE染色、Masson特殊染色、Mallory特殊染色、电镜超微结构对大鼠尿道横纹肌性括约肌的显微解剖进行研究。5、将建模成功的33只大鼠分成ADSCs移植治疗组和培养基注射对照组,以5×106个细胞／只的细胞数移植注射到大鼠尿道中下段3、9、12点。移植2周后进行尿动力学检测,评价动物尿动力学变化情况。通过HE染色、Masson染色、Mallory染色及对尿道评价细胞移植后尿道周围组织病理变化。同时对横纹肌性括约肌的超微结构的形态学变化观察及采用肌肉分割法评估干细胞治疗后控尿系统的修复。激光共聚焦扫描显微镜观察移植细胞存活情况,以及采用二次谐波(SHG)成像技术探测尿道周围SHG峰值信号,定量评价胶原蛋白含量的变化。
结果：①体外培养的ADSCs主要呈梭形和多角形,细胞表达CD44、CD90,不表达CD34、CD45、CD106;细胞成脂、成骨定向分化诱导后油红-0染色、茜素红染色阳性。倒置相差荧光显微镜下,GFP转染大鼠ADSCs发出绿色荧光,荧光强度随MOI值的增加而增强,但以MOI为8的病毒感染大鼠ADSCs荧光强度最强,传至P3时GFP阳性细胞达82%-86%。②ADSCs经5-Aza等诱导分化后其形态发生改变,细胞大小不一致；在透射电镜、激光共聚焦显微成像系统下观察到,逐渐由长梭形变为多角形及星形,细胞表面有许多微绒毛；细胞器丰富,胞质内可见丰富的线粒体、粗面内质网,胞浆内可见肌丝样结构。诱导后细胞表达结蛋白Desmin。RT—PCR、Western Blot结果证实,诱导细胞结蛋白Desmin基因的表达明显高于对照组(P＜0.05)。③雌性SD大鼠的尿道为一长约2.0 cm的管状结构。其组织结构由外向内依次为环形横纹肌层、环形平滑肌层、纵行平滑肌层、致密结缔组织层和上皮层。双侧阴部神经离断的方法能成功制造大鼠SUI模型。尿动力学检测结果：模型建立后大鼠膀胱最大容量增加(0.89±0.04)ml(P＜0.01),腹压漏尿点压下降(15.46±5.49)cmH2O(P<0.01),最大尿道闭合压和功能性尿道长度亦下降(7.97±3.25)cmH2O、(0.69±0.32)cm(P＜0.01)。建模后尿道及其周围组织形态学观察：括约肌排列松散,部分肌肉有断裂现象,肌层变薄；结缔组织所占比重增加,胶原纤维稀疏,排列紊乱。④尿动力学检测ADSCs移植后尿控功能变化,ADSCs治疗后大鼠膀胱最大容量下降(0.78±0.35)ml,腹压漏尿点压增加(15.06±4.36)cmH2O,最大尿道闭合压和功能性尿道长度亦增加(9.69±2.57)cmH2O、(0.69±0.32)cm,与对照组比较各指标均有显著性意义(P＜0.01)。ADSCs移植治疗后可以使注射局部尿道肌层得到明显的加强和增厚,括约肌形态及功能得到明显的改善,周围结缔组织紧密,支撑作用得到加强。横纹肌肌肉分割分析显示干细胞治疗4周横纹肌肌肉面积及其长短轴比值均得到明显增加。移植治疗后尿道周围的胶原蛋白含量明显高于对照组(P<0.01)。
结论：
1、通过消化分离,percoll密度梯度离心和preplate差速贴壁技术,可以获得纯度较高的SD大鼠ADSCs。本实验使用携带GFP基因重组的慢病毒成功高效地转染了ADSCs,该方法为长期观测ADSCs的体内状况创造了条件。
2、本研究提示：在特定诱导条件下ADSCs能被定向诱导分化为成肌细胞；并为SUI的干细胞移植治疗提供了新的思路。
3、本部分研究通过双侧阴部神经离断的方法,成功建立了SUI的动物模型,为后续实验奠定了基础。尿道横纹括约肌呈封闭环形分布于雌性大鼠尿道的中外段,提示此处为控尿机制的主要部位。
4、ADSCs移植至压力性尿失禁模型大鼠后能存活、分化而整合至宿主尿道括约肌,改善压力性尿失禁大鼠的尿控功能,并且主要可能通过在体内定向分化为肌细胞,增强尿道括约肌系统,并改善尿道粘膜闭合机制及尿道周围组织中胶原蛋白的含量而实现。组织工程学技术的发展为压力性尿失禁的治疗提供了新的思路和手段。
Objective:ADSCs were purified through percoll density gradient centrifugation and preplate technique,and to differentiate rat ADSCs into myoblasts in vitro.Labeled ADSCs will be transplatated into the SUI rats around the posterior urethral.By urodynamics,morphological,transmission electron microscope and multiphoton microscopic imaging changes of urethra and its surrounding tissue.Our objective is to observe whether ADSCs transplant in the treatment of SUI rats is effective and feasible or not and its possible mechanism.Rat ADSCs transplantation can be provided a new way for cell transplantation therapy of stress urinary incontinence.
Methods:1. ADSCs were purified through percoll density gradient centrifugation and preplate technique. ADSCs were further identified by flow cytometry technology for CD44、CD90, CD34、CD45、CD106 antibodies and by adipogenesis,osteogenesis cellular differentiation.2. A lentiviral vector with green fluorescent protein(GFP) was be used to transfection ADSCs.3. The twice passage ADSCs were treated for 24 h with 5-azacytidine (5-Aza, 10μmol-L-1) to induce cellular differentiation. The morphology and ultrastructure of treated ADSCs were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging. The expression of desmin identified by immunohistochemistry, was used to identify myoblasts differentiation.The sarcomeric and Desmin expression were detected with RT-PCR and Western Blot.4. Bilateral pudendal nerve transaction was preparation of rat animal model of SUI. Simulated human body urodynamic method of the maximum bladder capacity of experimental animals (MBV), abdominal leak point pressure (ALPP), maximum urethral closure pressure (MUCP), and the functional length of the urethra (FUL);and HE staining, Masson special stain, Mallory staining and special nature of the urethral sphincter striated muscle ultrastructure in transmission electron microscopy studies confirmed the successful production of animal models of SUI.5. Rats were randomly assigned to transplant Group I (n=16) injected with ADSCs and control GroupⅡ(n=9) injected with culture medium without serum. Urodynamic testing, evaluation of dynamic changes in animal,morphology and ultrastructure of were observed by inverted microscope, transmission electron microscope and multiphoton microscopic imaging to evaluation the effective and feasible of ADSCs transplantation.
Results:1. Method combining improved two-step enzymatic digestion with preplating technique is a kind of easy and practicable in some degree to obtain relative high purity of ADSCs.The purified ADSCs by limited-dilution appeared fibroblast-like shapes and proliferated rapidly in vitro. Results of flow cytometry showed that ADSCs were positive for CD44 and CD90, and negative for CD34, CD45 and CD106. Florescence intensity increased was companied with MOIs increased,but MOI of 8 fluorescence intensity was the strongest.82%-86%of successively passaged P3 ADSCs were GFP-positive.2. The morphology of ADSCs treated with 5-Aza under inverted microscope and multiphoton microscopic imaging changed from fusiform to polygon or astrocyte. With transmission electron microscope, the ultrastructure of the treated ADSCs was visible.The magnitude of cells was not identical, the surface of some cells had abundance of microvilli. Intracytoplasm had rich organelles, mitochondrium and rough endoplasmic reticulum.The induced ADSCs demonstrated positive staining for desmin by immunohistochemistry.The outcome of RT-PCR and Western Blot manifested that the gene expression of Desmin was up regulated in induced ADSCs.3.The urethral of female SD rat is a tubular structure about 2.0 cm. Its structure includes circular striated muscle layer,circular smooth muscle layer,longitudinal smooth muscle layer,dense connective tissue and epithelium from outside to inside. Bilateral pudendal nerve transection of the method can be successfully produced in rat model of SUI.Compared with those at baseline, the average quantity of maximum bladder volume (MBV) were increased by (0.89±0.04)ml (P<0.01); Abdomen pressure leak point pressure (ALPP) were significantly decreased by (15.46±5.49)cmH2O, respectively (P<0.01).Maximum urethra closure pressure (MUCP) and functional length of the urethra (FUL) were decreased by (7.97±3.25) cmH2O and (0.69±0.32)cm,respectively (P<0.01). Modeling of the posterior urethra and surrounding tissue morphology:sphincter loose arrangement of some muscles fracture phenomenon, muscle thinning; the proportion of connective tissue increase in sparse collagen fibers arranged in disorder.
4. Two weeks after transplantation, by urodynamic testing, compared with the control group all parameters of MBV, ALPP, MUCP, FUL were statistically significant (P <0.01). By ADSCs transplantation,the urethral circular striated muscle layer can be significantly strengthened and thickened, sphincter morphology and function was significantly improved, the surrounding connective tissue close, supportive role to be strengthened. Striated muscle segmentation analysis revealed that ADSCs therapy for 4 weeks the ratio of muscle area and the length of the shaft were to be increased significantly. Collagen contents around the urethra and its surrounding was significantly higher compared with control group(P<0.01).
Conclusion
1. This part study manifests that high purify ADSCs can be obtained through digestion,percoll density gradient centrifugation and preplate technique. Lentiviral vector with green fluorescent protein(GFP) was be successfully used to transfection ADSCs, which creates good condition for long time observation of ADSCs in vivo.
2. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.
3. In this part study,rats models of SUI were obtained by transaction of bilateral pudendal nerve,which established the foundation of extending experiment. The striated urethral sphincter of female rats was considered as a closed ring surrounding the urethral from the middle to the distal. It suggests that this may be the main site of urinary continence mechanism.
4. ADSCs transplanted into rats with stress urinary incontinence can survive, divide and integrated into the host urethral sphincter,and it can be improved the control of urinary function. The possible mechanism of ADSCs may be adopted to differentiate into myoblasts, increased urinary sphincter system, and to improve urethral closure mechanism mucosal achieved. With the development of tissue engineering technology, it provided a new concept and method to treat stress urinary incontinence.
引文
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