细胞间接触在诱导骨髓间充质干细胞向平滑肌细胞分化中作用的实验研究
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摘要
尿失禁是女性的常见病,据报道全世界患者有2亿以上,其中又以压力性尿失禁为主,已成为重要的妇女健康问题,影响生活质量。导致SUI的关键机制主要有两种,尿道固有括约肌缺陷(intrinsic sphincter deficiency,ISD)和膀胱颈支撑功能障碍。目前,治疗SUI的方法包括手术治疗和非手术治疗,但这些方法均只能解决膀胱颈支撑功能障碍,不能纠正ISD,因此长期疗效不理想,且并发症多。利用干细胞的再生潜能,修复括约肌缺陷,治疗SUI已经成为目前尿失禁研究的热点。
间充质干细胞(mesenchymal stem cells, MSCs)为成体干细胞的一种,是一群来源于中胚层的具有自我更新和多向分化潜能的多能干细胞,它们可以在体外经诱导后最终分化为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、肌管、神经细胞与支持造血干细胞的基质。MSCs具有取材方便、扩增迅速、可自体移植等特点,因此,利用MSCs的多向分化的特性及通过基因修饰能在体外表达多种外源目的基因的特性,将其作为种子细胞用于细胞治疗和基因治疗的研究已成为当前研究的热点。但是目前的研究的中,体外诱导它分化产生的平滑肌细胞在数量和功能上均难以达到改善治疗压力性尿失禁的目的。现阶段的研究已证实移植的MSCs能在组织微环境中分化成与其周围细胞生物学特性相似的细胞,认为干细胞能够对环境中的调节信号做出适应性改变,而其分子机制尚不清楚,因此本研究模仿体内细胞生存的微环境,将其向SMCs诱导,初步探讨细胞间接触在MSCs向SMCs分化中的作用,为MSCs作为种子细胞用于组织工程研究提供理论基础。
本研究以SD大鼠为研究对象,分别利用密度梯度离心法和酶消化法,分离培养了BMSCs和BSMCs;模仿体内细胞生长的微环境,建立大鼠膀胱平滑肌细胞诱导BMSCs向SMCs分化的体外共培养模型,在共培养前后,检测膀胱平滑肌细胞诱导BMSCs的成肌分化标志蛋白表达变化;在共培养的基础上阻断细胞间缝隙连接,检测抑制细胞间连接后对BMSCs向SMCs分化情况的影响。主要实验方法及结果如下:
一、骨髓间充质干细胞和膀胱平滑肌细胞分离培养及鉴定
1.无菌条件下取一月龄雌性SD大鼠双侧股骨,冲出骨髓,采用密度梯度离心法分离单个核细胞,DMEM-12培养,获得贴壁细胞,监测细胞生长曲线,流式细胞仪检测其免疫表型,采用不同条件培养基诱导细胞向成脂、成骨分化。和生长情况。结果:分离的骨髓单核细胞培养后呈纺锤体样,其分子免疫表型为: CD44、CD90阳性;CD31、CD45阴性。在不同的诱导条件下,BMSCs可形成脂滴、骨结节结构,成功分离并培养了BMSCs。
2.无菌条件下取一月龄雌性SD大鼠膀胱,采用胰酶消化法分离单个核细胞,HG-DMEM培养,获得贴壁细胞,监测细胞生长曲线,免疫荧光法鉴定其标志蛋白的表达。结果:平滑肌特异性的a-SMA, Calponin和SM-MHC表达阳性,证实分离并培养了膀胱平滑肌细胞。
二、BMSCs向SMCs分化的体外共培养模型的建立与鉴定
1.建立大鼠BSMCs诱导BMSCs向SMCs分化的体外共培养模型,在共培养前后,检测BSMCs诱导BMSCs的成肌分化标志蛋白表达变化。以悬挂式细胞培养小室transwell底层的多孔膜(1um)作为BMSCs和BSMCs之间的间隔,细胞分泌的细胞因子,胶原等物质可通过膜上的孔相流通,多孔膜上下两面的细胞可通过膜上的孔进行细胞间接触,但细胞不能通过。将BMSCs和BSMCs分别接种于多孔膜两侧作为接触共培养实验组,将BMSCs接种于多孔膜上,BSMCs接种于六孔板底,使得两种细胞仍处在一个培养环境中,之间距离<900μm,两种细胞分泌的细胞因子仍可以通过多孔膜膜孔相互影响,但是不发生接触作为非接触共培养实验组,以单独培养的BMSCs作为对照组,在共培养的第4天和第8天的免疫荧光法和Western blot法检测BMSCs中平滑肌特异性蛋白的表达情况,在共培养1,2,3天检测实验组中BMSCs的平滑肌特异性蛋白基因的表达情况。结果:免疫荧光法、Western blot法和qRT-PCR均显示随共培养时间的延长,接触组的BMSCs中SMC特异的蛋白的表达量增加,而非接触组与共培养前无明显变化,提示细胞间的接触在诱导BMSCs向SMCs分化方面起了重要的作用。
2.正庚醇阻断细胞间缝隙连接对BMSCs向SMCs分化的影响。初步实验证明细胞间接触共培养可以很好的诱导BMSCs向SMCs分化,正庚醇阻断缝隙连接后可以有明显的抑制BMSCs向SMCs分化,进一步证明缝隙连接在在诱导BMSCs向SMCs分化方面起了重要的作用。
结论:本研究通过模拟体内细胞生长分化的微环境环境,建立了大鼠膀胱平滑肌细胞诱导BMSCs向SMCs分化的体外共培养模型,证明了BMSCs与平滑肌细胞间的直接接触是诱导分化的重要因素之一,利用这种方法可进一步深入研究MSCs的分化机制,提高分化效率,为BMSCs移植治疗压力性尿失禁提供理论支持。
There are over 200 million people worldwide with incontinence, a condition that is associated with social impact and a reduced quality of life. Stress urinary incontinence (SUI) is one of the most common forms of incontinence and it can be grouped into two major categories: urethral hyper mobility and intrinsic sphincter deficiency (ISD). The periurethral injection of bμlking agents including polytetrafluoroethylene, bovine collagen, silicone particles, carbon beads, and autologous ear chondrocytes has yielded short-term success in the treatment of SUI. However, use of bulking agents has leading to chronic inflammatory reactions, foreign body giant cell responses, periurethral abscess, and particle migration, erosion of the urinary bladder or the urethra, obstruction of the lower urinary tract with urinary retention, severe voiding dysfunction, and pulmonary embolism. Smooth muscle cells (SMCs) is an essential component of sphincter muscle in urinary system. It plays a key role in SUI, so the ideal substance for such a procedure is considered to be one that is improve sphincter muscle function, readily available, safe, economic, efficacious, and durable, the effecting improvements sphincter contractility is critical for treatment SUI. A major obstacle for such an approach has been finding a reliable source of healthy SMC that can be safely harvested and that requires minimal wound. Cellular injection therapy displays an ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. Mesenchymal stem cells (MSCs) have been reported to differentiation toward SMCs; In addition, stem cells may release neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. Bladder tissue engineering pointed out that using MSCs show better results than by using differentiated cells, MSCs may migrate to the bladder grafts and differentiate into smooth muscle cells.
Although the Bone marrow cells containing MSCs were most widely studied in fundamental and clinical trials; however, the underlying mechanisms have not been fully clearly, the differentiation of MSCs into smooth muscle cells (SMCs) might not completely explain. Stem cells microenvironment or‘niche’is one current line of research have been put forward to explain stem cell lineage determination. Adult stem cells that are implanted into a totally different niche (different germ layer) can potentially differentiate into cell types similar to those found in the new environment. A niche consists of signaling molecules, intercellular communication and the interaction between stem cells and their neighboring extracellular matrix. Gap junctions provide a pathway for direct intercellular communication. As numerous physiological processes are mediated by regulatory molecules that are exchanged via gap junctions, gap junction is considered a key mechanism in the control of virtually all aspects of the cellular life cycle.
In this study, we conducted co-culture experiments to investigate whether BMSCs differentiation to SMCs is the effect of soluble chemical factors signal on BMSCs or direct contact between BMSCs and SMCs, meanwhile elucidating the potential role of gap junction in differentiation of BMSCs.
Results
1. The characteristics of Cultured BMSC
Passage 3rd rat BMSC cell-surface antigen profile was ascertained by staining with rat-specific monoclonal antibodies by flow cytometry. CD90, CD44 expression was positive , but CD31 and CD45 were negative. This result suggests that these BMSCs are undifferentiated stem cells and they different from hematopoietic stem cells and endothelial cell. In the experiment of osteogenic, and adipogenic differentiation. they were assessed after induced 3 weeks, osteogenic differentiation show calcium deposition, and BMSCs formation adipose in adipogenic differentiation. From the results of experiment can be seen that these BMSCs had potential of Multilineage Differentiation.
2. Co-culture induce MSC differentiate into SMC
2.1 Immunofluorescence analysis
In CON. group, NO-CON. group, and CON.+H group, after co-culture 8days,the rat BMSCs expression of a-SMA, Calponin and SM-MHC of SMCs was examined by immunofluorescence. Untreated rat BMSCs and adult SMCs were used as negative and positive controls, respectively. we observed a-SMA expression on three groups. Only in direct contacted culture group revealed an increased expression of Calponin but all of three groups have no expression of SM-MHC.
2.2 Western analysis
Total protein was isolated from rat BMSCs of CON. group, NO-CON. group, and CON.+H group, on co-culture 4 days and 8days respectively. At the same time , a total protein was isolated from untreated rat BMSCs as control. This experiment confirmed the immunofluorescence Data, NO-CON. group and CON.+H group have no discrepancy by statistical evaluation after co-culture. BMSCs in CON. group with increases in the expression of ASMA and Calponin compared to untreated rat BMSCs , and expression of SM-MHC only in cells grown in CON. group at 8 days. In CON.+H group which present the increase of a-SMA and decrease of Calponin.
2.3 Quantitative Real-Time PCR
RT-PCR analyses of CON. group were performed as previously describe.[1] The expression of ASMA, Calponin, and SM-MHC was quantified for differentiation conditions described above. mRNA of a-SMA, Calponin and SM-MHC increases in the expression were confirmed with real-time PCR.
Discussion
Cell transplantation therapy offers hope for the treatment of the regenerative repair of the intrinsic sphincter deficiency (ISD) of urinary incontinence. The use of autologous multipotent stem cells becomes the first choice of Cell-based therapies and tissue engineering. Stem cell therapy might replace, repair, or enhance the biological function of damaged tissue or organs. One commonly described source of SMCs is the muscle derived stem cells (MDSC). Although muscle stem cells and satellite cells can be isolated from biopsy of adult, the number of cells that can be harvested may be limited. Bone marrow stem cells are an idea source of autologous adult stem cells. BMSCs are easy to isolate and expand rapidly from patients without leading to major ethical and technical problems; they have great potential as therapeutic agents. But the application to ISD depends on the ability to control their differentiation into SMCs with high efficiency and purity.
In our study we conduct a direct co-culture system via used a semi- permeable membrane to investigate the effect of local environment on BMSCs differentiation. We sowed the SMCs under the membrane, and sowed BMSCs in the upper of membrane when SMCs confused. The semi- permeable membrane has more 1um diameter pores which would not permit cell through the pores of semi- permeable membrane but can form cell-cell contact and allow chemical factors to diffuse. Through this method to establish whether rat BMSCs is differentiation to SMC after direct co-culture with adult rat SMCs, we assessed the expression of certain unique markers of SMCs, include a-SMA, Calponin and SM-MHC by real-time PCR, confocal laser microarray scanner and western blot. We not only detected the expression of a-SMA and Calponin which are early and middle marker of developing smooth muscle respectively, but also tested the present of SM-MHC, a highly restricted to differentiated smooth muscle at western blot experiment. In this study we didn't add any exogenous cytokine or signaling molecules, only rely on SMCs secreted. signaling molecules and interaction between stem cells with their neighboring cell are two important factor of local environment, Chiu et al. have been suggested that milieu-induced differentiation play an important role in induced myoblasts to acquire a cardiac-like phenotype. The‘‘milieu-induced differentiation’’hypothesis focuses on the local microenvironment. Our experiment demonstrated local environment and resident cellular populations are likely to be major factors in determining MSCs fates.
In NO-CON. group, We sowed the SMCs up the six well culture plate, and sowed BMSCs in the upper of membrane when SMCs confused. through this method, BMSCs wouldn’t contact with SMCs, but the SMCs might secreted cytokine or signaling molecules diffuse into DMEM and permeate the semi- permeable membrane affect the BMSCs. after co-culture ,We found that there was no expression of a-SMA, Calponin and SM-MHC of BMSCs. This means cytokine or signaling molecules released by SMCs can not induce BMSCs to differentiate into SMCs. This result suggested that the cell-cell contact is dominant in the differentiation of BMSCs at the condition without artificially add exogenous cytokine or signaling molecules.
Our data suggest that contact with SMCs would render BMSCs differentiate into SMCs lineage. There has been report that differentiation of bone marrow stromal cells into cells with cardiac phenotype requires intercellular communication with myocytes. so, whether intercellular communication is essential for SMCs differentiation is unknown. To confirm that whether gap junction contribute to stem cell differentiation, on the basis of the direct contact culture experiment we added the gap junction uncoupler heptanol which is a selectively block gap junction communication. The data showed, the inductive effect in direct contact culture group was inhibition by heptanol. The expression of Calponin decrease, and not expression SM-MHC the late marker of SMCs .However, in this group the expression of a-SMA was increased indicates that gap junction is not inherently and invariably linked to the differentiation of BMSCs. This suggests that the relationship between gap junction and BMSCs differentiation depends upon the specific cascades activated by the particular growth factor involved. It has also been reported that the signals in local environment are likely to be major factors determining stem cells fates, which exert influence through cell surface receptors and ligands, the formation of structures. The possibility that other singnal path way reported in differerntiation process might also be involved at the transcript or protein level in BMSCs. Gap junctions allow exchange of small molecules, including secondary messengers between adjacent cells might be one of potential route for BMSCs to differentiation.
In this study, we found that the direct contact co-culture with SMCs facilitate BMSCs to SMCs lineage. This inductive effect mainly mediated through gap junction. The direct contact co-culture system serves as an important tool for the study of the communications between cells, because the method is simple and easy to master and the material is easily obtainable. Treatment of SUI with MSCs might be a long-term application toward cell replacement therapies in the future. it can increase the function of deficient rhabdosphincter , which has a significant clinical impact.
间充质干细胞(mesenchymal stem cells, MSCs)为成体干细胞的一种,是一群来源于中胚层的具有自我更新和多向分化潜能的多能干细胞,它们可以在体外经诱导后最终分化为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、肌管、神经细胞与支持造血干细胞的基质。MSCs具有取材方便、扩增迅速、可自体移植等特点,因此,利用MSCs的多向分化的特性及通过基因修饰能在体外表达多种外源目的基因的特性,将其作为种子细胞用于细胞治疗和基因治疗的研究已成为当前研究的热点。但是目前的研究的中,体外诱导它分化产生的平滑肌细胞在数量和功能上均难以达到改善治疗压力性尿失禁的目的。现阶段的研究已证实移植的MSCs能在组织微环境中分化成与其周围细胞生物学特性相似的细胞,认为干细胞能够对环境中的调节信号做出适应性改变,而其分子机制尚不清楚,因此本研究模仿体内细胞生存的微环境,将其向SMCs诱导,初步探讨细胞间接触在MSCs向SMCs分化中的作用,为MSCs作为种子细胞用于组织工程研究提供理论基础。
本研究以SD大鼠为研究对象,分别利用密度梯度离心法和酶消化法,分离培养了BMSCs和BSMCs;模仿体内细胞生长的微环境,建立大鼠膀胱平滑肌细胞诱导BMSCs向SMCs分化的体外共培养模型,在共培养前后,检测膀胱平滑肌细胞诱导BMSCs的成肌分化标志蛋白表达变化;在共培养的基础上阻断细胞间缝隙连接,检测抑制细胞间连接后对BMSCs向SMCs分化情况的影响。主要实验方法及结果如下:
一、骨髓间充质干细胞和膀胱平滑肌细胞分离培养及鉴定
1.无菌条件下取一月龄雌性SD大鼠双侧股骨,冲出骨髓,采用密度梯度离心法分离单个核细胞,DMEM-12培养,获得贴壁细胞,监测细胞生长曲线,流式细胞仪检测其免疫表型,采用不同条件培养基诱导细胞向成脂、成骨分化。和生长情况。结果:分离的骨髓单核细胞培养后呈纺锤体样,其分子免疫表型为: CD44、CD90阳性;CD31、CD45阴性。在不同的诱导条件下,BMSCs可形成脂滴、骨结节结构,成功分离并培养了BMSCs。
2.无菌条件下取一月龄雌性SD大鼠膀胱,采用胰酶消化法分离单个核细胞,HG-DMEM培养,获得贴壁细胞,监测细胞生长曲线,免疫荧光法鉴定其标志蛋白的表达。结果:平滑肌特异性的a-SMA, Calponin和SM-MHC表达阳性,证实分离并培养了膀胱平滑肌细胞。
二、BMSCs向SMCs分化的体外共培养模型的建立与鉴定
1.建立大鼠BSMCs诱导BMSCs向SMCs分化的体外共培养模型,在共培养前后,检测BSMCs诱导BMSCs的成肌分化标志蛋白表达变化。以悬挂式细胞培养小室transwell底层的多孔膜(1um)作为BMSCs和BSMCs之间的间隔,细胞分泌的细胞因子,胶原等物质可通过膜上的孔相流通,多孔膜上下两面的细胞可通过膜上的孔进行细胞间接触,但细胞不能通过。将BMSCs和BSMCs分别接种于多孔膜两侧作为接触共培养实验组,将BMSCs接种于多孔膜上,BSMCs接种于六孔板底,使得两种细胞仍处在一个培养环境中,之间距离<900μm,两种细胞分泌的细胞因子仍可以通过多孔膜膜孔相互影响,但是不发生接触作为非接触共培养实验组,以单独培养的BMSCs作为对照组,在共培养的第4天和第8天的免疫荧光法和Western blot法检测BMSCs中平滑肌特异性蛋白的表达情况,在共培养1,2,3天检测实验组中BMSCs的平滑肌特异性蛋白基因的表达情况。结果:免疫荧光法、Western blot法和qRT-PCR均显示随共培养时间的延长,接触组的BMSCs中SMC特异的蛋白的表达量增加,而非接触组与共培养前无明显变化,提示细胞间的接触在诱导BMSCs向SMCs分化方面起了重要的作用。
2.正庚醇阻断细胞间缝隙连接对BMSCs向SMCs分化的影响。初步实验证明细胞间接触共培养可以很好的诱导BMSCs向SMCs分化,正庚醇阻断缝隙连接后可以有明显的抑制BMSCs向SMCs分化,进一步证明缝隙连接在在诱导BMSCs向SMCs分化方面起了重要的作用。
结论:本研究通过模拟体内细胞生长分化的微环境环境,建立了大鼠膀胱平滑肌细胞诱导BMSCs向SMCs分化的体外共培养模型,证明了BMSCs与平滑肌细胞间的直接接触是诱导分化的重要因素之一,利用这种方法可进一步深入研究MSCs的分化机制,提高分化效率,为BMSCs移植治疗压力性尿失禁提供理论支持。
There are over 200 million people worldwide with incontinence, a condition that is associated with social impact and a reduced quality of life. Stress urinary incontinence (SUI) is one of the most common forms of incontinence and it can be grouped into two major categories: urethral hyper mobility and intrinsic sphincter deficiency (ISD). The periurethral injection of bμlking agents including polytetrafluoroethylene, bovine collagen, silicone particles, carbon beads, and autologous ear chondrocytes has yielded short-term success in the treatment of SUI. However, use of bulking agents has leading to chronic inflammatory reactions, foreign body giant cell responses, periurethral abscess, and particle migration, erosion of the urinary bladder or the urethra, obstruction of the lower urinary tract with urinary retention, severe voiding dysfunction, and pulmonary embolism. Smooth muscle cells (SMCs) is an essential component of sphincter muscle in urinary system. It plays a key role in SUI, so the ideal substance for such a procedure is considered to be one that is improve sphincter muscle function, readily available, safe, economic, efficacious, and durable, the effecting improvements sphincter contractility is critical for treatment SUI. A major obstacle for such an approach has been finding a reliable source of healthy SMC that can be safely harvested and that requires minimal wound. Cellular injection therapy displays an ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. Mesenchymal stem cells (MSCs) have been reported to differentiation toward SMCs; In addition, stem cells may release neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. Bladder tissue engineering pointed out that using MSCs show better results than by using differentiated cells, MSCs may migrate to the bladder grafts and differentiate into smooth muscle cells.
Although the Bone marrow cells containing MSCs were most widely studied in fundamental and clinical trials; however, the underlying mechanisms have not been fully clearly, the differentiation of MSCs into smooth muscle cells (SMCs) might not completely explain. Stem cells microenvironment or‘niche’is one current line of research have been put forward to explain stem cell lineage determination. Adult stem cells that are implanted into a totally different niche (different germ layer) can potentially differentiate into cell types similar to those found in the new environment. A niche consists of signaling molecules, intercellular communication and the interaction between stem cells and their neighboring extracellular matrix. Gap junctions provide a pathway for direct intercellular communication. As numerous physiological processes are mediated by regulatory molecules that are exchanged via gap junctions, gap junction is considered a key mechanism in the control of virtually all aspects of the cellular life cycle.
In this study, we conducted co-culture experiments to investigate whether BMSCs differentiation to SMCs is the effect of soluble chemical factors signal on BMSCs or direct contact between BMSCs and SMCs, meanwhile elucidating the potential role of gap junction in differentiation of BMSCs.
Results
1. The characteristics of Cultured BMSC
Passage 3rd rat BMSC cell-surface antigen profile was ascertained by staining with rat-specific monoclonal antibodies by flow cytometry. CD90, CD44 expression was positive , but CD31 and CD45 were negative. This result suggests that these BMSCs are undifferentiated stem cells and they different from hematopoietic stem cells and endothelial cell. In the experiment of osteogenic, and adipogenic differentiation. they were assessed after induced 3 weeks, osteogenic differentiation show calcium deposition, and BMSCs formation adipose in adipogenic differentiation. From the results of experiment can be seen that these BMSCs had potential of Multilineage Differentiation.
2. Co-culture induce MSC differentiate into SMC
2.1 Immunofluorescence analysis
In CON. group, NO-CON. group, and CON.+H group, after co-culture 8days,the rat BMSCs expression of a-SMA, Calponin and SM-MHC of SMCs was examined by immunofluorescence. Untreated rat BMSCs and adult SMCs were used as negative and positive controls, respectively. we observed a-SMA expression on three groups. Only in direct contacted culture group revealed an increased expression of Calponin but all of three groups have no expression of SM-MHC.
2.2 Western analysis
Total protein was isolated from rat BMSCs of CON. group, NO-CON. group, and CON.+H group, on co-culture 4 days and 8days respectively. At the same time , a total protein was isolated from untreated rat BMSCs as control. This experiment confirmed the immunofluorescence Data, NO-CON. group and CON.+H group have no discrepancy by statistical evaluation after co-culture. BMSCs in CON. group with increases in the expression of ASMA and Calponin compared to untreated rat BMSCs , and expression of SM-MHC only in cells grown in CON. group at 8 days. In CON.+H group which present the increase of a-SMA and decrease of Calponin.
2.3 Quantitative Real-Time PCR
RT-PCR analyses of CON. group were performed as previously describe.[1] The expression of ASMA, Calponin, and SM-MHC was quantified for differentiation conditions described above. mRNA of a-SMA, Calponin and SM-MHC increases in the expression were confirmed with real-time PCR.
Discussion
Cell transplantation therapy offers hope for the treatment of the regenerative repair of the intrinsic sphincter deficiency (ISD) of urinary incontinence. The use of autologous multipotent stem cells becomes the first choice of Cell-based therapies and tissue engineering. Stem cell therapy might replace, repair, or enhance the biological function of damaged tissue or organs. One commonly described source of SMCs is the muscle derived stem cells (MDSC). Although muscle stem cells and satellite cells can be isolated from biopsy of adult, the number of cells that can be harvested may be limited. Bone marrow stem cells are an idea source of autologous adult stem cells. BMSCs are easy to isolate and expand rapidly from patients without leading to major ethical and technical problems; they have great potential as therapeutic agents. But the application to ISD depends on the ability to control their differentiation into SMCs with high efficiency and purity.
In our study we conduct a direct co-culture system via used a semi- permeable membrane to investigate the effect of local environment on BMSCs differentiation. We sowed the SMCs under the membrane, and sowed BMSCs in the upper of membrane when SMCs confused. The semi- permeable membrane has more 1um diameter pores which would not permit cell through the pores of semi- permeable membrane but can form cell-cell contact and allow chemical factors to diffuse. Through this method to establish whether rat BMSCs is differentiation to SMC after direct co-culture with adult rat SMCs, we assessed the expression of certain unique markers of SMCs, include a-SMA, Calponin and SM-MHC by real-time PCR, confocal laser microarray scanner and western blot. We not only detected the expression of a-SMA and Calponin which are early and middle marker of developing smooth muscle respectively, but also tested the present of SM-MHC, a highly restricted to differentiated smooth muscle at western blot experiment. In this study we didn't add any exogenous cytokine or signaling molecules, only rely on SMCs secreted. signaling molecules and interaction between stem cells with their neighboring cell are two important factor of local environment, Chiu et al. have been suggested that milieu-induced differentiation play an important role in induced myoblasts to acquire a cardiac-like phenotype. The‘‘milieu-induced differentiation’’hypothesis focuses on the local microenvironment. Our experiment demonstrated local environment and resident cellular populations are likely to be major factors in determining MSCs fates.
In NO-CON. group, We sowed the SMCs up the six well culture plate, and sowed BMSCs in the upper of membrane when SMCs confused. through this method, BMSCs wouldn’t contact with SMCs, but the SMCs might secreted cytokine or signaling molecules diffuse into DMEM and permeate the semi- permeable membrane affect the BMSCs. after co-culture ,We found that there was no expression of a-SMA, Calponin and SM-MHC of BMSCs. This means cytokine or signaling molecules released by SMCs can not induce BMSCs to differentiate into SMCs. This result suggested that the cell-cell contact is dominant in the differentiation of BMSCs at the condition without artificially add exogenous cytokine or signaling molecules.
Our data suggest that contact with SMCs would render BMSCs differentiate into SMCs lineage. There has been report that differentiation of bone marrow stromal cells into cells with cardiac phenotype requires intercellular communication with myocytes. so, whether intercellular communication is essential for SMCs differentiation is unknown. To confirm that whether gap junction contribute to stem cell differentiation, on the basis of the direct contact culture experiment we added the gap junction uncoupler heptanol which is a selectively block gap junction communication. The data showed, the inductive effect in direct contact culture group was inhibition by heptanol. The expression of Calponin decrease, and not expression SM-MHC the late marker of SMCs .However, in this group the expression of a-SMA was increased indicates that gap junction is not inherently and invariably linked to the differentiation of BMSCs. This suggests that the relationship between gap junction and BMSCs differentiation depends upon the specific cascades activated by the particular growth factor involved. It has also been reported that the signals in local environment are likely to be major factors determining stem cells fates, which exert influence through cell surface receptors and ligands, the formation of structures. The possibility that other singnal path way reported in differerntiation process might also be involved at the transcript or protein level in BMSCs. Gap junctions allow exchange of small molecules, including secondary messengers between adjacent cells might be one of potential route for BMSCs to differentiation.
In this study, we found that the direct contact co-culture with SMCs facilitate BMSCs to SMCs lineage. This inductive effect mainly mediated through gap junction. The direct contact co-culture system serves as an important tool for the study of the communications between cells, because the method is simple and easy to master and the material is easily obtainable. Treatment of SUI with MSCs might be a long-term application toward cell replacement therapies in the future. it can increase the function of deficient rhabdosphincter , which has a significant clinical impact.
引文
1.段继宏,杨勇,吴士良.北京地区尿失禁发病率调查.北京医科大学学报, 2002, 32(1): 74-75.
2. Perucchini D, Delancey J O, Ashton-Miller J A, et al. Age effects on urethral striated muscle. i. changes in number and diameter of striated muscle fibers in the ventral urethra. Am J Obstet Gynecol, 2002, 186(3): 351-355.
3. Perucchini D, Delancey J O, Ashton-Miller J A, et al. Age effects on urethral striated muscle. ii. anatomic location of muscle loss. Am J Obstet Gynecol, 2002, 186(3): 356-360.
4. Hampel C, Wienhold D, Benken N, et al. Prevalence and natural history of female incontinence. Eur Urol, 1997, 32 Suppl 2: 3-12.
5. Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the international continence society. Urology, 2003, 61(1): 37-49.
6. Kevorkian R. Physiology of incontinence. Clin Geriatr Med, 2004, 20(3): 409-425.
7. Karram M M. Pathophysiology of genuine stress incontinence: what do we really know?. Int Urogynecol J Pelvic Floor Dysfunct, 2003, 14(2): 77.
8. Appell R A, Dmochowski R R, Herschorn S. Urethral injections for female stress incontinence. Bju Int, 2006, 98 Suppl 1: 27-30, 31.
9. Mccrery R J, Smith P P, Appell R A. The emergence of new drugs for overactive bladder. Expert Opin Emerg Drugs, 2006, 11(1): 125-136.
10. Takahashi S, Homma Y, Fujishiro T, et al. Electromyographic study of the striated urethral sphincter in type 3 stress incontinence: evidence of myogenic-dominant damages. Urology, 2000, 56(6): 946-950.
11. Kang S B, Lee H N, Lee J Y, et al. Sphincter contractility after muscle-derived stem cells autograft into the cryoinjured anal sphincters of rats. Dis Colon Rectum, 2008, 51(9): 1367-1373.
12. Smaldone M C, Chancellor M B. Muscle derived stem cell therapy for stress urinary incontinence. World J Urol, 2008, 26(4): 327-332.
13. Strasser H, Marksteiner R, Margreiter E, et al. [stem cell therapy for urinaryincontinence]. Urologe A, 2004, 43(10): 1237-1241.
14. Dominici M, Le B K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. the international society for cellμlar therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
15. Mimeaμlt M, Hauke R, Batra S K. Stem cells: a revolution in therapeutics-recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin Pharmacol Ther, 2007, 82(3): 252-264.
16. Howell J C, Lee W H, Morrison P, et al. Pluripotent stem cells identified in mμltiple murine tissues. Ann N Y Acad Sci, 2003, 996: 158-173.
17. Lee J Y, Paik S Y, Yuk S H, et al. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells, 2004, 18(3): 309-313.
18. Chermansky C J, Tarin T, Kwon D D, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology, 2004, 63(4): 780-785.
19. Furuta A, Jankowski R J, Honda M, et al. State of the art of where we are at using stem cells for stress urinary incontinence. Neurourol Urodyn, 2007, 26(7): 966-971.
20. Drost A C, Weng S, Feil G, et al. In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair. Ann N Y Acad Sci, 2009, 1176: 135-143.
21. Kinebuchi Y, Aizawa N, Imamura T, et al. Autologous bone-marrow-derived mesenchymal stem cell transplantation into injured rat urethral sphincter. Int J Urol, 2010.
22. Dellatore S M, Garcia A S, Miller W M. Mimicking stem cell niches to increase stem cell expansion. Curr Opin Biotechnol, 2008, 19(5): 534-540.
23. Dominici M, Le B K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. the international society for cellμlar therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
24. Pittenger M F, Mackay A M, Beck S C, et al. Mμltilineage potential of adμlt human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
25. Liechty K W, Mackenzie T C, Shaaban A F, et al. Human mesenchymal stem cellsengraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000, 6(11): 1282-1286.
26. Tμli R, Tμli S, Nandi S, et al. Characterization of mμltipotential mesenchymal progenitor cells derived from human trabecμlar bone. Stem Cells, 2003, 21(6): 681-693.
27. Tsai M S, Lee J L, Chang Y J, et al. Isolation of human mμltipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod, 2004, 19(6): 1450-1456.
28. Trubiani O, di Primio R, Traini T, et al. Morphological and cytofluorimetric analysis of adμlt mesenchymal stem cells expanded ex vivo from periodontal ligament. Int J Immunopathol Pharmacol, 2005, 18(2): 213-221.
29. Shi Y Y, Nacamμli R P, Salim A, et al. The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging. Plast Reconstr Surg, 2005, 116(6): 1686-1696.
30. Mauck R L, Yuan X, Tuan R S. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage, 2006, 14(2): 179-189.
31. Choi Y S, Park S N, Suh H. The effect of plga sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering. J Mater Sci Mater Med, 2008, 19(5): 2165-2171.
32. Peerani R, Zandstra P W. Enabling stem cell therapies through synthetic stem cell-niche engineering. J Clin Invest, 2010, 120(1): 60-70.
33. Vazin T, Schaffer D V. Engineering strategies to emμlate the stem cell niche. Trends Biotechnol, 2010, 28(3): 117-124.
34. Morrison S J, Spradling A C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell, 2008, 132(4): 598-611.
35. Ball S G, Shuttleworth A C, Kielty C M. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol, 2004, 36(4): 714-727.
36. Chen X D. Extracellμlar matrix provides an optimal niche for the maintenance and propagation of mesenchymal stem cells. Birth Defects Res C Embryo Today, 2010, 90(1): 45-54.
37. Deregibus M C, Tetta C, Camussi G. The dynamic stem cell microenvironment is orchestrated by microvesicle-mediated transfer of genetic information. Histol Histopathol, 2010, 25(3): 397-404.
38. Saunders K B, D'Amore P A. An in vitro model for cell-cell interactions. In Vitro Cell Dev Biol, 1992, 28A(7-8): 521-528.
39. Fillinger M F, Sampson L N, Cronenwett J L, et al. Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models. J Surg Res, 1997, 67(2): 169-178.
40. Owens G K. Regμlation of differentiation of vascμlar smooth muscle cells. Physiol Rev, 1995, 75(3): 487-517.
41. Owens G K, Kumar M S, Wamhoff B R. Molecμlar regμlation of vascμlar smooth muscle cell differentiation in development and disease. Physiol Rev, 2004, 84(3): 767-801.
42. de Ruiter M C, Poelmann R E, van Iperen L, et al. The early development of the tunica media in the vascμlar system of rat embryos. Anat Embryol (Berl), 1990, 181(4): 341-349.
43. Sugi Y, Lough J. Onset of expression and regional deposition of alpha-smooth and sarcomeric actin during avian heart development. Dev Dyn, 1992, 193(2): 116-124.
44. Perry T E, Kaushal S, Sutherland F W, et al. Thoracic surgery directors association award. bone marrow as a cell source for tissue engineering heart valves. Ann Thorac Surg, 2003, 75(3): 761-767, 767.
45. Kuro-O M, Nagai R, Tsuchimochi H, et al. Developmentally regμlated expression of vascμlar smooth muscle myosin heavy chain isoforms. J Biol Chem, 1989, 264(31): 18272-18275.
46. Aikawa M, Kim H S, Kuro-O M, et al. Phenotypic modμlation of smooth muscle cells during progression of human atherosclerosis as determined by altered expression of myosin heavy chain isoforms. Ann N Y Acad Sci, 1995, 748: 578-585.
47. Miano J M, Cserjesi P, Ligon K L, et al. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res, 1994, 75(5): 803-812.
48. Hackney J A, Charbord P, Brunk B P, et al. A molecμlar profile of a hematopoieticstem cell niche. Proc Natl Acad Sci U S A, 2002, 99(20): 13061-13066.
49. Rodriguez L V, Alfonso Z, Zhang R, et al. Clonogenic mμltipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells. Proc Natl Acad Sci U S A, 2006, 103(32): 12167-12172.
50. Vinken M, Vanhaecke T, Papeleu P, et al. Connexins and their channels in cell growth and cell death. Cell Signal, 2006, 18(5): 592-600.
51. Keevil V L, Huang C L, Chau P L, et al. The effect of heptanol on the electrical and contractile function of the isolated, perfused rabbit heart. Pflugers Arch, 2000, 440(2): 275-282.
2. Perucchini D, Delancey J O, Ashton-Miller J A, et al. Age effects on urethral striated muscle. i. changes in number and diameter of striated muscle fibers in the ventral urethra. Am J Obstet Gynecol, 2002, 186(3): 351-355.
3. Perucchini D, Delancey J O, Ashton-Miller J A, et al. Age effects on urethral striated muscle. ii. anatomic location of muscle loss. Am J Obstet Gynecol, 2002, 186(3): 356-360.
4. Hampel C, Wienhold D, Benken N, et al. Prevalence and natural history of female incontinence. Eur Urol, 1997, 32 Suppl 2: 3-12.
5. Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the international continence society. Urology, 2003, 61(1): 37-49.
6. Kevorkian R. Physiology of incontinence. Clin Geriatr Med, 2004, 20(3): 409-425.
7. Karram M M. Pathophysiology of genuine stress incontinence: what do we really know?. Int Urogynecol J Pelvic Floor Dysfunct, 2003, 14(2): 77.
8. Appell R A, Dmochowski R R, Herschorn S. Urethral injections for female stress incontinence. Bju Int, 2006, 98 Suppl 1: 27-30, 31.
9. Mccrery R J, Smith P P, Appell R A. The emergence of new drugs for overactive bladder. Expert Opin Emerg Drugs, 2006, 11(1): 125-136.
10. Takahashi S, Homma Y, Fujishiro T, et al. Electromyographic study of the striated urethral sphincter in type 3 stress incontinence: evidence of myogenic-dominant damages. Urology, 2000, 56(6): 946-950.
11. Kang S B, Lee H N, Lee J Y, et al. Sphincter contractility after muscle-derived stem cells autograft into the cryoinjured anal sphincters of rats. Dis Colon Rectum, 2008, 51(9): 1367-1373.
12. Smaldone M C, Chancellor M B. Muscle derived stem cell therapy for stress urinary incontinence. World J Urol, 2008, 26(4): 327-332.
13. Strasser H, Marksteiner R, Margreiter E, et al. [stem cell therapy for urinaryincontinence]. Urologe A, 2004, 43(10): 1237-1241.
14. Dominici M, Le B K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. the international society for cellμlar therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
15. Mimeaμlt M, Hauke R, Batra S K. Stem cells: a revolution in therapeutics-recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies. Clin Pharmacol Ther, 2007, 82(3): 252-264.
16. Howell J C, Lee W H, Morrison P, et al. Pluripotent stem cells identified in mμltiple murine tissues. Ann N Y Acad Sci, 2003, 996: 158-173.
17. Lee J Y, Paik S Y, Yuk S H, et al. Long term effects of muscle-derived stem cells on leak point pressure and closing pressure in rats with transected pudendal nerves. Mol Cells, 2004, 18(3): 309-313.
18. Chermansky C J, Tarin T, Kwon D D, et al. Intraurethral muscle-derived cell injections increase leak point pressure in a rat model of intrinsic sphincter deficiency. Urology, 2004, 63(4): 780-785.
19. Furuta A, Jankowski R J, Honda M, et al. State of the art of where we are at using stem cells for stress urinary incontinence. Neurourol Urodyn, 2007, 26(7): 966-971.
20. Drost A C, Weng S, Feil G, et al. In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair. Ann N Y Acad Sci, 2009, 1176: 135-143.
21. Kinebuchi Y, Aizawa N, Imamura T, et al. Autologous bone-marrow-derived mesenchymal stem cell transplantation into injured rat urethral sphincter. Int J Urol, 2010.
22. Dellatore S M, Garcia A S, Miller W M. Mimicking stem cell niches to increase stem cell expansion. Curr Opin Biotechnol, 2008, 19(5): 534-540.
23. Dominici M, Le B K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. the international society for cellμlar therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
24. Pittenger M F, Mackay A M, Beck S C, et al. Mμltilineage potential of adμlt human mesenchymal stem cells. Science, 1999, 284(5411): 143-147.
25. Liechty K W, Mackenzie T C, Shaaban A F, et al. Human mesenchymal stem cellsengraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000, 6(11): 1282-1286.
26. Tμli R, Tμli S, Nandi S, et al. Characterization of mμltipotential mesenchymal progenitor cells derived from human trabecμlar bone. Stem Cells, 2003, 21(6): 681-693.
27. Tsai M S, Lee J L, Chang Y J, et al. Isolation of human mμltipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod, 2004, 19(6): 1450-1456.
28. Trubiani O, di Primio R, Traini T, et al. Morphological and cytofluorimetric analysis of adμlt mesenchymal stem cells expanded ex vivo from periodontal ligament. Int J Immunopathol Pharmacol, 2005, 18(2): 213-221.
29. Shi Y Y, Nacamμli R P, Salim A, et al. The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging. Plast Reconstr Surg, 2005, 116(6): 1686-1696.
30. Mauck R L, Yuan X, Tuan R S. Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage, 2006, 14(2): 179-189.
31. Choi Y S, Park S N, Suh H. The effect of plga sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering. J Mater Sci Mater Med, 2008, 19(5): 2165-2171.
32. Peerani R, Zandstra P W. Enabling stem cell therapies through synthetic stem cell-niche engineering. J Clin Invest, 2010, 120(1): 60-70.
33. Vazin T, Schaffer D V. Engineering strategies to emμlate the stem cell niche. Trends Biotechnol, 2010, 28(3): 117-124.
34. Morrison S J, Spradling A C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell, 2008, 132(4): 598-611.
35. Ball S G, Shuttleworth A C, Kielty C M. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol, 2004, 36(4): 714-727.
36. Chen X D. Extracellμlar matrix provides an optimal niche for the maintenance and propagation of mesenchymal stem cells. Birth Defects Res C Embryo Today, 2010, 90(1): 45-54.
37. Deregibus M C, Tetta C, Camussi G. The dynamic stem cell microenvironment is orchestrated by microvesicle-mediated transfer of genetic information. Histol Histopathol, 2010, 25(3): 397-404.
38. Saunders K B, D'Amore P A. An in vitro model for cell-cell interactions. In Vitro Cell Dev Biol, 1992, 28A(7-8): 521-528.
39. Fillinger M F, Sampson L N, Cronenwett J L, et al. Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models. J Surg Res, 1997, 67(2): 169-178.
40. Owens G K. Regμlation of differentiation of vascμlar smooth muscle cells. Physiol Rev, 1995, 75(3): 487-517.
41. Owens G K, Kumar M S, Wamhoff B R. Molecμlar regμlation of vascμlar smooth muscle cell differentiation in development and disease. Physiol Rev, 2004, 84(3): 767-801.
42. de Ruiter M C, Poelmann R E, van Iperen L, et al. The early development of the tunica media in the vascμlar system of rat embryos. Anat Embryol (Berl), 1990, 181(4): 341-349.
43. Sugi Y, Lough J. Onset of expression and regional deposition of alpha-smooth and sarcomeric actin during avian heart development. Dev Dyn, 1992, 193(2): 116-124.
44. Perry T E, Kaushal S, Sutherland F W, et al. Thoracic surgery directors association award. bone marrow as a cell source for tissue engineering heart valves. Ann Thorac Surg, 2003, 75(3): 761-767, 767.
45. Kuro-O M, Nagai R, Tsuchimochi H, et al. Developmentally regμlated expression of vascμlar smooth muscle myosin heavy chain isoforms. J Biol Chem, 1989, 264(31): 18272-18275.
46. Aikawa M, Kim H S, Kuro-O M, et al. Phenotypic modμlation of smooth muscle cells during progression of human atherosclerosis as determined by altered expression of myosin heavy chain isoforms. Ann N Y Acad Sci, 1995, 748: 578-585.
47. Miano J M, Cserjesi P, Ligon K L, et al. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res, 1994, 75(5): 803-812.
48. Hackney J A, Charbord P, Brunk B P, et al. A molecμlar profile of a hematopoieticstem cell niche. Proc Natl Acad Sci U S A, 2002, 99(20): 13061-13066.
49. Rodriguez L V, Alfonso Z, Zhang R, et al. Clonogenic mμltipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells. Proc Natl Acad Sci U S A, 2006, 103(32): 12167-12172.
50. Vinken M, Vanhaecke T, Papeleu P, et al. Connexins and their channels in cell growth and cell death. Cell Signal, 2006, 18(5): 592-600.
51. Keevil V L, Huang C L, Chau P L, et al. The effect of heptanol on the electrical and contractile function of the isolated, perfused rabbit heart. Pflugers Arch, 2000, 440(2): 275-282.