CD34及Sca-1蛋白在不同月龄大鼠阴茎海绵体中的表达

目的 检测CD34和干细胞抗原-1(Sca-1)在不同月龄组SD大鼠阴茎海绵体组织中的表达,对阴茎海绵体干细胞进行初步的观察.方法 随机选取2月龄、5月龄、20月龄不同窝别SD大鼠各10只分别作为幼龄组、壮龄组和老龄组,乙醚麻醉下取阴茎海绵体组织,分别采用免疫组织化学和RT-PCR技术对CD34和Sca-1进行测定.结果 阴茎海绵体组织中存在CD34和Sca-1这两种干细胞抗原标志物的表达.CD34表达以血管及血窦内皮为主,并可见部分散在表达;Sca-1散在表达,主要沿血窦及动脉血管分布;亦可见极少量双阳性染色细胞(CD34/Sca-1),主要表达于海绵体血窦周围.CD34、Sca-1、CD34/Sca-1表达量于组间两两比较均有显著统计学差异(P＜0.01),且均与月龄呈明显负相关(P＜0.01).结论 SD大鼠阴茎海绵体内CD34及Sca-l蛋白表达随月龄增长明显下降,并有双阳性染色细胞的存在.海绵体组织中有内源性干细胞存在的可能.     

一种新的脱细胞阴茎海绵体基质的制备方法

目的探索一种新的脱细胞阴茎海绵体基质的制备方法。方法取健康壮年兔完整阴茎海绵体组织，以Triton-X100与NH3·H2O（氨水）联合提取法进行脱细胞处理。标本作HE染色，组织学观察分析脱细胞效果。结果脱细胞处理25天后，成功获得脱细胞海绵体基质。所得基质外观良好。HE染色观察无细胞存在，弹力纤维排列规整，间隙较大，结构无破坏。结论利用Triton-X100与NH3·H2O联合提取法可成功制备完整无细胞阴茎海绵体基质。     

骨髓CD34+干细胞与单核细胞应用于组织工程小血管内皮化的比较

目的 探讨骨髓CD34+干细胞与骨髓单核细胞(MNCs)应用于组织工程小血管内皮化的效果,并作比较.方法 采集犬骨髓,经免疫磁珠分离出CD34+细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,行细胞免疫荧光、免疫组织化学和体外成血管实验鉴定;将培养后的细胞和未经分离的MNCs分别种植于人工小血管支架,扫描电镜观察,比较两组组织工程小血管的内皮化.结果 经流式细胞仪测定,分离后的细胞中CD34+细胞含量为(78.46±6.37)％.CD34+细胞培养2周后细胞基本铺满培养瓶底面,细胞呈“铺路石”状排列,CD31和Ⅷ因子免疫细胞化学染色均为阳性,体外成血管实验显示6h后CD34+细胞组出现较多毛细血管样网状结构.CD34+细胞组人工血管内膜化面积为(68.4±2.3)％,明显高于MNCs组人工血管内膜化面积(41.3±3.4)％,差异有统计学意义(P＜0.01).结论 通过免疫磁珠方法可分离得到高纯度的骨髓CD34+细胞,经体外培养VEGF诱导后可定向分化为内皮细胞,种植于人工血管内表面,较MNCs组内皮化效果理想.     

兔骨髓间充质干细胞修复膝关节软骨缺损的实验研究

目的研究由同种异体兔骨髓间充质干细胞(MSCs)诱导分化的软骨细胞和聚乳酸-聚乙醇酸共聚物(PLGA)双层支架构建复合体对兔软骨缺损的修复作用。方法用密度梯度离心法和贴壁培养法获得5月龄兔骨髓来源间充质干细胞,在体外培养并进行分化诱导后作为种子细胞复合双层PLGA构建成复合体。36只兔在股骨髁间制造骨软骨缺损模型,分成三组,A组植入复合体,B组植入单纯双层PLGA支架,C组植入自体骨软骨。第24周取材进行大体观察、组织学检查和Wakitani评分。结果 A组及C组植入物愈合良好,组织学检查见Ⅰ、Ⅱ型胶原纤维形成。A组可见透明软骨修复。Wakitani评分A组2.75,B组7.00,C组1.98,A、C组与B组间统计学分析单项指标得分差异有统计学意义(P0.05),A组与C组间差异无统计学意义(P0.05)。结论诱导兔MSCs+PLGA双层支架能较好地修复兔膝关节骨软骨损伤。     

种植自体脂肪干细胞的可吸收性细胞支架移植行兔阴茎增粗术的安全有效性研究

目的 观察自体脂肪干细胞(adipose derived stem cell,ADSC)种植可吸收性细胞支架对小阴茎增粗安全性和有效性.方法 45只雄性成年新西兰白兔随机分为假手术对照组(A组)、单纯Maxpol-T移植组(B组)和种植自体ADSC的Maxpol-T移植组(C组).静脉麻醉后取腹膜后脂肪组织,体外分离、鉴定和扩增ADSC并种植于可吸收性细胞支架Maxpol-T后,移植入兔阴茎皮下深筋膜和白膜之间.术后6个月评估阴茎增粗的效果及其安全性和有效性.结果 ADSC培养后流式鉴定结果CD34(+),CD90(+),CD105(+),CD133(+),CD13(-),CD45(-).移植术后6个月,三组均未发生支架排斥及炎症反应,A组无明显组织再生变化(P＞0.05),B组阴茎直径较A组无明显差异(P＞0.05),C组阴茎直径较A组和B组增粗效果显著(P＜0.001).局部组织病理学结果可发现增生组织及新生血管.结论 应用自体ADSC种植可吸收性细胞支架后植入成年新西兰白兔阴茎皮下,可安全有效地增生阴茎皮下局部组织,无明显排斥和炎症反应.     

兔再造阴茎模型内构建组织工程化阴茎假体的实验研究

目的 探讨于家兔再造阴茎模型内构建带内支撑组织工程化阴茎假体的可行性及其特点.方法 取成年兔耳软骨分离培养软骨细胞,制备以医用多孔高密度聚乙烯(HDPE)为内支撑,外裹聚羟基乙酸(PGA)的支架(直径3mm、长20mm柱状HDPE,外裹厚2 mm的PGA),细胞-支架复合物体外培养4周.以兔一侧腹壁浅血管为轴型血管的皮瓣形成再造阴茎模型,将经体外培养的细胞-支架复合物分别植入兔即时再造的阴茎模型内和对侧腹壁皮下,在对侧腹壁下同时植入一单纯支架.将植入体分为A组:细胞-支架复合物植入再造阴茎模型内;B组:细胞-支架复合物植入腹壁下;C组:单纯支架植入腹壁下.术后3个月将兔处死取材,标本行大体观察、组织学、组织化学、免疫组化、糖胺聚糖(GAG)含量及生物力学检测.结果 A组与B组取材标本的体积、外形与植入时相近,外层新生组织与内部HDPE结合紧密,经HE染色、Safranin O染色、Masson trichrome染色和Ⅱ型胶原免疫组化检测证实,新生组织为软骨组织,且分布相对均一完整,纤维组织长人较少,少量炎性细胞浸润;C组无软骨组织形成.A组与B组标本各项检测指标(湿重、GAG含量、抗压强度、弹性模量)差异无统计学意义(P>0.05).结论 自体软骨细胞接种于HDPE-PGA复合支架,经体外培养4周后,植于家兔即时再造阴茎模型内继续培养,可成功地构建出具有预制形态、体积、结构与组织学良好的软骨-HDPE复合体(阴茎假体).     

干细胞治疗阴茎勃起功能障碍的研究进展

阴茎勃起功能障碍(ED)是指男性反复或者持续性的难以达到和维持充分的阴茎勃起,无法完成性交或满意性活动的病理现象。海绵体神经(CN)损伤引起的勃起神经反射中断,是患者出现ED的直接原因,此外,CN损伤后,阴茎海绵体组织平滑肌细胞和内皮细胞凋亡增加,海绵体平滑肌纤维数量减少加重了ED的发生。因此,尽早干预CN损伤的病理过程,促进CN再生是治疗CN损伤性ED的关键。近年来,干细胞在ED治疗中的应用日益成为临床研究热点。现对胚胎干细胞(ESC)、间充质干细胞(MSCs)、肌源性干细胞(MDSCs)、脂肪干细胞(ADSCs)在ED治疗中的研究综述如下。     

阴茎包埋对海绵体形态结构的影响

目的:探讨阴茎包埋对大鼠阴茎海绵体形态结构的影响。方法:通过建立隐匿阴茎大鼠模型获得实验标本,分2月组、4月组进行观测,每组25只大鼠。各阶段又分包埋组(15只)、正常组(10只),光镜和电镜下观察海绵体形态结构的改变。结果:阴茎包埋2月组海绵体形态结构无明显变化,4月组病理改变较为明显,光镜下见大鼠阴茎海绵体平滑肌细胞,内皮细胞分布杂乱,细胞间大量间质组织增生,海绵窦狭窄;电镜下见阴茎海绵体平滑肌细胞及内皮细胞萎缩、线粒体退变、内质网扩张,致密体及收缩纤维减少,脂滴增加,空泡形成。包埋组与正常组阴茎海绵体在外观和重量上无明显差异(P>0.05)。结论:阴茎包埋对海绵体外观和重量无明显影响,但随着包埋时间的延长,海绵体组织发生超微结构上的病理改变。     

人脐动脉平滑肌细胞复合脱细胞基质构建海绵体平滑肌的动物实验研究

目的 探讨脐动脉平滑肌细胞(HUASMC)与阴茎海绵体脱细胞基质(ACCM)复合构建海绵体平滑肌的可行性.方法 以1%的Triton-X100与0.1%NH3H2O混合液对兔阴茎海绵体进行脱细胞处理,制备ACCM.采用贴块法分离、培养、扩增HUASMC.HUASMC以30×10<'6>/ml密度接种ACCM,细胞-ACCM体外复合10 d后将复合物植入9只5周龄BALB/C裸鼠背部皮下,术后10、20和40 d分别对移植物进行HE染色、免疫组织化学染色和器官浴槽实验,评价其裸鼠体内构建情况.结果 ACCM为白色圆柱状,镜下为富含胶原的疏松多孔结构,不含细胞成分.HUA-SMC与ACCM相容性良好,HUASMC在与ACCM接触部位充分伸展,并沿ACCM窦隙活跃生长.9只裸鼠均存活,植入部位无感染,无植入物排斥发生.随培育时间延长,裸鼠体内ACCM逐渐降解,植入的HUASMC分化形成结构良好、交错排列的平滑肌组织.器官浴槽实验显示,构建组织对去氧肾上腺素和电刺激均表现出收缩功能,去氧肾上腺素和电刺激所诱导的最大收缩力分别为(3.64±0.18)和(2.50±0.21)g.结论 HUASMC作为种子细胞与ACCM复合可构建出具有一定形态和功能的组织工程海绵体平滑肌.     

转基因骨髓间质干细胞在组织工程承载体中生长及成骨转化的研究

[目的]观察经携带人骨形态发生蛋白-2(BMP-2)基因的复制缺陷重组腺病毒(Ad-BMP-2)转染的兔骨髓间质干细胞(MSCs)在以兔脱细胞脱钙骨基质作为组织工程承载体中的生长情况及转基因前后细胞成骨能力的变化。[方法]用携带有人BMP-2基因片段的复制缺陷重组腺病毒(Ad-BMP-2)转染兔骨髓间质干细胞并将其种植在兔脱细胞脱钙骨基质支架中,扫描电镜观察转基因细胞在支架中的黏附生长情况;并通过检测培养上清中的碱性磷酸酶(ALP)、骨钙素(BGP)含量及Ⅰ型胶原(CollagenⅠ)的分泌量来评估转基因对MSCs成骨能力的影响。[结果]转基因骨髓间质干细胞在组织工程支架中黏附生长良好,转基因组细胞的ALP、BGP含量及Ⅰ型胶原的分泌量与对照组比较差异有显著性意义。[结论]转基因骨髓间质干细胞在兔脱细胞脱钙骨基质支架中能很好的黏附并立体生长,转基因细胞在目的基因所表达的BMP-2蛋白作用下成骨能力明显增强。     

骨髓间充质干细胞复合脱细胞血管基质构建组织工程血管的初步研究

目的：探讨骨髓间充质干细胞复合脱细胞血管基质构建组织工程血管的可行性。方法：采用胰蛋白酶、乙二胺四乙酸和曲拉通X-100对兔主动脉进行脱细胞处理，制备成脱细胞血管基质；体外分离和扩增人骨髓间充质千细胞，并将其作为种子细胞种植于脱细胞血管基质上，复合构建组织工程化人工血管。结果：制备的脱细胞血管基质由胶原、弹力纤维等组成，未见细胞成分残留；体外扩增的人骨髓间充质干细胞种植于脱细胞血管基质上可生长增殖。结论：骨髓间充质干细胞与脱细胞血管基质支架材料复合可成功构建组织工程血管，有望为血管缺损的修复提供一种全新的技术方法和手段。     

Construction of tissue‐engineered corpus cavernosum with muscle‐derived stem cells and transplantation in vivo

Study Type – Therapy (case series)
Level of Evidence 4 What’s known on the subject? and What does the study add? The tissue‐engineered research of corpus cavernosum has been studied, but an ideal method was not carried out. In the study, muscle‐derived stem cells were used as seeding cells to construct tissue‐engineered corpus cavernosums. The result demonstrated MDSCs could be seeded on three‐dimensional scaffolds of acellular corporal collagen matrices and developed into tissues similar to native corpus cavernosum in vivo.

OBJECTIVE

• ? To investigate the feasibility of tissue‐engineered corpus cavernosum (TECC) with muscle‐derived stem cells (MDSCs) as seed cells and determine the growth potential in vivo.

MATERIALS AND METHODS

• ? Acellular corporal collagen matrices (ACCMs) were obtained from adult rabbit penis by a cell removal procedure. MDSCs were separated and purified using a digestion method and Preplate technique, then seeded on ACCMs at a concentration of 30 × 10⁶ cells/mL to construct TECCs. After 5 days of culture, seeded ACCMs were implanted with albuginea of rabbits. The implants were retrieved at 2, 4 and 6 months after implantation.
• ? Histochemistry, immunohistochemisry and scanning electron microscopy were performed to analyse the morphological characteristics of the TECCs.

RESULTS

• ? The decellularization process successfully extracted all cellular components while preserving the original collagen fibres.
• ? Histological analyses of the explants at all time points in the experimental group had more cells and better arranged growth than the control group. α‐Smooth muscle actin and endothelial nitric oxide synthase‐positive cells were more prevalent in the experimental group.

CONCLUSION

• ? Our study showed that MDSCs can be seeded on three‐dimensional ACCM scaffolds and develop tissues that are similar to native normal corpus cavernosum.

     

内皮祖细胞复合膀胱无细胞基质的生物相容性

目的 应用内皮祖细胞复合膀胱无细胞基质构建组织工程学膀胱.方法 分离培养猪外周血内皮祖细胞,制备膀胱无细胞基质.体外检测细胞与膀胱无细胞基质的生物相容性.观察细胞与膀胱无细胞基质的体外复合.结果 成功分离培养猪外周血内皮祖细胞,其表面标志分别为CD133 25.1%、CD34 55.9%、flk-1 97.7%、CD31 82.0%、CDl44 95.4%.成功制备膀胱无细胞基质.生物相容性检测证明膀胱无细胞基质对细胞无明显的细胞毒性,细胞活力保持在90%以上.体外观察细胞可以复合于无细胞基质,并在其上增殖、生长.结论 可以应用内皮祖细胞作为种子细胞,膀胱无细胞基质作为支架材料构建组织工程学膀胱,可能成为提高体内组织工程膀胱血管化的一种新方法.     

脱细胞骨软骨支架复合自体骨髓间充质干细胞修复羊骨软骨缺损的研究

目的探讨脱细胞骨软骨支架接种自体骨髓间充质干细胞(BMSCs)修复羊骨软骨缺损效果,探索骨软骨缺损新的修复方式。方法制备直径为8mm骨软骨脱细胞支架,培养羊BMSCs,接种于骨软骨支架,制备羊负重区骨软骨缺损模型,分空白、空白支架及细胞支架复合物3组,每组4只羊,3个月后处死动物取标本行大体及组织学检测。结果修复羊负重区骨软骨缺损模型实验结果显示细胞支架复合修复组骨软骨有较好修复,空白支架组软骨下骨基本修复、软骨侧无明显修复,空白对照组未见明显修复,缺损边缘软骨退变。结论含骨软骨连接结构的脱细胞骨软骨支架接种种子细胞能较好的修复羊负重区骨软骨缺损。     

毛囊干细胞结合电纺纳米丝素纤维构建组织工程尿道膜片的体外研究

目的：探索毛囊干细胞结合电纺纳米丝素纤维,构建组织工程尿道膜片的可行性。方法使用酶消化法分离兔毛囊细胞,利用流式细胞仪分选毛囊干细胞,体外原代培养扩增,计算细胞的最大扩增倍数及克隆形成率;使用流式细胞仪检测细胞K19、p63、CD29的阳性表达率。将毛囊干细胞接种在电纺纳米丝素纤维支架上,构建组织工程尿道。应用组织学、荧光染色法观察组织工程尿道的体外构建情况。结果兔毛囊干细胞与兔毛囊细胞的最大扩增倍数分别为（5.92±0.77）×10^4倍和（6.61±3.62）×10^3倍;兔毛囊干细胞与兔毛囊细胞的克隆形成率分别为（9.07±1.18）%和（3.95±1.01）%。K19、p63、CD29在毛囊干细胞中表达率分别为（89.36±6.69）%、（92.10±5.49）%和（89.98±6.89）%;在兔毛囊细胞中分别为（39.38±2.20）%、（40.78±2.61）%和（40.57±2.79）%;毛囊干细胞在支架上能形成复层上皮样结构,AE1/AE3染色阳性。结论兔毛囊干细胞结合电纺纳米丝素纤维,能成功构建组织工程尿道,可用于体内修复的进一步研究。     

Engineering Vascularized Bone Graft With Osteogenic and Angiogenic Lineage Differentiated Bone Marrow Mesenchymal Stem Cells

Tissue‐engineered bone provides a promising method for the rehabilitation of acquired bone defects and congenital deformities. However, generating a vascular supply to the engineered graft remains a major challenge. We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells (MSCs). MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet–EPC complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro‐CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. The results showed that cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary‐like structures in vitro. The osteogenic cell sheet–EPC complexes yielded well‐vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet–EPC complex group than in the control group. The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet‐based bone engineering. These findings suggest that the strategy of engineering bone grafts with osteogenic and angiogenic lineage differentiated MSCs has great potential for clinical applications to repair large bone defects.     

力学刺激促进软骨细胞与骨髓基质干细胞共培养体外软骨分化

目的：研究不同的应力刺激对软骨细胞与骨髓基质干细胞（BMSCs）共培养体外构建组织工程化软骨的影响。方法：分离、培养、扩传兔MSCs及软骨细胞,二者按7：3比例混和,以5．0×107／ml的细胞密度接种于聚羟基乙酸（PGA）支架上,一周后根据不同的施加力分为4组：离心组、摇床组、搅拌组,静止培养作为对照组。6周后取材行相关检测。结果：三受力组形成的细胞材料复合物基本保持原来的体积与外形。HE染色结果显示大量成熟软骨陷窝形成,细胞外基质沉积均匀;Safranin-O及甲苯胺兰染色显示有大量的GAG形成,免疫组化检测II型胶原表达强阳性。三受力组标本组织湿重、体积、GAG含量等指标均优于对照组。结论：力学刺激有利于促进少量软骨细胞与BMSCs共培养体外软骨分化;并在三维支架材料上构建组织工程化软骨。     

Neural‐differentiated mesenchymal stem cells incorporated into muscle stuffed vein scaffold forms a stable living nerve conduit

Autologous nerve grafts to bridge nerve gaps have donor site morbidity and possible neuroma formation resulting in development of various methods of bridging nerve gaps without using autologous nerve grafts. We have fabricated an acellular muscle stuffed vein seeded with differentiated mesenchymal stem cells (MSCs) as a substitute for nerve autografts. Human vein and muscle were both decellularized by liquid nitrogen immersion with subsequent hydrolysis in hydrochloric acid. Human MSCs were subjected to a series of treatments with a reducing agent, retinoic acid, and a combination of trophic factors. The differentiated MSCs were seeded on the surface of acellular muscle tissue and then stuffed into the vein. Our study showed that 35–75% of the cells expressed neural markers such as S100b, glial fibrillary acidic protein (GFAP), p75 NGF receptor, and Nestin after differentiation. Histological and ultra structural analyses of muscle stuffed veins showed attachment of cells onto the surface of the acellular muscle and penetration of the cells into the hydrolyzed fraction of muscle fibers. We implanted these muscle stuffed veins into athymic mice and at 8 weeks post‐implantation, the acellular muscle tissue had fully degraded and replaced with new matrix produced by the seeded cells. The vein was still intact and no inflammatory reactions were observed proving the biocompatibility and biodegradability of the conduit. In conclusion, we have successfully formed a stable living nerve conduit which may serve as a substitute for autologous nerves. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1674–1681, 2012     

组织工程心脏瓣膜裸鼠体内移植的实验研究

目的将人血管混合细胞体外动态种植于脱细胞牛心包构建的组织工程心脏瓣膜（Tissue engineered hean valves，TEHVs）并移植入裸鼠体内，观察其生物学特征变化。方法将体外动态构建的TEHVs移植入裸鼠体内，30d时取材，观察组织、细胞的超微结构变化，种子细胞的生长行为和生物学特征变化，种子细胞与支架材料的黏附性，TEHVs的生物相容性等参数。结果组织学观察见：细胞数量、分层数较移植前增多，并有大量细胞浸润入TEHVs深层组织内：免疫组化显示：TEHVs上细胞的内皮细胞Ⅷ因子、平滑肌细胞α-肌动蛋白、成纤维细胞Fibronectin相关抗原呈阳性表达；扫描电镜见：裸鼠体内移植后细胞数量较移植前增多，细胞表面和细胞之间的网状胶原结构较埋置前增多明显；透射电镜可见：W—P小体、质膜小泡、密斑、密体、微丝等特征性结构。结论TEHVs细胞能够在裸鼠体内定向分化、增殖，生物学特征无改变，TEHVs生物相容性良好。