摘要
第一部分:大鼠骨髓间充质干细胞的分离、培养和鉴定
目的:建立一套简单高效,重复性好的体外分离培养大鼠骨髓间充质干细胞(MSCs)的方法,为阴道组织工程的研究作准备。
方法:全骨髓贴壁培养法培养MSCs。分离大鼠双侧胫、股骨,剪去两侧骨骺端,无菌条件下用DMEM/F12培养基冲洗骨髓腔。获得的细胞悬液移入离心管200g,5min离心。弃上清,DMEM/F12完全培养基重悬,70μm细胞筛过滤细胞悬液,计数,调整细胞浓度为7.5×107/ml,以1.5×107个/cm~2密度铺于25cm~2细胞培养瓶中,即每瓶5ml。置细胞培养箱中培养。48小时首次半量换液,之后3-4天换液一次。待细胞70%-80%融合时使用胰蛋白酶消化传代。弃去培养液,37℃预热的PBS液冲洗约2-3次,加入0.5ml胰蛋白酶溶液,25℃消化约2min,加入DMEM/F12完全培养基终止消化。弯头吸管沿瓶底轻轻吹打,收集细胞悬液,以1×104个/cm~2的密度接种于新的培养瓶内进行扩增培养。MTT法评价第1至5代细胞增殖情况。经传代纯化后第三至五代MSCs使用流式细胞学分析CD29,CD90及CD45表达。成骨成脂诱导鉴定MSCs的多向分化能力。成骨诱导液诱导14天后,按照试剂盒说明做碱性磷酸酶(ALP)活力测定,不溶的红色颗粒沉淀指示ALP的活性位点。诱导21-28天时,用茜素红染色评价大鼠骨髓间充质干细胞的矿化能力。成脂诱导液诱导14天后油红O染色确定是否有脂滴生成。
结果:原代培养7-10天左右细胞呈集落生长,逐渐融合成片,融合约70%-80%,可传代培养。传代活细胞24h完全贴壁生长。传代后细胞生长更快,三至五天后细胞即可融合,呈梭形漩涡样生长,可再次传代。传代MSCs在接种第1-2天为细胞适应期,第3-4天进入对数生长期。第5天后曲线逐渐变得平缓,细胞增殖明显减慢,进入平台期。MSCs经流式细胞分析高表达CD29,CD90,阳性率分别为99.91%、99.95%;而CD45呈阴性,阳性率为0.03%。成骨分化诱导2周后ALP活性增高,4周后茜素红矿化结节染色阳性。成脂分化诱导2周后细胞中出现油红O染色阳性的脂滴。
结论:使用全骨髓贴壁培养法可以获得高纯度,具有多向分化能力的MSCs。
第二部分:大鼠阴道上皮细胞原代培养方法的改进
目的:探讨大鼠阴道上皮细胞(VECs)体外培养的影响因素,对现有的VECs体外分离培养方法进行改进,建立大鼠阴道上皮细胞稳定的体外分离培养方法以用于组织工程。
方法:酶消化法分离消化大鼠阴道上皮细胞。取阴道全层组织,修剪成1×0.5cm大小,移入超净台。组织块经4℃PBS清洗液彻底清洗6遍,加入中性蛋白酶Ⅱ型(DispaseⅡ),放4℃冰箱过夜消化18h。取出后经PBS冲洗,分离上皮层。上皮层加入0.25%胰蛋白酶+0.02%EDTA后37℃消化。轻轻吹打,DMEM/F12完全培养基终止消化,吸管吹打成单细胞悬液,200g,5min离心,KBM完全培养液重悬,细胞计数板计活细胞数量,细胞悬液以1.0×105个/cm~2的密度接种于培养板中,置细胞培养箱中静置培养。第2天首次换液,以后每两天换液一次。在此方法基础上比较大鼠不同动情周期(动情前期,动情期,动情后期),不同DispaseⅡ配置(PBS配置与KBM配制),不同DispaseⅡ浓度(0.6U/ml与1.2U/ml),不同胰蛋白酶消化方法(30min消化一次与10min重复3次),培养表面不同处理(FN/C包被及去离子水包被),不同培养基(KBM培养基与含6%FBS的KBM培养基)对大鼠阴道上皮细胞体外培养的影响,对上皮分离的结果进行评分,并观察48h首次换液时细胞贴壁情况。免疫细胞化学染色及免疫荧光检测广谱角蛋白AE1/AE3表达。扫描电镜观察细胞表面超微结构。
结果:动情前期的大鼠阴道上皮层消化后可获得更多大鼠阴道上皮细胞(P=0.0346);上皮层用KBM配制的DispaseⅡ消化后48h贴壁细胞多于用PBS配制的DispaseⅡ;1.2U/mlDispaseⅡ的分离结果评分及可获活细胞数高于0.6U/mlDispaseⅡ(P=0.025; P=0.034);三步胰酶消化方法相比一步胰酶消化方法可获得更多的贴壁细胞;FN/C包被液处理的培养表面相比去离子水可以获得更多的贴壁细胞;大鼠阴道上皮细胞在含血清培养基中生长快,但易受成纤维细胞污染。经改良方法培养的VECs免疫细胞化学染色及免疫荧光显示广谱角蛋白AE1/AE3阳性。扫描电镜显示VECs呈镶嵌排列,细胞之间界限清晰,表面可见褶曲和微绒毛,呈不规则疏网状。
结论:使用改良的原代培养方法可获得更多的大鼠阴道上皮细胞,细胞显示上皮细胞特征。
第三部分:大鼠阴道上皮细胞诱导骨髓间充质干细胞向上皮细胞的分化
目的:探索体外诱导骨髓间充质干细胞(MSCs)向上皮细胞分化的可行性。
方法:将阴道上皮细胞(VECs)铺于事先包被FN/C的0.4μm Transwellinsert共培养小室内,与铺有MSCs的6孔板建立间接共培养体系,诱导MSCs向上皮细胞分化。分别于0天、7天及14天时,移去小室,倒置显微镜下观察MSCs的形态变化。细胞爬片使用免疫细胞化学染色对诱导后的MSCs进行上皮细胞标志物广谱角蛋白AE1/AE3的检测。VECs和MSCs分别用作阳性及阴性对照。Western Blot检测上皮细胞标志物广谱角蛋白AE1/AE3的表达水平。GAPDH用作内参照物。
结果:MSCs与VECs共培养诱导分化7天时,MSCs的形态由0天时长梭形开始变为上皮样的多边形。共培养14天时,更多MSCs变为多边形。免疫细胞化学染色表明经诱导7天及14天的MSCs广谱角蛋白AE1/AE3表达阳性;Western blot检测诱导后的MSCs表达AE1/AE3,诱导7天组AE1/AE3的蛋白水平高于0天组(36.28±1.49vs.1.00±0.00,P<0.01),诱导14天组AE1/AE3的蛋白水平高于0天组(48.80±3.17vs.1.00±0.00,P<0.01)及7天组(48.80±3.17vs.36.28±1.49,P=0.0232)。
结论:VECs可以通过间接共培养的方式诱导MSCs向上皮细胞分化,上皮细胞标志物广谱角蛋白AE1/AE3的表达量随时间延长而增高。
第四部分:骨髓间充质干细胞向上皮细胞分化中Wnt/β-catenin信号通路的调控作用
目的:探讨骨髓间充质干细胞(MSCs)向上皮细胞分化过程中Wnt/β-catenin信号通路的可能作用。
方法: MSCs和VECs间接共培养诱导分化。分别于0天、7天及14天时,使用Western Blot对诱导后的MSCs及VECs进行Wnt/β-catenin信号通路的重要成分β-catenin,GSK-3β,pi-GSK-3β,TCF-3表达水平的测定。为研究Wnt信号通路在MSCs向上皮细胞分化中的调控作用,分三组进行MSCs和VECs的间接共培养诱导:(1)对照组加入含3%FBS的KBM培养液;(2) Wnt通路激动剂组加入含20μM氯化锂(LiCl)的3%FBS-KBM培养液;(3) Wnt通路抑制剂组加入含20ng/ml Dickkopf-1(DKK-1)的3%FBS-KBM培养液。于共培养第7天移去培养小室,使用Western Blot对诱导后的MSCs进行Wnt/β-catenin信号通路的重要成分β-catenin,GSK-3β,TCF-3及广谱角蛋白AE1/AE3的表达水平测定。GAPDH用作内参照物。
结果:MSCs与VECs共培养7天时,Western blot检测诱导后的MSCs其β-catenin (1.50±0.11vs.1.00±0.00, P=0.009), pi-GSK-3β (1.50±0.23vs.1.00±0.00, P=0.09)及TCF-3(1.25±0.16vs.1.00±0.00, P=0.19)的表达量逐渐增加,GSK-3β的表达量(0.69±0.04vs.1.00±0.00, P=0.002)逐渐减少。共培养14天与共培养7天相比,β-catenin (1.85±0.23vs.1.50±0.11,P=0.24)与pi-GSK-3β (2.31±0.33vs1.50±0.23, P=0.114)的表达水平增高,GSK-3β (0.34±0.03vs.0.69±0.04, P=0.0021)及TCF-3(0.98±0.17vs.1.25±0.16, P=0.3144)的表达水平下降。GSK-3β的表达与β-catenin呈现出相反的趋势。pi-GSK-3β的表达与GSK-3β也呈现出相反的趋势。诱导7天时,20μM LiCl组β-catenin (1.24±0.07vs.1.00±0.00, P=0.02),TCF-3(2.15±0.24vs.1.00±0.00, P=0.14)表达增加, GSK-3β(0.60±0.07vs.1.00±0.00, P<0.01)表达减少;20ng/ml DKK-1组β-catenin(0.92±0.08vs.1.00±0.00, P=0.38),TCF-3(0.77±0.09vs.1.00±0.00, P=0.06)表达减少,GSK-3β (1.11±0.03vs.1.00±0.00, P=0.02)表达增加。检测AE1/AE3的表达水平发现相比对照组,LiCl可以增加AE1/AE3的表达水平(1.20±0.18vs.1.00±0.00, P=0.32),而DKK-1则降低AE1/AE3的表达(0.83±0.11vs.1.00±0.00, P=0.19)。
结论:Wnt/β-catenin信号通路可能参与调控VECs诱导MSCs向上皮细胞分化的过程,使用LiCl激活Wnt/β-catenin信号通路可以加速分化。
Part one:Isolation, culture and characterization of rat bone marrowmesenchymal stem cells in vitro
Objective: To establish a simple, high-yield, and repeatable protocol forisolation and culture of mesenchymal stem cells in vitro, as the foundation ofthe vaginal tissue engineering.
Methods: Bone marrow cells were isolated and cultured following thewhole bone marrow adherent protocol. Bone marrow from the bilateral femursand tibias were flushed out using DMEM/F12medium under asepticconditions. The DMEM/F12marrow suspension was then filtered through a70μm cell strainer, collected, and centrifuged for5minutes at200g. Afterremoval of the supernatant, the resulting pellet was resuspended inDMEM/F12supplemented with10%fetal bovine serum,2mM–glutamine,1%penicillin, and streptomycin and plated at a density of1.5×107cells/cm~2.It was then maintained in a humidified atmosphere of95%air and5%CO2at37°C. Nonadherent cells were discarded after48h. The cells were fed every3–4days and passaged when70-80%confluent with0.25%trypsin/0.02%EDTA. After discarded the culture medium, wash the cells with37℃preheating phosphate-buffered saline and lift cells by incubation in0.5ml of0.25%trypsin/0.02%EDTA for2min at room temperature. Neutralize thetrypsin by adding complete medium, and culture all lifted cells in a25cm~2flask at a density of1×104cells/cm~2. The proliferation of MSCs at passage1to passage5was evaluated by MTT protocol. After several subculturingpurification,the MSCs at passage three to five were tested for the markersCD45, CD29, and CD90by flow cytometry analysis. Osteoblast and adipocytedifferentiation of MSCs were conducted to determine whether the cells hadmulti-potential properties. After14d of osteoblast differentiation, the alkaline phosphatase (ALP) activity of the cells was assessed using an ALP Kitfollowing the manufacturer’s instructions. The resulting red, insoluble,granular dye deposit indicated sites of ALP activity. Following an additional7-14d culture, the wells were stained with Alizarin Red to evaluate themineralization capacity. After14days of adipogenic differentiation, Oil Red Ostaining was conducted to assess lipid accumulation.
Results: After7–10days of initial culture, distinct colonies offibroblastic cells formed in various sizes. When cell confluence achieved70%-80%, the cells were subcultured to passage one. After the subcultures, allthe living cells could adherent within24h. Cells grew more rapidly afterpassage. The cells were confluent and had a spiral whorl-like outlook after3-5days, which indicating another passaging. After the subculture, MSCs were inadaptive phase at1-2days, and went into logarithmic phase at3-4days. After5days, the proliferation of MSCs slow down and the curve flattened gradually,which suggesting they were in plateau phase. Flow cytometry analysis showedthat MSCs expressed CD29and CD90at99.91%and99.95%separately, butnot CD45. For osteogenic differentiation, ALP activity increased after14d ofculture, and mineralized nodules formed after4weeks of induction. Foradipogenic differentiation, intracellular Oil-red-O-stained lipids appeared after2weeks of culture.
Conclusions: Whole bone marrow adherent protocol is a simple, efficientmethod to yield MSCs with high purification and multi-potential properties.
Part two:Improved protocol for primary culture of rat vaginal epithelialcells in vitro
Objective: To explore factors impacting on primary culture of rat vaginalepithelial cells (VECs) in vitro, to improve the current protocol of VECsseparation and culture, and establish a reproducible protocol for VECsseparation and culture that could be applied in tissue engineering.
Methods: VECs were harvested by enzyme digestion protocol. Thevaginal tissues were dissected out, cleaned of as much connective tissue aspossible, minced into small pieces (10×5mm~2), and rinsed three times with PBS supplemented with penicillin/streptomycin (100units/ml,100mg/mL) at4℃. Vaginal tissues were incubated in Dispase II overnight at4°C. VECs wereisolated from the epithelium and peeled away from vaginal pieces by threerounds of enzymatic digestion with0.25%trypsin/0.02EDTA at37°C. Withgentle pipette suction and neutralize the trypsin by adding complete medium,the cell-fluid suspension was centrifuged at200g for5min. The pellet wasresuspended in keratinocyte growth medium with growth supplement,distributed into culture dishes with a density of1.0×105cells/cm~2, andmaintained in KGM with medium changes every48h. Based on the protocolmentioned above, we compared influence of rats in different estrous cycles(preoestrus, oestrus, metaoestrus, and anestrus), different Dispase Ⅱpreparation (0.6U/ml DispaseⅡsolved in PBS and0.6U/ml DispaseⅡsolvedin KBM), different Dispase Ⅱ concentration (0.6U/ml DispaseⅡsolved inKBM and1.2U/ml DispaseⅡsolved in KBM), different trypsine digestion(30min×1and10min×3), different coating solution (FN/C and deionizedwater), and different culture medium (KBM and KBM supplemented with6%FBS) on VECs culture. Detach scores of vaginal tissue and available live cellswere evaluated. At the first feed, the attachments of VECs on48h after theinitial culture were also observed. Epithelial cell phenotypes were confirmedby morphology, immunohistochemical and immunoflurorescence staining withantibodies to pan cytokeratins (AE1/AE3). The ultrastructure was oberservedby scanning electron microscope.
Results: Epithelial layer of rat in preoestrus was more abundant thanother phases (P=0.0346). More VECs could attach after digestion withDispase Ⅱ solving in KBM than in PBS. Detach scores of epithelium fromunderlying connective tissues with1.2U/ml Dispase and available live cellsamout were higher than that of0.6U/ml Dispase (P=0.025; P=0.034). MoreVECs could attach after digestion with trypsine by3steps than that by onestep, and more VECs could attach after seeding on Fibronectin/collagen (FN/C)coating plastic culture vessels. VECs grew fast in medium containing fetalbovine serum, but were vulnerable to fibroblast cells contamination. The expression of AE1/AE3staining of the cultured cells was positive. Microvilliridges were observed on cells' surface with scanning electron microscope.
Conclutions: More VECs demonstrating epithelial characterization canbe obtained by improved protocol.
Part three: Differentiation of bone marrow mesenchymal stem cells intoepithelial cells
Objective: To explore the feasibility of differentiation of bone marrowmesenchymal stem cells (MSCs) into epithelial cells in vitro.
Methods: MSCs were gathered following the whole bone marrowadherent culture protocol. Vaginal epithelial cells (VECs) were collected bydispase and trypsin digestion protocol. For differentiation, MSCs wereco-cultured with VECs using cell culture transwell inserts (0.4μm pore,4.5cm~2) for14days. Fibronectin/collagen type I (FN/C)-coated inserts weredehydrated, loaded with VECs in KGM and incubated for48h. Then, MSCswere dispensed into the culture well or coverglass and incubated for24h. Atthe end of the incubation period, VECs in the top chamber and MSCs in thebottom chamber were washed, and3ml of growth medium was added to theculture wells. VECs in the top chamber and MSCs in the bottom chamberwere co-cultured using cell culture transwell inserts. The culture system wasassembled and incubated with KGM with growth supplement and FBS at afinal concentration of3%for indirect co-culture. The cells were feed every2-3days. Then,7–14days after the initial plating, morphological analysis(microscopy) and protein expression of pan-cytokeratin AE1/AE3(Westernblotting and immunocytochemistry) were assessed. VECs and MSCs wereused as positive and negative controls, respectively. Western Blot:Cells werewashed twice with PBS and lysed in RIPA extraction buffer for30min on ice.After centrifugation at12,000rpm for30minutes, the supernatants werecollected and the protein concentration was estimated using NanodropND-1000Spectrophotometer. Proteins were separated on8%SDS-PAGE gels,and electroblotted onto polyvinylidene difluoride (PVDF) membrane at4°C.The blots were blocked with blocking buffer (5%non-fat dry milk in PBS) for 1hour. The blots were incubated with anti-pan-cytokeratins (AE1/AE3), orGAPDH antibodies overnight in TBS-T at4°C. After washing3times inTBS-T for10minutes each, blots were incubated with fluorescent labeledIRDye800secondary antibody diluted in TBS-T in the dark for2h at roomtemperature. After thorough washing in TBS-T, the blots were scanned usingthe LI-COR Odyssey Infrared Imaging System using the appropriate channels.The integral optical density of bands was detected with Gelpro Analyzer. Theexpression levels of AE1/AE3were determinated with GAPDH as innercontrol.
Results: The morphology of MSCs changed from spindle shapes to apolygonal epithelium-like shape when co-cultured with VECs fordifferentiation on day7and day14. Immunocytochemical staining of MSCsco-cultured with vaginal epithelial cells for AE1/AE3revealed expression ofepithelial marker in induced cells. Western blot analysis showed atime-dependent increase in protein expression of epithelial marker AE1/AE3in induced MSCs for0,7, and14days (1.00±0.00;36.28±1.49,7days vs.0day, P<0.01;48.80±3.17,14days vs.0day, P<0.01). Cells at14days showedhigher levels of AE1/AE3protein than cells at7days (48.80±3.17vs.36.28±1.49, P=0.0232).
Conclusions: VECs can trigger epithelial differentiation of MSCs byindirect co-culture. The expression level of epithelial marker AE1/AE3increased with the co-culture time prolonged.
Part four: Role of Wnt/β-catenin signaling pathway in the differentiationof bone marrow mesenchymal stem cells into epithelial cells
Objective: To explore the role of the Wnt/β-catenin signaling pathway inthe process of differentiation of bone marrow mesenchymal stem cells (MSCs)into epithelial cells.
Methods: For differentiation, VECs in the top chamber and MSCs in thebottom chamber were co-cultured using0.4μm cell culture transwell inserts.Then,7–14days after the initial plating, Western blot was conducted to detectthe expression of β-catenin,GSK-3β,pi-GSK-3β, TCF-3, and pan-cytokeratin AE1/AE3. To evaluate the role of Wnt/β-catenin signaling, the co-culturemedium of MSCs and VECs were devided into three groups:(1) control group,KBM supplemented with3%FBS;(2) Wnt signaling activator group, KBMsupplemented with3%FBS and20μM lithium chloride (LiCl);(3) Wntsignaling inhibitor group, KBM supplemented with3%FBS and20ng/mlDickkopf-1(DKK-1). Western blotting was used to show the expression levelsof β-catenin, GSK-3β, TCF-3, and pan-cytokeratin AE1/AE3under co-cultureconditions with LiCl and DKK-1for7days. GAPDH was used as the innercontrol.
Results: Western blot analysis demonstrated more expression ofβ-catenin (1.50±0.11vs.1.00±0.00, P=0.009), pi-GSK-3β (1.50±0.23vs.1.00±0.00, P=0.09) and TCF-3(1.25±0.16vs.1.00±0.00, P=0.19) and lessexpression of GSK-3β (0.69±0.04vs.1.00±0.00, P=0.002) in co-culturedMSCs on day7than on day0. After14days of co-culture, the expressionlevels of β-catenin (1.85±0.23vs.1.50±0.11, P=0.24) and pi-GSK-3β (2.31±0.33vs1.50±0.23, P=0.114) were higher than after7days, and GSK-3βlevels were lower (0.34±0.03vs.0.69±0.04, P=0.0021). However, theexpression level of TCF-3was lower than at7days (0.98±0.17vs.1.25±0.16, P=0.3144). The expression levels of GSK-3β in co-cultured MSCspresented trend opposite that of β-catenin levels. In addition, thephosphorylation levels of GSK-3β in induced MSCs tended to oppose thelevels of GSK-3β, corresponding to their each protein levels.20μM LiClincreased the expression levels of β-catenin (1.24±0.07vs.1.00±0.00, P=0.02)and TCF-3(2.15±0.24vs.1.00±0.00, P=0.14) and decreased expression ofGSK-3β (0.60±0.07vs.1.00±0.00, P<0.01) after7days of co-culture.Conversely, β-catenin (0.92±0.08vs.1.00±0.00, P=0.38) and TCF-3(0.77±0.09vs.1.00±0.00, P=0.06) expression decreased after DKK-1exposure, and GSK-3β (1.11±0.03vs.1.00±0.00, P=0.02) expressionincreased. Moreover, LiCl caused an increase in the levels of AE1/AE3expression relative to controls (1.20±0.18vs.1.00±0.00, P=0.32). Cellsexposed to DKK-1showed less AE1/AE3expression than those in control group (0.83±0.11vs.1.00±0.00, P=0.19).
Conclusions: Wnt/β-catenin signaling might participate in the regulationof MSC differentiation into epithelial cells under co-culture conditions andthat up-regulation of Wnt/β-catenin with LiCl might speed up thedifferentiation process.
引文
1Kimberley N, Hutson JM, Southwell BR, et al. Well-being and sexualfunction outcomes in women with vaginal agenesis. Fertility and Sterility,2011,95(1):238-241
2zkan, Erman Akar M, zkan, et al. Reconstruction of VaginalAgenesis. Annals of Plastic Surgery,2011,66(6):673-678
3Lima M, Ruggeri G, Randi B, et al. Vaginal replacement in the pediatricage group: a34-year experience of intestinal vaginoplasty in children andyoung girls. Journal of Pediatric Surgery,2010,45(10):2087-2091
4Dorin RP, Atala A, Defilippo RE. Bioengineering a vaginal replacementusing a small biopsy of autologous tissue. Semin Reprod Med,2011,29(1):38-44
5De Filippo RE, Yoo JJ, Atala A. Engineering of vaginal tissue in vivo.Tissue Engineering,2003,9(2):301-306
6De Filippo RE, Bishop CE, Filho LF, et al. Tissue engineering a completevaginal replacement from a small biopsy of autologous tissue.Transplantation,2008,86(2):208-214
7Panici PB, Bellati F, Boni T, et al. Vaginoplasty using autologous in vitrocultured vaginal tissue in a patient withMayer-von-Rokitansky-Kuster-Hauser syndrome. Human Reproduction,2007,22(7):2025-2028
8周慧梅.组织工程医用生物补片重建阴道的实验及临床研究.北京,中国协和医科大学;2008
9朱兰,周慧梅,郎景和.组织工程医用补片在人工阴道成形术中的应用.中国实用妇科与产科杂志,2007,22(12):953-954
10翁慧男,王沂峰,刘风华.建立人阴道黏膜干细胞构建人工阴道的动物模型研究.中国实用妇科与产科杂志,2006,22(9):669-672
11张明乐,黄向华,李雅钗,等.小鼠阴道上皮细胞与猪脱细胞真皮基质-纤维蛋白凝胶构建组织工程阴道的初步研究.实用妇产科杂志,2012,28(8):639-642
12P unescu V, Deak E, Herman D, et al. In vitro differentiation of humanmesenchymal stem cells to epithelial lineage. Journal of Cellular andMolecular Medicine,2007,11(3):502-508
13Gu S, Xing C, Han J, et al. Differentiation of rabbit bone marrowmesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.Molecular Vision,2009,15:99-107
14Liu A, Liu L, Chen S, et al. Activation of canonical wnt pathwaypromotes differentiation of mouse bone marrow-derived MSCs into typeII alveolar epithelial cells, confers resistance to oxidative stress, andpromotes their migration to injured lung tissue in vitro. Journal ofCellular Physiology,2012,228(6):1270-1283
15谢林海,刘伟.骨髓间充质干细胞在阴道组织工程应用.江西医药,2009,44(2):176-179
16Atala A. Tissue engineering of reproductive tissues and organs. Fertilityand Sterility,2012,98(1):21-29
17薛龙,刘伟,李文芳,等. BMSCs分化为阴道上皮细胞的可行性探索.江西医药,2010,45(009):941-944
18薛龙.体外诱导兔骨髓间充质干细胞向上皮细胞分化的实验研究,南昌大学;2009
19张明乐,李雅钗,黄向华.组织工程技术在阴道重建中的应用.中国修复重建外科杂志,2011,25(7):864
20Valkenburg KC, Graveel CR, Zylstra-Diegel CR, et al. Wnt/β-cateninSignaling in Normal and Cancer Stem Cells. Cancers,2011,3(2):2050-2079
21Ling L, Nurcombe V, Cool SM. Wnt signaling controls the fate ofmesenchymal stem cells. Gene,2009,433(1-2):1-7
22Friedenstein A, Chailakhjan R, Lalykina K. The development offibroblast colonies in monolayer cultures of guinea‐pig bone marrowand spleen cells. Cell Proliferation,1970,3(4):393-403
23da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stemcells reside in virtually all post-natal organs and tissues. Journal of CellScience,2006,119(11):2204-2213
24Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential ofadult human mesenchymal stem cells. Science,1999,284(5411):143-147
25Arthur A, Zannettino A, Gronthos S. The therapeutic applications ofmultipotential mesenchymal/stromal stem cells in skeletal tissue repair.Journal of Cellular Physiology,2009,218(2):237-245
26Hare JM, Fishman JE, Gerstenblith G, et al. Comparison of Allogeneic vsAutologous Bone Marrow–Derived Mesenchymal Stem Cells Deliveredby Transendocardial Injection in Patients With Ischemic CardiomyopathyThe POSEIDON Randomized Trial Mesenchymal Stem Cells andIschemic Cardiomyopathy. JAMA,2012,308(22):2369-2379
27Elliott MJ, De Coppi P, Speggiorin S, et al. Stem-cell-based, tissueengineered tracheal replacement in a child: a2-year follow-up study. TheLancet,2012,380(9846):994-1000
28Lee JS, Hong JM, Moon GJ, et al. A Long-Term Follow-Up Study ofIntravenous Autologous Mesenchymal Stem Cell Transplantation inPatients With Ischemic Stroke. Stem Cells,2010,28(6):1099-1106
29Jiang R, Han Z, Zhuo G, et al. Transplantation of placenta-derivedmesenchymal stem cells in type2diabetes: a pilot study. Front. Med.,2011,5(1):94-100
30Si YL, Zhao YL, Hao HJ, et al. MSCs: Biological characteristics, clinicalapplications and their outstanding concerns. Ageing Research Reviews,2011,10(1):93-103
31Martin DR, Cox NR, Hathcock TL, et al. Isolation and characterization ofmultipotential mesenchymal stem cells from feline bone marrow.Experimental Hematology,2002,30(8):879-886
32Bosnakovski D, Mizuno M, Kim G, et al. Isolation and multilineagedifferentiation of bovine bone marrow mesenchymal stem cells. CellTissue Res,2005,319(2):243-253
33Izadpanah R, Joswig T, Tsien F, et al. Characterization of multipotentmesenchymal stem cells from the bone marrow of rhesus macaques. StemCells and Development,2005,14(4):440-451
34Fau LD, Caplan AI. Isolation of rat marrow-derived mesenchymal stemcells. Experimental Hematology,2006,34(11):1606-1607
35McCarty RC, Gronthos S, Zannettino AC, et al. Characterisation anddevelopmental potential of ovine bone marrow derived mesenchymalstem cells. Journal of Cellular Physiology,2009,219(2):324-333
36Kadiyala S, Young RG, Thiede MA, et al. Culture expanded caninemesenchymal stem cells possess osteochondrogenic potential in vivo andin vitro. Cell Transplantation,1997,6(2):125-134
37林楚伟,周胜华,杜优优.全骨髓贴壁并差速传代分离纯化大鼠骨髓间充质干细胞:与密度梯度离心法的比较.中国组织工程研究与临床康复,2010,14(14):2508-2512
38Soleimani M, Nadri S. A protocol for isolation and culture ofmesenchymal stem cells from mouse bone marrow. Nature Protocols,2009,4(1):102-106
39Pertoft H, Laurent TC, L s T, et al. Density gradients prepared fromcolloidal silica particles coated by polyvinylpyrrolidone (Percoll).Analytical Biochemistry,1978,88(1):271-282
40English D, Andersen BR. Single-step separation of red blood cells,granulocytes and mononuclear leukocytes on discontinuous densitygradients of Ficoll-Hypaque. Journal of Immunological Methods,1974,5(3):249-252
41Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation ofrapidly self-renewing and multipotential adult stem cells in colonies ofhuman marrow stromal cells. Proc Natl Acad Sci U S A,2001,98(14):7841-7845
42李艳菊,李宁,胡亮杉,等.大鼠骨髓间充质干细胞体外培养方法的改进.中国组织工程研究与临床康复,2009,13(1):71-74
43王佳南,张晓刚,汤为学,等.大鼠骨髓间充质干细胞的原代培养条件.中国组织工程研究与临床康复,2009,13(14):2740-2745
44彭德新,陈自谦,白伟,等.不同首次换液模式对大鼠骨髓间充质干细胞培养的影响.临床军医杂志,2011,39(3):403-406
45Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for definingmultipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy,2006,8(4):315-317
46赵玉金,李瑶琛,赖洁娟,等.大鼠骨髓间充质干细胞体外培养条件的优化及生物学特性.第三军医大学学报,2010,32(20):2163-2167
47Hanley PJ, Mei Z, da Graca Cabreira-Hansen M, et al. Manufacturingmesenchymal stromal cells for phase I clinical trials. Cytotherapy,2013,15(4):416-422
48Kretlow JD, Jin Y-Q, Liu W, et al. Donor age and cell passage affectsdifferentiation potential of murine bone marrow-derived stem cells. BMCCell Biology,2008,9(1):60
49Mizuno H. Adipose-derived stem cells for tissue repair and regeneration:ten years of research and a literature review. Journal of Nippon MedicalSchool,2009,76(2):56-66
50Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymalstem cells from bone marrow, umbilical cord blood, or adipose tissue.Stem Cells,2006,24(5):1294-1301
51Markov V, Kusumi K, Tadesse MG, et al. Identification of cordblood-derived mesenchymal stem/stromal cell populations with distinctgrowth kinetics, differentiation potentials, and gene expression profiles.Stem Cells and Development,2007,16(1):53-74
52Minguell JJ. Umbilical Cord-Derived Mesenchymal Stem Cells.Regenerative Medicine Using Pregnancy-Specific Biological Substances.London: Springer;2011:249-253
53Nekanti U, Rao VB, Bahirvani AG, et al. Long-term expansion andpluripotent marker array analysis of Wharton’s jelly-derivedmesenchymal stem cells. Stem Cells and Development,2010,19(1):117-130
54Wang L, Tran I, Seshareddy K, et al. A comparison of human bonemarrow–derived mesenchymal stem cells and human umbilicalcord–derived mesenchymal stromal cells for cartilage tissue engineering.Tissue Engineering Part A,2009,15(8):2259-2266
55Insausti CL, Blanquer M, Bleda P, et al. The amniotic membrane as asource of stem cells. Histology and Histopathology,2010,25(1):91
56Lindenmair A, Hatlapatka T, Kollwig G, et al. Mesenchymal Stem orStromal Cells from Amnion and Umbilical Cord Tissue and TheirPotential for Clinical Applications. Cells,2012,1(4):1061-1088
57Miao Z, Jin J, Chen L, et al. Isolation of mesenchymal stem cells fromhuman placenta: comparison with human bone marrow mesenchymalstem cells. Cell Biology International,2006,30(9):681-687
58Chang Y-J, Hwang S-M, Tseng C-P, et al. Isolation of mesenchymal stemcells with neurogenic potential from the mesoderm of the amnioticmembrane. Cells Tissues Organs,2010,192(2):93-105
59Corrao S, La Rocca G, Iacono L, et al. New frontiers in regenerativemedicine in cardiology: the potential of Wharton's jelly mesenchymalstem cells. Current Stem Cell Research&Therapy,2012:
60Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4identifiesmesenchymal stem cells from bone marrow. Blood,2007,109(4):1743-1751
1李雅钗,黄向华,宋淑霞.组织块法和酶消化法体外培养原代鼠阴道上皮细胞的研究.中国细胞生物学学报,2010,32(5):758-761
2李雅钗,黄向华,张明乐.分步消化法原代培养鼠阴道黏膜上皮细胞的研究.中国组织化学与细胞化学杂志,2010,19(5):463-466
3何秋明,肖尚杰,夏慧敏.大鼠阴道涂片的观察.广州医学院学报,2007,35(4):54-56
4Rheinwatd JG, Green H. Seria cultivation of strains of human epidemalkeratinocytes: the formation keratinizin colonies from single cell is. Cell,1975,6(3):331-343
5De Philippo RE, Bishop CE, Filho LF, et al. Tissue Engineering aComplete Vaginal Replacement From a Small Biopsy of AutologousTissue. Transplantation,2008,86(2):208-214
6王伟,陈建苏,陈玲.改良的兔口腔黏膜上皮体外培养方法.安徽医科大学学报,2011,46(12):1249-1252
7Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectinextracellular matrix. Annual Review of Cell and Developmental Biology,2010,26:397-419
8Williams CM, Engler AJ, Slone RD, et al. Fibronectin expressionmodulates mammary epithelial cell proliferation during acinardifferentiation. Cancer Research,2008,68(9):3185-3192
9Schwarzbauer RO, Hynes JE, Tamkun JW. Fibronectin: a versatile genefor a versatile protein. Basement Membranesand Cell Movement,2009,838:75
10Freshney RI, Freshney MG. Culture of epithelial cells. Vol6: Wiley-Liss;2002
11谭波,魏人前,杨志明,等.口腔黏膜上皮细胞与猪小肠黏膜下层体外复合培养的实验研究.华西口腔医学杂志,2010,28(1):76-80
1Kimberley N, Hutson JM, Southwell BR, et al. Well-being and sexualfunction outcomes in women with vaginal agenesis. Fertility and Sterility,2011,95(1):238-241
2zkan, Erman Akar M, zkan, et al. Reconstruction of VaginalAgenesis. Annals of Plastic Surgery,2011,66(6):673-678
3Lima M, Ruggeri G, Randi B, et al. Vaginal replacement in the pediatricage group: a34-year experience of intestinal vaginoplasty in children andyoung girls. Journal of Pediatric Surgery,2010,45(10):2087-2091
4Badylak SF, Weiss DJ, Caplan A, et al. Engineered whole organs andcomplex tissues. The Lancet,2012,379(9819):943-952
5Raya-Rivera A, Esquiliano DR, Yoo JJ, et al. Tissue-engineeredautologous urethras for patients who need reconstruction: anobservational study. The Lancet,2011,377(9772):1175-1182
6De Filippo RE, Bishop CE, Filho LF, et al. Tissue engineering acomplete vaginal replacement from a small biopsy of autologous tissue.Transplantation,2008,86(2):208-214
7周慧梅.组织工程医用生物补片重建阴道的实验及临床研究[博士].北京,中国协和医科大学;2008
8Aldahmash A, Zaher W, Al-Nbaheen M, et al. Human stromal(mesenchymal) stem cells: basic biology and current clinical use fortissue regeneration. Annals of Saudi Medicine,2012,32(1):68-77
9Gu S, Xing C, Han J, et al. Differentiation of rabbit bone marrowmesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.Molecular Vision,2009,15:99-107
10Liu A, Liu L, Chen S, et al. Activation of canonical wnt pathwaypromotes differentiation of mouse bone marrow-derived MSCs into typeII alveolar epithelial cells, confers resistance to oxidative stress, andpromotes their migration to injured lung tissue in vitro. Journal ofCellular Physiology,2012,228(6):1270-1283
11Ning J, Li C, Li H, et al. Bone marrow mesenchymal stem cellsdifferentiate into urothelial cells and the implications for reconstructingurinary bladder mucosa. Cytotechnology,2011,63(5):531-539
12Elliott MJ, De Coppi P, Speggiorin S, et al. Stem-cell-based, tissueengineered tracheal replacement in a child: a2-year follow-up study. TheLancet,2012,380(9846):994-1000
13Wang LD, Wagers AJ. Dynamic niches in the origination anddifferentiation of haematopoietic stem cells. Nature Reviews MolecularCell Biology,2011,12(10):643-655
14Chen L, Kasai T, Li Y, et al. A model of cancer stem cells derived frommouse induced pluripotent stem cells. PLoS One,2012,7(4): e33544
15Voog J, Jones DL. Stem Cells and the Niche: A Dynamic Duo. Cell StemCell,2010,6(2):103-115
16Tian H, Bharadwaj S, Liu Y, et al. Differentiation of human bone marrowmesenchymal stem cells into bladder cells: potential for urological tissueengineering. Tissue Engineering Part A,2010,16(5):1769-1779
17Jiang TS, Cai L, Ji WY, et al. Reconstruction of the corneal epitheliumwith induced marrow mesenchymal stem cells in rats. Molecular Vision,2010,16:1304-1316
18Popov BV, Serikov VB, Petrov NS, et al. Lung Epithelial Cells InduceEndodermal Differentiation in Mouse Mesenchymal Bone Marrow StemCells by Paracrine Mechanism. Tissue Engineering,2007,13(10):2441-2450
19P unescu V, Deak E, Herman D, et al. In vitro differentiation of humanmesenchymal stem cells to epithelial lineage. Journal of Cellular andMolecular Medicine,2007,11(3):502-508
20薛龙.体外诱导兔骨髓间充质干细胞向上皮细胞分化的实验研究.南昌,南昌大学;2009
1Saraswati S, Bastakoty D, Young PP. Molecular and Signaling PathwaysThat Modulate Mesenchymal Stem Cell Self-renewal. Stem Cells andCancer Stem Cells. Netherlands: Springer,2012:131-141
2Ling L, Nurcombe V, Cool SM. Wnt signaling controls the fate ofmesenchymal stem cells. Gene,2009,433(1-2):1-7
3Wang Y, Sun Z, Qiu X, et al. Roles of Wnt/β-catenin signaling inepithelial differentiation of mesenchymal stem cells. Biochemical andBiophysical Research Communications,2009,390(4):1309-1314
4Pinto D, Clevers H. Wnt control of stem cells and differentiation in theintestinal epithelium. Experimental Cell Research,2005,306(2):357-363
5薛龙.体外诱导兔骨髓间充质干细胞向上皮细胞分化的实验研究.南昌,南昌大学;2009
6Scheel C, Eaton EN, Li SHJ, et al. Paracrine and autocrine signals induceand maintain mesenchymal and stem cell states in the breast. Cell,2011,145(6):926-940
7Nusse R. Wnt signaling and stem cell control. Cell Research,2008,18(5):523-527
8Zhang B, Ma J. Wnt pathway antagonists and angiogenesis. Protein&Cell,2010,1(10):898-906
9Du Y, Ling J, Wei X, et al. Wnt/β-Catenin Signaling Participates inCementoblast/Osteoblast Differentiation of Dental Follicle Cells.Connective Tissue Research,2012,53(5):390-397
10Brunt KR, Zhang Y, Mihic A, et al. Role of WNT/β-Catenin Signalingin Rejuvenating Myogenic Differentiation of Aged Mesenchymal StemCells from Cardiac Patients. The American Journal of Pathology,2012,181(6):2067-2078
11Fathke C, Wilson L, Shah K, et al. Wnt signaling induces epithelialdifferentiation during cutaneous wound healing. BMC Cell Biology,2006,7(1):4
12Lim KT, Gupta MK, Lee SH, et al. Possible involvement ofWnt/β-catenin signaling pathway in hatching and trophectodermdifferentiation of pig blastocysts. Theriogenology,2013,79(2):284-290.e282
13Li J, Khavandgar Z, Lin SH, et al. Lithium chloride attenuates BMP-2signaling and inhibits osteogenic differentiation through a novelWNT/GSK3-independent mechanism. Bone,2011,48(2):321-331
14Boland GM, Perkins G, Hall DJ, et al. Wnt3a promotes proliferation andsuppresses osteogenic differentiation of adult human mesenchymal stemcells. Journal of Cellular Biochemistry,2004,93(6):1210-1230
15Ke Z, Zhou F, Wang L, et al. Down-regulation of Wnt signaling couldpromote bone marrow-derived mesenchymal stem cells to differentiateinto hepatocytes. Biochemical and Biophysical ResearchCommunications,2008,367(2):342-348
16Day TF, Guo X, Garrett-Beal L, et al. Wnt/β-Catenin Signaling inMesenchymal Progenitors Controls Osteoblast and ChondrocyteDifferentiation during Vertebrate Skeletogenesis. Developmental Cell,2005,8(5):739-750
17Li H, Luo X, Liu R, et al. Roles of Wnt/beta-catenin signaling inadipogenic differentiation potential of adipose-derived mesenchymalstem cells. Mol Cell Endocrinol,2008,291(1-2):116-124
18Cawthorn WP, Bree AJ, Yao Y, et al. Wnt6, Wnt10a and Wnt10b inhibitadipogenesis and stimulate osteoblastogenesis through aβ-catenin-dependent mechanism. Bone,2012,50(2):477-489
19Rabbani P, Takeo M, Chou W, et al. Coordinated Activation of Wnt inEpithelial and Melanocyte Stem Cells Initiates Pigmented HairRegeneration. Cell,2011,145(6):941-955
20Popov BV, Serikov VB, Petrov NS, et al. Lung Epithelial Cells InduceEndodermal Differentiation in Mouse Mesenchymal Bone Marrow StemCells by Paracrine Mechanism. Tissue Engineering,2007,13(10):2441-2450
1Gargollo PC, Cannon Jr GM, Diamond DA, et al. Should ProgressivePerineal Dilation be Considered First Line Therapy for Vaginal Agenesis?The Journal of Urology,2009,182(4, Supplement):1882-1891
2Edmonds DK, Rose GL, Lipton MG, et al. Mayer-Rokitansky-Küster-Hauser syndrome: a review of245consecutive cases managed by amultidisciplinary approach with vaginal dilators. Fertility and Sterility,2012,97(3):686-690
3Calcagno M, Pastore M, Bellati F, et al. Early prolapse of a neovaginacreated with self-dilatation and treated with sacrospinous ligamentsuspension in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome:a case report. Fertil Steril,2010,93(1):267e261-264
4Fedele L, Bianchi S, Frontino G, et al. The laparoscopic Vecchietti’smodified technique in Rokitansky syndrome: anatomic, functional, andsexual long-term results. American Journal of Obstetrics and Gynecology,2008,198(4):377. e371-377. e376
5Csermely T, Halvax L, Sarkany A, et al. Sexual function after modifiedlaparoscopic Vecchietti's vaginoplasty. J Pediatr Adolesc Gynecol,2011,24(3):147-152
6Abramowicz S, Oden S, Joutel N, et al. Laparoscopic creation of aneovagina by Vecchietti's technique: anatomic and functional results.Gynecol Obstet Fertil. Vol41.2013/01/05ed2013:4-9
7Allen LM, Lucco KL, Brown CM, et al. Psychosexual and functionaloutcomes after creation of a neovagina with laparoscopic Davydov inpatients with vaginal agenesis. Fertil Steril,2010,94(6):2272-2276
8Fedele L, Frontino G, Restelli E, et al. Creation of a neovagina byDavydov's laparoscopic modified technique in patients with Rokitanskysyndrome. American Journal of Obstetrics and Gynecology,2010,202(1):33. e31-33. e36
9Bianchi S, Frontino G, Ciappina N, et al. Creation of a neovagina inRokitansky syndrome: comparison between two laparoscopic techniques.Fertil Steril,2011,95(3):1098-1100e1091-1093
10刘茂华.应用FSFI量表评估腹腔镜下腹膜代阴道成形术后患者性功能状态[硕士].上海,复旦大学;2010
11Acién P, Acién M, Quereda F, et al. Cervicovaginal agenesis: spontaneousgestation at term after previous reimplantation of the uterine corpus in aneovagina: Case Report. Human Reproduction,2008,23(3):548-553
12Creatsas G, Deligeoroglou E, Christopoulos P. Creation of a neovaginaafter Creatsas modification of Williams vaginoplasty for the treatment of
200patients with Mayer-Rokitansky-Kuster-Hauser syndrome. FertilSteril,2010,94(5):1848-1852
13Kuhn A, Neukomm C, Dreher EF, et al. Prolapse and sexual function8years after neovagina according to Shears: a study of43cases withMayer-von Rokitansky-Kuster-Hauser syndrome. Int Urogynecol J,2012:[Epub ahead of print]
14Walch K, Kowarik E, Leithner K, et al. Functional and anatomic resultsafter creation of a neovagina according to Wharton-Sheares-George inpatients with Mayer-Rokitansky-Kuster-Hauser syndrome-long-termfollow-up. Fertil Steril,2011,96(2):492-497e491
15Karina K, Zbigniew F. Morphology of the neovagina and sexualfunctioning of patients with Mayer-Rokitansky-Küster-Hauser syndromewho underwent modified Wharton vaginoplasty. Ginekologia Polska,2012,83(4):255
16Kajikawa MM, Jarmy-Di Bella ZI, Focchi GR, et al. Role of estrogenreceptor alpha on vaginal epithelialization of patients withMayer-Rokitansky-Kuster-Hauser syndrome submitted toneovaginoplasty using oxidized regenerated cellulose. Int Urogynecol J,2012,23(4):467-472
17Dornelas J, Jármy-Di Bella ZI, Heinke T, et al. Vaginoplasty withoxidized cellulose: anatomical, functional and histological evaluation.European Journal of Obstetrics&Gynecology and Reproductive Biology,2012:163(2):204-9
18Fotopoulou C, Sehouli J, Gehrmann N, et al. Functional and anatomicresults of amnion vaginoplasty in young women withMayer-Rokitansky-Kuster-Hauser syndrome. Fertil Steril,2010,94(1):317-323
19Sarwar I, Sultana R, Nisa RU, et al. Vaginoplasty by using amnion graftin patients of vaginal agenesis associated withMayor-Rokitansky-Kuster-Hauser syndrome. J Ayub Med CollAbbottabad,2010,22(1):7-10
20Borkowski A, Czaplicki M, Dobronski P. Twenty years of experiencewith Krzeski's cystovaginoplasty for vaginal agenesis in Mayer-Rokitansky-Kuster-Hauser syndrome: anatomical, histological,cytological and functional results. BJU Int,2008,101(11):1433-1440
21Panici PB, Bellati F, Boni T, et al. Vaginoplasty using autologous in vitrocultured vaginal tissue in a patient with Mayer-von-Rokitansky-Kuster-Hauser syndrome. Human Reproduction,2007,22(7):2025-2028
22Panici PB, Ruscito I, Gasparri ML, et al. Vaginal Reconstruction with theAbbè-McIndoe Technique: From Dermal Grafts to Autologous in VitroCultured Vaginal Tissue Transplant. Semin Reprod Med,2011,29(01):045-054
23Zhu L, Zhou H, Sun Z, et al. Anatomic and Sexual Outcomes afterVaginoplasty Using Tissue-Engineered Biomaterial Graft in Patients withMayer-Rokitansky-Kuster-Hauser Syndrome: A New Minimally Invasiveand Effective Surgery. J Sex Med,2013:
24Stany MP, Sunde J, Bidus MA, et al. The Use of Acellular DermalAllograft for Vulvovaginal Reconstruction. International Journal ofGynecological Cancer,2010,20(6):1079-1081
25Schneider W, Nguyen-Thanh P, Dralle H, et al. Ileal J-pouch vaginoplasty:reconstruction of a physiologic vagina with an ileal J-pouch. AmericanJournal of Obstetrics and Gynecology,2009,200(6):694. e691-694. e694
26Wu JX, Li B, Liu T, et al. Eighty-six cases of laparoscopic vaginoplastyusing an ileal segment. Chin Med J (Engl),2009,122(16):1862
27Zhong CX, Wu JX, Liang JX, et al. Laparoscopic and gaslesslaparoscopic sigmoid colon vaginoplasty in women with vaginal agenesis.Chin Med J (Engl),2012,125(2):203-208
28Carrard C, Chevret-Measson M, Lunel A, et al. Sexuality after sigmoidvaginoplasty in patients with Mayer-Rokitansky-Kuster-Hausersyndrome. Fertil Steril,2012,97(3):691-696
29Labus LD, Djordjevic ML, Stanojevic DS, et al. Rectosigmoidvaginoplasty in patients with vaginal agenesis: sexual and psychosocialoutcomes. Sex Health,2011,8(3):427-430
30Rawat J, Ahmed I, Pandey A, et al. Vaginal agenesis: Experience withsigmoid colon neovaginoplasty. J Indian Assoc Pediatr Surg,2010,15(1):19-22
31黄向华,高志敏,张琳,等.乙状结肠代阴道成形术后黏膜的形态学改变及神经型一氧化氮合酶表达的研究.中华医学杂志,2007,(41):2905-2908
32黄向华,张琳,高志敏,等.乙状结肠代阴道成形术后阴道黏膜细胞形态及闭锁蛋白,带状闭合蛋白1表达的变化.中华妇产科杂志,2009,44(008):588-592
33李雅钗.乙状结肠阴道黏膜神经分布与性生活质量的关系及组织工程阴道动物模型构建的初步研究[博士],河北医科大学;2011
34Zhu L, Chen N, Lang J. Vault prolapse of sigmoid neovagina26yearsafter vaginoplasty in Mayer-Rokitansky-Kuster-Hauser syndrome: a casereport. Int Urogynecol J,2013,24(1):179-180
35Schober JM. Cancer of the neovagina. Journal of Pediatric Urology,2007,3(3):167-170
36Reichman DE, Laufer MR. Mayer-Rokitansky-Küster-Hauser syndrome:fertility counseling and treatment. Fertility and Sterility,2010,94(5):1941-1943
37Brannstrom M, Diaz-Garcia C. Transplantation of female genital organs.J Obstet Gynaecol Res,2011,37(4):271-291
38Del Priore G, Schlatt S, Wagner R, et al. Uterus transplantation: on theedge. Semin Reprod Med,2011,29(1):55-60
39Diaz-Garcia C, Akhi SN, Wallin A, et al. First report on fertility afterallogeneic uterus transplantation. Acta Obstet Gynecol Scand,2010,89(11):1491-1494
40Enskog A, Johannesson L, Chai D, et al. Uterus transplantation in thebaboon: methodology and long-term function after auto-transplantation.Human Reproduction,2010,25(8):1980-1987
41Wranning CA, Akhi SN, Diaz-Garcia C, et al. Pregnancy after syngeneicuterus transplantation and spontaneous mating in the rat. Hum Reprod,2011,26(3):553-558
42Dittrich R, Maltaris T, Mueller A, et al. Uterus cryopreservation in thesheep: one step closer to uterus transplantation. In Vivo,2010,24(5):629-634
43Avison DL, DeFaria W, Tryphonopoulos P, et al. Heterotopic uterustransplantation in a swine model. Transplantation,2009,88(4):465-469
44Hanafy A, Diaz-Garcia C, Olausson M, et al. Uterine transplantation: onehuman case followed by a decade of experimental research in animalmodels. Aust N Z J Obstet Gynaecol,2011,51(3):199-203
45Brannstrom M. Uterus transplantation. Fertil Steril,2013,99(2):348-349
46Hansen A. Swedish surgeons report world's first uterus transplantationsfrom mother to daughter. BMJ,2012,345: e6357
47Ozkan O, Akar ME, Erdogan O, et al. Preliminary results of the firsthuman uterus transplantation from a multiorgan donor. Fertil Steril,2013,99(2):470-476
48Del Priore G, Saso S, Meslin EM, et al. Uterine transplantation--a realpossibility? The Indianapolis consensus. Hum Reprod,2013,28(2):288-291
49Catsanos R, Rogers W, Lotz M. The ethics of uterus transplantation.Bioethics,2013,27(2):65-73
50Brosens I, Ghaem-Maghami S, Pijnenborg R. Uterus transplantation inthe human: a complex surgical, medical and ethical challenge. HumReprod,2013,28(2):292-293
1Rogers RG. Urinary Stress Incontinence in Women. New England Journalof Medicine,2008,358(10):1029-1036
2DeLancey JO. Why do women have stress urinary incontinence?Neurourology and Urodynamics,2010,29(S1): S13-S17
3Saaby M-L, Klarskov N, Lose G. Urethral pressure reflectometry duringintra-abdominal pressure increase—an improved technique tocharacterize the urethral closure function in continent and stress urinaryincontinent women. Neurourology and Urodynamics,2013: n/a-n/a
4Chermansky CJ, Winters JC. Complications of vaginal mesh surgery.Current Opinion In Urology,2012,22(4):287-291
5Kirchin V, Page T, Keegan PE, et al. Urethral injection therapy forurinary incontinence in women. Cochrane Database Syst Rev,2012,2
6Rodrigo S, van Ramshorst J, Beeres S, et al. Intramyocardial injection ofbone marrow derived, ex vivo expanded, mesenchymal stem cells inacute myocardial infarction patients is safe and improves myocardialperfusion at3years follow up. Journal of the American College ofCardiology,2012,59(13s1): E478-E478
7Jiang R, Han Z, Zhuo G, et al. Transplantation of placenta-derivedmesenchymal stem cells in type2diabetes: a pilot study. Front Med,2011,5(1):94-100
8Lee JS, Hong JM, Moon GJ, et al. A Long-Term Follow-Up Study ofIntravenous Autologous Mesenchymal Stem Cell Transplantation inPatients With Ischemic Stroke. Stem Cells,2010,28(6):1099-1106
9Voog J, Jones DL. Stem Cells and the Niche: A Dynamic Duo. Cell StemCell,2010,6(2):103-115
10Biswas A, Hutchins R. Embryonic stem cells. Stem Cells andDevelopment,2007,16(2):213-222
11Fong CY, Gauthaman K, Bongso A. Teratomas from pluripotent stemcells: a clinical hurdle. Journal of Cellular Biochemistry,2010,111(4):769-781
12Daley GQ. Stem cells: roadmap to the clinic. The Journal of ClinicalInvestigation,2010,120(1):8
13Stangel-Wojcikiewicz K, Majka M, Basta A, et al. Adult stem cellstherapy for urine incontinence in women. Ginekol Pol,2010,81(5):378-381
14Tedesco FS, Dellavalle A, Diaz-Manera J, et al. Repairing skeletalmuscle: regenerative potential of skeletal muscle stem cells. The Journalof Clinical Investigation,2010,120(1):11
15Lindroos B, Suuronen R, Miettinen S. The potential of adipose stem cellsin regenerative medicine. Stem Cell Reviews and Reports,2011,7(2):269-291
16Si YL, Zhao YL, Hao HJ, et al. MSCs: Biological characteristics, clinicalapplications and their outstanding concerns. Ageing Research Reviews,2011,10(1):93-103
17Wagner JE, Gluckman E. Umbilical cord blood transplantation: the first20years. Paper presented at: Seminars in hematology2010
18Yu Z, Pestell TG, Lisanti MP, et al. Cancer stem cells. The InternationalJournal of Biochemistry&Cell Biology,2012,44(12):2144-2151
19Singh A, Settleman J. EMT, cancer stem cells and drug resistance: anemerging axis of evil in the war on cancer. Oncogene,2010,29(34):4741-4751
20Shackleton M. Normal stem cells and cancer stem cells: similar anddifferent. Paper presented at: Seminars in Cancer Biology2010
21Bomken S, Fi er K, Heidenreich O, et al. Understanding the cancer stemcell. British Journal of Cancer,2010,103(4):439-445
22Sharifiaghdas F, Taheri M, Moghadasali R. Isolation of human adult stemcells from muscle biopsy for future treatment of urinary incontinence.Urology Journal,2011,8(1):54-59
23Sumino Y, Hirata Y, Hanada M, et al. Long‐term cryopreservation ofpyramidalis muscle specimens as a source of striated muscle stem cellsfor treatment of post‐prostatectomy stress urinary incontinence. TheProstate,2011,71(11):1225-1230
24Roche R, Festy F, Fritel X. Stem cells for stress urinary incontinence: theadipose promise. J Cell Mol Med,2010,14(1-2):135-142
25Boregowda SV, Phinney DG. Therapeutic Applications of MesenchymalStem Cells. BioDrugs,2012,26(4):201-208
26Lim J-J, Jang J-B, Kim J-Y, et al. Human umbilical cord bloodmononuclear cell transplantation in rats with intrinsic sphincterdeficiency. Journal of Korean Medical Science,2010,25(5):663-670
27Wu S, Wang Z, Bharadwaj S, et al. Implantation of autologous urinederived stem cells expressing vascular endothelial growth factor forpotential use in genitourinary reconstruction. The Journal of Urology,2011,186(2):640-647
28Lee CN, Jang JB, Kim JY, et al. Human cord blood stem cell therapy fortreatment of stress urinary incontinence. J Korean Med Sci,2010,25(6):813-816
29Dissaranan C, Cruz MA, Couri BM, et al. Stem cell therapy forincontinence: where are we now? What is the realistic potential? CurrUrol Rep,2011,12(5):336-344
30Lin C-S, Lue TF. Stem cell therapy for stress urinary incontinence: acritical review. Stem Cells and Development,2011,21(6):834-843
31Nikolavasky D, Stangel-Wójcikiewicz K, Stec M, et al. Stem cell therapy:a future treatment of stress urinary incontinence. Paper presented at:Seminars in Reproductive Medicine2011
32Nitta M, Tamaki T, Tono K, et al. Reconstitution of experimentalneurogenic bladder dysfunction using skeletal muscle-derivedmultipotent stem cells. Transplantation,2010,89(9):1043
33Xu Y, Song Y, Lin Z. Transplantation of muscle-derived stem cells plusbiodegradable fibrin glue restores the urethral sphincter in a pudendalnerve-transected rat model. Brazilian Journal of Medical and BiologicalResearch,2010,43(11):1076-1083
34Fu Q, Song X-F, Liao G-L, et al. Myoblasts differentiated fromadipose-derived stem cells to treat stress urinary incontinence. Urology,2010,75(3):718-723
35Wu G, Song Y, Zheng X, et al. Adipose-derived stromal celltransplantation for treatment of stress urinary incontinence. Tissue andCell,2011,43(4):246-253
36Zhao W, Zhang C, Jin C, et al. Periurethral injection of autologousadipose-derived stem cells with controlled-release nerve growth factorfor the treatment of stress urinary incontinence in a rat model. EuropeanUrology,2011,59(1):155-163
37Drost AC, Weng S, Feil G, et al. In vitro Myogenic Differentiation ofHuman Bone Marrow–Derived Mesenchymal Stem Cells as a PotentialTreatment for Urethral Sphincter Muscle Repair. Annals of the New YorkAcademy of Sciences,2009,1176(1):135-143
38Kinebuchi Y, Aizawa N, Imamura T, et al. Autologousbone‐marrow‐derived mesenchymal stem cell transplantation intoinjured rat urethral sphincter. International Journal of Urology,2010,17(4):359-368
39Kim S-O, Na HS, Kwon D, et al. Bone-marrow-derived mesenchymalstem cell transplantation enhances closing pressure and leak pointpressure in a female urinary incontinence rat model. UrologiaInternationalis,2010,86(1):110-116
40Corcos J, Loutochin O, Campeau L, et al. Bone marrow mesenchymalstromal cell therapy for external urethral sphincter restoration in a ratmodel of stress urinary incontinence. Neurourology and Urodynamics,2011,30(3):447-455
41Imamura T, Ishizuka O, Kinebuchi Y, et al. Implantation of autologousbone-marrow-derived cells reconstructs functional urethral sphincters inrabbits. Tissue Engineering Part A,2011,17(7-8):1069-1081
42Zou XH, Zhi YL, Chen X, et al. Mesenchymal stem cell seeded knittedsilk sling for the treatment of stress urinary incontinence. Biomaterials,2010,31(18):4872-4879
43Cruz M, Dissaranan C, Cotleur A, et al. Pelvic organ distribution ofmesenchymal stem cells injected intravenously after simulated childbirthinjury in female rats. Obstetrics and Gynecology International,2012:612946
44Sebe P, Doucet C, Cornu JN, et al. Intrasphincteric injections ofautologous muscular cells in women with refractory stress urinaryincontinence: a prospective study. Int Urogynecol J,2011,22(2):183-189
45Peters K, Kaufman M, Dmochowski R, et al. Autologous muscle derivedcell therapy for the treatment of female stress urinary incontinence: Amulti-center experience. The Journal of Urology,2011,185(4):e535-e536
46翁慧男,王沂峰,刘风华.建立人阴道黏膜干细胞构建人工阴道的动物模型研究.中国实用妇科与产科杂志,2006,(09):669-672
47翁慧男,王沂峰,刘风华.人阴道黏膜干细胞的筛选与培养.现代妇产科进展,2007,(01):50-53+87
48薛龙.体外诱导兔骨髓间充质干细胞向上皮细胞分化的实验研究[硕士].南昌,南昌大学;2009
49谢林海.体外诱导兔骨髓间充质干细胞向平滑肌细胞分化的研究[硕士].南昌,南昌大学;2008
50Yang X, Zhang M, Zhang Y, et al. Mesenchymal stem cells derived fromWharton jelly of the human umbilical cord ameliorate damage to humanendometrial stromal cells. Fertility and Sterility,2011,96(4):1029-1036.e1024