阴道上皮细胞体外诱导骨髓间充质干细胞向上皮细胞分化的研究
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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.
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
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