Yes相关蛋白通过Wnt/β-catenin信号通路调控软骨细胞Sox9表达的研究

目的研究Yes相关蛋白（Yes?associated protein, YAP）通过Wnt/β?catenin信号通路调控软骨细胞Sox9表达的机制。方法用siRNA分别沉默YAP基因和Lats1基因来调控YAP的活性,通过Real?time PCR检测软骨特异性基因和Wnt/β?catenin信号通路下游基因的表达;通过Western Blot检测GSK3β的磷酸化水平、活化β?catenin的蛋白水平以及Sox9的蛋白水平;并通过阿利新蓝染色检测软骨细胞外基质的分泌情况。用siRNA沉默YAP基因后,再用氯化锂干预细胞,通过Western Blot检测Sox9的表达和GSK3β的磷酸化水平。结果沉默YAP基因后,磷酸化GSK3β和活化β?catenin的蛋白水平减少,c?Myc和Nanog基因表达减少,Sox9、Col2和Aggrecan基因表达升高,并且软骨细胞分泌基质增多;沉默Lats1基因后,磷酸化GSK3β和活化β?catenin的蛋白水平增加,c?Myc和Nanog基因表达上调,Sox9、Col2和Aggre?can基因表达减少,并且软骨细胞分泌基质减少。此外,氯化锂可以阻断沉默YAP引起的Sox9表达上调。结论 YAP通过Wnt/β?catenin信号通路调控软骨细胞Sox9的表达。     

KLD-12多肽/重组人BMP-2凝胶诱导兔BMSCs成骨活性的实验研究

目的探讨KLD-12多肽复合重组人BMP-2(recombinant human BMP-2,rhBMP-2)诱导兔BMSCs成骨活性的影响。方法取3月龄新西兰大白兔骨髓,采用密度梯度法分离培养BMSCs。取第3代BMSCs分别采用KLD-12多肽/rhBMP-2凝胶三维培养(实验组)和KLD-12多肽凝胶培养(对照组)。培养7 d倒置相差显微镜下观察实验组细胞在凝胶内的形态;3、7、10、14、21 d检测两组细胞培养基中ALP及骨钙蛋白含量;培养14 d两组行Ⅰ型胶原免疫荧光染色观察,实时荧光定量PCR检测Ⅰ型胶原和骨钙蛋白基因相对表达量。结果倒置相差显微镜下观察,培养7 d实验组及对照组凝胶内BMSCs呈圆形,分布均匀。两组培养3、7 d时ALP表达量及骨钙蛋白含量比较差异无统计学意义(P0.05),10~21 d实验组以上指标均明显高于对照组(P0.05)。培养14 d,激光共聚焦显微镜观察示,实验组Ⅰ型胶原免疫荧光染色阳性,且荧光强度较对照组高;实时荧光定量PCR检测实验组Ⅰ型胶原、骨钙蛋白基因相对表达量均显著高于对照组,比较差异有统计学意义(t=15.902,P=0.000;t=12.998,P=0.000)。结论 BMSCs在KLD-12多肽内正常生长并增殖,KLD-12多肽/rhBMP-2凝胶诱导BMSCs向成骨细胞分化的生物活性良好。     

改良免疫磁珠结合克隆法培养SD大鼠阴茎海绵体内皮细胞

目的:探索大鼠阴茎海绵体内皮细胞(CCECs)分离、纯化和培养的方法,观察其生长特性,并为勃起功能障碍的研究提供种子细胞。方法:采用0.1%弹性蛋白酶消化SD大鼠阴茎海绵体组织,分离原代SD大鼠CCECs,然后采用免疫磁珠纯化,进一步扩增,利用克隆环筛选出单克隆CCECs,并观察其形态及增殖特性,免疫荧光染色鉴定CCECs血管假性血友病因子(VWF)、免疫组化染色鉴定CCECs CD31分子,流式细胞仪测定CCECs纯度,CCK-8法检测CCECs细胞增殖并绘制生长曲线。结果:经克隆环筛选纯化培养1周后倒置相差显微镜下观察到CCECs生长汇合呈铺路石样改变。生长曲线显示CCECs最初1~2 d处于潜伏期,生长缓慢,3~4 d处于对数生长期,生长迅速,第6天进入平台期。免疫荧光染色检测CCECs VWF呈阳性,荧光显微镜下胞质内可见大量绿色荧光。免疫组化检测CCECs CD31分子呈阳性,倒置显微镜下胞质内可见大量黄褐色颗粒。流式细胞仪检测经磁珠联合克隆环纯化VWF标记的CCECs细胞阳性率可达(91.9±3.75)%。结论:免疫磁珠联合克隆环纯化法可成功培养出纯度高SD大鼠CCECs,并可在内皮细胞培养液中稳定生长及传代。     

自体PRP促进人脂肪来源干细胞成骨分化的体外实验研究

目的 探讨自体PRP对体外培养人脂肪来源干细胞(adipose-derived stem cells,ADSCs)增殖及成骨分化的影响.方法 取自愿捐献吸脂术获取的脂肪组织进行分离培养ADSCs,倒置显微镜下观察细胞生长情况.将第3代ADSCs分别行成脂、成软骨定向诱导培养鉴定,并行CD29及CD44免疫荧光染色观察.取第3代ADSCs分别采用含10 mL/L PRP的成骨诱导培养基(PRP组)和不含PRP的成骨诱导培养基(对照组)进行培养,倒置显微镜观察细胞生长情况,培养后1、2、3、4、5 d采用MTT法检测细胞增殖活性;7、14、21、28 d行ALP活性检测,另取培养7、14 d的PRP组细胞行ALP染色观察;14 d时行PRP组茜素红染色检测钙结节形成情况.结果 倒置显微镜下第3代ADSCs多为梭形,倍增时间约35 h.成脂及成软骨诱导鉴定均为阳性,免疫荧光染色CD29和CD44呈阳性.MTT法示PRP组1、2、3、4、5 d的吸光度值分别为0.137±0.015、0.219±0.023、0.367±0.031、0.586±0.039、0.948±0.046,对照组分别为0.081±0.009、0.115±0.012、0.162±0.017、0.242±0.025、0.356±0.032,两组比较差异均有统计学意义(P＜0.01).成骨诱导7 d后,PRP组ALP染色阳性,细胞胞浆呈灰黑色,可见黑色沉淀:14 d后阳性细胞增多.ALP活性检测示PRP组7、14、21、28 d细胞活性值分别为23.96±2.05、41.26±3.38、38.12±3.03、35.89±2.24,对照组分别为17.83±1.62、26.64±2.37、23.85±1.99、20.78±1.81,两组比较差异均有统计学意义(P＜0.01).成骨诱导14 d后,茜索红染色示PRP组钙结节形成.结论 体外培养时,自体PRP可促进人ADSCs的增殖并诱导成骨分化,为骨组织工程提供一种新的种子细胞来源.     

转染特异性报导基因成骨细胞生物学性状及其与骨支架材料生物相容性的研究

目的研究转染特异性报导基因成骨细胞的生物学性状，并观察该细胞在骨支架材料上的生长特性和黏附性。方法常规复苏培养转染12×SBE—OC—Luc报导基因的前成骨细胞株OCTl细胞，通过倒置相差显微镜、HE染色、I型胶原免疫组化法染色、碱性磷酸酶（ALP）偶氮偶联法染色、矿化结节茜素红法染色及四环素法染色观察该转基因成骨细胞的生物学性状；并将其与纳米磷酸钙复合骨支架进行体外复合培养，通过扫描电镜观察该转基因成骨细胞在骨支架材料上的生长特性和黏附性。结果转染12×SBE—OC—Luc报导基因的前成骨细胞株OCTl细胞经培养后能贴壁生长，形态和成纤维细胞相似，能分泌胶原基质和ALP，经I型胶原免疫组化法染色及ALP偶氮偶联法染色呈强阳性；并能够形成矿化结节，茜素红法染色及四环素法染色呈阳性；该转基因成骨细胞在骨支架材料上具有良好的生长特性和黏附性，并能正常分化、增殖和成熟，分泌大量胶原基质和钙结节。结论转染12×SBE—OC—Luc报导基因后成骨细胞生物学性状未发生改变，其与骨支架材料亦具有良好的生物相容性。     

脂肪基质干细胞培养及其定向分化为成骨细胞、软骨细胞特性的研究

目的研究脂肪基质干细胞（adipose—derived stem cells，ADSCs）分离培养的方法，探讨大鼠ADSCs在体外向成骨细胞、软骨细胞分化的能力。方法从成年SD大鼠腹股沟处无菌获取脂肪组织，胶原酶消化分离，培养出ADSCs。基础培养基传至第二代时改换诱导培养基，分别诱导向成骨、软骨细胞分化，培养2～4周，碱性磷酸酶（alkaline phosphatase，ALP）、Vonkossa染色鉴定向成骨细胞分化能力；阿新蓝染色鉴定向软骨细胞分化能力。RT—PCR检测成骨细胞、软骨细胞标志基因。结果大鼠脂肪能够分离培养出生长旺盛的ADSCs；向成骨细胞诱导，ALP、Vonkossa染色阳性，RT—PCR检测有ALP、Osteocalcin及Osteopontin表达；向软骨细胞诱导，阿新蓝染色阳性，RT—PCR检测有Ⅱ型胶原、X型胶原和Aggreean表达。结论大鼠脂肪组织可以分离培养出ADSCs，生物学特性与骨髓基质干细胞（mesenchymal stemcells，MSCs）相似，能够向成骨细胞、软骨细胞分化，有希望成为组织工程理想的种子细胞来源。     

小鼠睾丸gdnf基因的克隆及其在支持细胞中的表达

目的：从小鼠睾丸中克隆胶质细胞源神经营养因子基因gdnf,构建真核表达载体,并转染支持细胞,以便用作培养精原干细胞（SSCs）的滋养层。方法：以正常成年昆明鼠为材料,提取小鼠睾丸组织中总RNA后,以RT-PCR技术克隆小鼠睾丸gdnf基因,构建真核表达载体,并转染TM4细胞（睾丸支持细胞株）,在转染后40h进行免疫荧光鉴定。结果：成功克隆小鼠睾丸gdnf基因的cDNA,测序正确,免疫荧光细胞染色显示转染后的支持细胞中有GDNF蛋白表达。结论：本研究为以转染了gdnf基因的支持细胞作饲养层培养SSCs奠定了基础。     

人GDNF基因的腺病毒载体构建及其在人神经干细胞中的表达

目的：观察构建的含人胶质细胞源性生长因子（GDNF）基因腺病毒载体对人神经干细胞的感染及其基因表达情况,为脊髓损伤的基因及神经干细胞治疗提供前期实验依据。方法：从12周龄流产人新鲜胚胎中提取人神经干细胞并进行培养,行Nestin免疫荧光染色进行检验。将全长558bp编码人GDNF的cDNA克隆到重组腺病毒载体质粒,并在人胚肾细胞（HEK293细胞）中包装出含有目的基因hGDNF的腺病毒,然后用该腺病毒感染人神经干细胞,应用荧光显微镜和Western-blot检测病毒感染及外源基因的表达情况,并进行GFAP和Tubulin免疫荧光染色检测神经干细胞向神经细胞分化情况。结果：Nestin免疫荧光染色显示所培养细胞为阳性染色红色,表明其具有神经干细胞性状;感染48h后观察到神经干细胞中有大量的增强绿色荧光蛋白（EGFP）表达,以及hGDNF蛋白高表达;感染后的神经干细胞有长的伪足伸出,呈GFAP和Tubulin染色阳性,表明促进了神经干细胞向神经元的分化。结论：腺病毒对神经干细胞具有较高感染效率,可作为一种良好的基因导入载体,实现外源基因hGDNF在神经干细胞内的有效表达,并可为神经干细胞分化为神经元提供更有利条件。     

成骨细胞与血管内皮细胞联合培养的生物学特性

目的探讨成骨细胞与血管内皮细胞联合培养的生物学特性. 方法取2周龄乳兔颅盖骨及肾脏皮质传代培养制备成骨细胞(A组)、血管内皮细胞(B组)及成骨细胞与血管内皮细胞联合培养(C组),用Ⅰ型胶原和血管Ⅷ因子免疫细胞化学染色鉴定成骨细胞和血管内皮细胞,倒置相差显微镜和组织学染色观察细胞的生长特性和细胞相容性,检测碱性磷酸酶 (alkaline phosphatase,ALP)活性,观察血管内皮细胞对成骨细胞产生的ALP活性有无影响,MTT法检测细胞活力,分析细胞生长和增殖情况. 结果免疫细胞化学染色证实,培养的细胞为成骨细胞和血管内皮细胞.倒置相差显微镜、HE和Masson染色均显示两种细胞混合生长良好.ALP检测结果:C组ALP活性明显高于A组和B组(P＜0.01),A组高于B组(P＜0.05).MTT检测结果表明:C组细胞早期增殖较慢,而后期增殖较快. 结论成骨细胞与血管内皮细胞具有良好的相容性,血管内皮细胞能够增强成骨细胞的ALP活性,提高成骨细胞的增殖能力.联合培养细胞具有很强的增殖潜能.     

人正常腹膜间皮细胞原代培养方法

目的探讨人正常腹膜间皮细胞原代培养方法。方法以胰酶和乙二胺四乙酸磁性搅拌分离人正常腹膜间皮细胞,倒置相差显微镜观察细胞形态结构和大体生长过程,Calretinin免疫荧光染色鉴定间皮细胞,初步观察传代细胞的贴壁生长情况。结果培养第4天细胞贴壁良好,第15天呈铺路石状、生长良好,1个月时细胞密集铺满皿壁;腹膜间皮细胞Calretinin表达都呈阳性;传代腹膜间皮细胞数量显著减少。结论磁性搅拌酶分离培养可得到数量多、纯度高的原代人正常腹膜间皮细胞,能够满足一般实验要求。     

人卵泡壁层颗粒细胞源性非整合诱导多能干细胞的制备

目的探索将卵泡液中壁层颗粒细胞诱导为非整合的诱导多能干细胞(iPS细胞),并检测其颗粒细胞方向的分化能力。方法在取卵的操作过程中,收集废弃的壁层颗粒细胞,在颗粒细胞培养至第4天时,加入iPS仙台病毒,经过20余天的维持,挑取iPS细胞克隆,并扩增培养。对其多能基因表达情况、外源基因整合情况、自然分化能力、颗粒细胞定向分化能力进行鉴定,并与皮肤细胞来源的iPSC系进行平行分化能力比较。结果成功建立了壁层颗粒细胞来源的iPS细胞系,其通过了类似胚胎干细胞的多能性检测及体外三胚层的分化能力检测,尤其在定向分化为颗粒细胞时,分化出了大量FOXL2、CYP19A1和FSHR阳性的细胞,经ELISA试剂盒检测,发现该分化细胞可以分泌AMH并且能够将雄激素转化成雌激素;且颗粒细胞源iPS系较皮肤细胞源的iPS系在颗粒细胞方向的分化效率更高。结论提供了一种从人颗粒细胞建立iPSC的方法,并验证了其颗粒细胞方向分化的优势。该系统不仅可以用于建立生殖不孕疾病的iPSC库,还为颗粒细胞功能障碍不孕的患者提供了一种细胞治疗的新思路。     

Long-term culture of Japanese human embryonic stem cells in feeder-free conditions

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.     

A Novel Model of Urinary Tract Differentiation,Tissue Regeneration,and Disease: Reprogramming Human Prostate and Bladder Cells into Induced Pluripotent Stem Cells

Background

Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.

Objective

To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.

Design, setting, and participants

Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.

Outcome measurements and statistical analysis

The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.

Results and limitations

Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.

Conclusions

IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.     

Induced pluripotent stem cells attenuate chronic allogeneic vasculopathy in an integrin beta‐1‐dependent manner

This study aimed to determine the mechanism of isogeneic‐induced pluripotent stem cells (iPSCs) homing to vascular transplants and their therapeutic effect on chronic allogeneic vasculopathy. We found that integrin β1 (Intgβ1) was the dominant integrin β unit in iPSCs that mediates the adhesion of circulatory and endothelial cells (ECs). Intgβ1 knockout or Intgβ1‐siRNAs inhibit iPSC adhesion and migration across activated endothelial monolayers. The therapeutic effects of the following were examined: iPSCs, Intgβ1‐knockout iPSCs, iPSCs transfected with Intgβ1‐siRNAs or nontargeting siRNAs, iPSC‐derived ECs, iPSC‐derived ECs simultaneously overexpressing Intgα4 and Intgβ1, iPSCs precultured in endothelial medium for 3 days (endothelial‐prone stem cells), primary aortic ECs, mouse embryonic fibroblasts, and phosphate‐buffered saline (control). The cells were administered every 3 days for a period of 8 weeks. iPSCs, iPSCs transfected with nontargeting siRNAs, and endothelial‐prone stem cells selectively homed on the luminal surface of the allografts, differentiated into ECs, and decreased neointimal proliferation. Through a single administration, we found that iPSCs trafficked to allograft lesions, differentiated into ECs within 1 week, and survived for 4‐8 weeks. The therapeutic effect of a single administration was moderate. Thus, Intgβ1 and pluripotency are essential for iPSCs to treat allogeneic vasculopathy.     

Genetic Insulin Resistance Is a Potent Regulator of Gene Expression and Proliferation in Human iPS Cells

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation, we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling, paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types, indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus, iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover, altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations.