骨髓间充质干细胞对豚鼠皮肤光老化的修复作用

目的研究骨髓间充质干细胞(MSCs)对皮肤光老化的修复作用,为皮肤光老化的治疗提供新途径。方法每天UV-A和UV-B紫外线灯同时照射2 h,连续照射50 d,制备豚鼠皮肤光老化模型;体外分离培养豚鼠乳鼠MSCs;经DAPI标记后,局部多点皮内注入豚鼠皮肤紫外线照射区域,激光共聚焦显微镜下分别观察注入后24 h、5 d及10 d,DAPI标记的MSCs在紫外线照射区域皮肤组织的迁徙及定居;肉眼及HE染色切片观察MSCs对豚鼠光老化皮肤的修复作用。结果DAPI标记的MSCs经紫外线照射皮肤区域局部皮内多点注射24 h后,即出现在真皮层,5 d后出现在真皮层及毛囊组织,10 d后广泛弥散在皮肤各层。肉眼及皮肤HE染色切片镜下观察均显示,治疗组豚鼠较模型组明显延缓光老化进程,且在停止照射后,治疗组光老化症状比自然恢复组有更为显著的改善。结论MSCs对皮肤光老化有明显的延缓及修复作用。     

大鼠骨髓间充质干细胞向成骨与成脂分化过程中相关基因的表达变化

目的探讨大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨与成脂分化过程中相关基因表达的变化。方法采用全贴壁法分离培养大鼠BMSCs,并观察其形态学特征的变化,MTT法检测其生长状况,并绘制生长曲线。分别采用成骨和成脂诱导剂对第4代BMSCs进行诱导分化,应用碱性磷酸酶试剂盒、茜素红和油红O染色液检测其ALP活性、成骨和成脂分化能力;RT-QPCR检测诱导0、7、14和21 d的成骨分化相关基因Runt相关转录因子2(Runx2)、骨钙素(osteocalcin,OCN)、碱性磷酸酶(ALP)及成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxidase proliferator activated receptor gamma,PPARγ)和脂肪酸结合蛋白(FABP4)的表达变化。结果全骨髓贴壁法能成功分离培养BMSCs,传代细胞生长增殖迅速,以长梭形细胞生长为主,细胞生长曲线呈S形。第4代BMSCs分别经成骨和成脂诱导剂诱导后,ALP、茜素红和油红O染色均呈阳性;诱导7、14和21 d后,Runx2、OCN、ALP、PPARγ和FABP4基因mRNA的表达量均显著高于0 d(P0.05);成骨分化过程中,Runx2和ALP在第7天时表达量最高,之后呈下降趋势,OCN的表达量呈稳定上升趋势;成脂分化过程中,PPARγ在第7天时表达量最高,FABP4始终高表达。结论 BMSCs具有易于体外分离培养、扩增和经诱导后具有多向分化潜能等特点,成骨和成脂分化相关基因的表达量随诱导时间延长而变化,呈明显的时序性表达差异,提示分别在成骨与成脂分化过程中起重要调控作用,为BMSCs在骨、细胞和基因等工程中的机制研究提供了实验依据。     

骨髓间充质干细胞对大鼠胰腺损伤组织的修复作用

目的观察骨髓间充质干细胞(MSCs)对大鼠胰腺损伤组织的修复作用。方法应用贴壁法分离、纯化、扩增大鼠MSCs,经流式细胞仪检测其细胞周期及表面标志后,用DAPI标记,经尾静脉注入胰腺损伤模型大鼠体内,15d后,在激光共聚焦显微镜下观察MSCs在大鼠胰腺组织的定位,组织病理切片观察胰腺损伤组织的病理改变,PCR检测胰腺损伤区组织的Sry基因。结果体外纯化、扩增、富集的MSCs经流式细胞仪检测,86.67%的细胞处于G0/G1期,细胞表面CD34呈阴性表达,CD44呈阳性表达。DAPI标记的MSCs移植治疗15d后,激光共聚焦显微镜下可见,MSCs在损伤的胰腺组织中多见,在正常胰腺组织中偶见;组织病理切片可见损伤的胰腺组织结构开始恢复,胰岛再建;PCR结果显示,治疗组胰腺组织可扩增出Sry基因。结论MSCs对大鼠胰腺损伤组织可能具有修复作用。     

β-catenin对骨形态发生蛋白9诱导间充质干细胞成骨分化的影响

目的探讨经典Wnt信号通路关键节点β-catenin对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导间充质干细胞(mesenchymal stem cells,MSCs)成骨分化的影响。方法用重组腺病毒介导BMP9在C3H10T1/2细胞中过表达,联用β-catenin重组腺病毒上调β-catenin的表达,并通过RNA干扰抑制β-catenin的表达。分析C3H10T1/2细胞碱性磷酸酶(alkaline phosphatase,ALP)活性的变化;RT-PCR检测细胞成骨分化相关基因骨桥蛋白(osteopontin,OPN)和骨钙蛋白(osteocalcin,OC)基因mRNA的转录水平;茜素红S染色检测细胞的钙盐沉积。结果 BMP9单独作用能诱导C3H10T1/2细胞向成骨方向分化,并增强细胞ALP活性;单独的β-catenin无成骨诱导作用,但可剂量依赖性地增强BMP9诱导的C3H10T1/2细胞的ALP活性,并促进BMP9诱导的细胞OPN和OC基因mRNA的转录水平及钙盐沉积;抑制β-catenin表达可显著降低BMP9诱导的C3H1OT1/2细胞的ALP活性(P0.05),下调OPN和OC基因mRNA的转录水平,并抑制钙盐沉积。结论经典Wnt信号通路可能通过β-catenin协同BMP9诱导C3H10T1/2细胞成骨分化,且BMP9诱导的成骨分化可能需要通过Wnt/β-catenin途径来实现。     

cAMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用

目的探讨cAMP-PKA-CREB信号通路在骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠间充质干细胞(mesenchymal stem cells,MSCs)C3H10T1/2成骨分化过程中的作用及其机制。方法将C3H10T1/2细胞分别加入不同浓度的cAMP-PKA-CREB信号通路抑制剂H89(1、2.5、5和10μmol/L),检测其对碱性磷酸酶(alkaline phosphatase,ALP)活性的影响;通过ALP定量和钙盐沉积试验分别检测H89对BMP9诱导C3H10T1/2细胞早期和晚期成骨分化的影响;经Western blot法检测H89对C3H10T1/2细胞中磷酸化CREB、骨钙素(Osteocalcin,OCN)和成骨关键转录因子Runx2表达水平的影响;通过Wentern blot及荧光素酶活性的检测,观察H89对经典信号通路BMPs-smad1/5/8的影响。结果随着H89浓度的增加,对BMP9诱导的C3H10T1/2细胞ALP的抑制作用明显增强(P0.05),且呈剂量依赖性;ALP定量和钙盐沉积试验结果表明,H89可明显抑制BMP9诱导的C3H10T1/2细胞早期及晚期成骨分化;H89可显著抑制BMP9诱导的C3H10T1/2细胞中磷酸化CREB、OCN及Runx2蛋白的表达(P0.05),与AdBMP9组比较,H89对经典BMPs-smad1/5/8信号通路无明显影响(P0.05)。结论阻断cAMP-PKA-CREB信号通路可抑制BMP9诱导的MSCs C3H10T1/2的成骨分化,为BMP9的临床应用奠定了理论基础。     

人胎盘来源的造血干细胞和间充质干细胞的分离及鉴定

目的建立人胎盘来源的造血干细胞(placenta hematopoietic stem cells,hP-HSCs)和间充质干细胞(placentaderived mesenchymal stem cells,hP-MSCs)的分离方法,并进行鉴定和组分分析。方法选取10份新生健康婴儿胎盘组织,采用机械破碎法联合磁珠分选法分离h P-HSCs,胎盘绒毛膜组织块贴壁法分离hP-MSCs,利用形态学观察、集落培养、流式细胞术等进行鉴定。结果 hP-HSCs:分离后有核细胞数(total nucleated cell number,TNC)为(11. 82±2. 46)×10~8个,TNC回收率≥80%;细胞活性为(99. 7±0. 3)%;细胞表面抗体CD34~+CD45dim表达率为(8. 69±0. 36)%,CD34~+总数为(108. 0±6. 48)×10~6个;集落形成总数为(1. 88±1. 07)×10~6。hP-MSCs:冻存细胞总数为(40. 78±9. 35)×10~7个;细胞活性为(99. 0±1. 5)%;细胞表面抗体CD34~+CD45~+表达率为(0. 1±0. 1)%,CD44~+CD105~+为(99. 6±0. 2)%,CD14~+CD19~+为(0. 1±0. 1)%,CD90~+CD73+为(98. 9±0. 2)%;且具有良好成脂、成骨分化潜能。结论成功建立了hP-HSCs和hP-MSCs体外分离培养方法,为胎盘的临床应用奠定了基础,并提供了细胞种子资源。     

脐带间充质干细胞促进烫伤创面愈合的作用机制

目的探讨人脐带间充质干细胞(umbilical cord-derived mesenchymal stem cells,UCMSCs)多点局部注射后,修复烫伤皮肤创面的潜在机制。方法机械消化法分离人UCMSCs,培养至第3代后,进行形态观察、免疫表型和多向分化潜能鉴定。将第3代UCMSCs与碱性成纤维细胞因子(basic fibroblast growth factor,b FGF)共培养14 d,免疫荧光法检测成纤维细胞标志物波形蛋白的表达;对比小鼠Ⅲ度烫伤模型多点局部注射UCMSCs后创面愈合率及创面愈合时间;Dil标记UCMSCs,局部注射后检测UCMSCs在创面局部的分布情况。结果分离培养的UCMSCs的形态、表面抗原表达及体外诱导分化能力均符合间充质干细胞特性;经b FGF诱导后可显著提高UCMSCs中成纤维细胞标志物波形蛋白的表达量;局部注射UCMSCs可显著提高烫伤小鼠的创面愈合率,UCMSCs可定植于烫伤创面下。结论 UCMSCs具有较强的成纤维样细胞分化能力,经局部注射后,可通过定植于烫伤创面而显著提高创面愈合率。     

成骨生长肽对鼠骨髓间充质干细胞增殖及成骨分化的作用

目的探讨成骨生长肽(osteogenic growth peptide,OGP)对鼠骨髓间充质干细胞(mouse bone marrow mesenchymal stem cell,mBMSC)增殖及成骨分化的作用及其分子机制。方法采用乳鼠骨片法分离mBMSC,流式细胞术测定mBMSC表面特征性分子。以10-5 mol/L OGP10-14作用于mBMSC,以不含OGP10-14的成骨诱导培养基作为对照组,MTS法检测OGP10-14对mBMSC增殖的影响;茜素红染色评价OGP10-14对mBMSC成骨分化的作用;qPCR及Western blot法检测OGP10-14干预下mBMSC骨分化相关因子β-catenin、RUNX2、BSP和细胞周期相关因子cyclin B1、CDK2、c-myc mRNA及蛋白表达水平。结果原代分离培养的mBMSC高表达CD29和CD90,低表达CD45和CD11b/c,符合BMSC的表型特征。与对照组相比,OGP10-14组mBMSC培养24、48及72 h时均促进细胞增殖(P <0.05)。茜素红染色显示,对照组部分细胞呈聚集生长,集落状,随诱...     

小鼠骨髓间充质干细胞对异基因肝移植模型大鼠移植区域组织损伤的修复作用

目的观察小鼠骨髓间充质干细胞(MSCs)注入后在异基因肝移植模型大鼠移植区域的迁徙、定居及组织修复作用,探讨MSCs诱导异基因移植免疫耐受的可能性。方法将昆明小鼠肝组织块埋入Wister大鼠肝脏切口,制备异基因肝移植大鼠模型;体外分离、纯化并培养昆明小鼠乳鼠MSCs;经DAPI标记后,尾静脉注入模型大鼠,通过激光共聚焦显微镜,分别在24h、5d及10d观察MSCs在肝脏移植区域的迁徙及定居;采用HE染色观察肝移植区域组织损伤情况。结果MSCs尾静脉注入后,24h即出现在肝移植区域,5d时分布于血管周围,10d时扩散至移植区域血管及周围组织;MSCs治疗5d,HE染色显示移植区域浸润的炎性细胞减少,可见肝组织结构。结论小鼠MSCs可在大鼠体内存活,并参与损伤区血管及组织的修复。     

间充质干细胞来源外泌体修复炎症损伤的研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是一种具有自我更新和多向分化潜力的多能干细胞,能促进组织修复、再生和血管生成,还具有修复炎症损伤的潜力。MSCs来源外泌体（mesenchymal stem cell-derived exosomes,MSC-Exos）具有与MSCs相似的免疫调节和组织修复特性,并具有更高的安全性,可作为一种无细胞疗法替代MSCs治疗炎症损伤相关疾病。本文对MSC-Exos治疗炎症损伤的机制作一综述。     

Comparison of the Translational Potential of Human Mesenchymal Progenitor Cells from Different Bone Entities for Autologous 3D Bioprinted Bone Grafts

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.     

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein–substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.     

Osteogenic Differentiation of Human Adipose-Derived Stem Cells Seeded on a Biomimetic Spongiosa-like Scaffold: Bone Morphogenetic Protein-2 Delivery by Overexpressing Fascia

Human adipose-derived stem cells (hADSCs) have the capacity for osteogenic differentiation and, in combination with suitable biomaterials and growth factors, the regeneration of bone defects. In order to differentiate hADSCs into the osteogenic lineage, bone morphogenetic proteins (BMPs) have been proven to be highly effective, especially when expressed locally by route of gene transfer, providing a constant stimulus over an extended period of time. However, the creation of genetically modified hADSCs is laborious and time-consuming, which hinders clinical translation of the approach. Instead, expedited single-surgery gene therapy strategies must be developed. Therefore, in an in vitro experiment, we evaluated a novel growth factor delivery system, comprising adenoviral BMP-2 transduced fascia tissue in terms of BMP-2 release kinetics and osteogenic effects, on hADSCs seeded on an innovative biomimetic spongiosa-like scaffold. As compared to direct BMP-2 transduction of hADSCs or addition of recombinant BMP-2, overexpressing fascia provided a more uniform, constant level of BMP-2 over 30 days. Despite considerably higher BMP-2 peak levels in the comparison groups, delivery by overexpressing fascia led to a strong osteogenic response of hADSCs. The use of BMP-2 transduced fascia in combination with hADSCs may evolve into an expedited single-surgery gene transfer approach to bone repair.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

A New Perspective for Bone Tissue Engineering: Human Mesenchymal Stromal Cells Well-Survive Cryopreservation on β-TCP Scaffold and Show Increased Ability for Osteogenic Differentiation

The clinical breakthrough of bone tissue engineering (BTE) depends on the ability to provide patients routinely with BTE products of consistent pharmacological quality. The bottleneck of this approach is the availability of stem cells. To avoid this, we suggest immobilization of random-donor-derived heterologous osteoinductive MSCs onto osteoconductive matrices. Such BTE products could then be frozen and, after thawing, could be released as ready-to-use products for permanent implantation during surgery. For this purpose, we developed a simple protocol for cryopreservation of BTE constructs and evaluated the effects of this procedure on human MSC (hMSCs) metabolic and osteogenic activity in vitro. Our findings show that hMSCs can be freeze-thawed on a β-TCP scaffold through a technically simple procedure. Treated cells sustained their metabolic activity and showed favorable osteogenic potential. Mechanistically, HIF1α and YBX1 genes were activated after freeze-thawing, and supposed to be linked to enhanced osteogenesis. However, the detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs remains to be evaluated. Additionally, we demonstrated that our BTE products could be stored for 3 days on dry ice; this could facilitate the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room.