Activin-Directed Differentiation of Human Embryonic Stem Cells Differentially Modulates Alveolar Epithelial Wound Repair via Paracrine Mechanism

Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models;therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro?wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.

Applications of stem cells in orthodontics and dentofacial orthopedics:Current trends and future perspectives

A simple overview of daily orthodontic practice involves use of brackets, wires and elastomeric modules. However, investigating the underlying effect of orthodontic forces shows various molecular and cellular changes. Also, orthodontics is in close relation with dentofacial orthopedics which involves bone regeneration. In this review current and future applications of stem cells(SCs) in orthodontics and dentofacial orthopedics have been discussed. For craniofacial anomalies, SCs have been applied to regenerate hard tissue(such as treatment of alveolar cleft) and soft tissue(such as treatment of hemifacial macrosomia). Several attempts have been done to reconstruct impaired temporomandibular joint. Also, SCs with or without bone scaffolds and growth factors have been used to regenerate bone following distraction osteogenesis of mandibular bone or maxillary expansion. Current evidence shows that SCs also have potential to be used to regenerate infrabony alveolar defects and move the teeth into regenerated areas. Future application of SCs in orthodontics could involve accelerating tooth movement, regenerating resorbed roots and expanding tooth movement limitations. However, evidence supporting these roles is weak and further studies are required to evaluate the possibility of these ideas.

In Vitro Targeted Magnetic Delivery and Tracking of Superparamagnetic Iron Oxide Particles Labeled Stem Cells for Articular Cartilage Defect Repair

To assess a novel cell manipulation technique of tissue engineering with respect to its ability to augment superparamagnetic iron oxide particles （SPIO） labeled mesenchymal stem cells （MSCs） density at a localized cartilage defect site in an in vitro phantom by applying magnetic force. Meanwhile, non-invasive imaging techniques were use to track SPIO-labeled MSCs by magnetic resonance imaging （MRI）. Human bone marrow MSCs were cultured and labeled with SPIO. Fresh degenerated human osteochondral fragments were obtained during total knee arthroplasty and a cartilage defect was created at the center. Then, the osteochondral fragments were attached to the sidewalls of culture flasks filled with phosphate-buffered saline （PBS） to mimic the human joint cavity. The SPIO-labeled MSCs were injected into the culture flasks in the presence of a 0.57 Tesla （T） magnetic force. Before and 90 min after cell targeting, the specimens underwent T2-weighted turbo spin-echo （SET2WI） sequence of 3.0 T MRI. MRI results were compared with histological findings. Macroscopic observation showed that SPIO-labeled MSCs were steered to the target region of cartilage defect. MRI revealed significant changes in signal intensity （P0.01）. HE staining exibited that a great number of MSCs formed a three-dimensional （3D） cell ＂sheet＂ structure at the chondral defect site. It was concluded that 0.57 T magnetic force permits spatial delivery of magnetically labeled MSCs to the target region in vitro. High-field MRI can serve as an very sensitive non-invasive technique for the visualization of SPIO-labeled MSCs.

Targeting mesenchymal stem cell therapy for severe pneumonia patients

Pneumonia is the inflammation of the lungs and it is the world’s leading cause of death for children under 5 years of age.The latest coronavirus disease 2019(COVID-19)virus is a prominent culprit to severe pneumonia.With the pandemic running rampant for the past year,more than 1590000 deaths has occurred worldwide up to December 2020 and are substantially attributable to severe pneumonia and induced cytokine storm.Effective therapeutic approaches in addition to the vaccines and drugs under development are hence greatly sought after.Therapies harnessing stem cells and their derivatives have been established by basic research for their versatile capacity to specifically inhibit inflammation due to pneumonia and prevent alveolar/pulmonary fibrosis while enhancing antibacterial/antiviral immunity,thus significantly alleviating the severe clinical conditions of pneumonia.In recent clinical trials,mesenchymal stem cells have shown effectiveness in reducing COVID-19-associated pneumonia morbidity and mortality;positioning these cells as worthy candidates for combating one of the greatest challenges of our time and shedding light on their prospects as a nextgeneration therapy to counter future challenges.

Improved guided bone regeneration by combined application of unmodified, fresh autologous adipose derived regenerative cells and plasma rich in growth factors:A first-in-human case report and literature review

BACKGROUND Novel strategies are needed for improving guided bone regeneration(GBR) in oral surgery prior to implant placement, particularly in maxillary sinus augmentation(GBR-MSA) and in lateral alveolar ridge augmentation(LRA). This study tested the hypothesis that the combination of freshly isolated, unmodified autologous adipose-derived regenerative cells(UA-ADRCs), fraction 2 of plasma rich in growth factors(PRGF-2) and an osteoinductive scaffold(OIS)(UAADRC/PRGF-2/OIS) is superior to the combination of PRGF-2 and the same OIS alone(PRGF-2/OIS) in GBR-MSA/LRA.CASE SUMMARY A 79-year-old patient was treated with a bilateral external sinus lift procedure as well as a bilateral lateral alveolar ridge augmentation. GBR-MSA/LRA was performed with UA-ADRC/PRGF-2/OIS on the right side, and with PRGF-2/OIS on the left side. Biopsies were collected at 6 wk and 34 wk after GBRMSA/LRA. At the latter time point implants were placed. Radiographs(32 mo follow-up time) demonstrated excellent bone healing. No radiological or histological signs of inflammation were observed. Detailed histologic,histomorphometric, and immunohistochemical analysis of the biopsies evidenced that UA-ADRC/PRGF-2/OIS resulted in better and faster bone regeneration than PRGF-2/OIS.CONCLUSION GBR-MSA with UA-ADRCs, PRGF-2, and an OIS shows effectiveness without adverse effects.

Stem cell therapy for idiopathic pulmonary fibrosis: How far are we from the bench to the bedside?

Idiopathic pulmonary fibrosis (IPF) is characterized by exuberant apoptosis and inadequate regeneration of lung parenchyma cells. Intratracheal alveolar type II epithelial cell instillation alleviates lung inflammation and fibrosis. Resident lung epithelial stem cells, as well as exogenous mesenchymal stem cells, are capable of differentiating into lung epithelial cells and repair the injured lung. It is thus supposed that, either engraftment of exogenous stem cells, or methods facilitating endogenous lung stem cell proliferation, are promising treatments for IPF, a devastating disease. Arrays of cellular and animal studies have shown the potential of stem cells in alleviating experimental lung fibrosis. Moreover, clinical trials have been launched to investigate the potentials of cell-based therapy in IPF patients. We intend to discuss the newest advances on stem cell therapy in pulmonary fibrosis, particularly the advantages, promises, and possible hurdles to pass from the successes in laboratory experiments to the eventual clinical applications.

Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization

Alveolar bone regeneration has been strongly linked to macrophage polarization.M1 macrophages aggravate alveolar bone loss,whereas M2 macrophages reverse this process.Berberine(BBR),a natural alkaloid isolated and refined from Chinese medicinal plants,has shown therapeutic effects in treating metabolic disorders.In this study,we first discovered that culture supernatant(CS)collected from BBR-treated human bone marrow mesenchymal stem cells(HBMSCs)ameliorated periodontal alveolar bone loss.CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro.To clarify the underlying mechanism,the bioactive materials were applied to different animal models.We discovered macrophage colony-stimulating factor(M-CSF),which regulates macrophage polarization and promotes bone formation,a key macromolecule in the CS.Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats.Colony-stimulating factor 1 receptor(CSF1R)inhibitor or anti-human M-CSF(M-CSF neutralizing antibody,Nab)abolished the therapeutic effects of the CS of BBR-treated HBMSCs.Moreover,AKT phosphorylation in macrophages was activated by the CS,and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab.These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis.Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets.Overall,our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

Single-cell RNA sequencing profiling of the effects of aging on alveolar stem cells

The aging of alveolar stem cells has been linked to many chronic lung diseases, including pulmonary fibrosis. However, the effects of aging on alveolar stem cells during homeostasis and post-injury alveolar repair have not been well characterized. Here we conducted a single-cell RNA sequencing(scRNA-seq) analysis of alveolar stem cells of 3-month-old and 12-month-old mice to characterize the aging effect on alveolar stem cells. Our results have shown that the transcriptomes of alveolar stem cells of 3-month-old and 12-month-old mice are not significantly different under the steady condition. However, after a bleomycin-induced lung injury, the alveolar stem cells of 12-month-old mice show enhanced inflammatory responses and decreased lipid metabolism. Our study suggests a close relationship among aging, lipid metabolism, inflammatory responses and chronic lung diseases.

脂肪间充质干细胞过表达骨形态发生蛋白2促进骨质疏松大鼠牙槽骨缺损修复

背景:颌骨在骨质疏松症中最容易受累,脂肪间充质干细胞和骨形态发生蛋白2具有促进骨质疏松症骨再生的效果,然而骨形态发生蛋白2修饰的脂肪间充质干细胞对骨质疏松症牙槽骨缺损的修复作用鲜有报道。目的:探究过表达骨形态发生蛋白2的脂肪间充质干细胞对骨质疏松大鼠牙槽骨缺损的修复作用。方法:①将过表达骨形态发生蛋白2基因的慢病毒感染大鼠脂肪间充质干细胞,通过检测绿色荧光蛋白和骨形态发生蛋白2表达进行鉴定;②切除卵巢建立骨质疏松大鼠模型,于上颌两侧第一磨牙位置制备3 mm×3 mm×3 mm的圆柱形缺损;③假手术组和骨质疏松组大鼠植入明胶海绵,脂肪间充质干细胞组植入空载体慢病毒感染的脂肪间充质干细胞与明胶海绵复合体,过表达骨形态发生蛋白2的脂肪间充质干细胞组植入过表达骨形态发生蛋白2的脂肪间充质干细胞与明胶海绵复合体,1个月后进行相关指标检测。结果与结论:①脂肪间充质干细胞组和过表达骨形态发生蛋白2的脂肪间充质干细胞组转染效率均达到70%以上;与脂肪间充质干细胞组相比,过表达骨形态发生蛋白2的脂肪间充质干细胞组骨形态发生蛋白2蛋白表达水平明显升高(P<0.05);②假手术组骨缺损区可见大量新骨生成;与假手术组相比,骨质疏松组有少量新骨生成,新骨面积明显减小,碱性磷酸酶、骨钙素及骨形态发生蛋白2 mRNA和蛋白水平明显降低;与骨质疏松组相比,脂肪间充质干细胞组和过表达骨形态发生蛋白2的脂肪间充质干细胞组有大量新骨生成,新骨面积明显增加,碱性磷酸酶、骨钙素及骨形态发生蛋白2 mRNA和蛋白水平明显升高,且过表达骨形态发生蛋白2的脂肪间充质干细胞组优于脂肪间充质干细胞组(均P<0.05);③结果表明,骨形态发生蛋白2在骨质疏松大鼠牙槽骨表达较少,过表达骨形态发生蛋白2的脂肪间充质干细胞能够促进骨质疏松大鼠牙槽骨缺损的成骨再生。

间充质干细胞移植对降植烷诱导狼疮小鼠肺泡出血的影响及可能机制

目的:探讨间充质干细胞(mesenchymal stem cells,MSCs)移植对降植烷诱导狼疮小鼠肺泡出血的作用及可能机制。方法:10周龄雌性C57BL/6(B6)小鼠20只腹腔注射0.5 mL降植烷,3只同周龄雌性B6小鼠腹腔注射PBS作为正常对照组。1周后将20只注射降植烷小鼠随机分为MSC组和PBS组,分别给予尾静脉注射1×10^(6)人脐带来源的MSCs和PBS;观察1周后,计算造模小鼠生存率并称量小鼠体重,处死所有小鼠,取出肺脏,观察肺脏大体及肺脏HE染色评估造模小鼠肺出血严重程度。酶消化法制备获取3组小鼠肺脏单细胞悬液,流式细胞术检测3组小鼠肺部CD4^(+)、CD8^(+)T淋巴细胞,CD19^(+)B淋巴细胞,CD11b^(+)Gr1^(+)中性粒细胞,F4/80^(+)肺间质巨噬细胞的绝对数以及抗炎型CD206^(+)肺间质巨噬细胞的百分率。结果:MSC组小鼠生存率高于PBS组,但差异无统计学意义(P=0.07);体重明显高于PBS组小鼠(P<0.01);MSC组小鼠完全弥漫性肺泡出血(diffuse alveolar hemorrhage,DAH)及部分DAH的发生率低于PBS组。与PBS组相比,MSC组肺脏单细胞总数显著减少(P<0.01),肺脏CD4^(+)T细胞数量有下降趋势,而CD8^(+)T细胞及CD19^(+)B细胞的数量则明显下降(P<0.05),肺脏CD11b^(+)Gr1^(+)中性粒细胞及F4/80^(+)巨噬细胞的绝对数也均显著降低(P<0.01和P<0.05);此外,肺出血小鼠CD206^(+)抗炎型巨噬细胞的百分率显著降低(P<0.01),而MSC移植显著提高CD206^(+)抗炎型巨噬细胞的百分率(P<0.01)。结论:MSC移植可显著降低狼疮小鼠肺泡出血的发生率,其机制可能是对肺脏免疫细胞的调控及诱导巨噬细胞CD206抗炎表型的产生。

电离辐射对不同发育阶段肺泡类器官的损伤及机制

为了研究电离辐射对不同发育阶段人肺泡的损伤程度及机制,以人源肺泡类器官为模型,通过qRT-PCR、免疫荧光、Western blot、流式细胞术等生物学方法,比较辐射对不同发育阶段人肺类器官的形态、发育潜能、细胞死亡以及细胞类型的影响.结果表明:(1)定型内胚层阶段的细胞较干细胞更耐辐射,辐射引起其更少的细胞死亡;(2)芽尖祖类器官辐射后形态学发生明显变化,出现细胞向外迁移现象,同时随着辐射剂量增加,其逐渐丧失诱导形成肺泡类器官的能力;(3)肺泡类器官辐射后仍保持着原有形态学特征,但7天后,Ⅰ型肺泡细胞和Ⅱ型肺泡细胞的比例均显著下调,同时恶性转化相关基因也出现显著性变化,说明辐射对肺泡类器官的功能产生较长期的影响.总体而言,电离辐射对肺的发育有显著性影响,该研究为临床放射性肺损伤及修复提供了一定的理论依据.

人羊膜间充质干细胞对松木屑烟雾溶液染毒大鼠肺泡Ⅱ型上皮细胞增殖、凋亡及炎症因子影响的研究

背景烟雾吸入性肺损伤在烧伤患者中较为常见,吸入的有毒气体、颗粒等易引起急性肺损伤(acute lung injury,ALI),目前无特异性治疗方法。目的研究人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)对松木屑烟雾溶液染毒大鼠肺泡Ⅱ型上皮细胞(alveolar typeⅡepithelial cells,AT-Ⅱ)增殖、凋亡及炎症因子的影响。方法采用酶消化法分离培养hAMSCs和AT-Ⅱ,采用流式细胞术和免疫荧光分别鉴定hAMSCs和AT-Ⅱ;松木屑烟雾溶液染毒AT-Ⅱ复制烟雾诱导的细胞损伤。实验细胞分为对照组(无血清DMEM/F12)、致伤组(0.75‰松木屑烟雾溶液染毒)和治疗组(0.75‰松木屑烟雾溶液染毒,hAMSCs与AT-Ⅱ共培养)。采用CCK-8检测细胞的增殖活性;Annexin V-FITC/PI染色检测细胞凋亡;Elisa检测炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6和IL-10的表达。结果与对照组比较,致伤组AT-Ⅱ的活性降低(P<0.01),经hAMSCs治疗后,与致伤组比较,AT-Ⅱ细胞活性增加(P<0.01);致伤组细胞的凋亡率在12 h和24 h均增加(P<0.01),hAMSCs治疗后细胞凋亡率降低(P<0.01);细胞致伤后炎症因子TNF-α、IL-6和IL-10的含量增加(P<0.05),经hAMSCs共培养12 h和24 h后,促炎因子TNF-α和IL-6的表达均下调(P<0.05),IL-10含量增加(P<0.05)。结论hAMSCs可以促进松木屑烟雾溶液诱导的AT-Ⅱ增殖和抑制细胞凋亡,可能与调节炎症因子的表达有关,为探究hAMSCs治疗烟雾引起的急性肺损伤提供理论依据。

牙槽骨骨髓间充质干细胞膜片复合PCL/HA支架具有良好早期成骨效果:基于动物模型研究

目的评估牙槽骨骨髓间充质干细胞膜片复合PCL/HA支架的骨缺损再生修复效果。方法培养牙槽骨骨髓间充质干细胞(Al-BMSCs)膜片和长骨骨髓间充质干细胞(Lon-BMSCs)膜片,熔融沉积成型技术制作PCL/HA支架。取新西兰大白兔共9只,建立双侧下颌骨缺损模型,按植入材料将其分为PCL/HA支架组(A组)、Lon-BMSCs膜片复合PCL/HA支架组(B组)、Al-BMSCs膜片复合PCL/HA支架组(C组),3组共得到18个样本,6个/组。术后4周处死动物,取下颌骨组织,行大体观察、锥形束CT分析、HE染色和Masson染色,对各组成骨情况进行分析。结果锥形束CT结果显示C组骨体积分数、骨小梁厚度和骨小梁数量均高于A组和B组,组间差异有统计学意义(P<0.05)。HE和Masson染色显示C组成骨最为活跃,有较多的新生骨和血管形成。结论牙槽骨骨髓间充质干细胞膜片复合PCL/HA支架具有良好的早期成骨效果,为颌面部骨缺损再生修复提供了一种新的策略。

PF-127水凝胶联合骨髓间充质干细胞外泌体来源miR-132诱导成骨分化修复牙槽骨缺损

目的:探究PF-127水凝胶联合骨髓间充质干细胞外泌体来源miR-132诱导成骨分化对牙槽骨缺损的修复作用。方法:培养骨髓间充质干细胞,(bone mesenchymal stem cells,BMSCs),并转染anti-miR-132、anti-miR-NC质粒。制备BMSCs来源外泌体(bone mesenchymal stem cells-exosomes,BMSCs-exo),蛋白质印迹法鉴定表达标志物。制备PF-127水凝胶和BMSCs-Exo复合物,PKH67标记法检测BMSCs的摄取;茜素红染色检测BMSCs的成骨能力;逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)检测细胞中miR-132表达和碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)mRNA表达。建立牙槽骨缺损大鼠模型,将大鼠随机分为PF-127水凝胶组、PF-127水凝胶+BMSCs-exo-anti-miR-NC组和PF-127水凝胶+BMSCs-exo-anti-miR-132组。微型计算机断层扫描(Micro-computed tomography,micro-CT)评估牙槽骨缺损情况;蛋白质印迹法检测牙槽骨组织中ALP、OCN、Runx2蛋白表达。结果:anti-miR-132组BMSCs中miR-132表达明显低于anti-miR-NC组(P<0.05),提示anti-miR-132成功转染至BMSCs中。BMSCs-exo-anti-miR-NC组和BMSCs-exo-anti-miR-132组中外泌体标志物CD9和CD63显著表达。和BMSCs-exo-anti-miR-NC组相比,BMSCs-exo-anti-miR-132组中miR-132表达明显降低(P<0.05)。和PF127水凝胶组相比,BMSC-exo-anti-miR-NC组、PF127水凝胶+BMSC-exo-anti-miR-NC组、PF127水凝胶+BMSC-exo-anti-miR-132组中miR-132表达均明显降低,茜素红染色相对活性、细胞中Runx2、ALP和OCN mRNA表达均明显增加,且PF127水凝胶+BMSC-exo-anti-miR-132组变化最显著(P<0.05)。和Control组相比,PF-127水凝胶+BMSCs-exo-anti-miR-NC组和PF-127水凝胶+BMSCs-exo-anti-miR-132组BV/TV比值、BMD、Tb.N和Tb.Th及细胞中Runx2、ALP、OCN蛋白表达均明显升高,Tb.Sp明显降低(P<0.05),且PF-127水凝胶+BMSCs-exo-anti-miR-132组变化最显著。结论:PF-127水凝胶联合骨髓间充质干细胞来源外泌体来源干扰miR-132表达可促进骨髓间充质干细胞的成骨分化,从而促进牙槽骨缺损大鼠的修复。

拔牙窝炎性肉芽组织的转归和调控

牙周炎、根尖周炎常导致牙槽骨进行性吸收,是牙齿松动和缺失的主要原因。在局部炎性因素的长期作用下,毛细血管和成纤维细胞增生,中性粒细胞、淋巴细胞等炎细胞浸润,炎性肉芽组织替代周围骨组织,传统观念认为炎性肉芽组织属于病理性组织,应在拔除患牙的同时将其彻底刮除,以避免拔牙术后出血、感染、骨组织愈合不良等问题。虽然炎性肉芽组织的再生修复能力降低,但当机体抵抗力增强或病原刺激消除(拔除患牙、根管治疗等)后,炎性肉芽组织中纤维成分逐渐增多,浸润的炎细胞逐渐减少,最终转化为修复性肉芽组织进而成骨,且即刻种植中利用拔牙窝的炎性肉芽组织进行创口关闭或软组织重建亦获得良好的临床效果,同时组织学研究证实炎性肉芽组织含有间充质干细胞群体,因此炎性肉芽组织需要彻底刮除的传统观念需要改变。炎性肉芽组织在合适的干预措施下可进行成骨转化,调控炎性肉芽组织转化为修复性肉芽组织成骨再生成为牙槽骨炎性病损再生修复的新策略,具有广阔的临床应用前景,亦是未来重要的研究方向。针对拔牙窝炎性肉芽组织转化为修复性肉芽组织的关键调控因素如活性氧、NOD样受体热蛋白结构域相关蛋白3、组织蛋白酶K等,以及修复性肉芽组织成骨的关键调控因素如骨形态发生蛋白2、血管内皮生长因子等的分子机制研究将有助于筛选促进牙槽骨炎性病损修复的合适靶点以进行相关生物治疗技术和药物材料的研发,以期能为牙周炎等导致的牙槽骨炎性病损提供更微创、更有效的治疗方式,但目前该方面研究仍处于起步阶段,距离临床转化应用尚有距离。

OPG过表达的ADSC对大鼠牙槽后槽骨缺损的修复作用

目的:探究骨保护素(Osteoprotegerin,OPG)过表达的脂肪间充质干细胞(Adipose-derived stem cell,ADSC)对大鼠牙槽后槽骨缺损修复的影响。方法:选择SD雄性大鼠38只,其中6只用于获得ADSC及ADSCOPG过表达转染,鉴定转染情况并观察不同ADSC的形态、成骨分化、成脂分化及增殖能力;其中8只作为对照组,另外24只建立牙槽后槽骨缺损大鼠模型,随机分为模型组、正常ADSC组和OPG过表达的ADSC组。6周后,Micro-CT扫描观察骨缺损愈合情况;苏木素-伊红(Hematoxylin-eosin,HE)染色观察骨缺损组织病理改变;免疫组化分析组织中碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(Osteocalcin,OCN)的表达;Westernblot分析组织中OPG/核因子-κB受体活化因子配体(Receptor activator of nuclearfactor-κB ligand,RANKL)/核因子-κB受体活化因子(Receptor activator of nuclear factor-κB,RANK)通路蛋白表达。结果:携带空载体与OPG过表达慢病毒的转染效率分别为75.23%和79.08%;两种ADSC均为长梭形;OPG过表达ADSC中的OPG蛋白表达水平、增殖能力、成骨能力明显高于正常ADSC,成脂能力明显低于正常ADSC(P<0.05);与对照组相比,模型组的牙槽后槽骨缺损体积、RANKL、RANK蛋白表达水平明显升高,Lane-Sandhu组织学评分、ALP、OCN阳性表达率、OPG蛋白表达水平明显降低(P<0.05);与模型组相比,正常ADSC组获得与上述相反的结果;OPG过表达的ADSC组上述指标的变化幅度较正常ADSC组更显著。结论:OPG过表达的ADSC能够促进大鼠牙槽后槽骨缺损的修复。

骨髓间充质干细胞外泌体调控NLRP3炎性小体对糖尿病大鼠牙槽骨缺损的机制研究

目的 探究骨髓间充质干细胞外泌体通过调控NLRP3炎性小体信号通路影响糖尿病大鼠牙槽骨缺损愈合相关分子机制。方法 选取50只SD大鼠纳入研究,随机选取10只大鼠作为对照组,其余大鼠构建糖尿病+双侧上颌牙槽骨缺损模型,设置模型组、骨髓间充质干细胞外泌体组(BMMSC-Exos组)、MCC950组(NLRP3抑制剂)、BMMSC-Exos+MCC950组,其中BMMSC-Exos组、MCC950组、BMMSC-Exos+MCC950组SD大鼠分别尾静脉注射BMMSC-Exos及MCC950试剂,模型组注射等量盐水。模型构建28d后取大鼠上颌骨组织。TEM检测BMMSC-Exos形态,Westernblot检测BMMSC-Exos标志性蛋白CD9、CD63、CD81表达。检测各组大鼠空腹血糖水平、HE染色分析各组大鼠牙槽骨炎性浸润及Lance-Sandhu评分;Western blot检测各组大鼠牙槽骨组织内NLRP3炎性小体信号通路关键蛋白(NLRP3、caspase-1、IL-1β)、骨代谢关键蛋白(OPG、ALP、Collal)表达。结果 BMMSC-Exos直径大小在100 nm左右,呈双凹圆盘状态,膜结构完整;与纯BMMSCs相比,BMMSC-Exos标志物蛋白CD9、CD63、CD81的含量表达明显提升(P <0.05)。糖尿病大鼠模型建立成功,BMMSC-Exos干预、NLRP3抑制剂(MCC950)下调大鼠血糖水平(P <0.05);两者联合可以进一步下调大鼠血糖水平(P <0.05);与对照组相比,糖尿病牙槽骨缺损模型建立可以下调骨再生Lane-Sandhu评分、OPG、ALP、Collal的表达(P <0.05),上调NLRP3、caspase-1、IL-1β表达(P <0.05);BMMSC-Exos干预可以再次上调Lane-Sandhu评分及OPG、ALP、Collal蛋白表达(P<0.05),下调NLRP3、caspase-1、IL-1β的表达(P <0.05)。与模型组相比,BMMSC-Exos及MCC950干预后大鼠骨再生Lane-Sandhu评分、骨代谢关键蛋白(OPG、ALP、Collal)的表达提升(P<0.05);两者联合可进一步上调大鼠骨再生Lane-Sandhu评分及骨代谢关键蛋白表达(P <0.05)。结论 骨髓间充质干细胞外泌体通过抑制NLRP3炎症小体活性来改善糖尿病牙槽骨缺损大鼠骨代谢,降低炎症反应,促进骨缺损的愈合。

肺癌干细胞的球体形成与致瘤性分析

目的：从人肺腺癌细胞株SPC—A1中诱导球体形成，对其中的肺癌干细胞进行鉴定并评估其致瘤能力。方法：无血清条件下培养肺癌细胞，采用表皮生长因子（epidermal growth factor，EGF）、重组人胰岛素样生长因子-1（insulin—like growth factor-1，IGF-1）和重组人成纤维细胞生长因子-10（fibroblast growth factor—10，FGF-10）等细胞生长因子诱导球体形成，球体细胞采用RT—PCR及免疫荧光技术检测干细胞相关标志的表达，并通过NOD—SCID小鼠移植评估其致瘤性。结果：SPC—A1肺腺癌细胞经培养5—10d后，即可形成悬浮生长的细胞球体（肺球体）。RT—PCR检测显示肺球体细胞表达肺癌干细胞表面标志CD24和CD221．细支气管肺泡干细胞标志Clara细胞分泌蛋白和肺泡活性蛋白C，胚胎干细胞主干基因OCT4、Nanog和Bmi-1，以及肺干细咆主干基因甲状腺转录因子-1。采用免疫荧光检测其中3个标志的蛋白表达，证实80％以上的肺球体细胞呈Clara细胞分泌蛋白、肺泡活性蛋白C和OCT4染色阳性。肺球体细胞具有高致瘤性，在NOD—SCID小鼠中移植5103个细胞即可形成肿瘤。结论：肺球体细胞中肺癌干细胞高度富集并具有致瘤性。

骨髓间充质干细胞在肺损伤大鼠肺组织的分化

目的探讨骨髓间充质干细胞(MSCs)分化为肺泡上皮细胞、支气管上皮细胞的可能性。方法将SD大鼠MSCs用DAPI标记后注入受体大鼠体内。75只受体大鼠随机分为5组(n=15):肺损伤组、MSCs治疗组、MSCs对照组、单个核细胞对照组和正常对照组。分别于MSCs、单个核细胞注入受体大鼠体内1、2、4周后观察肺组织结构变化,检测肺组织中植入细胞情况及广谱细胞角蛋白的表达水平。结果MSCs治疗组大鼠肺组织在各时间点均有少量植入的细胞,并且部分植入的细胞表达广谱细胞角蛋白。MSCs植入后2周大鼠细支气管壁有少量植入的细胞同时表达广谱细胞角蛋白。其余各组未发现植入细胞的标记。结论MSCs可在损伤的大鼠肺组织中分化为肺泡上皮细胞和支气管上皮细胞。

Notch和Wnt信号通路在自体骨髓间充质干细胞复合富血小板纤维蛋白修复兔牙槽骨缺损中的表达

目的通过建立自体骨髓间充质干细胞(BMSCs)复合富血小板纤维蛋白(PRF)修复兔拔牙窝骨缺损的动物模型,探讨Notch和Wnt信号通路在牙槽骨缺损修复过程中的表达及意义。方法选用36只健康雄性2月龄新西兰兔,随机分为4组,均于全身麻醉下微创拔除下颌左侧中切牙。A组植入BMSCs-PRF复合物,B、C组分别植入单一PRF和BMSCs,D组未植入任何材料作为空白对照。术后4、8、12周(每个时间点3只动物)处死动物,立即在骨缺损同一部位取材,制作骨标本,利用免疫组织化学和免疫荧光染色检测骨缺损修复过程中Notch1和Wnt3a的表达情况。结果免疫组织化学染色结果显示:第4、8周,A、B、C组Notch1和Wnt3a表达均高于D组(P<0.05);第12周,D组Notch1和Wnt3a表达高于其他3组(P<0.05)。免疫荧光染色显示:第4周,骨缺损区新生骨细胞Notch1和Wnt3a的表达最多,随着时间的延长,骨缺损修复完成,表达逐渐降低。结论 Notch1和Wnt3a信号分子在BMSCs复合PRF促进骨缺损修复过程中均有表达,可以通过调控BMSCs的增殖与分化加速牙槽骨缺损修复的愈合过程;二者表达情况相似,有可能存在串话机制。