骨髓间质干细胞通过影响PI3K/Akt信号通路抑制肝星状细胞的增殖

目的 探讨在体外非接触共培养条件下,大鼠骨髓间质干细胞(MSCs)如何调控肝星状细胞(HSCs)的增殖。方法 实验组将MSCs/HSCs按2×104/2×104(细胞/孔)的比例接种于Transwell共培养板上下层,建立非接触共培养体系;对照组为HSCs(2 ×104细胞/孔)单独培养;阳性对照组为加入抑制剂LY294002 (20 μmol/ml)观察抑制效果。共培养24、48、72h后应用流式细胞仪分析细胞周期,Western blot检测HSCs的p-Akt及Akt蛋白的表达。结果 共培养24、48、72 h后HSCs的S期细胞与对照组比较明显减少,且抑制程度呈时间依赖性(11.24±0.34 ＜15.73±0.76＜19.14±0.91 ＜23.16±1.80,P＜0.05);加入LY294002后可明显增加共培养组抑制效果,与单独使用抑制剂比较S期细胞减少更加明显(8.2±0.8＜11.7±1.6,P＜0.05);共培养组及共培养+抑制剂组p-Akt蛋白表达较单独培养组均显著下调,而Akt蛋白表达差异无统计学意义(P＞0.05);共培养+抑制剂组较共培养组p-Akt蛋白表达下调。结论 MSCs可明显抑制HSCs的增殖,可能是通过影响PI3K/Akt信号通路达到这一作用,磷酸化的Akt蛋白在其中起关键作用。     

Bone marrow-derived mesenchymal stromal cells accelerate wound healing in the rat

Bone marrow-derived mesenchymal stromal cells (BMSCs) are multipotential stem cells capable of differentiation into numerous cell types, including fibroblasts, cartilage, bone, muscle, and brain cells. BMSCs also secrete a large number of growth factors and cytokines that are critical to the repair of injured tissues. Because of the extraordinary plasticity and the ability of syngeneic or allogeneic BMSCs to secrete tissue-repair factors, we investigated the therapeutic efficacy of BMSCs for healing of fascial and cutaneous incisional wounds in Sprague-Dawley rats. Systemic administration of syngeneic BMSCs (2 x 10(6)) once daily for 4 days or a single treatment with 5 x 10(6) BMSCs 24 hours after wounding significantly increased the wound bursting strength of fascial and cutaneous wounds on days 7 and 14 postwounding. Wound healing was also significantly improved following injection of BMSCs locally at the wound site. Furthermore, allogeneic BMSCs were as efficient as syngeneic BMSCs in promoting wound healing. Administration of BMSCs labeled with iron oxides/1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescent dye revealed that systemically administered BMSCs engraft to the wound. The increase in the tensile strength of wounds treated with BMSCs was associated with increased production of collagen in the wound. In addition, BMSC treatment caused more rapid histologic maturation of wounds compared with untreated wounds. These data suggest that cell therapy with BMSCs has the potential to augment healing of surgical and cutaneous wounds.     

Bone marrow stem cells contribute to healing of the kidney

A variety of recent studies support the existence of pathways, in adult humans and rodents, that allow adult stem cells to be surprisingly flexible in their differentiation repertoires. Termed plasticity, this property allows adult stem cells, assumed until now to be committed to generating a fixed range of progeny, on relocation to switch to make other specialized sets of cells appropriate to their new niche. Cells normally present within the bone marrow seem particularly flexible and are able to contribute usefully to many recipient organs. In studies of the liver, bone marrow-derived cells are seen with specialized structural and metabolic adaptations commensurate with their new locations, and these may be abundant, even sufficient, to rescue recipient mice from genetic defects and with evidence that they have proliferated in situ. In the kidney, several studies provide evidence for the presence of "reprogrammed" cells, but in most, it remains possible that cells arrive and redifferentiate but are no longer stem cells. Nevertheless, that appropriately differentiated cells are delivered deep within organs simply by injection of bone marrow cells should make us think differently about the way organs regenerate and repair. Migratory pathways for multipotential cells could be exploited to effect repairs using an individual's own stem cells, perhaps after gene therapy. This concept makes it clear that a transplanted organ would in time become affected by the genetic susceptibilities of the recipient, because of phenotypes that are expressed when trafficking cells incorporate and differentiate.     

Denatured acellular dermal matrix seeded with bone marrow mesenchymal stem cells for wound healing in mice

It is the basic task of burn therapy to cover the wound with self-healthy skin timely and effectively. However, for patients with extensive burns, autologous skin is usually insufficient, and allogenic or heterogeneous skin leads to strong immune response. It is vital to choose an appropriate treatment for deep extensive burns. Nowadays, the dermal substitute combined with bone marrow mesenchymal stem cells (BM-MSCs) is a prospective strategy for burn wound healing. Denatured acellular dermal matrix (DADM), as one of dermal substitutes, which prepared by burn skin discarded in escharotomy, not only maintains a certain degree of 3D structure of collagen, but also has good biocompatibility. In this study, the preparation method of DADM was improved and DADM was seeded with BM-MSCs. Then BM-MSCs-seeded DADM (DADM/MSCs) was implanted into mice cutaneous wound, and the effect of DADM/MSCs dermal substitute was assessed on skin regeneration. As a result, BM-MSCs survived well and DADM/MSCs scaffolds significantly promoted wound healing in terms of angiogenesis, re-epithelialization and skin appendage regeneration. DADM/MSCs scaffold may represent an alternative promising therapy for wound healing in deep extensive burns.     

TLN1-PI3K/AKT通路调控骨髓间充质干细胞成骨分化

［摘要］ 目的 探讨Talin1在骨髓间充质干细胞（Mesenchymal stem cell, MSC）成骨分化中的作用及其调控机制。方法 诱导MSC成骨分化后通过qRT-PCR及Western blot检测TLN1的表达变化,在MSC中敲低TLN1后采用CCK-8检测细胞增殖能力改变,利用ALP活性检测及茜素红染色观察成骨分化变化情况。通过Western blot实验研究TLN1在MSC成骨分化过程中参与的分子机制。结果 qPCR及Western blot结果提示,TLN1在成骨分化过程中表达上升;siRNA敲低TLN1表达后,MSC增殖无明显差异,ALP活性及矿化结节定量较对照组下降（P<0.05）。Western blot结果显示敲低TLN1表达后p-AKT表达下降,回补实验证明激活PI3K-AKT表达可回复MSC的成骨分化能力。结论 诱导MSC成骨分化后TLN1表达逐渐上调,敲低TLN1后通过PI3K-AKT通路抑制骨髓间充质干细胞成骨分化。     

内皮祖细胞迁移与PI3K/Akt信号转导通路

PI3K/Akt信号转导通路作为细胞内重要信号转导通路之一,通过其上、下游多种效应分子的活化而影响内皮祖细胞迁移,在血管内皮损伤后修复和血管新生过程中起关键作用。该文就PI3K/Akt信号转导通路的上、下游信号通路的激活调节机制及其对内皮祖细胞迁移的影响,作一综述。     

Combination of PDGF-BB and adipose-derived stem cells accelerated wound healing through modulating PTEN/AKT pathway

Adipose-derived stem cells (ADSCs) have been widely proven to facilitate wound healing. Our study aimed to estimate the influence of combined ADSCs and platelet-derived growth factor-BB (PDGF-BB) on wound healing. We utilized 4 healthy SD rats to isolate ADSCs. Platelet-rich plasma (PRP) was acquired utilizing a two-step centrifugation technology. The role of PRP, PDGF-BB, and PDGF-BB combined with a PI3k inhibitor LY294002 on the viability, migration, and PTEN/AKT pathway in ADSCs were examined utilizing CCK-8, Transwell, and western blot assays. Then, we constructed an open trauma model in SD rats. Effects of ADSCs treated with PDGF-BB on pathological changes, CD31, and PTEN/AKT pathway of wound closure were assessed by hematoxylin & eosin (H&E) staining, Masson staining, immunohistochemical, and western blot assays, respectively. PRP and PDGF-BB intensified the viability and migration of ADSCs by modulating the PTEN/AKT pathway. Interestingly, LY294002 reversed the role of PDGF-BB on ADSCs. In vivo experiments, combined intervention with ADSCs plus PDGF-BB/PRP facilitated wound closure and ameliorated histological injury. Moreover, combined intervention with ADSCs and PDGF-BB attenuated the PTEN level and elevated the CD31 level as well as the ratio of p-AKT/AKT in the skin tissues. A combination of ADSCs and PDGF-BB facilitated wound healing might associate with the regulation of the PTEN/AKT pathway.     

骨髓间质干细胞及分化成骨的分子机制

骨髓间质干细胞(bone marrow mesenchymal sterncells，BMMSC)在骨髓中的含量极少，但其扩增能力很强，能诱导分化为多种间质组织，本文就BMMSC的生物学特性及其在骨损伤修复和造骨调控的作用机制进行综述。     

大鼠骨髓间充质干细胞对雄性生殖系统的修复作用

目的:探讨大鼠骨髓间充质干细胞(BMSCs)在无精子症大鼠模型中对睾丸内环境的修复能力。方法:将同种异体BMSCs移植入无精子症模型大鼠睾丸生精小管中,注射后30 d HE染色观察生精小管细胞组成和结构,免疫组化检测CD44、CD106和c-kit表达情况。结果:与正常大鼠相比,20 mg/kg白消安组中大鼠附睾中精子数量明显减少(P0.01)。分离得到的BMSCs表达CD44和CD106,而不表达c-kit。BMSCs注射移植30 d后,HE染色显示移植组生精小管内形成新的细胞,部分细胞表达CD106,部分细胞表达生殖细胞表面标记c-kit。结论:BMSCs在移植组无精子症大鼠生精小管中能够分化为生殖细胞,对受损的不育大鼠生精小管进行修复。     

Bone marrow-derived mesenchymal stem cells versus bone marrow nucleated cells in the treatment of chondral defects

Purpose  

The aim of this study was to compare bone marrow-derived mesenchymal stem cells (MSCs) with bone marrow nucleated cells (BNCs) as seed cells in the treatment of cartilage defects.     

Bone marrow mesenchymal stem cells from healthy donors and sporadic amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective therapies. Cell replacement therapy has been suggested as a promising therapeutic approach for multiple neurodegenerative diseases, including motor neuron disease. We analyzed expanded mesenchymal stem cells (MSCs) isolated from sporadic ALS patients and compared them with MSCs isolated from healthy donors. MSCs were isolated from bone marrow by Percoll gradient and maintained in culture in MSC Medium until the third passage. Growth kinetics, immunophenotype, telomere length, and karyotype were evaluated during in vitro expansion. Osteogenic, adipogenic, chondrogenic, and neurogenic differentiation potential were also evaluated. No morphological differences were observed in the MSCs isolated from donors or patients. The cellular expansion potential of MSCs from donors and patients was slightly different. After three passages, the MSCs isolated from donors reached a cumulative population doubling higher than from patients but the difference was not statistically significant. No significant differences between donors or patients were observed in the immunophenotype analysis. No chromosomal alteration or evidence of cellular senescence was observed in any samples. Both donor and patient MSCs, after exposure to specific conditioning media, differentiated into adipocytes, osteoblasts, chondrocytes, and neuron-like cells. These results suggest that extensive in vitro expansion of patient MSCs does not involve any functional modification of the cells, including chromosomal alterations or cellular senescence. Hence, there is a good chance that MSCs might be used as a cell-based therapy for ALS patients.     

人羊膜间充质干细胞促进创面修复及其作用机制

<正>严重创伤、大面积深度烧伤、慢性难愈性创面等的修复是一个复杂而重要的问题,随着间充质干细胞(MSCs)研究的不断深入,MSCs的应用为复杂创面修复提供了新的方法。表皮干细胞(ESCs)、骨髓间充质干细胞(BMSCs)、脂肪间充质干细胞(ADSCs)、人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)等多种MSCs被用于创面修复的研究,研究证明,这些MSCs有促     

骨髓间充质干细胞治疗脊髓损伤的研究进展

<正>骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)因具有取材方便、能够自体移植、无免疫源性、不受伦理学限制等特征成为治疗脊髓损伤的种子细胞之一[1、2],BMSCs治疗脊髓损伤的机制主要有分化为神经样细胞、抑制炎症反应、神经营养作用及促进轴突再生[3、4]。早期临床实验表明BMSCs移植治疗脊髓损伤安全、有一定疗效[5]。笔者针对目前BMSCs治疗脊髓损伤中的出现新观     

骨髓间充质干细胞移植治疗缺血性脑卒中恢复期患者的疗效观察

目的探讨骨髓间充质干细胞（BMSC）移植治疗缺血性脑卒中恢复期患者的临床疗效。方法选取2011年1月至2012年12月在湖州市第一人民医院康复医学科治疗的缺血性脑卒中恢复期患者为研究对象。18例干细胞组患者均在发生急性缺血性脑卒中后20 d左右在外院接受过BMSC移植治疗,连续3次,间隔时间为7 d,平均疗程（21.0±1.3）d;移植细胞数量为5×107~2×108,移植途径为静脉注射,移植后均未出现明显不良反应。选择同期在康复医学科治疗的18例未接受过干细胞移植的缺血性脑卒中恢复期患者为对照组。两组患者均给予常规药物治疗以及3个月康复治疗。两组患者在康复治疗前后均使用功能独立性评定（FIM）量表以及日常生活能力（ADL）量表进行评分。结果康复治疗前,干细胞组和对照组患者FIM和ADL评分差异均无统计学意义（t=1.21和1.17,P均〉0.05）。干细胞组患者康复治疗后FIM和ADL评分均高于康复治疗前,差异均有统计学意义（t=3.42和3.75,P均〈0.05）;对照组患者康复治疗后FIM和ADL评分均高于康复治疗前,差异均有统计学意义（t=2.87和3.24,P均〈0.05）。康复治疗后,两组患者FIM和ADL评分差异均无统计学意义（t=2.68和2.94,P均〉0.05）。结论 BMSC移植治疗缺血性脑卒中恢复期患者短期内观察疗效不确切。     

PI3K/AKT/mTOR信号通路在肝癌细胞自体吞噬中的作用研究

目的探讨在肝癌细胞中PI3K／AKT／mTOR信号通路在氨基酸缺乏诱导的自体吞噬中的作用。方法观察HCCLM3、MHCC97L及SMMC-7721肝癌细胞在氨基酸缺乏情况下及与P13K抑制剂3-MA、Wortmannin及LY294002，mTOR抑制剂雷帕霉素协同作用下细胞活力变化。GFP—LC3荧光法观察细胞内自噬体的形成。Western印迹检测LC3-Ⅱ蛋白的表达。结果经无氨基酸培养液EBSS处理后48h，HCCLM3、MHCC97L及SMMC-7721细胞存活率分别为（78．9±3．8）％、（57．2±13．1）％及（3．4±3．0）％（P〈0．01）。与HCCLM3、MHCC97L相比，细胞中的自噬体在SMMC-7721细胞中明显增多，分别为（13．1±3．5）％、（20．0±1．1）％及（48．9±4．5）％（P〈0．01）。Western印迹显示在EBSS处理后早期即有LC3—Ⅱ蛋白的明显表达。在EBSS基础上雷帕霉素20μmol／L处理24h，3种细胞株活力明显下降。3-MA、Wortmannin及LY294002处理也加速EBSS诱导的细胞死亡。结论高转移潜能肝癌细胞比较耐受自噬样细胞死亡。P13K／AKT／mTOR信号通路对自噬样细胞死亡可能起重要调节作用。     

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing

BackgroundType 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum.MethodsAn in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells.ResultsThe in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells.ConclusionRosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.     

Substance P promotes diabetic wound healing by modulating inflammation and restoring cellular activity of mesenchymal stem cells

Diabetic ulceration is one of the most debilitating complications of diabetes and is the main cause of amputation. The diabetic environment is characterized by prolonged inflammation and abnormal angiogenesis, leading to delayed wound healing. Thus, regulation of inflammation and neovascularization is considered a desirable target for diabetes. The critical purpose of this study was to determine whether systemically administered Substance P (SP) could promote wound healing in diabetic environments via suppression of inflammation, induction of angiogenesis, and mobilization of stem cells. The effect of SP was assessed by analyzing epidermal and dermal recovery, vessel formation, cytokine secretion profile, and the stem cell pool in the circulation and bone marrow. Compared with the vehicle‐treated group, the SP‐treated group exhibited more rapid wound coverage, reduced infiltration of leukocytes, suppression of injury‐mediated enlargement of the spleen and mesenteric lymph nodes, reduced tumor necrosis factor‐alpha levels, increased interleukin‐10, elevated pool of M2 monocytes and vascular endothelial growth factor levels in the blood. Moreover, the stem cell pool in the bone marrow, which is very low in diabetes, was markedly restored by SP to normal levels, which could provide a favorable environment to facilitate wound healing in diabetes. This result demonstrates, for the first time, a possible application of SP for the treatment of diabetic complications, including diabetic ulcers.     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

Inhibition of Notch signaling pathway promotes the differentiation of bone marrow mesenchymal stem cells into islet

Objective To investigate the effect of Notch signaling during bone marrow mesenchymal stem cells (BMSC) differentiating into islet in vitro. Methods The specific inhibitor of γ-secretase DAPT was used to inhibit the Notch signaling pathway. After induction, DTZ staining, indirect immunofluorescence staining, RT-PCR and Western blotting were used to detect the expression of insulin, glucagon, Pdx-1 and Ngn3. Results (1) Identification of BMSCs: Indirect immunofluorescence staining showed that BMSCs could express CD59 and CD90, which both were makrers of mesenchymal stem cells. Besides, BMSCs could express nerve culluar markers such as NSE, GFAP, suggesting multi-directional differentiation. (2) The result of MTT showed DAPT could inhibit the cell proliferation in a time-dependent manner and a dose-dependent mannar. Besides, DAPT could inhibit the expression of target gene of Notch signal pathway in a time-dependent manner and a dose-dependent mannar. After treated by 1, 5, 20 μmol DAPT, the expression of Hes1 had reached to 92.06%, 71.40% and 46.89% of controls respectively, suggesting efficiency of inhibition on Notch reached 7.94%, 28.6% and 53.11% respectively (all P＜0.05). (3) Indirect immunofluorescence staining showed the expression of pancreas-specific markers such as insulin and glucagon were much higher in DAPT treated BMSCs than that in controls, which was confirmed by RT-PCR and Western blotting analyses. The proportion of insulin-producing cells differentiated from DAPT treated BMSCs was (74.03±3.96)%, which was higher than that from controls[(36.49±3.24)%, P＜0.05]. (4) Furthermore, RT-PCR and Western blotting analysis showed that the expressions of Pdx-1 and Ngn3 were earlier than that of insulin and glucagon, and the expressions of Pdx-1 and Ngn3 were higher in DAPT treated BMSCs than that in controls. Conclusions Notch signaling pathway plays a role in the differentiation of BMSCs into islet in vitro. Pharmacological interference with Notch signaling pathway may provide a novel method to obtain islet for therapeutic use.