犬骨髓间充质干细胞定向分化为内皮细胞的研究

应用密度梯度离心法分离狗骨髓间充质干细胞,建立诱导分化内皮细胞的方法及条件。密度梯度离心法分离狗骨髓间充质干细胞后,在体外经血管内皮细胞生长因子(Vascular endothelial growth factor,VEGF)、内皮细胞生长因子(Endothelial growth factor,EGF)等诱导后,分化形成内皮样细胞。结果表明:光镜下细胞单层融合生长,呈铺路石样形态,单个核位于中央。电镜下可见到Weibel-Palade小体,在细胞胞浆vWF染色阳性。骨髓间质干细胞,在体外可诱导分化成内皮细胞。     

周期性牵张应变对大鼠骨髓间充质干细胞神经向分化的影响

目的 研究周期性牵张应变对大鼠骨髓间充质干细胞(rat bone marrow-derived mesenchymal stem cells, rBMSCs)向神经细胞分化的影响。方法 对rBMSCs加载不同幅度周期性应变24 h,然后继续培养5 d,检测神经细胞标志物表达和相关信号通路蛋白磷酸化水平。通过有限元分析牵张作用下细胞表面的应力分布。通过转录组测序分析周期性牵张应变引起差异表达的基因。结果 5%幅度、0.5 Hz周期性牵张应变可以显著促进神经细胞标志物的表达,提高细胞内胞外信号调节激酶(extracellular-signal-regulated kinase, ERK)、蛋白激酶B (AKT)和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)的磷酸化水平。KEGG通路富集分析发现,与细胞黏附、细胞外基质和受体相互作用相关的基因在周期性牵张作用后显著提高。结论 周期性牵张应变可以改变细胞与细胞外基质的相互作用,激活AKT/mTOR和ERK信号通路,从而促进rBMSCs向神经细胞分化。了解力学刺激对间充质干细胞分化的影响有望提高...     

丹参诱导骨髓基质干细胞向神经元样细胞分化及相关基因的表达

目的分析骨髓基质干细胞向神经元样细胞分化前后相关基因的表达。方法用丹参诱导骨髓基质干细胞向神经元样细胞分化,诱导后90mins,提取诱导前后的骨髓基质干细胞总RNA,RT-PCR检测ngn-1,mash-1的表达及观察其诱导前后的表达情况,免疫组化检测NSE和GFAP的表达情况。结果未经诱导的骨髓基质干细胞ngn-1,mash-1 mRNA为阴性,诱导后有表达。诱导后的细胞NSE和GFAP呈阳性反应。结论骨髓基质干细胞向神经元样的分化与ngn-1,mash-1可能有关。     

A Biological Pacemaker Restored by Autologous Transplantation of Bone Marrow Mesenchymal Stem Cells

Objective：To restore cardiac autonomic pace function by autologous trans- plantation and committed differentiation of hone marrow mesenchymal stern cells, and explore the technique for the treatment of sick sinus syndrome. Methods ： Mesenchymal stern cells isolated from canine bone marrow were culture-expanded and differentiated in vitro by 5-azacytidine. The models of sick sinus syndrome in canines were established by ablating sinus node with radio-frequency technique. Differentiated mesenchymal stem cells labeled by BrdU were autologously transplanted into sinus node area through direct injection. The effects of autologous transplantation of mesenchymal stern cells on cardiac autonomic pace function in sick sinus syndrome models were evaluated by electrocardiography, pathologic and immunohistochemical staining technique. Results：There was distinct improvement on pace function of sick sinus syndrome animal models while differentiated mesenchymal stem cells were auto-transplanted into sinus node area. Mesenchymal stern cells transplanted in sinus node area were differentiated into similar sinus node cells and endothelial cells in vivo, and established gap junction with native cardiomyocytes. Conclusion ： The committed- induced mesenchymal stern cells transplanted into sinus node area can differentiate into a- nalogous sinus node cells and improve pace function in canine sick sinus syndrome models.     

黄芪诱导大鼠骨髓间充质干细胞分化为神经样细胞的研究

目的研究大鼠骨髓间充质干细胞(MSCs)体外分离和培养方法,并用黄芪体外诱导骨髓间充质干细胞分化为神经细胞样细胞。方法利用梯度离心从大鼠骨髓中分离单核细胞进行培养,去除不贴壁的细胞,分离纯化,对其生长特性进行分析。采用含黄芪的无血清L-DMEM诱导分化为神经细胞样细胞,观察细胞形态变化,用免疫组织化学方法检测分化细胞中巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达。结果用含10%胎牛血清(FCS)的培养基培养,原代细胞贴壁生长,细胞形态均一,为纺锤形,有克隆团形成。传代培养时,形态变为成纤维细胞样,有较强的增殖能力。经黄芪诱导后,骨髓间充质干细胞形态发生改变,nestin、NSE和GFAP阳性,分化为神经元或胶质细胞样细胞。结论骨髓间充质干细胞可以体外分离、培养,在一定条件下向神经样细胞分化。     

Effects of Mesenchymal Stem Cells Treatment on Radiation-Induced Proctitis in Rats

PurposeThere are no effective treatment methods with which to control complications of radiation proctitis with fistula or recurrent bleeding following radiation treatment for prostate, cervical, or rectal cancer. Mesenchymal stem cells (MSCs) can induce immune modification, resulting in tissue repair and regeneration. Therefore, we used a rat model of radiation-induced proctitis and observed the effects of using human placenta-derived (PD) and adipose tissue-derived (AD) MSCs.Materials and MethodsFemale Sprague Dawley rats were irradiated at the pelvic area with 25 Gy. We injected 1×10⁶ cells of human PD-MSCs, human AD-MSCs, human foreskin fibroblasts, and control media into the rectal submucosa following irradiation. We sacrificed rats for pathologic evaluation.ResultsFibrosis on the rectum was reduced in both MSC groups, compared to the control group. Mucosal Ki-67 indices of both MSC injected groups were higher than those in the control group. Although caspase-3 positive cells in the mucosa gradually increased and decreased in the control group, those in both MSC injected groups increased rapidly and decreased thereafter.ConclusionWe demonstrated the effects of regional MSC injection treatment for radiation-induced proctitis in rats. MSC injection reduced fibrosis and increased proliferation in rat mucosa. Human AD-MSCs and PD-MSCs had similar effectiveness.     

Reparative Osteogenesis during Transplantation of Mesenchymal Stem Cells

Reparative osteogenesis was studied after xenotransplantation of suspension cell graft from human mesenchymal stem cells. A model of experimental damage to rat femoral diaphysis was developed. The state of animals was satisfactory and non-depressed in the early and late postoperation period. We revealed no local pathological reactions and complications. Administration of mesenchymal stem cells into the area of bone defect accelerated and improved regeneration. Unilateral transplantation of the cell graft stimulated regeneration in the contralateral limb due to acceleration of bone tissue maturation. On day 90 after treatment the bone regenerate was completely developed in the area of defect in animals of various groups. The newly formed bone tissue was well integrated into the bone organ. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 7, pp. 109–113, July, 2005     

Transplantation of Human Bone Marrow Mesenchymal Stem Cell Ameliorates the Autoimmune Pathogenesis in MRL/Ipr Mice

Recent evidence indicates that mesenchymal stem cells （MSC） possess immunosuppressive properties both in vitro and in vivo. We previously demonstrated the functional abnormality of bone marrow derived MSC in patients with systemic lupus erythematosus （SLE）. In this study, we aimed to investigate whether transplantation of human bone marrow derived MSC affects the autoimmune pathogenesis in MRL/Ipr mice. We found that human MSC from healthy donors reduced the proliferation of T lymphocytes from MRL/Ipr mice in a dose-dependent fashion. Two weeks after in vivo transfer of MSC, we detected significantly reduced serum levels of anti ds-DNA antibodies and 24 hour proteinuria in MRL/Ipr mice as compared with control groups without MSC transplantation. Moreover, flow cytometric analysis revealed markedly reduced number of CD4＋ T cells while increased Thl subpopulation in MSC group and MSC ＋ CTX group when compared with controls. Histopathological examination showed significantly reduced renal pathology in MSC-treated mice. Immunohistochemical studies further revealed reduced expression of TGF-~, FN, VEGF and the deposition of complement C3 in renal tissue after MSC and MSC ＋ CTX treatment. Taken together, we have demonstrated that transplantation of human MSC can significantly inhibit the autoimmune progression in MRL/Ipr mice. Cellular ＆ Molecular Immunology. 2008;5（6）：417-424.     

脑内移植间充质干细胞治疗大鼠帕金森病模型的研究

目的研究大鼠骨髓间充质干细胞(MSCs)脑内移植治疗帕金森病(PD)大鼠的可行性。方法贴壁培养法分离、培养大鼠骨髓MSCs。将BrdU标记的第3代MSCs移植到PD大鼠纹状体内,移植后1、2和4周检测PD大鼠的行为学变化,应用BrdU免疫荧光观察移植细胞在脑内的存活情况,应用免疫组化法检测酪氨酸羟化酶(TH)在移植细胞及黑质的表达。结果移植细胞后1周、2周和4周PD大鼠与移植前相比行为学有改善,旋转圈数明显减少(P<0.05);PD大鼠损毁侧和健侧黑质内TH阳性细胞数之比随移植时间的延长而增加,但未发现移植细胞呈现TH阳性表达。细胞移植后1周、2周和4周检测均可见纹状体内BrdU阳性细胞散在分布。结论植入脑内的MSCs能够生存和迁移,MSCs脑内移植对PD大鼠有一定的治疗作用。     

Transplantation of Human Bone Marrow Mesenchymal Stem Cell Ameliorates the Autoimmune Pathogenesis in MRL/lpr Mice

Recent evidence indicates that mesenchymal stem cells (MSC) possess immunosuppressive properties both in vitro and in vivo. We previously demonstrated the functional abnormality of bone marrow derived MSC in patients with systemic lupus erythematosus (SLE). In this study, we aimed to investigate whether transplantation of human bone marrow derived MSC affects the autoimmune pathogenesis in MRL/lpr mice. We found that human MSC from healthy donors reduced the proliferation of T lymphocytes from MRL/lpr mice ...     

Morphofunctional Study of the Therapeutic Efficacy of Human Mesenchymal and Neural Stem Cells in Rats with Diffuse Brain Injury

We studied the effect of transplantation of human stem cells from various tissues on reparative processes in the brain of rats with closed craniocerebral injury. Combined treatment with standard drugs and systemic administration of xenogeneic stem cells had a neuroprotective effect. The morphology of neurons rapidly returned to normal after administration of fetal neural stem cells. Fetal mesenchymal stem cells produced a prolonged effect on proliferative activity of progenitor cells in the subventricular zone of neurogenesis. Adult mesenchymal stem cells had a strong effect on recovery of the vascular bed in ischemic regions. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 23-37, 2009     

骨髓间充质干细胞静脉移植对扩张型心肌病大鼠毛细血管生成的影响

目的观察骨髓间充质干细胞（MSCs）经静脉途径移植对阿霉素诱导的扩张型心肌病（DCM）大鼠心功能的影响。方法建立大鼠阿霉素性心肌病模型,随机分为三组：DCM空白组（B组）、静脉移植组（C组）和静脉对照组（D组）,另设正常对照组（A组）。经尾静脉注射在体外扩增并DAPI标记的骨髓间充质干细胞和培养基至C组和D组大鼠,A组和B组大鼠不干预。移植后8周心脏彩超观察左室舒张末径（LVDD）、左室射血分数（LVEF）,多导生理记录仪观察左室最大压力上升速率（＋LVdp/dtmax）、左室最大压力下降速率（-LVdp/dtmax）,并分析组织病理学特征。结果细胞移植后8周,C组LVEF高于B组和D组;LVDD稍低于B组和D组;＋LVdp/dtmax明显高于B组,也显著高于D组;-LVdp/dtmax明显高于B组和D组;C组心肌中可见带有蓝色荧光的DAPI标记的骨髓MSCs,心肌胶原容积百分比低于B组和D组,毛细血管计数高于B组和D组;C组VEGF水平明显高于B组和D组。结论骨髓MSCs静脉移植治疗阿霉素诱导的扩张型心肌病大鼠,提高VEGF水平,促进心肌毛细血管新生,减少胶原纤维,改善心功能。     

转基因修饰后的自体骨髓间充质干细胞移植治疗mdx鼠后成肌调节因子的表达研究

目的探讨携带micro-dystrophin基因的自体骨髓间充质干细胞移植入mdx鼠后在移植鼠体内分化为肌细胞的可能机制。方法采用逆转录病毒介导micro-dystrophin基因转染mdx小鼠MSCs（mMSCs）,通过尾静脉注射移植治疗mdx鼠,在移植后免疫荧光检测micro-dystrophin的表达并在不同时间点检测MyoD的表达。结果移植后成功检测到micro-dystrophin,其表达随着移植时间的延长而增加;随移植时间延长MyoD阳性肌纤维比例增加,分别达到9%（4周时）、15%（8周时）、28%（12周时）。RT-PCR和Westernblot也发现,随着移植时间的延长,MyoD表达增加。结论自体mMSCs可携带外源性micro-dystrophin基因在受体鼠体内分化为micro-dystrophin阳性肌细胞,移植入的干细胞向肌细胞的分化是一个持久的、连续的过程,成肌调节因子在调节其分化过程中发挥了重大作用。     

Mesenchymal Stem Cells versus Mesenchymal Stem Cells Combined with Cord Blood for Engraftment Failure after Autologous Hematopoietic Stem Cell Transplantation: A Pilot Prospective,Open-Label,Randomized Trial

Engraftment failure (EF) after autologous hematopoietic stem cell transplantation is a serious complication. We prospectively evaluated the effects and safeties of mesenchymal stem cells (MSCs) alone and MSCs combined with cord blood (CB) for EF. Twenty-two patients were randomized to receive MSCs (MSC group; n = 11) or MSCs plus CB (CB group; n = 11). Patients with no response (NR) to MSCs received the therapeutic schedule in the CB group, and those patients with partial response (PR) in the MSC group and patients without complete remission (CR) in the CB group received another cycle of MSC treatment. Patients who did not achieve CR after 2 cycles of treatments received other treatments, including allogeneic HSCT. After the first treatment cycle, response was seen in 7 of 11 patients in the MSC group and in 9 of 11 in the CB group (P = .635), with a significant difference in neutrophil reconstruction between the 2 groups (P = .030). After 2 treatment cycles, 16 patients achieved CR, 3 achieved PR, and 3 had NR. No patient experienced graft-versus-host disease (GVHD). With a median follow-up of 345 d (range, 129 to 784 d) post-transplantation, 18 patients remained alive and 4 had died (3 from primary disease relapse and 1 from cytomegalovirus pneumonia). The 2-year overall survival, disease-free survival, and cumulative incidence of tumor relapse post-transplantation were 75.2% ± 12.0%, 79.5% ± 9.4%, and 20.5% ± 9.4%, respectively. Our data indicate that the 2 strategies are effective for EF and do not result in GVHD or increase the risk of tumor relapse, but the MSC plus CB regimen has a superior effect on neutrophil reconstruction.     

人脐带间充质干细胞的研究进展

脐带间充质干细胞是存在于脐带沃顿胶和血管周围组织中的一种干细胞,具有多向分化和自我更新的潜能。脐带间充质干细胞可分化为骨细胞、软骨细胞、脂肪细胞、肌细胞和神经细胞,并且具有免疫调节性;脐带作为医疗废弃物来源丰富,对供者无不利影响,无伦理问题的限制,这为细胞治疗和组织工程提供了新的种子细胞。     

Opposite Effects of Bone Marrow-Derived Cells Transplantation in MPTP-rat Model of Parkinson's Disease: A Comparison Study of Mononuclear and Mesenchymal Stem Cells

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model is a useful tool to study Parkinson''s disease (PD) and was used in the present study to investigate the potential beneficial as well as deleterious effects of systemic bone-marrow mononuclear cell (BMMC) or mesenchymal stem cell (BM-MSC) transplantation. MPTP administration resulted in a breakdown of the blood-brain barrier and motor impairment in the open field test 24 h after surgery. Three and 7 days after receiving the lesion, the injured animals showed remaining motor impairment compared to the sham groups along with a significant loss of tyrosine hydroxylase-immunoreactive (TH-ir) cells in the substantia nigra pars compacta (SNpc). The MPTP-lesioned rats treated with BMMCs immediately after lesioning exhibited motor impairment similar to the MPTP-saline group, though they presented a significantly higher loss of TH-ir cells in the SNpc compared to the MPTP-saline group. This increased loss of TH-ir cells in the SNpc was not observed when BMMC transplantation was performed 24 h after MPTP administration. In contrast, in the MPTP animals treated early with systemic BM-MSCs, no loss of TH-ir cells was observed. BMMCs and BM-MSCs previously labeled with CM-DiI cell tracker were found in brain sections of all transplanted animals. In addition, cells expressing CD45, an inflammatory white blood cell marker, were found in all brain sections analyzed and were more abundant in the MPTP-BMMC animals. In these animals, Iba1+ microglial cells showed also marked morphological changes indicating increased microglial activation. These results show that systemic BMMC transplantation did not ameliorate or prevent the lesion induced by MPTP. Instead, BMMC transplantation in MPTP-lesioned rats accelerated dopaminergic neuronal damage and induced motor impairment and immobility behavior. These findings suggest that caution should be taken when considering cell therapy using BMMCs to treat PD. However, systemic BM-MSC transplantation that reaches the injury site and prevents neuronal damage after an MPTP infusion could be considered as a potential treatment for PD during the early stage of disease development.     

力学因素对骨髓间充质干细胞成骨分化的影响

近年来,组织工程学领域发展突飞猛进,已被列为一种修复或再生许多组织和器官的方法。骨髓间充质干细胞具有良好的成骨向分化潜能,在骨组织工程领域中具有广阔的应用前景。骨髓间充质干细胞增殖和成骨向分化受多种力学因素的影响,且不同性质的力学刺激对其定向分化的调节作用不尽相同。目前,许多学者致力于深入探讨力学因素影响骨髓间充质干细胞成骨向分化的具体途径,但其调控机制尚未完全明确。本文综述及讨论力学因素对骨髓间充质干细胞所产生的增殖、定向成骨分化等生物学效应的影响及可能涉及的力化学信号转导通路作用机制,以期丰富研究思路     

外周血间充质干细胞移植对球囊损伤兔血管平滑肌细胞凋亡的影响

探讨外周血间充质干细胞(MSCs)移植对模型兔颈动脉球囊成形术后平滑肌细胞凋亡的影响。粒细胞集落刺激因子(G-CSF)动员5 d后采集外周血,用密度梯度离心法结合反复贴壁法分离培养获得纯化的MSCs,以增强型绿色荧光蛋白(GFP)报告基因标记备用。建立兔动脉粥样硬化狭窄模型,随机分为MSCs移植组及对照组,于颈动脉球囊成形术后分别静脉注入MSCs或等体积培养液。术后7、14、28 d取球囊损伤血管标本经TUNEL法(TdT-mediated d-UTP nick end labeling)测定血管平滑肌细胞凋亡率;同时进行归巢MSCs的鉴定;此外,测定术后28 d的内膜、中膜面积,再狭窄率及再内皮化情况。术后7、14、28 d,与对照组比较,移植组平滑肌细胞凋亡率明显增高;仅在移植组血管组织中有GFP阳性细胞分布;术后28 d,移植组内膜面积、内膜中膜面积之比及再狭窄率显著低于对照组,而再内皮化程度则明显优于对照组。静脉移植外周血MSCs能够促进模型兔球囊损伤血管快速内皮化,抑制新内膜增生、减少再狭窄率;这一作用可能与损伤血管局部平滑肌细胞凋亡增加有关。     

大鼠肝移植术后受体骨髓间充质干细胞输注肝内示踪与标记方法比较

目的采用羧基荧光素二醋酸盐琥珀酰亚胺酯（carboxyfluorescin diacetate succinimidyl ester,CFSE）与溴化脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine,Brdu）两种细胞标记物观测肝移植术后受体骨髓间充质干细胞（MSCs）肝内分布情况,并对两种方法进行比较。方法在已构建的DA-Lewis大鼠原位肝移植模型及提取培养Lewis大鼠MSCs的基础上分别用CFSE和Brdu对Lewis来源的MSCs进行标记．术中经门静脉输注．通过荧光显微镜和免疫组织化学法观测术后2mo内各时间点受体肝组织内MSCs的分布情况。结果CFSE细胞标记率约为（94．1±1．4）％。受体肝组织第1、7、14天内有CFSE标记的MSCs聚集,对照组第5天肝组织内发布的CFSE标记MSCs消失。Brdu细胞标记率约为（90．3±3．5）％。受体肝组织第14天、1个月、2个月可见Brdu阳性的MSCs分布,对照组相应时间点肝组织内未发现Brdu阳性的MSCs。结论CFSE和Brdu均为MSCs的良好标记物。CFSE标记细胞后荧光迅速减弱,适用于短期示踪。Brdu标记细胞后可维持较长时间．适用于长期示踪。两种标记方法均是MSCs较为简单有效的方法,实验显示受体MSCs大部分分布于移植肝脏。     

Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Cochlear Function in an Experimental Rat Model

Mesenchymal stem cell (MSC) therapy is an emerging treatment modality for various human diseases. Although induced pluripotent stem cells have been explored for the restoration of hearing, the potential of MSCs as a therapeutic strategy for various cochlear insults is not precisely known. MSCs possess anti-inflammatory, anti-apoptotic and neuroprotective properties, making them an attractive target for the treatment of inner ear disorders such as hair cell damage in response to inflammation. Most of the previous studies have used immunosuppression or the complex surgical techniques to deliver stem cells into the cochlea. However, no information is available regarding the biocompatibility and safety of MSCs in the inner ear in immunocompetent cochlea. The aim of the present study was to determine the effect of non-surgical administration of rodent bone marrow derived MSCs (BM-MSCs) through transtympanic delivery on the cochlear function and to assess any adverse effects on the auditory system employing a rat model without immunosuppression. We observed that the transtympanic administration of BM-MSCs has no significant effect on the hearing thresholds as determined by auditory brainstem response and distortion product otoacoustic emissions. Histopathological examination revealed no recruitment of inflammatory leukocytes and edema in the cochlea of BM-MSCs administrated rats. The results of this study suggest that transtympanic administration of BM-MSCs is safe and can be explored in providing otoprotection against cochlear insults. Anat Rec, 303:487–493, 2020. © 2019 American Association for Anatomy