Intravenous transplantation of bone marrow mesenchymal stem cells promotes neural regeneration after traumatic brain injury

To investigate the supplement of lost nerve cells in rats with traumatic brain injury by intravenous administration of allogenic bone marrow mesenchymal stem cells, this study established a Wistar rat model of traumatic brain injury by weight drop impact acceleration method and administered 3 × 106 rat bone marrow mesenchymal stem cells via the lateral tail vein. At 14 days after cell transplantation, bone marrow mesenchymal stem cells differentiated into neurons and astrocytes in injured rat cerebral cortex and rat neurological function was improved significantly. These findings suggest that intravenously administered bone marrow mesenchymal stem cells can promote nerve cell regeneration in injured cerebral cortex, which supplement the lost nerve cells.

The effect of neuropeptides on proliferation of rat bone marrow mesenchymal stem cells

Objective To investigate the effects and mechanism of calcitonin gene-related peptide(CGRP)and substance P (SP) on proliferation of rat bone marrow mesenchymal stem cells.Methods The rBMSCs were isolated using whole bone marrow

Improvement of neurological function in rats with spinal cord injury after the transplantation of neural stem cells directly differentiated from bone marrow mesenchymal stem cells

Objective To study the effect and mechanism of neurological function recovery in rats with spinal cord injury ( SCI) rats after transplantation of neural stem cells which are directly differentiated from bone marrow mesenchymal stem cells ( BMSC ) ,and to investigate the suitable engraftment time. Methods BMSC at 3rd passage were differentiated into neural stem cells ( NSC) , and immunofluorescence staining was used to

Cell multiplication, apoptosis and p-Akt protein expression of bone mesenchymal stem cells of rat under hypoxia environment

Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal Stem Cells（MSCs） of rats is influenced by a hypoxic environment ex vivo. Methods ：Passage 3 of bone marrow MSCs taken from Wistar rats,were cultured in a culturing chamber with 94%N2,1%O2,5%CO2 at 37℃. At different hypoxia time points ,0,0.5, 1,4 and 8 h, glucose uptake was assayed by using radiation isotope ^3H-G, Apoptotic Rate（AR） and dead rate（DR） were analyzed by flow cytometry（FCM） after Annexin V/PI staining, cell multiplication（by MTr methods） and p-Akt protein by immunocytochemistry and western blot. Results ：Assay for CD29^± ,CD44^± ,CD71^± ,CD34^-, Tn T^±（after 5-azacytidine agent inducing） and ALP^±（after bone differentiation agent inducing） suggested these bone-derived cells were MSCs. The ^3H-G intaking ratio （CPM/ flask value：157 ± 11,110 ± 11,107 ± 13,103 ± 10,100 ± 9 and 98 ± 10） of MSCs at different hypoxia time points, significantly decreased compared to that of normoxia（P 〈 0.01） and tended to descend slowly with hypoxia time duration, for which there was no statistical significance（P 〉 0.05）. The AR（0.09 ± 2.03%,12.9 ± 1.72%,13.7 ± 2.26%,13.8 ± 3.01%,14.1 ± 2.78% and 14.7 ± 4.01% at 0,0.5,1,4 and 8 h,respectively,P 〈 0.01） and DR （0.04, ± 1.79% ,0.93 ± 1.85% ,3.11 ± 2.14% ,4.09 ± 2.36% ,4.72 ± 2.05% and 4.91 ± 3.72% at 0,0.5,1,4 and 8 h, respectively, P 〈 0.05） at different hypoxia time points significantly increased compared to those time in normoxia; The AR further went up with time （P 〈 0.05）, however there was no statistical significance （P 〉 0.05） for the DR. Optical absorption value of MTr methods at different hypoxia time points significantly decreased compared to those with a corresponding normoxia time （P 〈 0.01） and degraded with time （in an hypoxic environment -P 〈 0.01）. IOD of p-Akt protein of MSCs at different hypoxia time points significantly increased （0.367 ± 0.031,0.556 ± 0.023,0.579 ± 0.013, 0.660 ± 0.024, 0.685 ± 0.039 and 0.685 ± 0.011, respectively） compared to their equivalents in normoxia （P〈0.05）, however, there was no statistical significance （P 〉 0.05） for different hypoxia time points. Hypoxia may result in ultramicrostructure changes, such as defluvium of Microvilli, apoptotic body, ＂margination＂ and so on and are further aggravated with hypoxia time stretching. Conclusion： Hypoxia may lead to a depression of MSCs intaldng glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.

Therapeutic Potential of Bone Marrow Derived Mesenchymal Stem Cells in Modulating Astroglyosis of Surgical Induced Experimental Spinal Cord Injury

Background: Spinal cord injury (SCI) unsuccessful regeneration was due to glial scar development. It was a major obstacle to axonal restoration. Safe therapeutic intervention by the use of bone marrow derived stem cells (BMMSCs) transplantation applied in the present study could reduce spinal disability. Material and methods: Forty male albino rats were divided into four groups: GI: negative control (n = 10 rats);GII: positive control after SCI (n = 10 rats);GIII: SCI + BM - MSCs intravenous injected and GIV: SCI + BM - MSCs intra lesion injected (n = 10 rats in each group). The samples were taken from spinal cord tissues around the region of injury and were subjected to histological, immunohistochemical assessment. RNA extraction and real time PCR for detection of nerve regeneration and astrocyte response to the injury were also performed. Results: Clinical improvement occurred by the enhancement in the Basso, Beattie and Bresnahan (BBB) score after SCI. Histological examinations showed positive regenerative responses in GIV compared to GIII. Conclusion: BM-MSCs transplantation has a promising role in enhancing the microenvironment for nerve regeneration through stumbling the glial scaring formation and inflammatory response after chronic spinal cord injury especially by using intra-lesion route injection.

Effect of SHU555A labeling on differentiation of bone marrow mesenchymal stem cells into neurocyte-like cells

Objective:To investigate the effect of SHU555A,a clinically approved iron nanoparticle,labeling on differentiation of bone marrow mesenchymal stem cells(BMSCs) into neurocyte-like cells in vitro.Methods:10 times dilution of 10μl,20μl,40μl and 80μl SHU555A were added to 2ml of culture medium containing rat BMSCs to obtain four experimental groups of SHU555A labeling of BMSCs with ferri ion concentrations of 14μg/ml,28μg/ml,56μg/ml and 112μg/ml,respectively.2ml of culture medium with rat BMSCs did not contain SHU555A served as control group.The BMSCs of all the groups were pre-induced by bFGF,and induced by DMSO/butylated hydroxyanisole(BHA) for six hours,subsequently reverse transcription polymerase chain reaction(RT-PCR) technique was employed to detect mRNA expression of nestin,neuronspecific analase(NSE) and glial fibrillary acid protein(GFAP).Western blot technique was used to detectprotein expression of nestin.Results:Quantitative-PCR revealed high mRNA expression of nestin,NSE and GFAP induced by DMSO/BHA in all the experimental groups,but the difference between the experimental groups and the control group was not significant(P>0.05).Western blot analysis demonstrated there was no statistically significant difference in nestin protein expression between the experimental groups and the control group(P>0.05).Conclusion:SHU555A labeling do not affect differentiation of rat BMSCs into neurocyte-like cells in vitro.

The effect of bone marrow mesenchymal stem cell transplantation on hypoxic pulmonary hypertension in rats

Objective:To study the influence of bone marrow mesenchymal stem cells(MSCs)transplantation on hypoxic pulmonary hypertension(HPH)in rats.Methods:MSCs in SD rats were separated,cultivated,identified in vitro,and labeled by the green fluorescence protein(GFP)adenovirus.Healthy male SD rats were randomly divided into four groups:normal control group(NC group)and HPH group,with eight rats in each group respectively;HPH+mesenchymal stem cell transplantation group(MSCs group)and HPH+vascular endothelial growth factor+mesenchymal stem cell transplantation group(VEGF+MSCs group),with twenty-four rats in each group respectively.In this experiment,intermittent normobaric hypoxia was employed to establish the pulmonary hypertension rat models,with stem cells transfected and transplanted.The mean pulmonary artery pressure(mPAP)was observed in rats to calculate the right ventricular hypertrophy index(RVHI);the morphological changes of pulmonary arterioles in each group of rats were observed under the microscope;the distribution and manifestation of MSCs fluorescently labeled by adenovirus transfection were observed in pulmonary arterioles under the fluorescence microscope at the set time points of 7 d,14 d and 28 d after the transplantation of stem cells.Results:For NC group,the mPAP(mmHg)was 15.5±1.5 at 28 d,while the mPAP in HPH,MSCs and VEGF+MSCs groups were 26.1±1.9,21.6±2.7 and 20.1±2.9 respectively which were apparently higher than that in NC group(p<.01).Compared with HPH group(p<.01),the mPAP was obviously decreased in MSCs and VEGF+MSCs groups.There was no significant difference between MSCs and VEGF+MSCs groups.At 28 d,RVHI for NC group was 0.28±0.02,while the RVHI in HPH,MSCs and VEGF+MSCs groups were 0.43±0.07,0.34±0.03 and 0.35±0.01 respectively which were apparently higher than that in NC group(p<.01).In comparison with HPH group,RVHI was significantly decreased in MSCs and VEGF+MSCs groups(p<.05).There was no significant difference between MSCs and VEGF+MSCs groups.For HPH group,at 28 d,pulmonary arterioles were apparently thickened,with luminal stenosis&obliteration and incomplete endothelial cells.Compared with HPH group,pulmonary arterioles in MSCs group became thinning,with the lumen unobstructed and the integrity of endothelial cells improved.The changes in the manifestation of MSCs and VEGF+MSCs groups were not significant.Conclusions:The transplantation of MSCs can improve the remodeling of pulmonary arterioles to partially reverse the progress of HPH;the combined transplantation of VEGF and MSCs doesn’t improve the effect of MSC transplantation.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

Human adipose-derived mesenchymal stem cells： a better cell source for nervous system regeneration

Background In order to suggest an ideal source of adult stem cells for the treatment of nervous system diseases,MSCs from human adipose tissue and bone marrow were isolated and studied to explore the differences with regard to cell morphology,surface markers,neuronal differentiation capacity,especially the synapse structure formation and the secretion of neurotrophic factors.Methods The neuronal differentiation capacity of human mesenchymal stem cells from adipose tissue （hADSCs） and bone marrow （hBMSCs） was determined based on nissl body and synapse structure formation,and neural factor secretion function.hADSCs and hBMSCs were isolated and differentiated into neuron-like cells with rat brain-conditioned medium,a potentially rich source of neuronal differentiation promoting signals.Specific neuronal proteins and neural factors were detected by immunohistochemistry and enzyme-linked immunosorbent assay analysis,respectively.Results Flow cytometric analysis showed that both cell types had similar phenotypes.Cell growth curves showed that hADSCs proliferated more quickly than hBMSCs.Both kinds of cells were capable of osteogenic and adipogenic differentiation.The morphology of hADSCs and hBMSCs changed during neuronal differentiation and displayed neuronlike cell appearance after 14 days＇ differentiation.Both hADSCs and hBMSCs were able to differentiate into neuron-like cells based on their production of neuron specific proteins including β-tubulin-Ⅲ,neuron-specific enolase （NSE）,nissl bodies,and their ability to secrete brain derived neurotrophic factor （BDNF） and nerve growth factor （NGF）.Assessment of synaptop hysin and growth-associated protein-43 （GAP-43） suggested synapse structure formation in differentiated hADSCs and hBMSCs.Conclusions Our results demonstrate that hADSCs have neuronal differentiation potential similar to hBMSC,but with a higher proliferation capacity than hBMSC.Adipose tissue is abundant,easily available and would be a potential ideal source of adult stem cells for neural-related clinical research and application.

Differentiation of marrow-derived islet-like cells and their effects on diabetic rats

In recent years, islet transplantation for diabetes has shown signs of the treatment efficacy, but its application is limited due to lack of donor organizations, sources and immune rejection. Bone marrow mesenchymal stem cells （BMSCs） have become a new resource of islet cell substitutes. One focus of the current research is the application of a specific inducing agent or a culture system to get directed differentiation of BMSCs, which may have part characteristics of islet cells and then be used in autologous transplantation for the treatment of diabetes.

丁苯酞对大鼠骨髓间充质干细胞炎性损伤的改善作用及机制

目的 研究丁苯酞对大鼠骨髓间充质干细胞(BMSCs)炎性损伤的改善作用及其可能机制。方法 将大鼠BMSCs分为对照组、模型组和丁苯酞低、中、高浓度组(10、20、50μmol/L)。体外培养BMSCs并采用脂多糖(终浓度为10 mg/L)建立炎性损伤模型,经丁苯酞干预后检测细胞存活率、凋亡率和细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)含量以及细胞中核因子κB(NF-κB)p65 mRNA和胱天蛋白酶3(caspase-3)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)和NF-κB p65蛋白表达。结果 与对照组比较,模型组细胞存活率和Bcl-2蛋白表达水平均显著降低(P<0.05);细胞凋亡率,TNF-α、IL-1β、IL-6含量,NF-κB p65 mRNA以及caspase-3、Bax、NF-κB p65蛋白表达水平均显著升高(P<0.05)。与模型组比较,丁苯酞各浓度组上述指标均显著逆转(P<0.05),且呈浓度依赖性。结论 丁苯酞对脂多糖诱导的BMSCs炎性损伤具有改善作用,其机制可能与抑制NF-κB信号通路有关。

骨髓间充质干细胞移植可提高骨质疏松大鼠骨代谢水平

背景:研究证实骨髓间充质干细胞具有增加骨保护素水平、促进骨代谢的作用。目的:探讨骨髓间充质干细胞对骨质疏松大鼠骨代谢的影响。方法:实验所用健康SD大鼠42只,随机分为假手术组、骨质疏松组、干细胞移植组,每组14只,后2组采用去卵巢法制备大鼠骨质疏松症模型,造模成功后干细胞移植组经尾静脉移植骨髓间充质干细胞,移植28 d后采用ELISA法检测各组大鼠血清骨保护素、碱性磷酸酶、抗酒石酸酸性磷酸酶、骨钙素、胰岛素样生长因子1水平;Western blot检测各组大鼠骨组织中FoxO1的表达;AG-IX生物力学万能试验机检测股骨最大载荷和断开裂载荷;苏木精-伊红染色观察大鼠骨形态学改变。结果与结论:①骨质疏松组骨保护素、碱性磷酸酶、胰岛素样生长因子1水平低于假手术组(P<0.05),抗酒石酸酸性磷酸酶、骨钙素水平高于假手术组(P<0.05);干细胞移植组骨保护素、碱性磷酸酶、胰岛素样生长因子1水平高于骨质疏松组(P<0.05),抗酒石酸酸性磷酸酶、骨钙素的表达水平低于骨质疏松组(P<0.05);②骨质疏松组最大载荷、断裂载荷值均低于假手术组(P<0.05);干细胞移植组最大载荷、断裂载荷值高于骨质疏松组(P<0.05);③干细胞移植组骨小梁明显增粗,排列较为有序,局部间隙较均匀,较骨质疏松组明显改善;④骨质疏松组大鼠骨髓组织中FoxO1蛋白表达水平低于假手术组(P<0.05);干细胞移植组大鼠骨髓组织中FoxO1蛋白表达水平高于骨质疏松组(P<0.05);⑤结果表明,骨髓间充质干细胞移植可提高骨质疏松大鼠骨代谢水平,其作用机制与促进骨保护素、碱性磷酸酶、FoxO1的表达有关。

慢病毒下调酪蛋白激酶2相互作用蛋白1表达的骨髓间充质干细胞修复骨质疏松大鼠牙槽骨缺损

背景:在口腔牙槽骨修复治疗过程中,患者的全身状况尤其是骨质疏松症等系统性疾病往往会影响治疗效果,近来发现酪蛋白激酶2相互作用蛋白1(casein kinase 2-interacting protein-1,CKIP-1)具有负调控成骨的作用,因此有希望作为新的靶点用于治疗骨质疏松。目的:探究CKIP-1表达下调的骨髓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)从1周龄SD乳鼠股骨及胫骨骨髓中分离培养出骨髓间充质干细胞,慢病毒转染降低骨髓间充质干细胞中的CKIP-1水平;(2)通过维甲酸[70 mg/(kg·d)]灌胃21 d的方式建立骨质疏松SD大鼠模型,将CKIP-1表达下调的骨髓间充质干细胞与明胶海绵复合体、空载体转染的骨髓间充质干细胞与明胶海绵复合体、明胶海绵分别植入骨质疏松大鼠模型牙槽骨缺损内,正常组将明胶海绵植入正常大鼠牙槽骨缺损内。术后正常喂养30 d后,取上颌牙槽骨进行苏木精-伊红染色并通过Lane-Sandhu组织学评分和新生骨百分比评估骨缺损愈合情况,通过q RTPCR技术和免疫组化染色检测各组术区牙槽骨中CKIP-1、碱性磷酸酶、骨钙素的m RNA相对表达量及阳性表达情况。结果与结论:(1)与空载体转染相比,CKIP-1下调后的骨髓间充质干细胞中CKIP-1的m RNA相对表达量减少(P <0.05);(2)与植入空载体转染的骨髓间充质干细胞与明胶海绵复合体组、明胶海绵组相比,植入CKIP-1表达下调的骨髓间充质干细胞与明胶海绵复合体组Lane-Sandhu组织学评分和新生骨百分比显著升高(P <0.05),与正常组相近;碱性磷酸酶、骨钙素表达显著升高(P <0.05);(3)结果说明,局部移植CKIP-1下调的骨髓间充质干细胞对骨质疏松大鼠牙槽骨缺损具有明显的修复作用。

酪蛋白激酶2相互作用蛋白1调控骨质疏松大鼠骨髓间充质干细胞的成骨能力

背景:目前对酪蛋白激酶2相互作用蛋白1(casein kinase 2-interaction protein-1,CKIP-1)分子机制的体外研究主要集中在基因敲除小鼠来源成骨细胞或骨髓间充质干细胞,鲜见报道骨质疏松模型大鼠来源骨髓间充质干细胞中CKIP-1表达的研究。目的:探讨下调CKIP-1基因前后骨质疏松状态骨髓间充质干细胞成骨分化能力的变化。方法:用维甲酸诱导雌性SD大鼠骨质疏松模型,采用全骨髓贴壁法体外培养骨质疏松组、正常组大鼠骨髓间充质干细胞。成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达,实时定量RT-PCR检测成骨诱导过程中2组细胞中CKIP-1的动态表达;通过基因转染沉默CKIP-1基因,成骨诱导后进行茜素红染色、碱性磷酸酶染色及实时定量RT-PCR检测骨桥蛋白、Runx2 mRNA的相对表达。结果与结论:①与正常组相比,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平降低(P<0.05),骨质疏松组骨髓间充质干细胞中CKIP-1基因动态表达水平总体偏高;②与未下调CKIP-1的骨质疏松组骨髓间充质干细胞相比,下调CKIP-1基因表达后,骨质疏松组骨髓间充质干细胞茜素红染色钙结节定量、碱性磷酸酶活性及骨桥蛋白、Runx2的mRNA水平明显升高(P<0.05);③结果表明,维甲酸诱导的骨质疏松大鼠骨髓间充质干细胞成骨能力降低,下调CKIP-1基因可以部分提高其成骨分化能力。

低氧低糖及血清剥夺联合处理抑制Nrf2信号通路诱发大鼠骨髓间充质干细胞氧化应激和凋亡

目的探讨低氧低糖血清剥夺(GSDH)处理对大鼠骨髓间充质干细胞(BMSCs)氧化应激及凋亡的影响。方法在体外对提取纯化后的原代BMSCs利用低氧(1%O_(2))、低糖(1.0 g/L)及血清剥夺联合处理建立BMSCs细胞损伤模型。采用集落形成实验、细胞周期测定以及CCK-8实验检测细胞增殖能力;划痕实验和Transwell实验检测细胞迁移能力;通过细胞凋亡试剂(AnnexinV-FITC/PI)、线粒体膜电位检测试剂(JC-1)染色和线粒体荧光探针(Mito-Trancker)检测细胞凋亡;细胞活性氧簇(ROS)和钙离子(Fluo-4AM)检测细胞氧化应激水平;Western blotting检测抗氧化应激相关分子谷胱甘肽过氧化物酶4(GPX4)等以及核因子红细胞系2相关因子2(Nrf2)信号通路中关键分子的蛋白表达水平。结果大鼠原代BMSCs表面标志物高表达CD29、CD71,低表达CD45、CD34;GSDH处理抑制BMSCs细胞增殖(P<0.05)和迁移(P<0.05),增加ROS和钙离子水平(P<0.05),且抑制抗氧化应激相关分子GPX4等蛋白表达(P<0.01);与空白对照组相比,GSDH处理后BMSCs凋亡显著升高(P<0.05),线粒体膜电位降低(P<0.01),网络化减少(P<0.01)。Nrf2信号通路中Nrf2蛋白及下游关键分子的蛋白表达水平均降低(P<0.05)。结论GSDH处理诱发的BMSCs氧化应激及进一步导致的凋亡损伤与Nrf2信号通路被抑制有关。

miR-145-5p在正常大鼠与2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较

背景糖尿病会导致骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)的成骨分化能力降低,miRNA在此过程中发挥重要作用,其中miR-145-5p对细胞成骨成软骨分化有重要的调节作用。然而miR-145-5p对糖尿病源BMMSCs成骨分化的影响尚不清楚。目的比较正常大鼠与2型糖尿病大鼠BMMSCs成骨分化能力,初步探讨在成骨分化中miR-145-5p及其靶基因SEMA3A和Wnt通路关键蛋白β-catenin表达的差异。方法12只GK大鼠采用高糖高脂饲料喂养构建2型糖尿病大鼠模型,12只Wistar大鼠常规饲养作为对照组。无菌条件下分离大鼠股骨,全骨髓培养法培养正常大鼠和2型糖尿病大鼠BMMSCs(分别对应WT-BMMSCs和GK-BMMSCs);CCK-8检测细胞增殖能力;结晶紫染色检测细胞集落形成能力;碱性磷酸酶染色及半定量分析检测碱性磷酸酶活性;茜素红染色检测矿化基质形成;qRT-PCR和Western blot检测成骨相关标志物、miRNA-145-5p、SEMA3A和β-catenin的表达。结果干细胞培养4~9 d时,GKBMMSCs的增殖水平低于WT-BMMSCs,差异有统计学意义(P<0.01);10 d时,其集落形成率显著低于WT-BMMSCs(P<0.01);成骨诱导7 d时,GK-BMMSCs碱性磷酸酶活性(P<0.001)、成骨相关标志物碱性磷酸酶基因(P<0.01)和蛋白(P<0.001)表达、骨钙素基因(P<0.001)和蛋白(P<0.001)表达均显著低于WT-BMMSCs,1型胶原蛋白(P<0.01)基因的表达显著低于WT-BMMSCs,而Runt相关转录因子2(P<0.001)蛋白表达显著高于WT-BMMSCs。在WT-BMMSCs成骨分化中,miR-145-5p表达下调,SEMA3A表达上调,而在GK-BMMSCs成骨分化中,miR-145-5p和SEMA3A表达均上调,β-catenin(P<0.001)在GK-BMMSCs中的表达显著降低。成骨诱导21 d时,WT-BMMSCs矿化基质染色较深。结论2型糖尿病大鼠BMMSCs细胞增殖、集落形成及成骨分化能力降低,推测在正常大鼠BMMSCs成骨分化中miR-145-5p起抑制作用,在2型糖尿病大鼠BMMSCs成骨分化中miR-145-5p起促进作用。

大耳白兔骨髓间充质干细胞的分离培养与生物学鉴定

骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源于发育早期的中胚层和外胚层,作为干细胞中的种子细胞,具有强大的自我复制和更新能力,并对治疗、修复、补充受损或缺损的肝、肌肉等组织有修复和治疗作用。为了研究BMSCs的生物学特性和多化潜能,对日本大耳白兔BMSCs进行体外分离培养及生物学鉴定研究。取日本大耳白胎兔骨髓组织,采用全骨髓贴壁法分离纯化、体外培养,鉴定日本大耳白兔BMSCs生物学特性,并进行细胞形态学观察、生长动力学检测及其成骨、成软骨、成脂肪能力鉴定。结果表明,采用全骨髓贴壁法成功从日本大耳白胎兔骨髓中分离得到生长状态良好的大耳白兔BMSCs,细胞形态为长梭形,生长曲线呈典型“S”形;免疫荧光和RT-PCR结果显示,大耳白兔BMSCs表达CD29、CD44、CD73、CD105间充质干细胞表面标记物基因,但不表达原始造血祖细胞标志物CD45;经特异性染色和RT-PCR分析表明,分离的细胞是间充质干细胞;体外诱导大耳白兔BMSCs能够分化为骨骼细胞、软骨细胞、脂肪细胞。建立了大耳白兔BMSCs体外分离培养体系,为利用骨髓间充质干细胞进行组织与工程学研究和动物遗传资源保存研究奠定了一定的理论基础。

干扰FoxO1慢病毒包装及稳转大鼠骨髓间充质干细胞株的建立

目的建立干扰叉头状转录因子O1(FoxO1)的大鼠骨髓间充质干细胞(BMSCs)稳转株,为研究BMSCs定向分化提供条件。方法提取大鼠原代BMSCs。构建FoxO1干扰载体,扩增后进行测序鉴定。FoxO1质粒瞬转293T细胞后,通过Western blot验证其干扰效果,并选择干扰效果最好的质粒载体。然后通过三因子慢病毒包装系统将FoxO1质粒和两个辅助质粒共转染293T细胞,包装慢病毒,组名为sh1,sh2,sh3,Vector。包装的干扰慢病毒和阴性对照病毒感染大鼠BMSCs,通过Western blot和RT-qPCR检测其表达效果。结果成功提取大鼠原代BMSCs;测序结果显示,重组FoxO1质粒构建成功;重组FoxO1质粒瞬时转染293T细胞48 h,细胞荧光明显,表明EGFP表达良好;Western blot结果显示,载体中标签蛋白表达成功,并筛选到干扰效果最佳的目的质粒sh1-FoxO1;干扰FoxO1慢病毒滴度为1×10^(8) TU·μL^(-1);包装完成的慢病毒感染BMSCs,经嘌呤霉素筛选后,Western blot结果显示,与Vector组相比,sh1组FoxO1的蛋白相对表达量降低(P<0.05);RT-qPCR结果显示,与Vector组相比,sh1组FoxO1的mRNA相对表达量降低(P<0.01),得到干扰FoxO1的大鼠BMSCs稳转株。结论成功构建了FoxO1干扰BMSCs稳转株,为进一步研究FoxO1在诱导BMSCs定向分化为IPCs过程中的作用及其潜在机制奠定了基础。

2型糖尿病对大鼠颌骨骨髓间充质干细胞生物学特性的影响及其机制研究

背景2型糖尿病患者骨代谢异常,颌骨骨质和骨量都发生明显改变,是牙周炎的全身促进因素之一。Wnt信号通路可以促进干细胞矿化并修复骨组织缺损。然而2型糖尿病影响下Wnt信号通路是否参与了对颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,JBMMSCs)的成骨分化调控仍未可知。目的研究2型糖尿病对大鼠JBMMSCs增殖及分化等生物学特性的影响,并对其机制进行初步探索。方法选取连续两周随机血糖≥16.7mmoL/L的13周龄GK大鼠为2型糖尿病组,相同周龄的Wistar大鼠作为对照组,两组各10只;无菌条件下采用骨髓冲洗法与骨片消化法相结合的方法分离培养两组JBMMSCs作为研究对象。CCK-8法检测并比较两组细胞增殖能力,流式细胞术检测细胞被诱导凋亡的能力。对JBMMSCs进行成骨、成脂诱导后,qRT-PCR评估成骨、成脂及Wnt信号通路相关基因的表达改变,碱性磷酸酶(alkaline phosphatase,ALP)染色检测成骨诱导后ALP的表达变化,茜素红染色比较钙结节形成能力,油红O染色检测成脂诱导后脂滴形成能力。结果与对照组相比,2型糖尿病组JBMMSCs的增殖及克隆形成能力降低,凋亡早期与晚期的细胞比例增加(P<0.05)。两组JBMMSCs成骨诱导后成骨相关基因ALP、OCN和Runx2 mRNA表达升高,但2型糖尿病组低于对照组(P<0.05),2型糖尿病组钙结节形成能力和ALP染色面积及密度较对照组低(P<0.05)。成脂诱导后,成脂相关基因的表达和脂滴的形成较对照组减少(P<0.05)。2型糖尿病组JBMMSCs成骨诱导7 d后Wnt信号通路相关分子Wnt4、Wnt5a、Wnt7b的mRNA表达较对照组升高,β-catenin的表达水平较对照组低。结论2型糖尿病影响大鼠颌骨骨髓间充质干细胞的增殖和克隆、抑制成骨及成脂分化能力,其成骨能力的降低可能与Wnt信号通路相关。

电针联合骨髓间充质干细胞治疗化疗致大鼠卵巢功能不全

背景:骨髓间充质干细胞治疗卵巢功能不全有不错的效果,但仍存在归巢效率低、效果欠佳等问题。目的:探讨电针疗法联合骨髓间充质干细胞治疗化疗型卵巢功能不全的效果。方法:从32只SD大鼠中随机选择8只做正常组,其余随机分为卵巢功能不全组、干细胞组、联合组,每组8只,采用腹腔注射环磷酰胺溶液制备卵巢功能不全动物模型。造模结束后,正常组不做处理,卵巢功能不全组尾静脉注射1 mL生理盐水,干细胞组与联合组尾静脉注射1 mL骨髓间充质干细胞悬液(细胞数为2×106),联合组大鼠进行电针治疗,30 min/d,持续7 d,治疗结束后取心脏血与卵巢组织观察疗效。结果与结论:①与卵巢功能不全组相比,干细胞组及联合组大鼠血清雌二醇激素水平明显升高,卵泡刺激素水平明显降低;②与卵巢功能不全组相比,干细胞组及联合组大鼠卵巢卵母细胞数量明显增加;③与干细胞组相比,联合组的骨髓间充质干细胞归巢水平明显增加(P=0.0043),CXCR4蛋白表达水平升高(P=0.036);④与卵巢功能不全组相比,干细胞组及联合组大鼠卵巢凋亡细胞明显减少,Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高;与干细胞组相比,联合组Bax表达水平明显降低(P=0.0284),Bcl-2表达水平明显升高(P=0.0364);⑤结果显示,电针能促进骨髓间充质干细胞归巢,并增强骨髓间充质干细胞的抗凋亡作用。