两份人脐血间充质干细胞混合培养及向神经元样细胞的分化

背景：脐血间充质干细胞可诱导分化为神经样细胞，但在临床中发现干细胞培养细胞增殖的数量不足。目的：观察两份脐血间充质干细胞混合培养的生长情况，以及经碱性成纤维细胞生长因子诱导脐血间充质干细胞向神经元样细胞的分化。设计、时间及地点：于2007—01/10在郑州大学基础医学院微生物与免疫学实验室完成以细胞为对象的对照观察性实验。材料：健康分娩产妇自愿捐献的脐血60～180mL。方法：应用Ficoll—Hypaque淋巴细胞分离液以密度梯度法获取脐血间充质干细胞，加入碱性成纤维细胞生长因子诱导分化。当细胞传至第3代时，随机取培养的6份脐血间充质干细胞（半量）为A组，6份脐血间充质干细胞（半量）为B组，将A、B组各剩余的半量脐血间充质干细胞混合为C组。3组细胞密度均为1．0×10^4L^-1。主要观察指标：以活细胞计数和MTT比色法比较3组细胞扩增数量，免疫组织化学检测脐血间充质干细胞神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白的表达。结果：活细胞计数和MTT比色检测显示，将两份不同脐血间充质干细胞混合后，细胞贴壁比单份脐血间充质干细胞快（P〈0．05），增殖也非常快（P〈0．05）。3组细胞经碱性成纤维细胞生长因子诱导后均表达神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白，并且巢蛋白阳性表达率无差别（P〉0．05）。结论：混合脐血间充质干细胞之间有互相促增殖作用，应用碱性成纤维细胞生长因子诱导的脐血间充质干细胞可表达神经胶质细胞标志物、神经元特异性烯醇化酶和巢蛋白。

骨髓间充质干细胞成骨性能的实验研究

目的研究骨髓间充质干细胞的生物学特性以及作为组织工程骨种子细胞的特点。方法分离培养兔骨髓间充质干细胞,与纳米晶羟基磷灰石胶原材料于体外联合培养,建立兔桡骨节段性缺损模型,分为空白组(n=8,不植入材料)、对照组(n=8,植入材料)、实验组(n=8,植入复合细胞的材料)。通过大体观察、组织学分析及X线摄片了解成骨情况。结果兔骨髓间充质干细胞在体外可以大量增殖,复合细胞的材料植入16周后,X线摄片可见桡骨缺损处连接良好。结论骨髓间充质干细胞具有很强的成骨作用,是一种较好的组织工程种子细胞。

神经营养因子3促进骨髓间充质干细胞向类神经元分化的信号通路

骨髓间充质干细胞在适当环境中，可被诱导分化为神经元，及神经胶质细胞，被认为是最好的种子细胞。神经营养因子3为一种多功能的神经营养因子，对神经系统的发育及维持其正常生理功能具有重要意义。文章旨在探讨神经营养因子3促进骨髓问充质干细胞向类神经细胞分化的作用及可能的信号传导通路。

Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化

目的探讨矮小同源盒基因亚型2(short stature homobox 2,Shox2)外源基因对犬骨髓间充质干细胞(canine mesenchymal stem cells,c MSCs)体外诱导分化的影响。方法分离培养犬骨髓间充质干细胞,用包装好的两种慢病毒载体p Lentis-m Shox2-RFP及p Lentis-RFP分别转染第3代c MSCs,收集转染阳性的c MSCs与乳鼠心肌细胞(rat ventricular myocytes,NRVMs)共培养。实验分4组:A组(RFP-c MSCs组)、B组(RFP-c MSCs+NRVMs共培养组)、C组(Shox2-RFP-c MSCs组)、D组(Shox2-RFPc MSCs+NRVMs共培养组)。共培养诱导分化5 d后,利用嘌呤霉素对c MSCs进行纯化,运用全细胞膜片钳检测诱导出内向电流的动力学特征,运用实时荧光定量PCR、Western blot检测HCN4、Tbx3、Cx45/43、Nkx2.5等标志性基因mRNA和蛋白的表达情况,并行免疫荧光检测。结果 Western blot及RTPCR检测结果显示,D组细胞Shox2、HCN4、Tbx3、Cx45窦房结细胞特异性基因的表达较A、B、C组明显增高(P<0.05),B组细胞Cx43及Nkx2.5较C、D组细胞明显增高(P<0.05);免疫荧光结果显示,在Shox2基因调控作用下,c MSCs有HCN4通道生成;膜片钳检测结果显示,C、D组细胞产生了具有明显时间-电压依赖性的内向电流,且对细胞外Cs+敏感,符合起搏离子流If的动力学特征;而A、B组未检测出此电流。结论经m Shox2基因修饰的c MSCs通过体外诱导,产生了起搏离子流If并高表达窦房结标志性基因,成功向具有一定自主搏动能力的窦房结样细胞分化。

低氧预处理在骨髓间充质干细胞缺氧再灌注损伤中的保护作用

目的:探讨低氧预处理在骨髓间充质干细胞(BMSCs)缺氧再灌注损伤中的保护作用。方法 :将体外正常培养的原代BMSCs随机分为4组,包括正常组(normal group)、低氧预处理组(hypoxia preconditioning,HP)、缺氧再灌注组(hypoxia-reoxygenation,HR)和低氧预处理-缺氧再灌注组(HP-HR)。分别对HP组和HP-HR组的BMSCs在原代、P1代和P2代连续3代进行低氧(1%O2)预处理30min;对HR组和HP-HR组的P3代BMSCs进行完全缺血缺氧3小时再复氧1天,然后用MTT法同时检测4组P3代BMSCs的细胞活力,并用Hoechst33342及Bax染色观察细胞的凋亡。结果:正常培养组与HP组P3代细胞的活力没有显著性差异,而HR组和HP-HR组的细胞活力均明显下降。但HP-HR组的细胞活力较HR组显著提高,差异有统计学意义(P<0.05),而且Hoechst33342及Bax染色显示细胞凋亡在HP-HR组较HR组减少。结论:低氧预处理能够减少细胞凋亡,保护骨髓间充质干细胞防止缺氧再灌注损伤。

骨髓间充质干细胞移植对急性肺水肿大鼠血清IL-6与SP-A的作用研究

目的:通过肺组织形态学及血清细胞因子(IL-6、SP-A)水平观察骨髓间充质干细胞对大鼠急性肺水肿的防治作用。方法:80只SD大鼠随机分为肺水肿组、干细胞治疗组、干细胞预防组、空白对照组、干细胞对照组(各16只)。腹腔注射氯化铵建立急性肺水肿组织模型,预防组及治疗组于模型建立前后移植MSCs1×105/只,移植后30min、2h、1d、7d光镜及电镜扫描观察肺组织病理,测定血IL-6、SP-A水平。结果:移植后2h时肺水肿组病变明显,治疗组肺水肿病变减轻。治疗组2h时IL-6明显低于肺水肿组(P<0.01),在30min SP-A水平明显高于肺水肿组(P<0.01),而预防组与肺水肿组无差异(P>0.05)。结论:MSCs移植治疗减轻急性肺水肿,预防性移植不减轻肺水肿。

Comprehensive Understanding of Immune Cells in The Pathogenesis of Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease,defined by several phases,ranging from benign fat accumulation to non-alcoholic steatohepatitis(NASH),which can lead to liver cancer and cirrhosis.Although NAFLD is a disease of disordered metabolism,it also involves several immune cell-mediated inflammatory processes,either promoting and/or suppressing hepatocyte inflammation through the secretion of pro-inflammatory and/or anti-inflammatory factors to influence the NAFLD process.However,the underlying disease mechanism and the role of immune cells in NAFLD are still under investigation,leaving many open-ended questions.In this review,we presented the recent concepts about the interplay of immune cells in the onset and pathogenesis of NAFLD.We also highlighted the specific non-immune cells exhibiting immunological properties of therapeutic significance in NAFLD.We hope that this review will help guide the development of future NAFLD therapeutics.

珍珠层生物性能及成骨诱导能力研究进展

珍珠层作为骨修复材料,具有诱导成骨作用、合适的降解性能以及良好的力学性能。文章综述了珍珠层的结构及成分、细胞相容性和生物降解性及对前成骨细胞和骨髓间充质干细胞的成骨诱导作用,同时展望了其作为骨缺损修复材料的应用前景。

A Review on the Biological Characteristics and Secretory Functions of Adipose-derived Mesenchymal Stem Cells

Adipose-derived mesenchymal stem cells （ADSCs） can be largely and easily obtained from a wide range of sources. Moreover, they have self-renewal ability, multi-differentiation potential, and an important role in immune regulation. They can secrete a variety of cytokines to regulate the in vivo micro-environment. Therefore, ADSCs are the ideal seed ceils for stem ceils application. This paper reviews the location, isolation, surface markers, proliferation, differentiation and other biological characteristics of ADSCs, as well as their secretory function and relative researches. ADSCs are expected to become excellent seed cells for cell therapy and tissue engineering through in-depth studies.

Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro

AIM: To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of celltypes for therapies of hepatic diseases. METHODS: MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured withFGF-4 and HGF, approximately 56.6% of cells became smallround and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5±1.4 μg/L (t = 2.31, P＜0.05) in MSCs cultured with FGF-4and HGF, and were higher (46.2±1.5 μg/L)ond 21 (t = 41.926, P＜0.01), then decreased to 24.8±2.2 μg/L on d 24 (t = 10.345, P＜0.01). Albumin increased significantly on d 21 (t= 3.325, P＜0.01) to 1.4±0.2 μg/mL,and to 2.1±0.7 μg/mL on d 24 (t= 3.646, P＜0.01). Urea(2.3±0.4 mmol/L) was first detected on d 21 (t = 6.739, P＜0.01), and continued to increase to 2.6±0.9 mmol/Lon d 24 (t= 4.753, P＜0.01). Glycogen storage was first seen on d 21.CONCLUSION: The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.

Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis

AIM: To compare the influence of different transplant sites in bone marrow mesenchymal stem cell (MSC)-based therapy for liver fibrosis. METHODS: MSCs isolated from Sprague Dawley (SD) rats were induced into hepatocyte-like cells. Liver fibrosis in SD rats was induced with carbon tetrachloride. Following hepatocyte induction in vitro, 4',6-diamidino- 2-phenylindole (DAPI)-labeled MSCs were transplanted by intravenous, intrahepatic, and intraperitoneal injection. Histopathological staining, immunohistochemistry, and biochemical analysis were used to compare the morphological and functional liver regeneration among different MSC injection modalities. The expression differences of interleukins, growth factor, extracellular matrix, matrix metalloproteinases, and tissue inhibitor of metalloproteinase were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) andenzyme linked immunosorbent assay (ELISA). RESULTS: Four days after exposure to hepatocyte differentiation medium, MSCs that did not express hepatocyte markers could express α-fetoprotein, albumin, and cytokeratin 18. The results of histopathological staining, immunohistochemistry, and biochemical analysis indicated that intravenous injection is more effective at rescuing liver failure than other injection modalities. DAPI-labeled cells were found around liver lobules in all three injection site groups, but the intravenous group had the highest number of cells. PCR and ELISA analysis indicated that interleukin-10 (IL-10) was highest in the intravenous group, whereas il1β, il6, tnfα and tgfβ, which can be regulated by IL10 and are promoters of liver fibrosis, were significantly lower than in the other groups. CONCLUSION: MSC administration is able to protect against liver fibrosis. Intravenous injection is the most favorable treatment modality through promotion of IL10 expression.

Bone marrow mesenchymal stem cell transplantation combined with perindopril treatment attenuates infarction remodelling in a rat model of acute myocardial infarction

Objective: This study was performed to evaluate whether implantation of mesenchymal stem cell (MSC) would reduce left ventricular remodelling from the molecular mechanisms compared with angiotensin-converting enzyme inhibitors (ACEIs) perindopril into ischemic myocardium after acute myocardial infarction. Methods: Forty rats were divided into four groups: control, MSC, ACEI, MSC+ACEI groups. Bone marrow stem cell derived rat was injected immediately into a zone made ischemic by coronary artery ligation in MSC group and MSC+ACEI group. Phosphate-buffered saline (PBS) was injected into control group. Perindopril was administered p.o. to ACEI group and MSC+ACEI group. Six weeks after implantation, the rats were killed and heart sample was collected. Fibrillar collagen was observed by meliorative Masson’s trichome stain. Western Blotting was employed to evaluate the protein expression of matrix metalloproteinase (MMP)-2, matrix metalloproteinase (MMP)-9 in infarction zone. The transcriptional level of MMP2, MMP9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in infarction area was detected by reverse transcriptase PCR (RT-PCR) analysis. Results: The fibrillar collagen area, the protein expression of MMP2, MMP9 and the transcriptional level of MMP2, MMP9 mRNA in infarction zone reduced in MSC group, ACEI group, and MSC+ACEI group. No significant difference was detected in the expression of TIMP1 mRNA among the 4 groups. Conclusion: Both MSC and ACEI could reduce infarction remodelling by altering collagen metabolism.

Culture and Identification of Human Amniotic Mesenchymal Stem Cells

Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.

Cognitive improvement following transvenous adipose-derived mesenchymal stem cell transplantation in a rat model of traumatic brain injury

The effects of adipose-derived mesenchymal stem cell (ADMSC) transplantation for the repair of traumatic brain injury remain poorly understood. The present study observed neurological functional changes in a rat model of traumatic brain injury following ADMSC transplantation via the tail vein. Cell transplants were observed in injured cerebral cortex, and expression of brain-derived nerve growth factor was significantly increased in the injured hippocampus following transplantation. Results demonstrated that transvenous ADMSC transplants migrated to the injured cerebral cortex and significantly improved cognitive function.

Comparison of human amniotic fluid-derived and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells： Characterization and myocardial differentiation capacity

Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

Mesenchymal stromal cells' role in tumor microenvironment:involvement of signaling pathways

Mesenchymal stromal cells(MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like "wounds that do not heal." In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed.

Rubidium-modified bioactive glass-ceramics with hydroxyapatite crystals for bone regeneration

In order to study the influence of rubidium(Rb)addition on the phase composition,microstructure,mechanical properties and cell response of bioactive glass-ceramics,CaO−SiO2−Na2O−B2O3−MgO−ZnO−P2O5 glass system was designed with and without addition of Rb.The results show that hydroxyapatite(HA)and Mg−whitelockite(Ca18Mg2H2(PO4)14)crystalline phases are formed in the glass matrix without Rb.After the addition of Rb,only HA phase is detected.The grain size of the crystals in the glass-ceramics is larger with the addition of Rb than that of samples without Rb.Rb addition can improve the bending strength of glass-ceramics.The cultivation of human bone marrow mesenchymal stem cells(hBMSCs)on Rb-containing glass-ceramics demonstrates enhanced cell adhesion,proliferation and ALP activity.In conclusion,Rb-modified glass-ceramics exhibit good mechanical property,excellent bioactivity and biocompatibility,which have potential for bone regeneration application.

Utility of co-transplanting mesenchymal stem cells in islet transplantation

Islet transplantation is characterized by the transplantation of isolated islets from donor pancreata into a diabetic recipient. Although it is a viable choice in the treatment of insulin dependent diabetes mellitus, most patients (approximately 90%) require insulin five years after transplantation. Recently, the co-transplantation of mesenchymal stem cells (MSCs) and islets in animal studies has revealed the effectiveness of MSCs co-transplantation for improving islet function. Themechanisms underlying the beneficial impact of MSCs include immunomodulation and the promotion of angiogenesis. In this review, we discuss MSCs and how they support improved graft survival and function.

Mesenchymal stromal cell-based therapy: Regulatory and translational aspects in gastroenterology

The past decade has witnessed an outstanding scientific production focused towards the possible clinical applications of mesenchymal stromal cells(MSCs) in autoimmune and chronic inflammatory diseases. This raised the need of novel standards to adequately address quality, efficacy and safety issues of this advanced therapy. The development of a streamlined regulation is currently hampered by the complexity of analyzing dynamic biological entities rather than chemicals. Although numerous pieces of evidence show efficacy in reducing intestinal inflammation, some inconsistencies between the mechanisms of action of rodent vs human MSCs suggest caution before assigning translational value to preclinical studies. Preliminary evidence from clinical trials showed efficacy of MSCs in the treatment of fistulizing Crohn's disease(CD), and preparations of heterologous MSCs for CD treatment are currently tested in ongoing clinical trials. However, safety issues, especially in longterm treatment, still require solid clinical data. In this regard, standardized guidelines for appropriate dosing and methods of infusion could enhance the likelihood to predict more accurately the number of responders and the duration of remission periods. In addition, elucidating MSC mechanisms of action could lead to novel and more reliable formulations such as those derived from the MSCs themselves(e.g., supernatants).

Effect of CXCR3/HO-1 genes modified bone marrow mesenchymal stem cells on small bowel transplant rejection

AIM To investigate whether bone marrow mesenchymal stem cells (BMMSCs) modified with the HO-1 and CXCR3 genes can augment the inhibitory effect of BMMSCs on small bowel transplant rejection. METHODS Lewis rat BMMSCs were cultured in vitro. Third-passage BMMSCs were transduced with the CXCR3 / HO-1 genes or the HO-1 gene alone. The rats were divided into six groups and rats in the experimental group were pretreated with BMMSCs 7 d prior to small bowel transplant. Six time points (instant, 1 d, 3 d, 7 d, 10 d, and 14 d) (n = 6) were chosen for each group. Hematoxylin-eosin staining was used to observe pathologic rejection, while immunohistochemistry and Western blot were used to detect protein expression. Flow cytometry was used to detect T lymphocytes and enzyme linked immunosorbent assay was used to detect cytokines. RESULTS The median survival time of BMMSCs from the CXCR3/HO-1 modified group (53 d) was significantly longer than that of the HO-1 modified BMMSCs group (39 d), the BMMSCs group (26 d), and the NS group (control group) (16 d) (P < 0.05). Compared with BMMSCs from the HO-1 modified BMMSCs, BMMSCs, and NS groups, rejection of the small bowel in the CXCR3 / HO-1 modified group was significantly reduced, while the weight of transplant recipients was also significantly decreased (P < 0.05). Furthermore, IL-2, IL-6, IL-17, IFN-gamma, and TNF-alpha levels were significantly decreased and the levels of IL-10 and TGF-beta were significantly increased (P < 0.05). CONCLUSION BMMSCs modified with the CXCR3 and HO-1 genes can abrogate the rejection of transplanted small bowel more effectively and significantly increase the survival time of rats that receive a small bowel transplant.