人肺成纤维母细胞表达骨髓间充质干细胞特性的研究

目的研究原代培养的人肺成纤维母细胞体外分化特性。方法于2006年3月至2006年10月在清华大学第一附属医院中心实验室,将原代培养的人肺成纤维母细胞分别在成骨细胞培养基(含地塞米松,维生素C和β-磷酸甘油)和成脂肪细胞培养基(含马血清,地塞米松和胰岛素)作用下进行分化诱导。组织化学方法行碱性磷酸酶和钙化斑块染色,Westernblotting法测定骨桥蛋白表达,油红染色鉴定脂肪细胞形成。结果在成骨细胞培养基诱导下,细胞内碱性磷酸酶表达增加,于第14天可见大量钙盐沉积和骨桥蛋白表达增加。在成脂肪细胞培养基作用第14天,部分细胞内出现脂肪小滴聚集。结论人肺成纤维母细胞具有多分化潜能,能够在一定条件下向成骨细胞和脂肪细胞分化,表现出骨髓间充质干细胞的特性。     

体外诱导间充质干细胞向心肌细胞方向分化的研究

目的证实来源于胎儿肝脏的间充质干细胞(FMSCs)具有向心肌细胞方向分化的潜能。方法取6~9代细胞,用不同浓度的诱导剂组合诱导细胞,分别置于不同的条件下进行培养,包括不同温度、不同氧浓度及不同培养液。结果在使用心肌分化培养液及常规培养条件下(37℃、20%O2),5-氮胞苷(50μmol/L)、维甲酸(10-3μmol/L)、二甲基亚砜(0.8%)的联合应用,诱导了FMSCs发生向心肌方向的分化。分化细胞呈小圆形细胞,具有相互聚集并形成球样细胞团结构的趋势,同时表达结蛋白及心肌肌钙蛋白Ⅰ。结论FMSCs具有向心肌细胞分化的潜能,FMSCs发生向心肌方向的分化所需条件与来源于其他动物种属的间充质干细胞的分化条件不同。     

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

AIM To investigate the effect of adipose-derived mesenchymal stem cells(ADMSCs)and their conditioned media(CM) on hepatocellular carcinoma(HCC) cell tumorigenesis.METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit8(commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.     

In vitro anti-myeloma activity of TRAIL-expressing adipose-derived mesenchymal stem cells

Recently, genetically modified mesenchymal stem cells (MSCs) have been exploited to deliver anti-cancer bio-drugs directly within the tumour mass. Here, we explored whether adipose-derived MSCs (AD-MSCs), engineered to express the pro-apoptotic ligand TRAIL (also known as TNFSF10), kill multiple myeloma (MM) cells and migrate towards MM cells in vitro. Different MM cell lines were assessed for their sensitivity to recombinant human (rh) TRAIL alone and in combination with the proteasome inhibitor bortezomib, which was shown to enhance the effect of rhTRAIL. TRAIL(+) -AD-MSCs were co-cultured with bortezomib-pretreated MM cells and their killing activity was evaluated in presence or absence of caspase inhibition. AD-MSC migration towards media conditioned by both myeloma cells and myeloma bone fragments was also investigated. Despite moderate MM cell sensitivity to rhTRAIL, TRAIL(+) -AD-MSCs in combination with bortezomib significantly induced myeloma cell death. This effect was associated with caspase-8 activation and abrogated by capsase inhibition. On the other hand, co-culture experiments were performed to evaluate whether unmodified AD-MSCs affect myeloma cell growth in vitro. AD-MSCs appeared ineffective on myeloma cell growth and showed migratory capacity towards MM cells in vitro. These data emphasize the anti-myeloma activity of TRAIL-engineered AD-MSCs and provide support for a future model of a cell-based approach against MM.     

Multipotent mesenchymal stromal cells in articular diseases

Although cartilage defects are common features of osteoarthritis and rheumatoid arthritis, current treatments can rarely restore the full function of native cartilage. Recent studies have provided new perspectives for cartilage engineering using multipotent mesenchymal stromal cells (MSC). Moreover, MSC have been used as immunosuppressant agents in autoimmune diseases and have tested successfully in animal models of arthritis. However, the sequential events occurring during chondrogenesis must be fully understood before we can reproduce the complex molecular events that lead to MSC differentiation and long-term maintenance of cartilage characteristics in the context of chronic joint inflammation. This chapter focuses on the potential of MSC to repair cartilage, with an emphasis on the factors that are known to be required in inducing chondrogenesis and on their immunosuppressive potential.     

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.The biophysical state of a cell is potentially a rich source of information indicative of cell identity and physiology. When the underlying biochemical activity that occurs as cells replicate, senesce, differentiate, become malignant, or undergo apoptosis is manifest as measurable changes in biophysical characteristics, then parameters such as cell size or mechanical stiffness (1–4) may serve as predictive markers of cellular fate. For example, the metastatic competence of cancer cell lines has been correlated with average mechanical creep compliance (5, 6), and the stiffness of malaria-infected (7) and sickle red blood cells (8) has been related to disease stage and severity.The successful application of mechanobiology to the analysis of human disease has prompted development of biophysical cytometry methods to study the functional multipotency of stem cells (9, 10). However, such efforts to predict multipotency or “stemness” are challenging due to potential coupling or plurality of biophysical changes in response to distinct cues. For example, changes in cell size are not only related to cell cycle events (11) and cell proliferation rates (12), but also to the reported differentiation capacity of progenitor cells derived from corneal epithelium (13), adipose tissue (14), or adult bone marrow (15, 16). It is thus evident that a more comprehensive physical profile of stem cells is required to consider whether biophysical markers are robust indicators of inducible function in vitro and in vivo. Herein, we demonstrate that multivariate biophysical analysis of cells can readily identify subpopulations of multipotent, as well as osteochondral progenitor, cells within in vitro culture-expanded mesenchymal stromal cells (MSCs).Although often referred to and treated as a uniform stem cell population, culture-expanded MSCs derived from adult bone marrow (aMSCs) are actually a heterogeneous cell mixture (16, 17). These cell populations exhibit reduced multilineage potential during in vitro culture expansion (18, 19). This decreased multipotency has been attributed to environmental cues (20–25) during in vitro culture and results in MSC subpopulations that render it difficult to study and engineer “stem cell behavior.” Such desirable behavior includes self-renewal and multilineage differentiation in vitro or production of uniform, robust therapeutic responses in vivo. However, the long-term and large-scale expansion of aMSCs is necessary to obtain a clinically relevant number of cells for many envisioned tissue regeneration therapies. Conventional high-throughput sorting of multipotent MSCs from this heterogeneous, putative MSC population via flow cytometry has proven insufficient, due to the lack of biomolecular surface markers that select specifically for multipotency (15, 26, 27); such molecular labeling approaches also restrict viability and use of such cells for therapeutic applications (28). Thus, it is common to verify the multipotency of MSC subpopulations or clones via in vitro experiments that directly quantify MSC capacity to form colonies and differentiate along multiple tissue lineages. These Schrodinger’s cat-like assessments of viable stem cell function are both retrospective and confer obvious limitations for robust studies of stem cell biology and for clinical applications of culture-expanded MSCs. Such considerations illustrate the need for alternative, multivariate, and functional cytometry platforms and methods that can identify marrow stromal cell subpopulations of predictable potency or progenitor status, without labeling or differentiating those cells.Here, we quantify several biophysical characteristics of MSCs subpopulations derived from human adult and fetal bone marrow. These potential multivariate biomarkers of MSC potency are as follows: (i) suspended cell diameter; (ii) adherent cell spread area; (iii) cell stiffness; (iv) nuclear to cytoplasmic ratio; and (v) relative nuclear membrane fluctuations. We correlated each property with molecular surface markers, in vitro multilineage differentiation potential, and in vivo regenerative potential (see SI Appendix for discussion of previous studies that noted one or more of these properties to be potential indicators of differentiation capacity or commitment). Of particular interest is whether any of these physical signatures, or combinations thereof, could prospectively identify and sort multipotent MSC subpopulations from precommitted progenitor cells. We find that cell size is a necessary but insufficient predictor of MSC multipotency: not all subpopulations of small diameter are multipotent, as might be inferred from previous in vitro studies that compared smaller and larger MSCs (16). Among the several other biophysical markers considered, we find that only cell stiffness and nuclear fluctuations correlated strongly with in vitro differentiation potential and in vivo bone and muscle regeneration capacity. Specifically, adult and fetal MSC subpopulations of sufficiently low mean diameter (D < 20 μm), low mechanical stiffness (E < 375 Pa), and high nuclear fluctuations (NF > 1.2%) consistently exhibited multipotency in vitro and in vivo. All other MSC subpopulations exhibited commitment toward the osteogenic lineage. Together these findings suggest a minimal set of biophysical markers exist for the identification of MSC and progenitor subpopulations toward clinical applications.     

Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure

Background and Aim:  Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo .
Methods:  In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum.
Results:  We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation.
Conclusions:  Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo . Thus, ASC may be a superior choice for the establishment of a therapy for injured liver.     

Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases

Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft-vs.-host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture-expanded MSCs and HSCs from the same human leukocyte antigen (HLA)-identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 ± 0.40) × 10⁶/kg (donor’s weight) MSCs were successfully expanded from 23.6 ± 5.96 ml of bone marrow samples. They had normal karyotypes with bi-lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II–IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29–57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.     

人骨髓间充质干细胞体外定向分化过程中心肌细胞特征表达及膜电位变化

目的:探讨体外诱导人骨髓间充质干细胞(hMSCs)分化过程中心肌细胞特征表达及膜电位变化的情况,为hMSCs临床移植治疗提供实验参考数据。方法:采用体外纯化、扩增后的第4代hMSCs在体外经5μmol/L5-杂氮胞苷诱导24h后继续培养8周,相差显微镜下观察细胞形态变化,免疫组化方法鉴定心肌特异性蛋白心房钠尿肽(ANP)和连接蛋白(CX43)的表达,应用RT-PCR技术分析肌球蛋白重链(β-MHC)和肌钙蛋白T(cTnT)等相关基因在分化过程中的动态表达,全细胞膜片钳技术检测不同时期细胞膜电位的变化。结果:hM-SCs诱导前为纺锤形,诱导后第2天部分细胞即开始发生形变,呈球形或短棒状,1周后细胞质中颗粒增多,20%~30%细胞呈毛刷样变化;ANP和CX43在诱导前无表达,诱导后第2周开始表达,且表达随时间逐渐增强,β-MHC和cTnT mRNA分别在诱导后第1和4周时表达开始增强,在第6周时均达到高峰,第8周时表达开始衰减,hMSCs经诱导后随心肌样细胞特征的表达膜静息电位、去极化幅值和去极化速率逐渐增高。结论:hNSCs在体外经纯化、扩增和诱导后可表现心肌样细胞的生物学特性,可为hMSCs临床移植治疗提供可靠细胞来源,但移植后有形成心律失常的潜在危险。     

大鼠脂肪间充质干细胞在部分肝切除模型中向肝细胞分化的研究

目的探讨脂肪间充质干细胞（ADMSCs）在部分肝切除模型中向肝细胞的分化。方法从大鼠脂肪组织中分离出干细胞，并进行体外扩增、传代，取第2代ADMSCs用PKH26标记，制作部分肝切除模型，将标记细胞经门静脉自体植入体内。2周后切下肝脏制成冰冻切片，荧光显微镜下观察标记细胞在肝脏的定位，进行免疫荧光染色检测标记细胞白蛋白的表达。结果从脂肪组织中分离出的ADMSCs能在体外大量扩增，PKH26标记后细胞在荧光显微镜下发红色荧光，细胞标记率约95％；荧光显微镜下可见肝脏冰冻切片中散在分布红色标记细胞，免疫荧光染色显示大多数标记细胞白蛋白染色阳性。结论 大鼠脂肪间充质干细胞在肝再生环境中能向肝细胞分化，有可能在肝部分切除后参与肝再生。     

肝孤核受体激动剂对间充质干细胞的抗缺氧复氧损伤作用及机制

目的探讨肝孤核受体(LXR)激动剂对脂肪间充质干细胞(AD-MSCs)缺氧损伤的保护作用及可能机制。方法酶消化法分离稳定表达萤火虫荧光素酶(Fluc)报告基因的小鼠AD-MSCs,流式细胞术检测CD90、CD44、CD34、CD45细胞表面标记物。3代AD-MSCs分为7组:对照组;缺氧6 h/复氧2 h组(缺氧复氧组);缺氧/复氧+DMSO组(DMSO组);缺氧复氧+不同浓度LXR激动剂T0901317干预组(1μmol/L组、5μmol/L组、10μmol/L组、15μmol/L组)。运用Fluc报告基因生物发光成像技术对AD-MSCs细胞增殖进行定量评价,免疫印迹法检测细胞NF-κB表达水平,ELISA法检测AD-MSCs的白细胞介素6(IL-6)、TNF-α分泌水平。结果流式细胞术结果显示.AD-MSCs呈CD44(+)、CD90(+)、CD34(-)、CD45(-)。光学成像结果显示,AD-MSCs细胞数与Fluc平均生物发光信号强度呈正相关(r~2=0.97)。与对照组比较,缺氧复氧组AD-MSCs分泌TNF-α、IL-6、NF-κB明显升高;与缺氧复氧组比较,10μmol/L组AD-MSCs分泌TNF-α、IL-6、NF-κB明显减少(P<0.05,P<0.01)。结论 LXR激动剂可以促进缺氧复氧损伤后AD-MSCs的存活,并能通过NF-κB信号途径抑制炎性因子IL-6、TNF-α的释放,可为干细胞移植治疗心肌缺血再灌注损伤提供新的细胞保护方法。     

Direct implantation versus platelet-rich fibrin-embedded adipose-derived mesenchymal stem cells in treating rat acute myocardial infarction

Background

This study tested whether adipose-derived mesenchymal stem cells (ADMSC) embedded in platelet-rich fibrin (PRF) scaffold is superior to direct ADMSC implantation in improving left ventricular (LV) performance and reducing LV remodeling in a rat acute myocardial infarction (AMI) model of left anterior descending coronary artery (LAD) ligation.

Methods

Twenty-eight male adult Sprague Dawley rats equally divided into group 1 [sham control], group 2 (AMI only), group 3 (AMI + direct ADMSC implantation), and group 4 (AMI + PRF-embedded autologous ADMSC) were sacrificed on day 42 after AMI.

Results

LV systolic and diastolic dimensions and volumes, and infarct/fibrotic areas were highest in group 2, lowest in group 1 and significantly higher in group 3 than in group 4, whereas LV performance and LV fractional shortening exhibited a reversed pattern (p < 0.005). Protein expressions of inflammation (oxidative stress, IL-1β, MMP-9), apoptosis (mitochondrial Bax, cleaved PARP), fibrosis (Smad3, TGF-β), and pressure-overload biomarkers (BNP, MHC-β) displayed a pattern similar to that of LV dimensions, whereas anti-inflammatory (IL-10), anti-apoptotic (Bcl-2), and anti-fibrotic (Smad1/5, BMP-2) indices showed a pattern similar to that of LV performance among the four groups (all p < 0.05). Angiogenesis biomarkers at protein (CXCR4, SDF-1α, VEGF), cellular (CD31 +, CXCR4 +, SDF-1α +), and immunohistochemical (small vessels) levels, and cardiac stem cell markers (C-kit +, Sca-1 +) in infarct myocardium were highest in group 4, lowest in group 1, and significantly higher in group 3 than in group 2 (all p < 0.005).

Conclusion

PRF-embedded ADMSC is superior to direct ADMSC implantation in preserving LV function and attenuating LV remodeling.     

5-氮胞苷浓度对体外骨髓间充质干细胞诱导分化为心肌样细胞的影响

目的探讨不同浓度5-氮胞苷(5-aza)体外诱导小型猪骨髓间充质干细胞(MSCs)向心肌样细胞分化的可能性及其对细胞扩增速度的影响.方法10ml骨髓,1.077g/ml Ficoll密度梯度离心法分离骨髓单个核细胞(MNCs),第2代传代细胞分别加入3,5,10μmol/L浓度5-aza化学诱导24h,与对照组连续培养4周,细胞爬片免疫荧光法鉴定结蛋白desmin及心肌特异性肌钙蛋白Ⅰ(cTnI)的表达,电镜观察诱导细胞的超微结构变化.结果5-aza化学诱导后MSCs形态变长、增大,排列更加紧密、整齐,5和10 μmol/L组可见较多的多核细胞,部分管状结构,诱导率分别为36%和31%,无显著性差异;3μmol/L组上述变化的细胞极少,3种浓度及对照组4周后细胞扩增数量分别为(3.81±0.40)×106、(3.76±0.62)×106、(3.67±0.80)×106及(2.90±0.21)×106,10μmol/L组增殖速度明显减慢(P＜0.05).各组诱导后的细胞均未见自发搏动.免疫组化提示desmin及cTnI表达阳性,5和10μmol/L组电镜下出现肌丝样结构、心房颗粒及细胞间的缝隙连接.结论小型猪MSCs经5、10 μmol/L 5-aza化学诱导可分化为心肌样细胞,其中5 μmol/L5-aza不仅可以诱导心肌样细胞而且对细胞增殖速度影响小.     

Therapeutic potential of mesenchymal stem cells overexpressing human forkhead box A2 gene in the regeneration of damaged liver tissues

Background and Aim: Although a liver transplantation is considered to be the only effective long‐term treatment in many cases of liver diseases, it is limited by a lack of donor organs and immune rejection. As an autologous stem cell approach, this study was conducted to assess whether forkhead box A2 (Foxa2) gene overexpression in bone marrow‐derived mesenchymal stem cells (MSC) could protect the liver from hepatic diseases by stimulating tissue regeneration after cell transplantation. Methods: Rat MSC (rMSC) were isolated, characterized, and induced to hepatocytes that expressed liver‐specific markers. Four different treatments (control [phosphate‐buffered saline], rMSC alone, rMSC/pIRES–enhanced green fluorescent protein (EGFP) vector, and rMSC/pIRES–EGFP/human Foxa2) were injected into the spleen of carbon tetrachloride‐injured rats. Biochemical and histological analyses on days 30, 60, and 90 post‐transplantation were performed to evaluate the therapeutic capacities of MSC overexpressing hFoxa2. Results: rMSC transfected with hFoxa2 were induced into hepatogenic linage and expressed several liver‐specific genes, such as, Foxa2, α‐fetoprotein, cytokeratin‐18, hepatocyte nuclear factor‐1α, and hepatocyte growth factor. A group of animals treated with MSC/hFoxa2 showed significant recovery of liver‐specific enzyme expressions to normal levels at the end of the study (90 days). Furthermore, when compared to the fibrotic areas of the samples treated with MSC alone or MSC/vector, the fibrotic area of the samples treated with rMSC/hFoxa2 for 90 days significantly decreased, until they were completely gone. Conclusions: Human Foxa2 efficiently promoted the incorporation of MSC into liver grafts, suggesting that hFoxa2 genes could be used for the structural or functional recovery of damaged liver cells.     

间充质干细胞分化为肝样细胞的研究进展

肝炎后肝硬化等慢性进展性肝病、急性肝功能衰竭、肝脏代谢性疾病及肝脏恶性肿瘤等终末期肝病的发病率日益上升,美国贝塞斯达回忆宣布肝移植是目前治疗终末期肝病最有效的方法,但由于肝源有限、移植后的免疫排斥反应以及高额费用等限制了这种治疗方法的开展。近年来,随着对干细胞研究的深入,发现间充质干细胞(MSCs)具有向肝样细胞分化的潜能,且安全性、可行性及疗效均显示了较好的应用前景,为终末期肝病的治疗提供了新的途径。本文就MSCs不同组织来源分化特点、体内外分化为肝样细胞研究、分化后的肝样细胞的生物学特性及MSCs分化为肝样细胞的机制、MSCs应用的一些问题作一概述。     

丁酸钠和5-aza诱导间充质干细胞向心肌细胞分化的研究

目的探讨组蛋白去乙酰化酶抑制剂丁酸钠（sodiumbutyrate）和DNA甲基化抑制剂5-aza对骨髓间充质干细胞（BMSCs）向心肌细胞分化的影响。方法常规培养大鼠BMSCs,细胞分为4组：依次加入丁酸钠0mM,0.5mM,1.0raM,2.0mM。MTT和流式细胞仪检测不同浓度丁酸钠对细胞增殖和凋亡的影响。4周后利用western blot和免疫荧光检测4组细胞的心肌细胞特异性蛋白的表达。从上述4组中选出诱导能力最强的丁酸钠组与5-aza组成4个实验组：阴性对照组,5-aza组,丁酸钠组,5-aza.L丁酸钠组。作用4周后检测心肌细胞特异性蛋白的表达。结果丁酸钠抑制细胞增殖,具有浓度依赖性,而对细胞凋亡没有明显影响。检测发现丁酸钠和5-a．za均能有效诱导BMSCs分化为心肌细胞,丁酸钠诱导分化最适浓度为1.0mM。同时1．0mM丁酸钠诱导分化效率高于5-aza。除此,5-aza和丁酸钠共同作用于BMSCs,其诱导分化能力明显高于其它组。结论丁酸钠能够有效诱导BMSCs向心肌细胞分化,丁酸钠浓度为1．0mM时诱导能力最强,同时诱导分化率高于5-aza。5-aza和丁酸钠一起作用诱导BMSCs向心肌细胞分化的能力明显高于其它组,间接证明DNA去甲基化和组蛋白乙酰化具有协同作用。丁酸钠在有效作用浓度范围内,虽然抑制BMSCs增殖,但是不影响细胞凋亡,表明丁酸钠具有低毒的特性,为以后丁酸钠应用于临床打下实验基础。     

体外诱导人羊膜间充质干细胞分化为胰岛素分泌细胞

目的 探讨体外诱导人羊膜间充质干细胞(hAD-MSCs)向胰岛素分泌细胞分化潜能.方法 采用胰蛋白酶-胶原酶消化法分离提取hAD-MSCs,流式细胞术分析和免疫细胞化学染色行表型鉴定.取第3代按2.5× 106个/ml或5×105个/ml细胞密度接种6孔培养板或预置盖玻片的24孔培养板,在含10mmol/L尼克酰胺和N2补充物的无血清HG-DMEM培养基培养,未诱导组为含10％胎牛血清的LG-DMEM基础培养基.分别于体外诱导第7、14、21天采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子1(PDX-1)mRNA的表达.结果 (1)hAD-MSCs高表达间充质干细胞表面标志CD29、CD44、CD73、CD166及波形蛋白;(2) hAD-MSCs诱导第7、14、21天胰岛素阳性细胞百分率分别为74.67％±1.53％、75.00％±1.00％、74.33％±1.53％,培养物上清液中胰岛素含量分别为(331.62±1.76)、(330.50±1.22)和(331.65±0.48) μIU/ml,各时点间比较无显著性差异(均P＞0.05),而未诱导组仍未见胰岛素阳性细胞;培养上清也未检测到胰岛素;(3) hAD-MSCs诱导前后均有PDX-1 mRNA和蛋白表达,胰岛素基因mRNA表达仅见于诱导组;(4) hAD-MSCs诱导组和未诱导组各时点均有β2微球蛋白表达,其阳性细胞百分率组间差异无显著性(均P＞0.05).结论 hAD-MSCs具有向胰岛素分泌细胞分化的能力,可能成为1型糖尿病细胞移植治疗的新的细胞供源.     

体外诱导骨髓间充质干细胞向神经元样细胞分化的研究

目的观察人骨髓间充质干细胞（BM-MSCs）体外扩增和定向诱导分化为神经元样细胞的变化,为其临床应用奠定基础。方法采用密度梯度离心法分离BM-MSCs细胞,用MesencultTM培养基和贴壁培养法培养、纯化和扩增细胞,流式细胞术检测细胞表面CD29和CD90分子表达率。采用20 ng/ml重组碱性成纤维细胞生长因子（bFGF）、20 ng/ml新型人表皮生长因子（hEGF）和20 g/L二甲基亚砜（DMSO）、5 mmol/Lβ-巯基乙醇（BME）分别诱导BM-MSCs;采用免疫组化法检测细胞的神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）、波形蛋白（VIM）表达情况。结果BM-MSCs细胞增殖迅速,3周可传代培养;BM-MSCs传十代扩增1－2×10^3倍。分离和扩增BM-MSCs CD29、CD90强表达率分别为95.02%、93.81%。药物诱导后的BM-MSCs发生轴突等神经元样细胞变化,阳性表达NSE、GFAP、VIM分子。结论人BM-MSCs能体外培养和扩增,并能定向诱导分化为神经元样细胞。     

过氧化氢预处理增强脂肪源间充质干细胞对小鼠急性心肌梗死疗效的研究

目的探讨H2O2体外预处理增强脂肪源间充质干细胞(ADMSC)对治疗急性心肌梗死(AMI)疗效的影响。方法制备小鼠皮下ADMSC,ELISA法分别检测上清液中干细胞生长因子(HGF)、血管内皮生长因子(VEGF)、白细胞介素6(IL-6)及TNF-α浓度。选取32只8～10周龄小鼠随机分为假手术组、AMI组、ADMSC组、H2O2预处理干细胞组(ADMSC-H2O2组),每组8只。移植术后第0、7、14、28天超声测量各组小鼠左心室舒张末内径(LVEDD)、左心室收缩末内径(LVESD)等。天狼星红染色检测心肌胶原含量,计算平均胶原容积分数(CVF)。结果与AMI组比较,ADMSC组和ADMSC-H2O2组LVESD和LVEDD明显下降,LVFS明显升高(P<0.01),全心重量/体重、左心室重量/体重、CVF明显降低(P<0.01);与ADMSC组比较,ADMSC-H2O2组小鼠LVESD和LVEDD明显下降,LVFS明显升高(P<0.01),全心重量/体重、左心室重量/体重、CVF明显降低(P<0.01)。结论体外H2O2预处理ADMSC增强了HGF、IL-6的分泌,减轻了AMI后心肌纤维化,心功能改善更为明显。     

骨髓间充质干细胞向肝细胞的诱导分化

目的:探索肝细胞生长因子(HGF)、制瘤素M(OSM)在体外诱导骨髓间充质干细胞(MSCs)向肝细胞分化的能力及效果.方法:分离、培养大鼠MSCs,取第3代按以下分组诱导其向肝细胞分化:A组:低糖杜氏改良培养基(DMEM-LG) 100 mL/L胎牛血清(fetal calf,serum,FCS);B组:肝细胞生长培养基(HGM);C组:HGM 20μg/L HGF;D组:HGM 20μg/L OSM;E组:HGM 20μg/L HGF 20μg/L OSM,于不同时间点用免疫细胞化学检测甲胎蛋白(AFP)、细胞角蛋白18(CK18)表达,高碘酸-希夫氏(PAS)染色检测糖原表达,谷氨酰胺脱氢酶法检测上清液尿素含量.结果:C,E组于诱导第7天出现AFP阳性表达,以后其阳性表达率随诱导时间延长逐渐降低,诱导第7天E组阳性表达率高于C组(x~2=6.322,P<0.05).C组、E组分别于诱导第7、14天出现CK18阳性表达,于诱导第7天开始出现糖原阳性表达,随诱导时间延长表达率逐渐增高,同一时间点E组CK18(14 d:x~2=4.811,P<0.05;21 d:x~2=6.902,P<0.01;28 d:x~2=5.771,P<0.05)及糖原(14 d:x~2=6.902,P<0.01;21 d:x~2=6.818,P<0.01;28 d:x~2=6.818,P<0.01)阳性表达率高于C组.C,E组培养上清液中尿素浓度随诱导时间延长逐渐增高,E组增长幅度比C组高.A,B,D组各时间点均未见AFP、CK18、糖原表达及尿素浓度变化.结论:MSCs具有向肝细胞分化的能力,HGF能够诱导MSCs分化肝样细胞,OSM与HGF联合使用,能促进MSCs的分化,明显提高分化率.