Application of multipotent mesenchymal stromal cells from human adipose tissue for compensation of neurological deficiency induced by 3-nitropropionic acid in rats

We evaluated possible therapeutic effect of multipotent mesenchymal stromal cells from human adipose tissue differentiated to neuronal phenotype with retinoic acid on Wistar rats subjected to toxic effect of 3-nitropropionic acid. Transplantation of mesenchymal stromal cells from human adipose tissue considerably decreased neurological symptoms, normalized exploratory activity (open field test) and long-term memory (Morris test), which correlated with normalization of pathomorphological manifestations in the brain. Destructive changes in the caudate nucleus caused by treatment with 3-nitropropionic acid (reduced size of neurons, changes in their shape, and cell edema) tended to decrease under the effect of multipotent mesenchymal stromal cells: the area of neurons increased 2-fold, the cells acquired typical round shape, cell edema decreased. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 2, pp. 83–89, April, 2008     

Chromosome variability of human multipotent mesenchymal stromal cells

We elaborated a method of preparing cytogenetic preparations of cultured multipotent mesenchymal stromal cells from the adipose tissue. It was found that karyotypic changes (monosomy, translocations) appear in some samples during culturing. Clones with changed karyotype were detected in 11–14-passage cultures from 2 of 7 individuals. The percent of aberrant cells in cultures from different individuals varied from 1.5 to 5.95 per 100 cells, which attested to karyotype instability. These data substantiate the need for cytogenetic control of cells before their transplantation into donor organism and further investigation of chromosome variability in stem cells. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 1, pp. 11–15, January, 2007     

Study of growth parameters and potentialities of differentiation of multipotent mesenchymal stromal cells from rat bone marrow in vitro

The dynamics of growth and proliferative activity of the population of multipotent mesenchymal stromal cells from rat bone marrow was studied during 7 passages. The efficiency of colony formation, the morphology of multipotent mesenchymal stromal cells, and the possibility of spontaneous and induced differentiation were studied. The rat bone marrow fibroblast-like multipotent mesenchymal stromal cells are capable of clonal growth; their proliferative activity and the yield remained high until passage 4, but then decreased. Induction of osteo-or adipogenic differentiation of bone marrow multipotent mesenchymal stromal cells increased the percentage of morphologically modified cells carrying specific markers. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 2, pp. 102–107, April, 2006     

Oestrogen and progesterone do not regulate the expression of retinoic acid receptors and retinoid 'X' receptors in human endometrial stromal cells in vitro

Elucidation of the gene structure for retinoic acid receptor-(RAR-) has suggested a potential role for oestrogen in regulatingthe expression of RAR-. We have previously shown that all threeRAR types are expressed in human endometrial stromal cells invitro and that RAR- expression is induced in response to retinoicacid. The aim of this study was to ask whether oestradiol andprogesterone could play a part in regulating the expressionof RARs in human endometrial stromal cells and to establishthe patterns of expression of a related group of nuclear retinoidreceptors, retinoid ‘X’ receptors (RXRs) and theirpotential for regulation by steroid hormones. The RAR expressionpatterns of endometrial stromal cells, grown in steroid-freemedium, did not change in response to the presence of steroidhormones. Furthermore, the retinoic acid-mediated inductionof RAR- was not affected by oestradiol or progesterone, andwas dependent on the continued presence of retinoic acid. Ofthe three RXR types, only RXR- was detectably expressed in stromalcells in vitro and the expression of RXR- did not change inresponse to steroid hormones or retinoic acid. These data indicatethat oestradiol and progesterone are not important in the regulationof RAR and RXR expression in human endometrial stromal cells.     

Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration

Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation.     

大鼠ADSCs的生物学特性及向成骨细胞分化的实验研究

目的探讨脂肪源性干细胞(ADSCs)的生长增殖活性以及成骨分化能力。方法取SD大鼠,无菌条件下取腹股沟部脂肪组织,制成单细胞悬液进行常规培养。倒置显微镜观察细胞生长情况,取第3代ADSCs,采用流式细胞技术检测细胞的增殖周期情况,茜素红染色观察诱导成骨情况。结果在倒置显微镜下所观察到的传代培养的ADSCs呈现长梭型及多角形的贴壁分布状态,形态大小比较均一,突起较少,并且增殖迅速,一般在3~4d即可生长汇合成单层、平行或者漩涡状排列。流式细胞仪检测细胞阳性率CD29为92.47%,CD44为90.05%,CD34为0.95%。成骨诱导21d,细胞钙结节形成明显,经茜素红染色出现红色结节的阳性染色,不加诱导剂的对照组染色为阴性。结论分离黏附细胞具有间充质干细胞的生物学特性,可以向成骨细胞分化。     

小分子化合物诱导大鼠脂肪基质干细胞向神经元的分化

目的:探讨小分子化合物诱导大鼠脂肪基质干细胞(ADSCs)向神经元的分化。方法:用Trichostatin A(TSA)、RG-108、8-BrcAMP和Rolipram 4种小分子物质对SD大鼠的ADSCs进行诱导分化,用免疫荧光形态学、免疫印迹和qRT-PCR等方法分别检测诱导前、后ADSCs神经元标志的表达。结果:诱导7 d时,ADSCs呈明显的神经元样结构,胞体呈圆形,具有多个突起,神经元样形态变化率高达95.12%±2.65%;诱导后的ADSCs nestin、β-tubulinⅢ、MAP2和ChAT在mRNA和蛋白水平的表达均有升高。结论:小分子化合物可将ADSCs诱导分化为神经元样细胞,其可作为治疗神经系统疾病及损伤的干细胞移植的种子细胞。     

人脂肪基质干细胞复合藻酸钙体外构建工程软骨的实验研究

目的 观察人脂肪基质干细胞(hADSC)复合藻酸钙凝胶体外构建工程软骨的可行性.方法 取临床整形外科超声乳化的成人脂肪组织溶液,经消化、分离、培养得到hADSC,经成软骨诱导培养后与藻酸钠凝胶复合,使细胞终浓度为5×10<'6>/mL,然后滴入浓度200mmol/L CaCl<,2>溶液中,固化15min形成藻酸钙凝珠...     

RA诱导P19细胞神经分化过程中相关蛋白分子的表达变化

目的:研究维甲酸(retinoic acid,RA)诱导P19细胞神经分化过程中与增殖、分化及凋亡相关分子表达变化情况。方法:P19细胞经RA处理4 d后进行分化培养,对分化培养细胞用MAP2抗体进行免疫组织化学染色。在P19细胞分化前、后不同时间点,Western Blot检测细胞周期蛋白(Cyclin D、Cyclin E、Cyclin A、p-CDK2)、生长分化相关蛋白(E2F1、PCNA、GAP43)、MAGE家族蛋白(Necdin、MAGE D1、MAGE H1)及凋亡效应分子(激活型Caspase 3)表达变化情况。结果:RA可诱导P19细胞向神经元分化;Necdin、GAP43和MAGE D1在RA处理后明显上调并在分化过程中保持高水平表达。E2F1、Cyclin D和Cyclin E表达随分化进程呈现逐步升高趋势,而Cyclin A、p-CDK2表达逐步下调。MAGE H1在分化第4 d到达顶峰后下降。PCNA在RA处理4 d时表达下降至最低,在分化早期渐上调后又降至低水平。RA处理后激活型Caspase 3表达明显上调,但在随后分化过程中又下调至对照组水平。结论:RA诱导P19细胞向神经元分化过程中,与细胞生长、分化、增殖及凋亡相关蛋白呈现动态表达变化,为进一步研究这些分子在神经分化中的作用奠定了基础。     

不同年龄段大鼠脂肪间充质干细胞增殖及成骨能力的研究

通过研究年龄对大鼠脂肪间充质干细胞增殖、成骨分化的影响,从而为临床寻找理想的种子细胞来源。用密度梯度离心法分离不同年龄段脂肪间充质干细胞进行培养,并保留贴壁细胞传代,观察细胞生长情况,检测其增殖活性,诱导后茜素红染色,钙离子浓度测定。结果显示,传代细胞的增殖速度比原代细胞快,但随着年龄的增长脂肪间充质干细胞的增殖能力下降。诱导条件下,各年龄组均出现矿化结节,但钙离子浓度随年龄增加而降低。本实验提示,脂肪间充质干细胞增殖和成骨分化能力随着年龄的增加而降低,但各年龄段脂肪间充质干细胞经过体外增殖、诱导后均可满足临床应用中不同患者的要求。     

人脂肪组织来源的神经干细胞移植治疗大鼠脑缺血再灌注损伤

目的探讨人脂肪组织来源的神经干细胞移植治疗大鼠脑缺血再灌注损伤的疗效。方法 线栓法制作大鼠大脑中动脉缺血(MCAO)2 h再灌注模型。体外培养脂肪基质细胞,诱导分化为神经干细胞。移植治疗组经尾静脉移植人脂肪组织来源的神经干细胞悬液(2×106/mL)。用MNSS量表测定神经功能,TTC、HE染色观察脑梗死体积及脑组织病理变化。结果移植治疗组再灌注14、21和28 d的神经功能评分低于缺血再灌组(P<0.05或P<0.01)。移植治疗组第14天脑梗死体积(40.20±9.52 mm3)小于缺血对照组(66.60±14.24 mm3)(P<0.01)。移植治疗组的神经细胞变性、坏死数量较缺血再灌组明显减少。结论 人脂肪组织来源的神经干细胞移植治疗可改善脑缺血再灌注损伤大鼠的神经功能,具有神经保护作用。     

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin α_E), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.     

Ⅱ型固有淋巴细胞在白色脂肪棕色化中的作用

正Ⅱ型固有淋巴细胞(typeⅡinnate lymphoid cells,ILC2s)于2001年被发现,由共同淋巴样祖细胞发育而来,广泛分布在血液、肠道、气管、肺脏、脾脏、肝脏、动物脂肪和皮肤等部位,经白细胞介素(interleukin,IL)-25或IL-33刺激后可产生IL-5和IL-13等2型辅助性T(type 2 helper T,Th2)细胞因子,在Th2     

明胶微冰胶支架有利于脂肪来源间充质干细胞体外干性维持并提高其体内应用价值

目的研究人脂肪来源间充质干细胞(ADSCs)与明胶微冰胶材料体外联合培养时,材料是否能维持间充质干细胞的生物学特性。方法 ADSCs种植于明胶微冰胶材料后进行Calcein-AM和PI活细胞染色检测细胞活力,细胞滴度蓝法检测细胞增殖能力,定量PCR检测干性基因OCT4、Nanog、SOX2表达情况,以及在成脂成骨诱导过程比较ADSCs在二维环境和种植于明胶微冰胶材料后的分化潜能。结果 ADSCs在三维明胶微冰胶支架材料中能保持较高活性,增殖能力不受影响,干性基因表达上调,成脂成骨分化相关基因表达水平比二维诱导环境低。结论明胶微冰胶可以为ADSCs提供一个较二维培养更好的微环境,有利于ADSCs体外干性维持,从而在干细胞移植法治疗相关疾病时以非侵入性的细胞传递方式发挥更长期的应用价值。     

肌糖蛋白TN-C促进人骨髓基质干细胞向成骨细胞的分化

目的 研究肌糖蛋白(Tenascin-c,TN-C)对人骨髓基质干细胞向成骨细胞分化的诱导作用,为TN-C在骨折愈合中的应用提供理论依据.方法 用全骨髓培养法获取人骨髓基质干细胞(hBMSCs),第6～10代的细胞用于本研究.体外培养的hBMSCs用自行制备的不同浓度重组TN-C (rTN-C)蛋白处理(0、1、10、50和100 nmol/L),于处理后不同时间进行细胞茜素红染色,检测细胞的矿化;或于不同时间收集细胞总RNA进行RT-PCR检测碱性磷酸酶(ALP)和骨钙素(OCN) mRNA水平的变化;同时利用ALP检测试剂盒和骨钙素放射性免疫试剂盒分别检测ALP活性和细胞培养基中OCN含量.结果 1)rTN-C处理hBMSCs 10 d后,rTN-C呈剂量依赖性促进成骨细胞的矿化(P＜0.05);2) rTN-C处理hBMSCs后,细胞ALP活性及培养基上清中OCN含量显著高于未处理对照组(P＜0.05).结论 肌糖蛋白对人BMSC向成骨细胞的分化有一定的促进作用.     

不同组织学类型人肺癌细胞系的维甲酸受体表达

目的：观察来源于不同组织学类型的人肺癌细胞系（肺巨细胞癌细胞系PLA－801，肺鳞状上皮细胞癌细胞系C－57，个旧肺腺癌细胞系GLC－82和肺腺癌细胞系PC－84045）的维甲酸受体的含量和分布特征。方法：用ELISA和免疫组化显色计算机图像分析方法，对4株具有不同侵袭潜能、不同组织学类型的肺癌细胞系的维甲酸受体（RARs、RARβ和RXRs)进行检测。结果：4株细胞系的RARβ表达极少，甚至缺如；RARs主要分布在癌细胞质中，但RXRs主要位于细胞核内，表达水平较高；RARs和RXRs在PLA－801细胞中表达最低。结论：肺癌中普遍存在着RARβ的缺失与RARs的表达分布异常，这些改变可能与肺癌的异常分化有关，也可能是肺癌细胞对维甲酸诱导分化不敏感的重要原因。RXRs较高水平的表达主要位于核内，提示用9－顺式维甲酸来诱导肺癌的分化效果可能会较好。     

肺癌和肺癌前病变组织中维甲酸受体的表达

目的： 探讨维甲酸受体（RAR）在肺癌癌前病变（支气管黏膜上皮不典型增生）和肺癌病变中的表达。方法: 在40例正常肺组织、41例支气管黏膜上皮不典型增生组织及143例肺癌患者手术标本中,用免疫组化的方法检测RAR的表达,分析RAR基因在肺癌变不同阶段间的变化。结果: 80.0%（32/40）的正常肺组织中可检测到RAR基因的蛋白表达;68.3%（28/41）的支气管黏膜上皮不典型增生组织和57.6%（83/143）的肺癌组织中可检出RAR（P<0.05）;且随着肺组织癌变过程的变化,RAR基因蛋白的表达呈逐渐降低的趋势（P<0.01）。吸烟者RAR蛋白检出率低于非吸烟者。结论: RAR表达下降与肺组织癌变过程密切相关,吸烟暴露可导致RAR基因表达降低,检测RAR基因表达下降可能为肺癌的早期临床诊断提供科学依据。     

Protective effects of quercetin against hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species that has been implicated in various neurodegenerative diseases. Quercetin, one of the plant flavonoids, has been reported to harbor various physiological properties including antioxidant activity. In this study, we investigated the neuroprotective effects of quercetin against H₂O₂-induced apoptosis in human neuronal SH-SY5Y cells. H₂O₂-mediated cytotoxicity and lactate dehydrogenase release were suppressed in a quercetin concentration-dependent manner. In addition, quercetin repressed the expression of the pro-apoptotic Bax gene and enhanced that of the anti-apoptotic Bcl-2 gene in SH-SY5Y cells. Moreover, quercetin effectively inhibited the activation of the caspase cascade that leads to DNA fragmentation, a key feature of apoptosis, and subsequent cell death. These results indicate the importance of quercetin in protecting against H₂O₂-mediated neuronal cell death. Thus, quercetin might potentially serve as an agent for prevention of neurodegenerative diseases caused by oxidative stress and apoptosis.