Proliferative capacity of stem/progenitor-like cells in the kidney may associate with the outcome of patients with acute tubular necrosis

Animal studies indicate that adult renal stem/progenitor cells can undergo rapid proliferation in response to renal injury, but whether the same is true in humans is largely unknown. To examine the profile of renal stem/progenitor cells responsible for acute tubular necrosis in human kidney, double and triple immunostaining was performed using proliferative marker and stem/progenitor protein markers on sections from 10 kidneys with acute tubular necrosis and 4 normal adult kidneys. The immunopositive cells were recorded using 2-photon confocal laser scanning microscopy. We found that dividing cells were present in the tubules of the cortex and medulla, as well as the glomerulus in normal human kidney. Proliferative cells in the parietal layer of Bowman capsule expressed CD133, and dividing cells in the tubules expressed immature cell protein markers paired box gene 2, vimentin, and nestin. After acute tubular necrosis, Ki67-positive cells in the cortex tubules significantly increased compared with normal adult kidney. These Ki67-positive cells expressed CD133 and paired box gene 2, but not the cell death marker, activated caspase-3. In addition, the number of dividing cells increased significantly in patients with acute tubular necrosis who subsequently recovered, compared with patients with acute tubular necrosis who consequently developed protracted acute tubular necrosis or died. Our data suggest that renal stem/progenitor cells may reside not only in the parietal layer of Bowman capsule but also in the cortex and medulla in normal human kidney, and the proliferative capacity of renal stem/progenitor cells after acute tubular necrosis may be an important determinant of a patient's outcome.     

Ovarian steroids and the human breast: regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor- (ER) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ER/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ER/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ER/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.     

Hedgehog signaling is involved in differentiation of normal colonic tissue rather than in tumor proliferation

The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.     

大鼠心肌匀浆上清液对骨髓间质干细胞诱导分化的影响

目的：探讨心肌匀浆上清液对大鼠骨髓间质干细胞（MSCs）诱导分化的影响。方法：体外分离培养大鼠MSCs,检测纯度。于原代培养第3d加入自体心肌匀浆上清液持续作用1周。3周后,观察细胞形态,采用免疫细胞化学检测肌球蛋白重链和心肌特异性肌钙蛋白-T,RT-PCR方法检测Nkx2.5、α-MHC和ANP基因的表达。结果：大鼠MSCs经诱导后,表达肌球蛋白重链、心肌特异性肌钙蛋白-T和Nkx2.5、α-MHC基因,但未表达ANP基因,也未观察到肌管和闰盘样结构。结论：心肌匀浆上清液对MSCs具有定向诱导分化作用,但诱导不完全,具体机制仍需深入研究。     

A graphene-based platform for induced pluripotent stem cells culture and differentiation

Induced pluripotent stem cells (iPSCs) hold great promise as a cell source for regenerative medicine yet its culture, maintenance of pluripotency and induction of differentiation remain challenging. Conversely, graphene (G) and graphene oxide (GO) have captured tremendous interests in the fields of materials science, physics, chemistry and nanotechnology. Here we report on that G and GO can support the mouse iPSCs culture and allow for spontaneous differentiation. Intriguingly, G and GO surfaces led to distinct cell proliferation and differentiation characteristics. In comparison with the glass surface, iPSCs cultured on the G surface exhibited similar degrees of cell adhesion and proliferation while iPSCs on the GO surface adhered and proliferated at a faster rate. Moreover, G favorably maintained the iPSCs in the undifferentiated state while GO expedited the differentiation. The iPSCs cultured on both G and GO surfaces spontaneously differentiated into ectodermal and mesodermal lineages without significant disparity, but G suppressed the iPSCs differentiation towards the endodermal lineage whereas GO augmented the endodermal differentiation. These data collectively demonstrated that the different surface properties of G and GO governed the iPSCs behavior and implicate the potentials of graphene-based materials as a platform for iPSCs culture and diverse applications.     

骨髓间充质干细胞对大鼠胶质瘤细胞增殖的影响

目的研究骨髓间充质干细胞（BMSC）对大鼠脑胶质瘤C6细胞增殖的影响,并且探讨其相关机制。方法提取SD大鼠BMSC,进行体外培养、扩增。应用MTt比色法检测不同浓度BMSC上清液对C6细胞系增殖的抑制作用。用Transwell小室将BMSC与C6细胞进行双层培养,以HE染色法检测C6细胞形态变化。用划痕实验检测细胞迁移情况。结果MTF结果显示BMSC上清液对C6细胞有抑制作用,120h后1：8、1：4、1：2的BMSC上清稀释液、BMSC上清原液培养组的生长抑率分别为17．1％、26．0％、39．9％、43．1％;与BMSC进行双层培养后的C6细胞其形态发生了明显改变,由圆形或多角形变为长梭形,细胞包体伸出长突起;划痕实验显示在36h时对照组迁移率为100％,划痕完全愈合,而BMSC上清组划痕未见全愈合,迁移率为82％。结论①BMSC上清液能抑制胶质瘤C6细胞的恶性增殖,而且抑制效应呈剂量依赖性;②BMSC对C6的运动和迁移产生了抑制作用,从而降低了其恶性侵袭程度。     

Gene expression in the peripheral leukocytes and association analysis of PDLIM5 gene in schizophrenia

The terminal mitosis of hair cells (HCs) and supporting cells (SCs) in mammalian cochlea occurred during middle embryonic development. Most hearing loss results from the incapacity of the cochlear sensory epithelium to replace lost hear cells. Deafness due to hair cells loss is normally irreversible. The present study showed that cells acutely dissociated from the cochlea of young rat, cultured with EGF and FGF2, developed into otospheres that showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). The subcultured otospheres maintained for up to 10 passages. In addition, the cochlea sphere-derivatives contributed to a variety of cell types. They were found to differentiate to neuron, glia, hair cell and supporting cell phenotypes. The results suggest that the young rat inner ear cells have self-renewal capability and multipotent differentiation potential. This work raises the possibility that inner ear cells in the early post-natal rat have the character of pluripotent stem cells and might be a source for cell replacement therapy in the inner ear.     

Histone deacetylase inhibitors increase neuronal differentiation in adult forebrain precursor cells

Chromatin modification plays a key role in fate decision of neural stem cells. Here, we explored the impact of epigenetic remodelling onto neuronal fate determination using specific inhibitors of histone deacetylases (iHDAC). Adult subventricular zone (SVZ) precursor cells were expanded as neurospheres and treated in vitro with second generation iHDAC MS-275, M344 and suberoylanilide hydroxamic acid (SAHA). All tested compounds revealed a significant increase of βIII-tubulin positive neurons (ranging from 258 to 431%) in a concentration-dependent manner. The number of oligodendrocytes was decreased by almost 50%, accompanied by a reduction of Olig2 mRNA expression. In contrast, astrocyte quantity remained unaffected after iHDAC treatment. Both control and iHDAC treated cells expressed markers of mature GABAergic and dopaminergic neurons. Increased expression levels of NeuroD, Cyclin D2 and B-lymphocyte translocation gene 3 (Btg3) point to a shift towards neuronal fate determination targeted by HDAC inhibitors.     

Constitutive production of regulators of stem cell proliferation in the hereditarily anaemic belgrade laboratory (b/b) rat

The Belgrade (b/b) rat has hereditary hypochromic microcytic anaemia as the consequence of intracellular iron deficiency together with decreased number and proliferative activity (a proliferative block) of progenitors and more mature stem cells (CFU-Sd8). In the present study, the investigations were extended to the activities influencing CFU-S proliferation in bone marrow of b/b rats. A stimulator of CFU-S proliferation (not associated with IL-1 and IL-6 activity), undetectable in steady-state haematopoiesis in bone marrow, was found in b/b rat. The inhibitor of CFU-S proliferation, present in the bone marrow of normal animals, was also detected in b/b rats, indicating impaired synchronisation of production of two opposite activities. The chronic treatment of b/b rats with iron increased proliferative activity of CFU-S, corrected anaemia whilst, only the activity of stimulator was detected indicating recovery of synchronisation in stimulator and inhibitor production. Complete correction of anaemia by chronic RBC transfusions of b/b rats normalised production of CFU-S proliferation regulators. Disturbances in production of regulators of CFU-S proliferation may result as a secondary consequence of the metabolic alterations associated with severe anaemia.     

体外培养的神经球中表达神经巢蛋白细胞的流式细胞术检测

采用体外培养不同时间的神经干细胞球 ,间接荧光法标记神经巢蛋白 (nestin) ,以流式细胞术检测神经干细胞球中表达nestin细胞的情况。结果显示 :在培养不同时间的细胞群体中均检出 nestin阳性细胞 ,培养 7d、1月、3月和 6月的细胞群含nestin阳性细胞数百分比分别为 (4 2 .2± 7.6) %、(4 1.7± 7.3 ) %、(3 3 .8± 12 .9) %和 (3 7.1± 5 .0 ) % ,几个时间点的细胞群体所含的 nestin阳性细胞数的百分比之间无显著性差异。提示 ,在体外培养条件下 ,神经干细胞群体可能通过自我更新和分化的调控 ,保持 nestin阳性细胞于一种较为恒定的比例。     

异丙酚促进体外培养的成年大鼠海马神经干细胞增殖

目的异丙酚对于成年神经干细胞增殖能力的影响目前尚不明确,本研究对异丙酚对体外培养的大鼠成年神经干细胞的作用及其可能的分子机制进行了研究。方法体外培养的大鼠成年神经干细胞给予BrdU后,分别用10、50、100μg/ml的异丙酚进行处理,并同时设立溶媒对照和空白对照。24h后,采用免疫染色显示BrdU阳性细胞,DAPI复染细胞核。细胞存活率采用细胞计数法和MTT法测定。人工计数BrdU阳性细胞和固缩细胞核的比例。用Fura2-AM检测胞浆内Ca2＋浓度。分别用免疫荧光和免疫印迹法检测细胞内CREB和磷酸化CREB的表达位置和水平。结果低浓度（10μg/ml）异丙酚不能促进大鼠成年神经干细胞的增殖,而中浓度（50μg/ml）和高浓度异丙酚（100μg/ml）则呈剂量依赖性的促进其增殖。异丙酚提高了BrdU阳性细胞的比例。细胞死亡率在处理前后维持在低水平上（〈2%）。异丙酚显著提高了大鼠成年神经干细胞胞浆内的游离Ca2＋浓度。异丙酚处理前后,大鼠成年神经干细胞均能表达CREB和磷酸化CREB,但是异丙酚干预后,大鼠成年神经干细胞内磷酸化CREB表达水平显著增高。结论异丙酚可促进体外培养的大鼠成年神经干细胞的增殖水平,这是细胞存活率升高的主要原因,可能与胞浆内Ca2＋浓度以及CREB磷酸化水平的升高有关。     

骨髓基质细胞促进神经干细胞增殖分化

目的：探讨骨髓基质细胞（BMSCs）对神经干细胞（NSCs）增殖分化的影响。方法:在体外比较NSCs在单独培养和在BMSCs条件培养液中培养下的分化和增殖情况。结果:应用BMSCs条件培养液培养NSCs,分化的神经元比例较显著高于NSCs单独培养（41.1%±3.2%vs23.3%±16.5%,P<0.05）,而分化的星形胶质细胞所占比例显著降低（33.8%±4.9%vs65.0%±10.4%,P<0.01）,同时增殖细胞所占比例也显著增高（74.7％±4.7％vs51.4％±12.3％,P<0.01）。结论:BMSCs对NSCs有促进其增殖和向神经元分化的作用。NSCs与BMSCs联合移植可能会增强NSCs移植的抗脑损伤作用。     

p53对体外神经干细胞增殖能力的影响

目的:探讨p53对神经干细胞(neural stem cells,NSCs)增殖能力的影响,寻求维持NSCs活力的有效途径。方法:分离出生后0 d(postnatal day 0,P0)和生后90 d(postnatal day 90,P90)小鼠前脑室管膜下区(subventricular zone,SVZ)组织,培养NSCs。比较P0和P90 NSCs增殖能力并通过q PCR和Western Blot检测衰老相关分子p53和p21在新生和成年NSCs的表达变化。应用p53抑制剂PFT-α(20μmol/L)连续作用于P90 NSCs 72 h,通过Brd U掺入实验和CCK-8实验,分析抑制p53对NSCs增殖能力的影响。结果:随年龄增加,NSCs的增殖能力降低,P90组神经球的数量和直径分别是P0组的22.9%±1.2%(P0.01)和63.5%±3.7%(P0.05)。P90NSCs p53和p21 mRNA表达水平分别较P0 NSCs显著增高了1.4±0.05和1.2±0.04(P0.01)。Western Blot结果证明P90 NSCs p53蛋白的表达水平比P0 NSCs增加了1.2±0.01倍(P0.05)。经p53抑制剂PFT-α连续处理P90 NSCs 72 h后,CCK-8结果显示PFT-α组吸光度值1.1±0.02高于DMSO组0.8±0.03(P0.05),Brd U掺入实验也显示PFT-α组Brd U阳性细胞率为43.3%±4.0%显著高于DMSO组24.8%±3.1%(P0.05)。结论:p53信号通路激活可能是导致成年小鼠NSCs增殖能力下降的重要因素,应用p53抑制剂PFT-α能够增加成年NSCs的增殖能力。     

影响神经干细胞增殖分化的因素

传统认为哺乳动物和人脑神经细胞一经发育成熟即不再进行增殖、分化 ,不具备再生能力 ,只有细胞的不断退化和死亡 ,因此中枢神经系统损伤后基本上不能恢复。近年来的研究发现 ,胚胎早期脑室和脑室下区、成年脑室区、纹状体、海马齿状回、脊髓等部位神经干细胞分布广泛 ,这些神经干细胞可以分化为神经元、星形胶质细胞和少突胶质细胞。随着对干细胞研究的深入 ,人们已能利用无血清培养、单克隆技术及免疫荧光化学技术对干细胞进行分离、培养和纯化。深入研究脑发育过程中的神经干细胞的增殖分化机制 ,搞清神经干细胞在成年脑内增殖、分化的影…     

胰岛素样生长因子对神经干细胞增殖分化的影响

神经干细胞 (ｎｅｕｒａｌｓｔｅｍｃｅｌｌｓ ,ＮＳＣｓ)能增殖成恰当数量的细胞 ,在脑部各个区域按正确顺序排列组合 ,分化为神经元 ,星形胶质细胞、少突胶质细胞 ,这是发育过程中必不可少的过程。众多研究提示细胞外因子参与此过程的调控并影响ＮＳＣｓ的分化启动和分化方向。在这些细胞外因子中有着显著影响作用的是众多的生长因子 ,如ＥＧＦ (表皮生长因子 )、ＦＧＦ (成纤维细胞生长因子 )、ＩＧＦ(胰岛素样生长因子Ｉｎｓｕｌｉｎ -ｌｉｋｅｇｒｏｗｔｈｆａｃｔｏｒ)等。深入了解生长因子对神经发生的作用有助于进一步认识细胞增殖…     

褪黑素促进体外缺氧的神经干细胞增殖及定向分化

目的:观察褪黑素对体外缺氧诱导的小鼠神经干细胞(neural stem cells,NSCs)增殖和分化的影响。方法:取孕12.5 d胚胎小鼠大脑皮质分离培养NSCs,建立缺氧模型。利用免疫荧光染色,检测分析褪黑素对缺氧诱导后不同时间点的NSCs增殖和分化的影响。结果:缺氧后NSCs的增殖能力明显下降,而褪黑素可显著改善这一现象,促进缺氧后NSCs的增殖。缺氧后NSCs向神经元的分化明显受阻,褪黑素处理组的神经元的分化率明显高于对照组,其中作用高峰期是分化的第七天。结论:体外缺氧的NSCs增殖和分化均明显受到抑制,而褪黑素的干预可明显改善干细胞的增殖,促进NSCs向神经元的分化,但对星形胶质细胞的分化没有显著影响。     

体外培养的神经干细胞球的超微结构

目的 探讨体外培养的神经干细胞球的超微结构。方法 采用常规透射电镜结合硝酸镧示踪染色、钌红染色和单宁酸染色等技术，观察体外培养的大鼠脑神经干细胞球的超微结构。结果 神经球中细胞之间连接较为松散，不存在紧密连接结构，神经干细胞增殖活跃，并可见神经球中有部分干细胞分化为具有突起和轴突样结构的细胞，甚至出现髓鞘样结构。结论 揭示了体外培养的神经干细胞球的超微结构。     

促红细胞生成素促进体外鼠胚脑皮质神经干细胞增殖

目的检测体外培养SD大鼠胚脑皮质神经干细胞(neural stem cells,NSCs)增殖和分化的生物学特性,为NSCs研究提供适宜的细胞模型;探讨促红细胞生成素(erythropoietin,EPO)对NSCs增殖的影响,为NSCs的相关研究提供实验依据。方法本研究对孕14d(E14)SD大鼠取鼠胚脑皮质悬浮培养、贴壁诱导分化。采用光电镜观察,以nestin免疫荧光染色鉴定NSCs,微管相关蛋白2(microtubule associated protein2,MAP2)和神经胶质原纤维酸性蛋白(glia fibrillary acid protein,GFAP)检测NSCs分化。取第三代(P3)NSCs向悬浮培养基中添加不同剂量的EPO,通过四甲基偶氮唑蓝(MTT)检测法检测NSCs的增殖情况。结果分离E14dSD大鼠胚脑皮质,在添加B27、bFGF、EGF的无血清培养基中培养,可形成大量悬浮的神经球并可进行体外扩增传代,神经球内的细胞均呈Nestin阳性、BrdU阳性。在添加10%胎牛血清的培养基中,神经干细胞可自然分化为神经元和神经胶质细胞。与对照组对比,加入≥5U/mlEPO后MTT检测NSCsOD值明显增高。结论 SD大鼠胚脑皮质体外培养可得到大量增殖的神经干细胞并能分化为神经元和神经胶质细胞,EPO可促进体外NSCs的增殖。     

养精胶囊提取液对精原干细胞增殖的影响

BACKGROUND: Chinese herbs for kidney nourishment can promote the proliferation and differentiation of spermatogonial stem cells.     

间质干细胞移植在大鼠缺血性脑卒中的实验研究

摘要目的:探索间质干细胞移植在脑梗塞大鼠的脑内分化及促进神经功能的修复作用。方法:从健康人的肋骨骨髓中分离、纯化而获得的间质干细胞,经体外培养、扩增、鉴定的同时制造大脑中动脉皮层梗塞的实验动物模型;并且,在梗塞后10d移植所鉴定的间质干细胞经免疫组化验证间质干细胞的脑内成活、及神经性分化后,在第2周和第6周进行神经功能评分。结果:间质干细胞在梗塞灶周边向神经元和神经胶质细胞的方向分化;移植的神经功能评分,修复作用与对照组有显著差异。结论:人间质干细胞为脑梗塞治疗提供了新的思路。