KLF2和KLF15基因在hBMSCs定向分化中的动态表达

目的研究KLF2和KLF15在人骨髓间充质干细胞(hBMSCs)成脂、成骨和成肌分化过程中的表达水平及变化趋势,探讨KLF2和KLF15在hBMSCs定向分化过程中可能的作用方式。方法密度梯度离心法分离hBMSCs,流式细胞仪检测细胞表面标志物,取第4代细胞分别进行成脂、成骨和成肌诱导并以油红O、茜素红及免疫荧光染色进行鉴定。通过实时定量PCR和免疫荧光分别从mRNA水平和蛋白水平检测相关标志物、KLF2、KLF15和GLUT4的表达。结果体外培养的hBMSCs表达间充质干细胞表面标志CD29、CD90,并在特定诱导剂作用下能够定向分化为脂肪细胞、成骨细胞和成肌细胞,染色鉴定结果为阳性,并分别检测到相关标志基因的表达;hBMSCs成脂、成骨和成肌过程中KLF2、KLF15和GLUT4的表达均呈动态变化。结论 KLF2和KLF15与hBMSCs成脂、成骨和成肌分化的启动和维持有关;KLF2和KLF15可能通过GLUT4调节能量代谢从而影响hBMSCs定向分化。     

miR-302对脂肪间充质干细胞向成脂和成骨分化的调控

背景：间充质干细胞具有自我更新能力,在一定条件下能够分化为一定谱系的细胞,但很多机制至今未明。 目的：探究miR-302b对脂肪间充质干细胞向成脂和成骨分化的调控作用。 方法：miR-302模拟物转染作用于脂肪间充质干细胞进行成骨成脂诱导,对照组转染miR-302阴性对照模拟物miR-NC。采用碱性磷酸酶染色及活性分析、茜素红染色、油红O染色和萃取实验观察miR-302上调对脂肪间充质干细胞成骨和成脂分化的影响,以及Western blot检测miR-302上调后成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在脂肪间充质干细胞中的表达。 结果与结论：①碱性磷酸酶沉淀物在miR-302过表达细胞中产生的量均明显少于对照组,进一步发现miR-302过表达实验组碱性磷酸酶活性明显低于对照组(P < 0.05)。②miR-302过表达明显抑制了矿物质沉积钙结节的形成,miR-302上调实验组橘红色的钙结节明显少于对照组。③miR-302过表达实验组油红O染色阳性的细胞数明显高于对照组,进一步表明实验组细胞萃取得到的油红O吸光度值明显上升(P < 0.05)。④成骨诱导第6天时成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在miR-302过表达的细胞中都有不同程度的下降。⑤以上结果表明miR-302的上调能够抑制脂肪间充质干细胞的成骨分化,同时促进其向成脂分化。miR-302在间充质干细胞向成脂和成骨分化平衡发挥了双向调控作用。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

长链非编码RNA AK089560在间质干细胞成骨与成脂分化中的表达

背景：最近研究发现众多长链非编码RNA调控干细胞多潜能性和分化。长链非编码RNA AK089560在干细胞多向分化中的表达变化及作用,目前尚不清楚。目的：观察间质干细胞C3H10T1/2成骨分化与成脂分化过程中AK089560的表达。方法：重组人骨形态发生蛋白2诱导间质干细胞C3H10T1/2成骨分化,碱性磷酸酶染色鉴定早期成骨分化。地塞米松、吲哚美辛和胰岛素三因子联合诱导C3H10T1/2成脂分化,油红O染色鉴定成脂分化结果。采用qRT-PCR检测诱导前后不同时间点AK089560表达的变化。采用RNAfold软件预测AK089560二级结构,UCSC基因组浏览器分析AK089560邻近编码蛋白基因,fancyGENE在线软件构件长链非编码RNA与编码基因关系图。结果与结论：C3H10T1/2经成骨诱导后,70%以上细胞碱性磷酸酶染色阳性;成脂分化诱导后,80%以上细胞油红O染色阳性。qRT-PCR结果显示,长链非编码RNA AK089560在成骨分化与成脂分化第2,4,6天表达均明显下降,成骨分化与成脂分化第2,4,6天与相应时间未分化组相比均差异有显著性意义(P < 0.05)。生物信息学分析表明,LncRNA AK089560具有复杂茎环结构,与编码基因Sema3a形成sense overlap关系。结果表明AK089560在间质干细胞成骨与成脂分化中下调表达,提示其可能参与调控间质干细胞多向分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

miR-424负向调节人脂肪源间充质干细胞向成脂细胞分化

目的研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响。方法用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化。在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导。用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达。结果 hAMSCs成脂分化后,miR-424表达下调。与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高。成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组。PPARγ和成脂相关标记物的表达量也出现下调。结论 miR-424可负向调节hAMSCs向脂肪细胞分化。     

miR-424 negatively regulates adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

目的 研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响.方法 用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化.在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导.用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达.结果 hAMSCs成脂分化后,miR-424表达下调.与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高.成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组.PPARγ和成脂相关标记物的表达量也出现下调.结论 miR-424可负向调节hAMSCs向脂肪细胞分化.     

间充质干细胞体外成骨及成脂肪分化中sortilin基因表达的研究

目的:研究间充质干细胞(MSC)体外成骨、成脂肪分化时sortilin基因表达变化。方法:从骨髓中分离培养人间充质干细胞,流式细胞术鉴定其表型,加入成骨和成脂肪诱导剂,RT-PCR检测成骨标志骨桥蛋白(Op)和成脂肪标志脂蛋白脂肪酶(LpL)表达,半定量RT-PCR分析sortilin基因随诱导时间的表达变化。结果:①体外分离培养的人间充质干细胞能向成骨细胞和脂肪细胞分化,分别表达Op和LpL。②在体外成骨分化过程中,sortilin基因表达第1d开始上调,持续约1周后恢复至原水平;在培养3d时,sortilin基因表达明显上调,与未诱导组相比,差异明显(P＜0.01)。③体外成脂肪分化时,sortilin基因表达无明显的改变(P＞0.05)。结论:sortilin基因可能参与间充质干细胞成骨分化的调节,而与成脂肪分化无关,通过调控sortilin基因的表达,有可能为成骨障碍性疾病的治疗,提供新的思想和策略。     

不同浓度细胞松弛素D对人脂肪源干细胞分化能力的影响

目的　观察不同浓度细胞松弛素D（Cyto D）对人脂肪源干细胞（hASC）成骨分化和成脂分化能力的影响。 方法　在生长培养基、成脂诱导培养基及成骨诱导培养基中分别加入0.05、0.1、0.2 μg/ml的Cyto D处理hASC,干扰肌动蛋白的延长,并在处理的第1、4、7 d时分别进行油红O染色、ALP染色及CCK8等实验检测细胞性能的改变。 结果　7 d时实验组0.2 μg/ml的存活率相比于对照组下降了1/3。Cyto D能抑制hASCs的存活和增殖,且浓度越大细胞的存活率越低。单纯的Cyto D既能促使hASC胞内脂滴形成,又能改变细胞内成骨的基础状态。Cyto D与成脂诱导液联合使用后能极大的促进成脂,且不同的浓度具有不同的成脂效果,低浓度使脂滴数量增加,高浓度促进脂滴成熟;与成骨诱导液联合使用后Cyto D用药浓度和成骨效果成反比。 结论　细胞松弛素D能增强成骨或成脂诱导液效果,且低浓度细胞松弛素D能够促进成骨,低浓度和高浓度细胞松弛素D都能促进成脂。本实验探究了细胞松弛素D引起的肌动蛋白解聚对脂肪源干细胞的分化能力影响,为研究脂肪源干细胞的分化机制提供了重要生物学信息。     

人脱落乳牙干细胞骨向分化相关分子Runx2的动态表达

背景:Runx2被认为是成骨基因表达的主要调节因子,是一种向成骨细胞分化的不可或缺的转移因子,在成骨细胞发育、分化、调控、骨钙化形成及骨修复过程中起着极其重要的作用。目的:观察人脱落乳牙干细胞生物学特征,探讨乳牙干细胞骨向分化潜能,以及不同时间点成骨相关转录因子Runx2的动态表达规律。方法:体外分离、培养人脱落乳牙干细胞。流式细胞仪检测乳牙干细胞表面标志。体外脂向诱导分化4周后进行油红O染色,检测脂滴形成情况;矿化诱导21 d进行茜素红染色,检测矿化结节形成情况。于成骨诱导的不同时间点,用RT-PCR技术检测Runx2的动态表达。结果与结论:通过酶消化法和有限稀释法获得了乳牙干细胞,流式细胞仪检测显示CD146和STRO-1均有不同程度的表达。成脂诱导油红O染色可见橙红色阳性颗粒。矿化诱导茜素红染色呈阳性。RT-PCR技术检测Runx2在成骨诱导第0天已有表达,0-6 d成明显增强趋势,6-12 d显著下降,12-18 d表达再次上升,以后相对平稳。结果表明乳牙干细胞可体外分离、培养,表达间充质干细胞表面标志,具有成脂和成骨向分化能力。Runx2在乳牙干细胞成骨分化各阶段均有表达,早期和晚期表达上升,中期表达有下降趋势。     

大鼠脂肪源干细胞中骨形态发生蛋白信号通路相关分子的表达

目的:探讨大鼠脂肪源干细胞(ADSCs)成骨和成脂肪分化能力及骨形态发生蛋白(BMP)信号通路相关分子的表达.方法:切取Wistar雌性大鼠腹股沟脂肪垫,用酶消化法培养ADSCs.将第4代ADSCs经成骨和成脂肪诱导分化培养后,进行相应的茜素红和油红O染色,以鉴定ADSCs的分化潜能.同时取培养的第4代ADSCs进行流...     

水通道蛋白AQP5对小鼠骨髓间充质干细胞分化能力的影响

目的探寻水通道蛋白5(Aquaporin 5,AQP5)对小鼠骨髓间充质干细胞分化能力的影响。方法采用RT-PCR和Western Blot方法检测AQP5在小鼠骨髓间充质干细胞(BMSCs)中的表达。定向诱导BMSCs后分别进行油红O染色(成脂),茜素红S和碱性磷酸酶染色(成骨),Collagen II和番红素O染色(成软骨)。检测钙离子、碱性磷酸酶和胞外糖胺多糖含量,Real-time PCR检测成脂、成骨和成软骨分化后的特异标记分子的相对+/+m RNA表达。结果 RT-PCR和Western Blot结果发现野生型(AQP5)BMSCs中有AQP5阳性条带。油红O、茜素红S、碱-/-性磷酸酶,Collagen II和番红素O染色阳性染色率在AQP5敲除型(AQP5)BMSCs明显高于野生型。Real-time PCR结果证-/-实AQP5 BMSCs在成脂、成骨和成软骨分化特异标记分子的相对m RNA表达水平显著提高。结论小鼠BMSCs中有AQP5表达,AQP5敲除显著提高BMSCs的成脂、成骨和成软骨分化能力。     

Electron-microscopic and electron-autoradiographic characteristics of embryonic lung cells in organ cultures

Electron-microscopic and electron-autoradiographic investigations of embryonic mouse lung cells were undertaken during the early stages of organotypical culture. The fate of undifferentiated cells could be traced by electron autoradiography.Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. N. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, vol. 88, No. 10, pp. 490–491, October, 1979.     

胸腺素β4的研究与开发进展

1966年Goldstein等首先从小牛胸腺中提取出促淋巴细胞生成因子，命名为“胸腺素（thymosin）”。1975年Hooper等又进一步提纯到胸腺素组分5，并证明其是30种以上9～50肽的混合物。从此关于胸腺素的研究得以快速发展。胸腺素根据等电点不同可分为α、β、γ三类，其中等电点位于5．0～7．0的为β族胸腺素（β-thymosins，Tβ）。     

Gastric carcinosarcoma with neuroendocrine differentiation as the carcinoma component and leiomyosarcomatous and myofibroblastic differentiation as the sarcomatous component

Gastric carcinosarcoma with neuroendocrine differentiation is a very rare neoplasm. In this article we present such a case. The gastroendoscopic examination of a 59-year-old Japanese man disclosed gastric cancer during follow-up after operation for rectal cancer. Subsequently, total gastrectomy was carried out because of gastric cancer. A large tumor measuring 9.2 x 8.4 cm was observed in the posterior wall of the upper portion of the stomach. The tumor was composed of carcinoma and sarcomatous cells, and the histological transition of both components was observed. Immunohistochemically, carcinoma and sarcomatous cells were positive for cytokeratin CAM5.2. The carcinoma contained adenocarcinoma and malignant cells with neuroendocrine differentiation. The sarcomatous component showed leiomyosarcomatous and myofibroblastic differentiation. The present tumor is the fifth case of gastric carcinosarcoma with neuroendocrine differentiation and the first case of gastric carcinosarcoma with myofibroblastic differentiation. Pathologists should bear in mind that gastric carcinosarcoma may show various types of differentiation.     

Bilateral aggressive malignant granulosa cell tumour with essentially different immunophenotypes in primary and metastatic lesions comprising predominantly sarcomatoid and fibrothecomatous patterns – looking for prognostic markers: a case report

We present an unusual case of a young woman with rare bilateral, very aggressive ovarian granulosa cell tumour (GCT), comprised of granulosa, sarcomatoid and fibrothecomatous fields with significantly different immunostaining of primary and metastatic tumours showing stronger WT1, Bcl2, fascin and EGFR expression in metastases. Despite radical surgery and chemotherapy the tumour recurred rapidly and the patient died 16 months later. Such results clearly demonstrate the usefulness of immunostaining for the above markers as prognostic/predictive factors and the need for careful assessment of the immunoprofile of both primary and metastatic tumours, which can be useful for therapy and follow-up planning in GCT cases.     

A study of 39 retinoblastomas with particular reference to morphology, cellular differentiation and tumour origin

A series of 39 retinoblastomas was studied using light microscopy and immunohistochemistry for localization of neurone-specific enolase and glial fibrillary acidic protein. Thirty-eight retinoblastomas (97.4%) occurred in children less than 6 years of age: one was in a 61-year-old man (2.6%); the mean age, excluding the 61-year-old, was 32.1 months. Unilateral tumours were found in 74.4% of patients and bilateral tumours in 25.6%; bilateral lesions occurred in a younger age group, mean age of 21.7 vs 35.9 months. The male to female ratio was 1.4:1. Morphologically, arrangement of tumour cells into trabeculae was noted in 69.2% of retinoblastomas and glomerulus-like structures in 43.6%. These two patterns of cellular arrangement were encountered where the choroid and sclera were invaded by neoplastic cells. Homer Wright rosettes were observed in all tumours. Flexner-Winstersteiner rosettes in 33.3% and fleurettes in 2.6%. Differentiation into neoplastic neurones as well as in the form of Flexner-Winstersteiner rosettes and fleurettes was noted in 46.1%, neoplastic ependymal cells in 100.0%, astrocytes in 58.9%, oligodendrocytes in 23.1%, and glioblastomas in 2.6%. These data suggest that retinoblastoma is a primitive stem cell neuroectodermal tumour with the capacity for differentiation in both neuronal and neuroglial directions.     

析中医辨证

整体观念和辨证论治是中医理论体系的核心,也是数千年中医临床实践经验的总结和理论上的升华、中医学术繁荣的重要标志,学术上形成了百花齐放、百家争鸣的松宽学术氛围,有效地促进了中医事业的发展。如汉代医圣张仲景将汉代以前中医临床经验和理论总结归纳为六经辨证;明清时期的温病学大家叶天士创立卫气营血辨证;温病学大家吴鞠通创立三焦辨证;后世医学家在此基础上还创立八纲辨证、脏腑辨证、气血精液辨证等等,但众多的辨证方法也容易使中医临床工作者在学习和选用辨证方法上产生困惑。现将常用的辨证方法之内在关系和辨证要点综合归纳、重新分类、执简驭繁,便于后学者学习和临床应用。     

Analysis of high and low responses toStaphylococcus aureus and interleukin 2 in human B lymphocytes

Fixed protein A-bearing staphylococci (SAC) stimulate human B cells via surface Ig, whereas IL-2 has been reported to provide a sufficient second signal for proliferation and differentiation. Using an ELISPOT assay to count cells secreting IgM, IgA, and IgG and flow cytometry with acridine orange to assess cell cycle progress, we have found that the purified B lymphocytes of a substantial minority (5/13) of healthy volunteers with normal serum Ig levels failed to differentiate to Ig secreting cells (ISC) in response to SAC + IL-2 (IgM, IgA, or IgG secreting cells, <5% of input B cells). High-responders generally formed 10–35% ISC. The proportions of B cells expressing IgG, IgA, IgM, or IgD were not different in the two groups. By average linkage cluster analysis, SAC/IL-2 high- and low-responders were shown to fall into two separate populations with respect to ISC. High- and low-responders tended to remain in the same group with repeated testing over several months, although some convergence was seen. The low-responders also showed significantly less advancement to late G1 and S phase than the high-responders, in the presence of SAC ± IL-2. Induction of IL-2 receptors on B cells by SAC + IL-2 was much greater in high-responders than in low-responders, as shown by flow cytometry with phycoerythrinconjugated IL-2. However, SAC + IL-2 induced transferrin receptors normally in low-responders, showing that some early activation steps occur in these cells. Low-responder B cells often improved their responses in the presence of macrophages and T cell supernatants. Finally, bypassing the surface Ig pathway using anti-CD3-activated T cells to stimulate B cells produced normal differentiation in low-responder B cells. Thus a subset of clinically normal individuals possesses B cells which fail to express IL-2 receptors, proliferate, and differentiate normallyin vitro in response to SAC + IL-2 yet can respond well to alternative activation pathways via T cells, monocytes, and their products.     

TLSFJM对同种异体抗原诱导的小鼠杀伤性T细胞分化及效应功能的影响

本文以小鼠混合淋巴细胞反应为模型，研究了ＪＭ 急性Ｔ 淋巴细胞白血病细胞分泌的抑制因子（ ＴＬＳＦＪＭ）在体外对同种异体抗原诱导小鼠Ｔ 细胞增殖和杀伤性Ｔ 细胞分化的影响。结果表明：（１） ＴＬＳＦＪＭ 可明显抑制同种异体抗原刺激的Ｔ 细胞增殖， 且具有剂量依赖性；（２） ＴＬＳＦＪＭ 能有效地抑制同种异体抗原诱导的小鼠ＣＴＬ 分化；（３）ＴＬＳＦＪＭ 对杀伤相ＣＴＬ 的杀伤活性没有抑制作用。     

Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound

The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD–OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results showed that RGD–OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.     

Isoform-specific functions of Ras in T-cell development and differentiation

Ras GTPases, well characterized for their role in oncogenesis, are the cells’ molecular switches that signal to maintain immune homeostasis through cellular development, proliferation, differentiation, survival, and apoptosis. In the immune system, T cells are the central players that cause autoimmunity if dysregulated. Antigen-specific T-cell receptor (TCR) stimulation activates Ras-isoforms, which exhibit isoform-specific activator and effector requirements, functional specificities, and a selective role in T-cell development and differentiation. Recent studies show the role of Ras in T-cell-mediated autoimmune diseases; however, there is a scarcity of knowledge about the role of Ras in T-cell development and differentiation. To date, limited studies have demonstrated Ras activation in response to positive and negative selection signals and Ras isoform-specific signaling, including subcellular signaling, in immune cells. The knowledge of isoform-specific functions of Ras in T cells is essential, but still inadequate to develop the T-cell-targeted Ras isoform-specific treatment strategies for the diseases caused by altered Ras-isoform expression and activation in T cells. In this review, we discuss the role of Ras in T-cell development and differentiation, critically analyzing the isoform-specific functions.