氯化锂诱导毛囊干细胞定向分化中Wnt信号通路介导基因的调控

背景：研究发现氯化锂有促进毛囊干细胞向毛囊上皮定向分化的作用,但其分子调节机制仍不清楚。 目的：通过外源性因子氯化锂干预人毛囊干细胞,观察Wnt信号通路中主要下游分子及目的基因的改变,探讨氯化锂对毛囊干细胞定向分化的诱导作用。 方法：取正常成人除皱术后的头皮,采用两步酶消化法结合手术显微镜分选毛囊干细胞,采用角蛋白10抗体-异硫氰酸荧光素鉴定毛囊干细胞。以外源性因子氯化锂干预人毛囊干细胞。 结果与结论：氯化锂干预5 d后可发现毛囊干细胞体积变大,核浆比减小,Wnt信号通路中β-catenin、cyclinD1表达增强,GSK3β、Tcf3表达减弱,c-myc在氯化锂浓度低时增强浓度高时减弱。提示氯化锂可影响毛囊干细胞中Wnt信号通路β-catenin、GSK3β、Tcf3、c-myc和cyclinD1的表达水平,可能对毛囊干细胞的分化起到重要作用。     

改良大鼠毛囊干细胞体外培养鉴定及诱导分化的研究

目的 探讨改良体外分离、培养、扩增、纯化SD大鼠毛囊干细胞(rHFSCs)及其免疫、超微结构的鉴定方法,观察rHFSCs的生物学特性及成脂、成骨分化能力。方法 2015年1—6月选取清洁级1周龄SD大鼠6只,在无菌条件下切取SD大鼠触须部皮肤,用 Dispase酶和Ⅳ型胶原酶混合液消化,显微镜下分离毛囊隆突部,用梯度加液组织块贴壁法培养rHFSCs,用两步酶消化法传代,用Ⅳ型胶原差速贴壁法纯化rHFSCs。收集第3代rHFSCs,采用流式细胞术、免疫荧光染色及透射电镜观察rHFSCs内部结构等方法鉴定rHFSCs。收集第1～10代rHFSCs检测细胞活力,第3、5、7、9代rHFSCs培养第1～8天时检测细胞增殖能力。收集第3代rHFSCs,按培养液的不同分为诱导组和对照组,对比两组目的基因PPAR-γ、C/EBPa、OPG、Runx2的相对表达量及染色后的光密度(OD)值变化情况,观察rHFSCs的成脂、成骨分化能力。结果 通过改良方法分离、培养、纯化的rHFSCs呈典型的铺路石状,生长曲线呈“S”形,生长增殖能力良好,克隆形成能力强。透射电镜显示细胞处于原始状态。流式细胞术检测显示Integrin β1、Integrin α6、P63呈高表达,CK15呈中等表达,符合rHFSCs标记鉴定特点。免疫荧光染色和透射电镜观察rHFSCs内部结构证明细胞的类型属于毛囊干细胞。rHFSCs成脂诱导培养7 d后RFqPCR定量结果显示,诱导组目的基因PPAR-γ、C/EBPa的相对表达量(5.598±0.168、4.757±0.416)均明显高于对照组(1.119±0.344、1.126±0.355),差异均有统计学意义(t=34.955、20.266,P值均＜0.01);诱导14 d后细胞油红O染色阳性,诱导组OD值(2.472±0.091)明显高于对照组(0.817±0.003),差异有统计学意义(t=114.641, P＜0.05)。rHFSCs成骨诱导培养14天后RFqPCR定量结果显示,诱导组目的基因OPG和Runx2的相对表达量(1.921±0.275,3.892±0.265)明显高于对照组(1.085±0.288,1.046±0.216),差异均有统计学意义(t=4.667、17.332, P值均＜0.01);诱导21 d后细胞茜素红染色阳性,诱导组OD值(0.716±0.016)明显高于对照组(0.076±0.002),差异有统计学意义(t=14.078, P＜0.01)。结论 采用改良体外分离、培养、扩增、纯化方法可成功建立rHFSCs体外培养体系,rHFSCs具有纯度大、增殖效率高、多向分化潜能强等特点,可为干细胞组织工程学相关发展提供良好的种子细胞。     

微环境调控毛囊干细胞增殖与分化的研究进展

毛囊干细胞生存的微环境改变使毛囊干细胞的增殖、分化也发生改变。微环境对干细胞的调控是通过信号转导通路及一些重要的信号分子实现的,如整合素家族、Wnt和Notch信号转导通路及c-myc基因和β-连环蛋白等。     

骨形态发生蛋白9体外诱导牙囊细胞的成骨分化

背景：有研究表明骨形态发生蛋白9能促进多种干细胞的成骨分化,但其是否具有诱导牙囊细胞成骨向分化的能力尚不清楚。目的：探讨骨形态发生蛋白9对大鼠牙囊细胞成骨分化的诱导作用。方法：以纯化的第3代大鼠牙囊细胞为研究对象,感染骨形态发生蛋白9腺病毒后,检测牙囊细胞中碱性磷酸酶活性、钙盐沉积以及矿化相关因子基因和蛋白的表达变化。结果与结论：感染骨形态发生蛋白9的牙囊细胞碱性磷酸酶活性持续增强,钙盐沉积明显增强。Real time PCR检测结果显示感染骨形态发生蛋白9的牙囊细胞中矿化相关因子碱性磷酸酶、骨钙素、骨涎蛋白、骨桥蛋白和核心结合因子mRNA表达增强。Western blot检测结果显示感染骨形态发生蛋白9的牙囊细胞中骨桥蛋白的表达增强。以上结果表明骨形态发生蛋白9可诱导牙囊细胞向成骨方向分化。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：     

干细胞诱导分化为毛囊及再生的研究进展

BACKGROUND:Stem cells have the potential to differentiate into various organs and tissues. In recent years, stem cells have been proved to differentiate into hair follicles under certain conditions. OBJECTIVE:To review the research progress and prospect of stem cells differentiating to hair follicles, thereby providing a reliable basis for clinical treatment of serious hair follicle injury. METHODS:A computer-based search of PubMed, EMBASE, WanFang, CNKI databases was performed for related articles published between 2013 and 2015, using the keywords of “cell hair, follicle stem, medicine regenerative, differentiation” in English and Chinese. A total of 207 articles were retrieved, and finally 34 articles were included in result analysis. RESULTS AND CONCLUSION:Stem cells from different sources all have the ability to differentiate into hair follicles under certain inductions. However, it is important to seek more scientific and rational methods for the differentiation of stem cells into hair follicles based on overcoming their own shortcomings. A great progress has been made in animal experiments and subclinical trials, and even a great breakthrough in some aspects. Further studies on combining the advantages and overcoming the shortcomings of various stem cells during differentiation are required for the clinical treatment of serious hair follicle injury.     

5-氮胞苷诱导体外培养的胚胎干细胞向心肌样细胞分化

目的研究5-氮胞苷(5-aza)体外诱导胚胎干细胞分化为心肌细胞的可行性。方法将P19细胞接种于培养板中或铺有琼脂的培养板中,5μmol/L5-aza培养7d后用不含5-aza的培养基继续培养。倒置显微镜观察细胞跳动情况,用免疫细胞化学、RT-PCR检测及电镜观察等方法鉴定细胞分化。结果5-aza暴露结合悬浮培养可诱导P19细胞分化为有节律跳动的心肌细胞。分化的细胞表达心肌特异的GATA-4、α-MHC mRNA及α-sarcomeric actin、cTnT蛋白,同时透射电镜观察到细胞质内有明显的肌丝。结论5-aza在体外可诱导胚胎干细胞向心肌样细胞分化,悬浮培养有助于细胞的心肌化过程。     

Directing stem cell differentiation into the chondrogenic lineage in vitro

A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage-related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.     

小鼠毛囊来源间充质干细胞体外诱导成软骨的细胞标记物

背景：小鼠毛囊来源的间充质干细胞有很强的多向诱导分化能力,但少有研究关注其在诱导分化为软骨细胞过程中的细胞表面标记物。 目的：观察小鼠毛囊来源的间充质干细胞分化为软骨细胞过程中的形态、细胞表面标记物和硫酸黏多糖含量的变化。 方法：体外分离培养ICR新生小鼠皮肤干细胞,用成软骨细胞诱导液诱导培养7,14 d观察各项指标。 结果与结论：经成软骨细胞诱导液处理后,小鼠毛囊来源的间充质干细胞中CD44(+)细胞数量无明显增加, 硫酸黏多糖含量无变化,CD54(+)细胞和CD166(+)细胞则显著增加,两者硫酸黏多糖含量亦明显升高。表明小鼠毛囊来源的间充质干细胞能诱导形成软骨样细胞;CD44不能作为小鼠毛囊来源的间充质干细胞向软骨细胞分化的细胞表面标记物;CD54和CD166以及硫酸黏多糖可作为对小鼠毛囊来源的间充质干细胞向软骨细胞分化的监测指标。     

Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy

Chemotherapy-induced alopecia represents one of the major unresolved problems of clinical oncology. The underlying molecular pathogenesis in humans is virtually unknown because of the lack of adequate research models. Therefore, we have explored whether microdissected, organ-cultured, human scalp hair follicles (HFs) in anagen VI can be exploited for dissecting and manipulating the impact of chemotherapy on human HFs. Here, we show that these organ-cultured HFs respond to a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC), in a manner that resembles chemotherapy-induced HF dystrophy as it occurs in vivo: namely, 4-HC induced melanin clumping and melanin incontinence, down-regulated keratinocyte proliferation, massively up-regulated apoptosis of hair matrix keratinocytes, prematurely induced catagen, and up-regulated p53. In addition, 4-HC induced DNA oxidation and the mitochondrial DNA common deletion. The organ culture system facilitated the identification of new molecular targets for chemotherapy-induced HF damage by microarray technology (eg, interleukin-8, fibroblast growth factor-18, and glypican 6). It was also used to explore candidate chemotherapy protectants, for which we used the cytoprotective cytokine keratinocyte growth factor as exemplary pilot agent. Thus, this novel system serves as a powerful yet pragmatic tool for dissecting and manipulating the impact of chemotherapy on the human HF.     

Kinetics of proliferation and differentiation in the hair follicle and epidermis in neonatally undernourished rats.

Cell population growth kinetics of hair follicle and epidermis were measured in rats undernourished during suckling by increasing the litter size. Animals reared in litters of normal size served as controls. There was a marked atrophy of epidermis and hair follicles, and the thickness and length of the hairs were markedly reduced in the undernourished animals. Autoradiography following pulse labeling with 3H TdR demonstrated prolongation of the cell generation cycle. The G2 and S phases were chiefly affected, whereas the G1 was not altered. The double pulse labeling technique indicated a hold-up in the S phase in the epidermis, although the labeling index was not altered in the undernourished rats. On the basis of available literature, it is porposed that G2 is more energy dependent, whereas protein deficiency affects predominantly the S phase. Unlike the alteration in cell generation cycle, the process of differentiation--measured histochemically and on the basis of rate of migration of 3H-TdR-labeled bulb cells--appeared to be unaffected in undernutrition. However, there was a lag in the movement of cells out of the germinative zone. It is proposed that in undernutrition, cells spend longer time in the germinative zone to complete a critical number of division which may be peculiar to every tissue.     

Lectin histochemistry of glycoconjugates in the feline hair follicle and hair

The distribution of glycoconjugate in the feline hair follicle and hair was studied by light and electron microscopic histochemical methods. The hair apparatus was found to contain considerable amounts of complex carbohydrates with different saccharide residues (alpha-D-mannose, beta-D-glucose, alpha-L-fucose, beta-N-acetyl-D-glucosamine). Variations of those were detected in the plasma membrane of the hair follicle cells during the course of their differentiation and keratinization, namely, alph-D-glucose, alpha-L-fucose and beta-N-acetyl-D-glucosamine in the suprabulbar and bulbar regions. The reaction level of sialic acid residues in the plasma membrane decreased in some cell layers during the course of differentiation. The results obtained from the present study indicated that interaction between saccharide residues of neutral carbohydrates and sialyl groups during the anagen phase might contribute to cell keratinization in hair follicles and hairs. It is discussed whether the existence of glycogen in outer root sheath cells might enable these cells to provide other hair apparatus cells with energy when necessary. Moreover, it became obvious from variations in sialyl residue distribution that cell differentiation processes terminate first of all in Huxley's and Henle's layers within the suprabulbar region of the hair follicle, as followed by the hair cortex.     

虫草多糖体外诱导大鼠骨髓间充质干细胞分化为类肝细胞样细胞

背景：如何促进骨髓间充质干细胞向肝细胞完全意义上的转化,以便更有效改善病变肝组织的结构和功能将成为未来相关研究的重中之重。 目的：探讨虫草多糖在体外诱导大鼠骨髓间充质干细胞分化为类肝细胞样细胞的可行性。 方法：采用贴壁法培养Wistar大鼠骨髓间充质干细胞,实验分3组对骨髓间充质干细胞进行诱导分化,虫草组培养液中加虫草多糖进行诱导,终末质量浓度为0.15 g/L;阳性对照组培养液中加肝细胞生长因子和表皮生长因子联合诱导,质量浓度分别为20 μg/L和10 μg/L;空白对照组仅用含体积分数为10%胎牛血清的DMEM培养液。 结果与结论：分离纯化的骨髓间充质干细胞经流式细胞仪检测显示CD34阴性表达,CD44阳性表达。诱导分化7 d时虫草组及阳性对照组细胞出现甲胎蛋白表达,14 d时表达增强,28 d时表达减弱,阳性对照组甲胎蛋白阳性率高于虫草组;诱导分化7 d时阳性对照组细胞角蛋白18出现表达,14 d时表达增强,虫草组则在14 d时出现表达,而后持续。诱导分化7 d时虫草组及阳性对照组白蛋白表达阴性,14 d时出现阳性。诱导分化14 d时虫草组及阳性对照组细胞糖原染色阳性,28 d时表达减弱。空白对照组结果皆为阴性。结果可见虫草多糖可以在体外诱导大鼠骨髓间充质干细胞分化为类肝细胞样细胞。     

大鼠骨髓源性血管内皮祖细胞体外培养分化及鉴定

目的：探索大鼠骨髓源性血管内皮祖细胞（EPCs）的培养、诱导分化及鉴定方法。方法：冲洗大鼠骨髓腔,梯度密度离心法获得单个核细胞,内皮细胞培养液EGM-2MV培养,通过细胞形态,免疫组化和免疫荧光检测CD34、VEGFR-2、vWF,CD133,摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,超微结构及染色体核型分析进行鉴定。结果：新分离的骨髓单个核细胞呈圆形,大小不一;48h后部分细胞开始贴壁,呈梭形、纺锤形或不规则形;4～8d呈细胞集落或线状排列;9～11d接近融合,呈典型鹅卵石样外观。贴壁细胞CD34、CD31、VEGFR-2、vWF、CD133均表达阳性并呈动态变化,能够摄取Dil-ac-LDL和结合FITC-Lectin-UEA-1,透射电镜见特征性Weible-Palade小体,能稳定保持二倍体核型。结论：本实验初步建立了一套大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定方法体系。     

毛囊真皮鞘细胞中过表达Noggin对皮肤及毛囊生长的作用

背景:Noggin蛋白是一个抑制BMP信号通路的重要分子,可以与BMP2/4结合形成复合物,从而阻断BMP信号,影响生物有机体的正常发育过程。研究认为真皮鞘细胞是一类能够长期自我更新的真皮干细胞,参与毛囊再生过程中真皮毛乳头和真皮鞘的形成。在毛囊发育过程中,真皮毛乳头中Noggin参与毛囊的起始,Noggin的缺失会导致毛囊数量的减少和毛囊生长缓慢,但人们对于Noggin蛋白在毛囊真皮鞘中的作用所知甚少。目的:研究Noggin蛋白在毛囊真皮鞘中的生物学功能。方法:利用真皮鞘特异的αSMA-Cre ERT2工具鼠在真皮鞘中特异过表达Noggin蛋白,分别在出生后8,9 d对实验组αSMA-CreER;pMES-Noggin小鼠和对照组αSMA-CreER小鼠注射4-羟基他莫昔芬,在出生后21,23,28 d获取皮肤组织,苏木精-伊红染色观察毛囊生长情况和皮下脂肪层厚度,免疫组化染色分析表型。结果与结论:通过苏木精-伊红染色结果发现毛囊生长并没有受到影响,但毛囊皮下脂肪组织生长发育受到严重影响,小鼠皮下脂肪层变薄。免疫组化结果说明BMP信号降低可能是导致毛囊脂肪层变薄的原因。这一发现拓展了人们对于真皮鞘细胞和Noggin蛋白的认识,也为人们更加准确理解毛囊再生和组织生长机制提供了重要依据。     

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.     

角质细胞无血清培养基促进大鼠毛囊干细胞的增殖

背景：以往研究培养毛囊干细胞多使用DMEM/F12+体积分数10%胎牛血清培养基,而进年来开发出的角质细胞无血清培养基也可应用于毛囊干细胞的培养。 目的：观察3种不同培养基对大鼠毛囊干细胞增殖情况及干细胞纯度的影响。 方法：取大鼠触须部皮肤组织,体式显微镜下分离出毛囊组织,中性蛋白酶Ⅱ与胰蛋白酶和乙二胺四乙酸混合液“两步酶法”消化。所得细胞悬液按细胞数平均分为3份,分别使用角质细胞无血清培养基、DMEM/F12+体积分数10%胎牛血清及角质细胞无血清培养基+体积分数10%胎牛血清共3种培养基培养,Ⅳ型胶原差速贴壁法筛选毛囊干细胞,进行传代培养。 结果与结论：培养毛囊干细胞传至第3代,锥虫蓝染色计数法检测结果显示,此3组间细胞活率差异无显著性意义(P > 0.05)。CCK8比色法检测细胞生长曲线显示,培养前2 d,此3组细胞生长均较缓慢;培养4 d,细胞生长进入对数生长期,3种培养基培养的细胞增殖活性：角质细胞无血清培养基+10%胎牛血清组>DMEM/F12+10%胎牛血清>角质细胞无血清培养基(P < 0.05)。流式细胞仪检测显示,角质细胞无血清培养基组CD34的表达高于角质细胞无血清培养基+10%胎牛血清组(P < 0.05)。DMEM/F12+10%胎牛血清组中CD34、β1-整合素(CD29)及CK15标记物的表达低于其他2组(P < 0.05)。结果表明,角质细胞无血清培养基较DMEM/F12+体积分数10%胎牛血清能培养出纯度更高的毛囊干细胞,并且在此培养基培养的基础上加入血清,能够促进毛囊干细胞的增殖。     

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.