单克隆人胰腺干细胞分离培养体系的建立

本研究探讨了建立单克隆人胰腺干细胞分离培养体系及单克隆人胰腺干细胞系．对一些影响干细胞增殖的因素进行了分析。无菌取人流产胎儿胰腺组织,切碎至1mm3,0．1％ Ⅳ型胶原酶消化,低糖DMEM、10％FBS、3．7g／LNaHCO3、0．08g／L青霉素及0．1g/L链霉素培养液贴壁培养细胞,2．5g／L胰蛋白酶＋0．4g／LEDTA消化传代。克隆环筛选单克隆干细胞,培养液中添加10ng／mLEGF．扩增单克隆干细胞。采用核型分析法检测干细胞染色体,MTT法测定干细胞生长曲线。胶原酶消化胰腺组织．获得单个细胞和细胞团。贴壁培养．原代上皮样胰腺干细胞克隆性生长。胰蛋白酶消化传代,上皮样胰腺干细胞逐渐被纯化。克隆环筛选,获得单克隆人胰腺干细胞。扩增培养,1例来源于4月龄男性流产胎儿胰腺组织干细胞建系,传50代。染色体核型分析．该干细胞系为正常的二倍体细胞。细胞生长曲线显示培养1—4d,干细胞生长缓慢,5-6d,进入倍增期。培养液中添加15％FBS,干细胞增殖较快。再添加15ng／mLEGF或10ng／mL IGF—Ⅱ．干细胞增殖更快。研究结果表明应用本实验建立的细胞分离培养体系获得了单克隆人胰腺干细胞系。     

中国仓鼠卵巢细胞的悬浮适应及代谢特征

目的：通过悬浮适应,使中国仓鼠卵巢细胞（CHO细胞）获得悬浮生长的特性,并可在悬浮培养条件下较快地生长。方法：将CHO细胞以3×10^5／mL接种于100mL的三角瓶内,培养时加入1％小牛血清、1g/LPIuronic F-68、25μg／mL硫酸葡聚糖,培养体积35mL,摇床转速90r／min,每24h离心换液,当细胞增殖为2×10^6／mL时传代。结果：经过悬浮适应,细胞的平均比生长速率由适应最初的0．27／d提高为适应后的0．48／d,最大总细胞密度由适应初期的2．5×10^6／mL提高为适应后的6．3×10^6／mL,目的蛋白活性也由适应前的2781U／mL提高为适应后的8878U／mL,适应后细胞的葡萄糖平均比消耗率为1．42μmol／（10^6细胞·d）,低于适应前的2．16μmol／（10^6细胞·d）。结论：贴壁生长的CHO细胞经过悬浮适应,不仅可以在悬浮培养条件下快速生长,而且细胞对葡萄糖的利用率也得到提高。     

不同浓度TGF-β1对肺泡II型细胞系MLE-12表型及功能的影响

探讨不同浓度及不同时间点TGF-β1对肺泡II型上皮细胞（AECII）表型及功能的影响。小鼠肺泡II型细胞系MLE-12,随机分为：对照组（0ng／mL）、低浓度组（0．1ng／mL）、中浓度组（1ng／mL）和高浓度组（10ng／mL）。应用细胞免疫荧光双标法及荧光定量PCR法观察各组12,24,48,72h细胞形态变化、AECII标记（肺表面活性物质蛋白B,SP—B）及成纤维细胞标记（成纤维细胞特异性蛋白1,FSP—1）蛋白及mRNA的表达情况。结果表明,随着TGF—β1干预时间的延长及浓度的升高,AECII逐渐由鹅卵石样变成纺锤体形状,获得成纤维细胞样外观。蛋白水平,AECII标记SP-B表达逐渐减弱,成纤维细胞标记FSP-1表达逐渐增强,48h中浓度组及24h高浓度组两者可见明显的共表达,同时,其SP-BmRNA表达较同时间对照组下调,而FSP1 mRNA表达较同时间对照组上调。低浓度组各时间点上述表现不明显。TGF—β1促使AECII向成纤维细胞转化（EMT）,且具有时间及浓度依赖性。     

恒河猴血清孕酮含量测定方法

目的建立恒河猴血清中孕酮含量测定方法。方法本文采用放射免疫测定技术。结果孕酮的回收率为94％,批内CV为5．1％～8．3％,批间CV为4．5％～7．7％,灵敏度为5～10Pg。说明该方法具有较高的灵敏度、特异性、准确性。分别测定了幼年组、成年组和老年组的雌性恒河猴的血清中的孕酮含量分别为：（0．20±0．04）ng／mL、（6．26±0．17）ng,mL和（0．35±0．06）ng/mL;成年雌性恒河猴月经周期孕酮的变化范围为：滤泡期为（1．10±0．12）ng／mL,排卵期（2．36±0．18）ng/mL,黄体期（6．17±0．15）ng/mL,妊娠期随着妊娠月份的增加,孕酮浓度也增加,最高可达50ng／mL。结论经实验验证,该方法灵敏、可靠、适用,可作为恒河猴血清中孕酮含量测定的一种方法。     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

芦竹内生真菌F0238细胞生长及其代谢调控

对芦竹内生真菌F0238的细胞生长和代谢产曲酸量进行了代谢调控。结果表明,F0238生长及产曲酸的营养和环境条件为：PDA培养基,8％淀粉为碳源,0．2％蛋白胨为N源,发酵温度28℃,初始pH为6．5,发酵时间5d／（120h）,装液量80mL/500mL三角瓶。在摇瓶试验的基础上,对该菌发酵过程作了初步放大试验（10L全自动发酵罐）,得到F0238发酵过程的动态曲线。动态曲线反映了在一个发酵周期内,发酵液的pH值、DO值及残糖的降低趋势和生物量与抗菌产物量的上升趋势。     

叶酸靶向载顺铂羧甲基-β-环糊精纳米复合物的制备及对鼻咽癌的体外抑制效应

为达到鼻咽癌（nasopharyngeal carcinoma,NeC）的靶向化疗,该研究通过酰胺化反应和配位偶联技术制备叶酸（folicacid,FA）分子靶向载川页铂（cisplatin,CDDP）羧甲基-β-环糊精（carboxymethyl-β-cyclodextrin,CM—β—CD）纳米复合物（FA-CM—β—CD—CDDP）,采用邻苯二胺（o-phenylenediamine,OPDA）比色法检测复合物中CDDP含量,紫外分光光谱检测FA含量,透射电镜观察复合物形态,激光粒度仪测定复合物粒径大小。荧光显微镜观察NPC叶酸受体（folatereceptor,FR）阳性HNE-1细胞及FR阴性CNE-2细胞对偶联FITC的复合物的吞噬及OPDA比色法检测细胞内CDDP的浓度。通过MTT法、集落形成实验和流式细胞术检测复合物对HNE-1细胞增殖能力和凋亡的影响。研究结果显示,复合物中偶联的FA和CDDP浓度分别为340gg／mL和2mg／mL,CDDP包封率达20．00％,复合物粒径均匀且大小为157．8nm。HNE-1细胞内见较多FITC,细胞内CDDP浓度为6．24ng／mL,而CNE-2细胞内FITC较少,细胞内CDDP浓度仅约2．01ng／mL。HNE-1生长抑制率在24h明显高于对照组（CM—β—CD—CDDP）,其IC50（4．80μg／mL）明显低于对照组（6．97μg／mL）,但当所载的CDDP终浓度达到16．00μg／mL时,两组抑制率均达到80％以上;作用48h两组抑制率无明显差异。在24h,当复合物的CDDP终浓度为1．00μg／mL时,HNE—1的集落形成率为33．21％,明显低于对照组（52．27％）。当复合物的CDDP终浓度为0．25μg／mL和1．00μg／mL时,HNE-1的凋亡率分别达12．65％和22．35％,明显高于对照组（6．91％和14.21％）。研究结果表明,成功构建的FA—CM—β—CD．cDDP纳米复合物能够靶向抑制FR阳性的NPC细胞增殖并促进其凋亡。     

多胺生物合成抑制剂D-精氨酸对拟南芥幼苗根系生长的影晌

为探讨多胺生物合成抑制剂D-精氨酸（D-arginine,D-Arg）对拟南芥根系生长的影响,首先用腐胺（0．1mmol‘L-1）和D—Arg（1．0mmol·L-1）处理种子萌发后生长2d的拟南芥幼苗。腐胺（0．1mmol·L-1）显著促进主根伸长,D-Arg（1．0mmol-L-1）显著抑制主根伸长,并对主根根尖的细胞形态有明显影响。为了进一步了解D—Arg影响拟南芥主根生长的机理,采用浓度梯度D．Arg处理幼苗根系。实验结果表明,随着D-Arg浓度增加（0．2～1．0mmol·L-1）,拟南芥幼苗主根生长受抑制的程度越严重。微分干涉观察主根根尖发现,外源施加D—Arg,引起拟南芥主根根尖分生区的细胞数目减少,使拟南芥幼苗表现出主根的伸长生长变缓。当分生区数目较少时,出现主根几乎不再仲长的现象。由此推测,多胺生物合成抑制剂D-Arg对拟南芥幼苗根生长的抑制作用机制,是D-Arg影响了其根尖分生区的细胞分裂活动,使分生区细胞数目减少,从而引起分生区长度减小,最终导致拟南芥主根仲长生长受到抑制。     

喉鳞癌患者血清中内皮抑素水平对喉鳞癌诊断价值的研究

目的：探讨喉鳞癌患者血清中内皮抑素水平的变化,及其与肿瘤临床分期及预后的关系。方法：（1）对50例喉鳞癌患者、30例喉息肉患者和30例健康人用ELISA方法检测血清中内皮抑素水平;（2）对喉鳞癌组不同临床分期的血清中内皮抑素的水平进行比较。结果：（1）喉鳞癌组血清中内皮抑素水平（51．45±19．83ng／mL）,显著高于喉息肉组（34．56±12．4ng／mL）和正常对照组（33．12±13．04ng／mL）,差别有统计学意义（P〈0．01）。喉息肉组与正常对照组之间差别无显著性（P〉0．05）。（2）Ⅱ期喉鳞癌患者血清内皮抑素的水平（66．22±10．89ng／mL）高于Ⅰ、Ⅱ期患者内皮抑素水平（39．31±14．42ng／mL,47．98±22．01ng／mL）,差别有统计学意义（P〈0．01）,Ⅰ期与Ⅱ期之间内皮抑素水平差别无显著性（P〉0．05）。结论：内皮抑素含量水平可以作为喉鳞癌的诊断及顸后判断的重要指标之一。     

矿质营养与其他生长物质对荷叶离褶伞菌丝生长的影响

1mg／mL KCl促进荷叶离褶伞菌丝生长;5或10mg／mL NaCl、5或10mg／mL MgSO4、5或10mg／mLKCl、10mg／mL H2PO4和1mg／mL CaSO4抑制菌丝生长;0．8mg／mL的MnSO4和CuSO4以及0．5mg／mL FeSO4、0．2或0．5mg／mL CoCl2和0．2、0．5或0．8mg／mL ZnSO4促进菌丝生长;0．5或0．2mg／mL CuSO4、0．2或0．5mg／mL MnSO4及0．8或0．2mg／mL FeSO4对菌丝生长的影响不显著;维生素B6、维生素C、维生素PP和维生素B1可促进菌丝生长,在含有10μg/L维生素B6的培养基上菌丝生长速度最快,但维生素C试用浓度较低（50μg／L）时对菌丝生长的影响不显著;吲哚丁酸、吲哚乙酸、奈乙酸对菌丝生长具有促进作用,但0．1、0．5或1．0μg／L赤霉素对菌丝生长的影响不显著。     

Sostdc1 Secreted from Cutaneous Lymphatic Vessels Acts as a Paracrine Factor for Hair Follicle Growth

In our previous study, we found that lymphatic vessels stimulate hair follicle growth through paracrine effects on dermal papilla cells. However, the paracrine factors secreted from cutaneous lymphatic vessels that can activate dermal papilla cells are still unknown. In this study, we investigated whether lymphatic endothelial cells might secrete paracrine factors that activate dermal papilla cells in vitro. We found that Sostdc1 was more expressed in lymphatic endothelial cells compared with blood vascular endothelial cells. In addition, Sostdc1 expression levels were significantly increased during the anagen phase in the back skin of C57BL/6J mice, as compared to the telogen phase. We also observed that incubation of dermal papilla cells with 200 ng/mL Sostdc1 for 72 h induced the expression levels of Lef-1, a downstream target of Wnt signaling. Taken together, our results reveal that Sostdc1, a BMP antagonist, secreted from cutaneous lymphatic vessels, may act as a paracrine factor for hair follicle growth.     

Functional role of beta 1 integrin-mediated signalling in the human hair follicle

Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, beta1 integrins and their ligands are of particular interest. It is not yet known whether beta1 integrins play any role in the regulation of human hair growth and the expression pattern of beta1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for beta1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of beta1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, beta1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two beta1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that beta1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of beta1 integrin-mediated signalling is a suitable candidate for human hair loss management.     

黄芪、何首乌、女贞子、菟丝子混合提取物对体外培养毛囊生长的影响及药理作用研究

目的:通过体内外实验探讨黄芪、何首乌、女贞子、菟丝子混合中药提取物对毛囊增殖的影响作用以及其作用机理。方法:通过体外培养的C57BL/6小鼠毛囊器官模型观察不同浓度中药提取物对毛囊生长的影响;采用MTT法测定不同浓度中药提取物对毛乳头增殖的影响;蛋白免疫印迹法(Western Blot)和ELISA检测中药提取物对毛乳头细胞分泌肝细胞生长因子(HGF)的影响。结果:中药提取物能够刺激体外培养的小鼠毛囊的生长,800μg/mL浓度的促进作用最强;160μg/mL中药提取物对毛乳头细胞的增殖作用最强,与米诺地尔、齐墩果酸阳性对照存在显著性差异(P0.05)。而且,药提取物促进了毛乳头细胞分泌HGF。结论:黄芪、何首乌、女贞子、菟丝子混合中药提取物在促进毛发生长中起到重要作用,促进毛乳头细胞增殖和分泌HGF是促进毛囊生长的可能性药理机制。     

Effect of growth factors on in vitro development of caprine preantral follicle oocytes

The objective of this work was to examine the effect of various growth factors including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), either individually or in association, in the presence of follicle stimulating hormone (FSH) on the in vitro growth and viability of caprine preantral follicle oocytes. Preantral follicles were disassociated enzymatically and mechanically from prepuberal caprine ovaries after the animals were anesthetically ovariectomized. In experiment, caprine preantral follicles in groups 1–4 were cultured in growth culture medium, growth culture medium + EGF, growth culture medium + IGF-I and growth culture medium + IGF-I + EGF, respectively, for 9 days. The results indicated that EGF (50 mg/l) increased the survival rate of oocytes, but decreased the growth rate of oocytes; IGF-I (100 mg/l) effectively maintained the survival of oocytes and stimulated their growth; IGF-I (100 mg/l) and EGF (50 mg/l) in combination produced a higher effect on both of the survival and the growth rate of oocytes than IGF-I or EGF alone. Conclusively, the growth factors can effectively maintain the survival of caprine preantral follicle oocytes and regulated their growth in culture. EGF and IGF-I in association could synergically meliorate the culture system of caprine preantral follicle oocytes.     

Establishment of an in vitro organoid model of dermal papilla of human hair follicle

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three‐dimensional (3D) DP spheroids (DPS) with or without a silk–gelatin (SG) microenvironment, which showed enhanced DP‐specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil‐treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell–cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial–mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.     

Stimulation of Human Hair Growth by the Recombinant Human Keratinocyte Growth Factor-2 (KGF-2)

Keratinocyte growth factor-2 (KGF-2) is found in dermal papilla fibroblasts and its receptor, fibroblast growth factor receptor 2 (FGFR2), in the neighboring outer root sheath of keratinocytes. Administration of recombinant human KGF-2 (rhKGF-2) at 10 ng ml⁻¹ significantly stimulated human hair-follicle cell proliferation in organ culture (26–35%). Thus, rhKGF-2 is a promising therapeutic agent to stimulate human hair growth.     

In vitro methodology, hormonal and nutritional effects and fibre production in isolated ovine and caprine anagen hair follicles

Mammalian hair follicles are complex multicellular structures in the skin, which produce hair fibre under the influence of locally produced and systemic signalling systems. Investigation to determine mechanisms of regulation, follicular responses and the importance of nutritional supply have utilised a number of in vivo and in vitro approaches. Included in these are studies on isolated intact anagen secondary follicles singly or in groups with incubation in culture medium. These utilise techniques developed for investigation of follicles from human skin. Results from selected studies reviewed here demonstrate differences in capacity for hair growth and protein synthesis between secondary follicles from Angora and cashmere-bearing goats. Mohair follicles were shown to exhibit faster hair shaft elongation both in vivo and in vitro, to have greater DNA content per follicle and to deposit significantly more protein per follicle and per unit of DNA. Incubation of anagen mohair and cashmere follicles in the presence of melatonin or prolactin showed positive responses in hair shaft growth and protein synthesis to both signalling molecules. This result indicated directly acting effects on the follicle in addition to any indirect effects arising at a whole animal level in response to, for example, variation in photoperiod. Similarly, epidermal growth factor was shown to alter elongation and protein synthesis in mohair follicles and to produce, at higher concentration, club hair structures similar to effects observed in other species. The vitamin biotin was shown to be important in maintaining viability of isolated sheep secondary hair follicles where supplementation increased the proportion continuing to grow. Effects on growth and apparent protein synthesis suggested comparatively lesser effects on follicles, which remained viable. Histology on follicles indicated effects of biotin deficiency in reducing proliferation of basal keratinocytes. The final study, included in this review, demonstrated that supply of the essential sulphur-containing amino acid l-methionine was necessary to maintain the viability and growth of mohair follicles. l-cysteine was not required in the presence of l-methionine, although there was evidence of an optimisation when both amino acids were present in adequate concentrations. Consideration is given to the importance of transport mechanisms and capacity to utilise absorbed nutrients when considering optimising nutritional supply to individual follicles. These may then provide targets for attainment in applied nutrition of animals in vivo.     

Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle

Alopecia areata (AA) is an autoimmune hair loss disease caused by a cell-mediated immune attack of the lower portion of the cycling hair follicle. Feeding mice 3–7 times the recommended level of dietary vitamin A accelerated the progression of AA in the graft-induced C3H/HeJ mouse model of AA. In this study, we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells (SCs) to induce the development and growth phase of the hair cycle (anagen), which may have made the hair follicle more susceptible to autoimmune attack. Our purpose here is to determine the mechanism by which dietary vitamin A regulates the hair cycle. We found that vitamin A in a dose-dependent manner increased nuclear localized beta-catenin (CTNNB1; a marker of canonical wingless-type Mouse Mammary Tumor Virus integration site family (WNT) signaling) and levels of WNT7A within the hair follicle bulge in these C3H/HeJ mice. These findings suggest that feeding mice high levels of dietary vitamin A increases WNT signaling to activate hair follicle SCs.     

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration

An increasing number of studies show that platelet‐rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15⁺ hair follicle stem cell proliferation in K14‐H2B‐GFP mice. Moreover, PSS promoted skin‐derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.     

Organotypic culture of outer root sheath cells from human hair follicles using a new culture device

Summary A new culture vessel for the growth of cells on biological substrata and under organotypic conditions is described. This device, named Combi-ring-dish (CRD), is composed of four concentric rings designed to take up one or several substrata on which cells can be grown either immersed in culture medium or exposed to air and fed from underneath. Using the CRD, outer root sheath cells, isolated from plucked human hair follicles and plated on growth-arrested 3T3 feeder layers, were grown on native collagen lattices populated with living human fibroblasts. After reaching confluence, the immersed cultures were recombined (in vitro) with pieces of freeze-killed dermis and grown further, exposed to air. Thus by mimicking epidermal growth conditions, differentiation was dramatically improved, compared to control cultures on plastic substratum. Virtually all morphologic features of interfollicular epidermis developed. This seems a suitable model to investigate the differentiation potential of human hair follicle cells.