小鼠精原干细胞体外培养体系建立与功能鉴定

目的构建小鼠精原干细胞(SSCs)体外培养体系,并对培养的SSCs进行功能鉴定。方法采用两步酶法消化小鼠睾丸组织制备单细胞悬液,CD90、CD49f免疫磁珠分选SSCs,采用Sertoli细胞共培养方法体外培养SSCs,并将培养的SSCs移植到受体小鼠曲细精管,定期观察移植后SSCs在受体小鼠曲细精管增殖分化情况。结果本次研究培养的SSCs在体外大量增殖,SSCs标记物检测显示,CD90、CD49f未分化SSCs标记物表达阳性率均99%,而CD117分化SSCs标记物表达阳性率1%,SSCs转染GFP慢病毒后移植到受体小鼠曲细精管内可以广泛定植并增殖分化,移植后12w在受体小鼠曲细精管内可见GFP~+精子细胞。结论本研究中建立的小鼠SSCs体外培养体系,不仅能够促进SSCs大量增殖,同时保持了其未分化SSCs干细胞特性。     

脑脊液诱导的骨髓源性神经样细胞移植的安全性研究

目的：研究脑脊液诱导的骨髓源性神经样细胞（BMSC-Ns）移植的安全性。方法将40只4~6周龄裸鼠采用随机数字表法分为5组（n=8）,其中致瘤性实验3组：阳性对照组（A组）,接种Hela细胞（2×107/0.2ml）;实验组（B组）,将BMSC-Ns按2×107/0.2 ml接种至裸鼠肋部皮下;阴性对照组（C组）,注射等体积生理盐水。接种后饲养1个月,大体观察肿瘤形成情况,1个月后处死动物取注射处局部皮肤、心脏、肝脏、肠道和肺组织,行H-E染色观察病理学改变。全身毒性实验2组：实验组（D组）,裸鼠尾静脉注射大鼠BMSC-Ns（5×106/0.2ml）;阴性对照组（E组）,注射等体积生理盐水。注射后即刻观察动物临床反应,以后每天观察1次,连续观察15天;将动物处死,采集血液标本行血液学检查。结果 BMSC-Ns接种于裸鼠肋部皮下1个月后未见接种部位肿瘤形成,局部皮肤、心脏、肝脏、肠道和肺组织病理学检查未见异常;尾静脉注射BMSC-Ns后动物一般情况无异常,至第15天裸鼠全部成活,血常规结果无异常。结论皮下接种或者尾静脉移植CSF诱导的BMSC-Ns对裸鼠是安全的,无致瘤性和全身毒性。     

鲜红斑痣内皮细胞的分离培养及鉴定

目的 探讨从鲜红斑痣分离培养内皮细胞的方法。方法 鲜红斑痣标本经胶原酶消化，CD31-免疫磁珠分选纯化细胞，接种于纤维连接蛋白铺层的培养皿；分别进行细胞形态学观察、细胞生长曲线的测定、培养细胞免疫荧光鉴定、低密度脂蛋白吸收试验。结果 从鲜红斑痣组织分离纯化出CD31＋细胞，在纤维连接蛋白铺层上6 h开始贴壁，细胞呈梭形，7 d生长融合呈铺路石状，CD31-免疫磁珠黏附于细胞上。鲜红斑痣内皮细胞CD31、vWF表达阳性，可摄取低密度脂蛋白。结论 建立鲜红斑痣内皮细胞的分离培养方法。     

单一核细胞条件培养液与细胞因子组合培养体系对骨髓HPP-CFC集落形成影响的研究

目的：观察两种培养体系对骨髓高增殖潜能集落形成细胞（high proliferative potential colony forming cell,HPP-CFC）集落形成的影响。方法：密度梯度离心法分离骨髓单一核细胞,分别在植物血凝素（PHA）刺激的单一核细胞条件培养液（phytohemagglutinin monocyte medium,PHA-MCM）及细胞因子组合支持下进行甲基纤维素半固体培养,14d时倒置显微镜下观察并计数两种培养体系中形成的集落数目及直径。结果：PHA-MCM与细胞因子组合均可支持HPP-CFC的集落形成,但细胞因子组合培养体系的集落数目明显高于PHA-MCM体系（P〈0.05）,而且集落直径明显大于后者。结论：在HPP-CFC的体外培养中,细胞因子组合培养体系明显优于PHA-MCM体系,是骨髓HPP-CFC较理想的体外培养体系,为银屑病及其它皮肤病的骨髓造血细胞研究奠定了一定的实验基础。     

鳞状细胞癌细胞系12F细胞中白介素1αmRNA的表达及对紫外线照射的反应

目的 了解人角质形成细胞(KC)起源的鳞状细胞癌细胞系(SCC)12F细胞自发表达白介素1α(IL-1α)mRNA的能力以及紫外线照射对IL-1α分泌水平的影响。方法 采用RNA印迹法检测IL-1αmRNA的表达水平,采用ELISA方法检测IL-1α蛋白的分泌水平。结果 SCC12F细胞可在正常KC培养体系中自发表达IL-1αmRNA,其表达水平随细胞培养时间的延长而增强,在120h达到高峰值;经中波紫外线(UVB)照射后,IL-1α蛋白的分泌水平既随照射时间增加,亦随照射剂量增加。结论 SCC12F细胞在正常KC培养体系中可自发表达IL-1αmRNA,其IL-1α蛋白的分泌水平对UVB照射呈时效及量效反应。     

不同培养条件对骨髓基质细胞贴壁及增殖的影响

银屑病是一种以T细胞为主、多种免疫细胞共同参与发病的慢性炎症性皮肤病.经过数年研究并结合国内外研究结果,推测骨髓造血细胞可能参与了银屑病的发病<'1-3>,为进一步研究骨髓造血细胞在银屑病发病中的作用,我们拟将银屑病患者和正常人骨髓移植于SCID鼠,用贴壁法培养骨髓基质细胞,并将其注射于经骨髓移植的SCID鼠腹腔内.在实际培养过程中发现一些问题,现将结果报道如下,供同仁们借鉴.     

白癜风自体培养黑素细胞移植疗效与白细胞介素17、Foxp3的相关性研究北大核心CSCD

目的 探讨白癜风自体培养黑素细胞移植疗效与白细胞介素17、Foxp3的相关性.方法 对40例稳定期寻常型白癜风患者进行自体培养黑素细胞移植,同时收集其外周血,6个月后根据移植效果将收集的血液分为成功组25例,失败组15例.用ELISA的方法测定移植成功与移植失败外周血中白细胞介素17及Foxp3水平,比较移植成功组与失败组血清中白细胞介素17及Foxp3水平的差异.用SPSS 17.0统计软件进行统计分析.结果 移植成功组血清中白细胞介素17（15.29±7.86） ng/L,明显低于移植失败组（43.88±13.02） ng/L,差异有统计学意义.血清中Foxp3的水平,移植成功组（6.08±2.03） ng/L,高于移植失贮组（3.37±1.81） ng/L,两者之间比较,差异有统计学意义.结论 血清白细胞介素17水平升高、Foxp3水平降低与白癜风患者的移植失败有相关性.     

Stem cell factor,a novel cutaneous growth factor for mast cells and melanocytes

Mechanisms affecting mast cell and melanocyte growth and function are still poorly understood. This report summarizes the current state of knowledge on a recently described growth factor for both these cell types and for primitive haematopoietic stem cells. Stem cell factor (SCF), also named mast cell growth factor or kit-ligand, has only recently been cloned and has been shown to be encoded on human chromosome 12. It may be of specific importance in cutaneous physiology and pathology since it is produced by several cell types in the skin (e.g. fibroblasts, keratinocytes, endothelial cells) and since it affects melanocyte and mast cell growth, survival, secretion and adhesion as well as migration into tissues. Defects in the genes encoding for the SCF receptor (c-kit-protein) have been shown to be responsible for human piebaldism. A pathogenetic role in mastocytosis has recently been proposed, but remains to be proven. SCF receptor expression is decreased on cells of some malignant cell lines compared to their physiological counterparts, making it unlikely that SCF is a key factor in malignant transformation and cellular hyperproliferation. In haematopoiesis, SCF acts primarily in concert with other growth factors, and we show here that alone in serum-free culture it has no effect on mast cell growth. Furthermore, there is evidence that besides SCF, additional mast cell growth factors are secreted by fibroblasts and keratinocytes, suggesting a complex orchestration of several growth factors in the regulation of cutaneous growth and differentiation in which SCF plays only one part.     

Regulation of mast cell characteristics by cytokines: divergent effects of interleukin-4 on immature mast cell lines versus mature human skin mast cells

Mast cells (MC) are of hematopoietic origin but complete their differentiation exclusively within tissues. The mediators that positively or negatively affect the maturation process are incompletely defined. Here, the human MC line HMC-1 (subclone 5C6) was used along with several treatments (IL-4, IL-6, NGF), either alone or in combination, and MC differentiation was monitored by flow-cytometric analysis of c-kit, tryptase, and FcRI expression. Of the different treatments, IL-4 displayed the clearest effects by suppressing the expression of the three markers and inhibiting cellular growth, while the other cytokines had no (NGF) or negligible (IL-6) effects only. The downregulating effects of IL-4 could not be overcome by any other treatment. There is some controversy in the literature as to the impact of IL-4 on the MC lineage. To determine whether the effects from IL-4 were differentiation stage dependent, two further human MC subsets (skin MC and LAD 2 cells) were investigated. No effects on c-kit and FcRI expression were noted when terminally differentiated skin MC were used as target cells, while a modest downregulation of c-kit was observed with intermediately matured LAD 2 cells. In sharp contrast to HMC-1 5C6 cells, the survival of skin MC was significantly enhanced by IL-4 treatment. Our data therefore imply that at a lower maturation stage, IL-4 acts as a negative regulator of the MC lineage, but that this property disappears or is even reversed upon terminal differentiation of the cell. Our study provides direct proof that the effects of IL-4 vary substantially in the course of MC maturation.     

诱导多潜能干细胞在皮肤病治疗中的应用

诱导多潜能干细胞拥有胚胎干细胞所有特征,包括多能性和生成各种体细胞.用皮肤细胞产生诱导多潜能干细胞,不仅起始细胞易获取,而且这些诱导多潜能干细胞更容易定向分化为角质形成细胞、黑素细胞和成纤维细胞等多种功能性皮肤细胞.患者自体来源的诱导多潜能干细胞是细胞疗法理想的细胞库,用诱导多潜能干细胞分化后的细胞治疗皮肤病,不仅细胞量充足,且可避免伦理问题和免疫排斥反应.利用回复突变体嵌合体,结合诱导多潜能干细胞技术,能获得充分的患者特异性功能性回复体细胞而用于治疗遗传性皮肤病.该技术可避免常规基因治疗中出现的免疫排斥和插入诱变.     

Effect of antianaphylactic agents on substance-P induced histamine release from rat peritoneal mast cells

Summary Substance P is known to be a potent histamine liberator for mast cells. The influence of antianaphylactic agents, disodium cromoglycate (DSCG), ketotifen, and tranilast was studied on substance-P and compound 48/80-induced histamine release from rat peritoneal mast cells. Substance-P induced histamine release was inhibited by these agents, while compound 48/80-induced histamine release was not inhibited by tranilast. Our findings suggest that these antianaphylactic agents are assumed to be effective for cutaneous diseases which might be concerned with substance P and histamine.     

Local injection of recombinant human stem cell factor promotes human skin mast cell survival and neurofibroma cell proliferation in the transplanted neurofibroma in nude mice

Abstract We studied the effects of stem cell factor (SCF) on human skin mast cell (HSMC) survival and the proliferation of neurofibroma (NF) cells in transplanted NF in nude mice. Small pieces of cutaneous NF from a patient with von Recklinghausen’s disease were transplanted subcutaneously into nude mice. Recombinant human SCF (10 or 100 ng) was injected six or seven times around the NF transplantation sites over 11 days (i.e. every other day). The number of HSMCs was reduced in vehicle-injected NF compared to the amount present before transplantation. In contrast, NF-transplanted animals that were injected with SCF (10 or 100 ng) showed preservation of mast cell numbers in the tissue. Using computerized image analysis, mast cell size in SCF-treated NF transplants was significantly altered (larger at the 10 ng dose, and smaller at the 100 ng dose) compared with the size before transplantation or in vehicle-injected tissue. Furthermore, at the higher SCF dose (100 ng) PCNA-positive NF cells showed a significant increase. These results indicate that HSMCs in transplanted NF tissue retain their capacity to respond to SCF in vivo, and that SCF contributes to the regulation of both HSMC survival and size in cutaneous NF. In addition, activated HSMCs induced by SCF may be involved in the growth of cutaneous NF in von Recklinghausen’s disease. Thus, this experimental model may be useful in the study of the cellular interactions between HSMCs and other stromal cells in cutaneous NF. Received: 9 June 1998 / Received after revision: 30 November 1998 / Accepted: 11 December 1998     

诱导多能干细胞在皮肤病研究的应用进展

诱导多能干细胞是将一系列转录因子转入动物或人多种分化成熟的体细胞,经过重组的程序诱导获得多能干细胞,其在形态、增殖能力、表面抗原、基因和蛋白表达、分化能力等方面与胚胎干细胞相似,因此,科研工作者利用诱导多能干细胞代替胚胎干细胞来研究皮肤病.因诱导多能干细胞除具有多能性还具有无限增殖的能力,这些特性能使研究者获得无限的特定细胞,进而建立遗传性皮肤病模型.将致病的突变基因在诱导多能干细胞水平矫正,矫正后的诱导多能干细胞能产生基因正常的健康皮肤组织细胞,从而可望治疗遗传性皮肤病.     

肥大细胞在慢性光损伤中的变化及研究进展

慢性光损伤是最常见的皮肤损害,由于长期接受紫外线照射所导致。研究表明,长期曝光部位皮肤中肥大细胞的数量较非曝光部位明显增多;肥大细胞在紫外线诱导的皮肤免疫抑制中发挥一定的作用。在慢性光损伤过程中,肥大细胞通过分泌基质金属蛋白酶和类胰蛋白酶等,参与光损伤中细胞外基质及基底膜的破坏;又通过分泌细胞因子如IL-10等,限制了小鼠皮肤慢性光损伤的病理过程。