中空纤维灌注培养模拟骨髓造血研究

采用细胞工程技术探索造血细胞体外扩增技术以维持其自我更新潜能,抑制过度分化。方法：首先建立微载体 基质细胞体外造血模型（G1组,即瓶培养模式）,设置单纯微载体 基质细胞培养（G2组）和单纯骨髓细胞液体悬浮培养（G3组）作对照。检测各组粒系 巨噬系造血祖细胞集落产率（CFU GM/105）。自行设计1对引物,以检测Balb/c小鼠原始造血细胞c kit基因mRNA表达水平。试用中空纤维模拟血管灌注功能（Gh组,即中空纤维灌注模式）,以G1、G2、G3作对照,并对各组培养效果进行评价。结果：微载体 基质细胞体外造血模型实验结果显示：小鼠骨髓细胞培养2周后,CFU GM/105检测G1组比G3组高7.7倍（P<0.05）,是2个对照组（G2+G3）集落产率总和的1.9倍。原始造血细胞c kit mRNA表达水平：模型G1组比G2组高3.7倍,比G3组高62.3倍,且差异均显著。在成功建立微载体 基质细胞体外造血模型基础上进行中空纤维灌注培养实验,CFU GM/105检测显示：Gh组比G3组高4.6倍,并且略高于G2组;Gh组与G1组集落产率差别不明显。在原始造血细胞c kit mRNA表达水平上Gh组最高,从Gh、G1、G2到G3依次呈下降趋势。结论：在没有外加细胞因子的条件下,微载体 基质细胞和中空纤维灌注造血模型可抑制造血干、祖细胞过度分化与耗竭,维持其c kit较高的表达水平。     

Effects of Mutations at the W Locus (c-kit) on Inner Ear Pigmentation and Function in the Mouse

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survivial of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (W^v/W^v) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (W^x, W^sh, and W⁴¹) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of W^sh/W^x, 25% of W^sh/W^sh, 60% of W⁴¹/W^x, and 69.2% of W⁴¹/W⁴¹ ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W⁴¹/W^x, W^sh/W^sh, and W⁴¹/W⁴¹ mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.     

花胫绿纹蝗和疣蝗配子发生时原癌基因c-kit特异性表达比较研究

应用免疫组织化学和生物统计分析相结合的方法,对花胫绿纹蝗Aiolopus tamulus (Fabricius)和疣蝗Trilophidia annulata (Thunberg)配子发生过程中原癌基因c-kit特异表达进行了比较研究。结果表明：（1）精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;（2）卵子发生过程中,第1～6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失;（3）2种蝗虫配子发生过程中c-kit表达存在种间差异。     

类c-kit原癌蛋白在多伊棺头蟋胚后精子发生中的表达和定位

为了了解类c-kit原癌蛋白在多伊棺头蟋Loxoblemmus doenitzi Stein胚后精子发生中的表达、定位及可能的调控作用,采用常规免疫组织化学方法进行了相关研究。结果表明：处于减数分裂中期Ⅰ至末期Ⅱ的初级精母细胞的细胞膜上有类c-kit原癌蛋白阳性颗粒;精巢或受精囊内成熟精子头部也具有类c-kit原癌蛋白阳性颗粒。结果反映了类c-kit蛋白对于维持动物精子发生过程中减数分裂、精子成熟及受精能力具有特殊功能。     

A Xenopus c-kit-related receptor tyrosine kinase expressed in migrating stem cells of the lateral line system

The mammalian c-kit receptor tyrosine kinase gene is required during embryogenesis for the survival and/or proliferation of three migrating stem cell populations: primordial germ cells, haematopoietic stem cells and neural crest-derived melanoblasts. We have cloned a Xenopus gene, XKrkl, whose closest relative is c-kit. Differences in the expression pattern suggest that XKrkl is not the Xenopus homologue of c-kit; however, it is expressed in a migrating stem cell population, the precursor cells for the mechanosensory lateral line system. XKrkl is the first reported marker for lateral line stem cells.     

c-kit和PDGFRA基因对胃肠间质瘤的影响

胃肠间质瘤(gastrointestinal stromal tumors,GIST)是较常见的人消化道间叶性肿瘤,多发于胃部.尽管有不同临床病理特征,但绝大多数GIST均存在c-kit或血小板衍生生长因子受体α(PDGFRA)基因突变. c-kit、PDGFRA的抑制剂—格列卫是目前主要应用于GIST治疗的分子靶向治疗药物,c-kit、PDGFRA的不同基因状态会对分子靶向治疗药物呈现不同的反应.c-kit基因外显子11发生突变的GIST对格列卫呈现良好的反应,而外显子 9突变对格列卫的反应略差.另外发现,c-kit、PDGFRA基因的二次突变会引起格列卫抗性.本文简要介绍c-kit、 PDGFRA基因与GIST的临床表现、分子靶向治疗之间的关系及其二次突变的特征.     

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit⁺ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit⁺ CSCs.     

Mesenchymal stem cells facilitate cardiac differentiation in Sox2-expressing cardiac C-kit cells in coculture

Stem cell therapy offers hope to reconstitute injured myocardium and salvage heart from failing. A recent approach using combinations of derived Cardiac-derived c-kit expressing cells (CCs) and mesenchymal stem cells (MSCs) in transplantation improved infarcted hearts with a greater functional outcome, but the effects of MSCs on CCs remain to be elucidated. We used a novel two-step protocol to clonogenically amplify colony forming c-kit expressing cells from 4- to 6-week-old C57BL/6N mice. This method yielded highly proliferative and clonogenic CCs with an average population doubling time of 17.2 ± 0.2, of which 80% were at the G1 phase. We identified two distinctly different CC populations based on its Sox2 expression, which was found to inversely related to their nkx2.5 and gata4 expression. To study CCs after MSC coculture, we developed micron-sized particles of iron oxide-based magnetic reisolation method to separate CCs from MSCs for subsequent analysis. Through validation using the sex and species mismatch CC-MSC coculture method, we confirmed that the purity of the reisolated cells was greater than 85%. In coculture experiment, we found that MSCs prominently enhanced Ctni and Mef2c expressions in Sox2 ^pos CCs after the induction of cardiac differentiation, and the level was higher than that of conditioned medium Sox2 ^pos CCs. However, these effects were not found in Sox2 ^neg CCs. Immunofluorescence labeling confirmed the presence of cardiac-like cells within Sox2 ^pos CCs after differentiation, identified by its cardiac troponin I and α-sarcomeric actinin expressions. In conclusion, this study shows that MSCs enhance CC differentiation toward cardiac myocytes. This enhancement is dependent on CC stemness state, which is determined by Sox2 expression.