P63在子宫内膜样腺癌的表达及意义

目的检测正常子宫内膜、子宫内膜增生症和子宫内膜样腺癌（endometriod adenocarcinoma,EC）组织中P63的表达,探讨P63与子宫内膜样腺癌发生、发展及预后的关系。方法采用免疫组化SP法检测正常子宫内膜（20例）,子宫内膜增生症（20例）,子宫内膜样腺癌（50例）组织中P63蛋白的表达。结果（1）正常子宫内膜中仅1例增生期内膜中有P63表达,阳性细胞零星分布于极个别腺体的基底部。子宫内膜增生症和子宫内膜样腺癌组织中,P63阳性细胞常相对集中分布于某一区域的腺体或实性巢,染色强。（2）EC组和子宫内膜增生症组P63的阳性率分别为46％和50％,与正常子宫内膜组（5．0％）比较差异有显著性护〈0．05）。子宫内膜增生症组P63的阳性率与EC组比较差异无显著性（P〉0．05）。（3）P63的表达与EC的分化程度无关（P〉0．05）。结论（1）EC组织中P63阳性细胞可能来源于胚胎时期的未分化细胞,具有多向分化的潜能。（2）P63与EC的发生发展有关,可能起癌基因的作用。（3）P63在子宫内膜增生症组织中高表达,表明P63与子宫内膜的异常增生相关。     

Musashi-1和β-catenin在子宫内膜中的表达及其在内膜异位病灶形成中的作用

目的:检测Musashi-1和β-连环蛋白(β-catenin)在子宫内膜异位症(EMs)患者的在位内膜和异位内膜中的表达,并初步探讨作用机制。方法:2016年9月至2018年9月,收集EMs患者的在位内膜(在位内膜组,28例)、异位内膜(异位内膜组,24例)和非EMs患者的正常内膜(正常内膜组,30例),采用实时荧光定量PCR(Real-time PCR)试验检测Musashi-1和β-catenin在各组内膜组织中的表达情况,并分析Musashi-1和β-catenin在各组内膜组织中的表达相关关系。结果:在位内膜组和异位内膜组中,Musashi-1和β-catenin的相对表达量均明显高于正常内膜组(P0.05),而在位内膜组与异位内膜组之间的差异无统计学意义(P0.05)。在位内膜组和正常内膜组中,增生期的Musashi-1和β-catenin相对表达量显著高于分泌期(P0.05),而异位内膜组中,在增生期和分泌期Musashi-1和β-catenin比较差异无统计学意义(P0.05)。在位内膜组和异位内膜组中,Musashi-1和β-catenin表达之间均呈正相关性(P0.05),而正常内膜组中,两者表达之间无明显相关性(P0.05)。结论:在EMs的发病过程中,干细胞标志物Musashi-1可能通过激活Wnt/β-catenin信号通路参与并促进子宫内膜异位病灶的形成。     

BrdU体内示踪骨髓基质干细胞生物学状态的效果分析

目的:探讨5-溴脱氧尿嘧啶核苷(Brd U)体内示踪骨髓基质干细胞(BMSCs)生物学状态的效果。方法:抽取健康成年比格狗骨髓,在传代培养中进行Brd U标记并鉴定,体外实验中测定细胞周期、凋亡率和细胞活力;在体内实验中将标记Brd U的骨髓基质干细胞植入自体股骨头缺损处,另一侧单纯植入自体骨作为对照,记录成骨量与分子标记物的表达情况。结果:骨髓基质干细胞的Brd U体外标记率为85.2%。Brd U组的细胞凋亡率为3.62±1.33%,未标记组为3.52±1.08%;Brd U组与未标记组的细胞成活率分别为96.31±1.39%和95.20±2.10%,两组对比差异均无统计学意义(P0.05)。移植侧Brd U标记的骨髓基质干细胞免疫组化观察可见Brd U免疫组化染色阳性,阳性率为81.6%。骨髓基质干细胞移植侧缺损区的骨钙素、Ⅰ型胶原阳性细胞表达数量与强度明显高于对照侧缺损区;骨髓基质干细胞移植侧成骨量为17.46±2.12%,对照侧为9.06±1.24%,两两对比差异有统计学意义(P0.05)。结论:Brd U在体外示踪骨髓基质干细胞能有效反映细胞的生物学状态,体内示踪显示移植的骨髓基质干细胞能成活,能促进骨组织形成和坏死骨修复。     

GnRHR-Ⅱ在子宫内膜的分布及表达变化规律研究

目的:研究促性腺激素释放激素Ⅱ型受体(GnRHR-Ⅱ)在子宫内膜的分布及表达变化规律。方法:选择2015年1月~2016年7月期间我院收治的100例女性不孕患者,经诊断性刮宫技术获取50例增生期子宫内膜组织与50例分泌期子宫内膜组织,分别作为研究组与对照组;采用免疫组织化学染色法检测两组子宫内膜基质细胞与腺上皮GnRHR-Ⅱ的表达。结果:增生期、分泌期子宫内膜均有GnRHR-Ⅱ分布;研究组子宫内膜基质细胞的GnRHR-Ⅱ表达明显高于对照组,而腺上皮则明显低于对照组,差异有统计学意义(P0.05)。结论:GnRHR-Ⅱ在子宫内膜增生期与分泌期均有表达,且在膜基质细胞与腺上皮均有分布,其分布于表达变化规律在一定程度上与子宫内膜容受性有关,有望成为评估子宫内膜容受性的标记物。     

大鼠脂肪间充质干细胞在体内向肝细胞样细胞方向的转化

目的:探讨大鼠脂肪间充质干细胞(Adipose tissue-derived mesenchymal stem cells,ADMSCs)在体内向肝细胞样细胞转化的可能性.方法:将从4周龄雄性SD大鼠腹股沟分离得到的原代脂肪间充质干细胞传至第3代,用5-溴脱氧尿嘧啶核苷(BrdU)在体外标记后,通过门静脉注射的方法移植入由四氯化碳造成慢性肝损伤的雄鼠体内.移植术后2周处死受体雄鼠,取其肝组织.通过免疫荧光双染色的方法观察BrdU标记细胞的存在和白蛋白的表达,以确定所注入的脂肪间充质干细胞在受鼠体内向肝细胞样细胞转化的情况.结果:在经门静脉注射法进行移植的实验组SD大鼠的肝组织内检测到同时表达BrdU和白蛋白的细胞.讨论:本研究证明了脂肪间充质干细胞在体内有向肝细胞样细胞转化的可能.     

PKH26标记的人羊膜间充质干细胞在宫腔粘连大鼠中的迁移示踪

为了观察PKH26标记的人羊膜间充质干细胞(hAMSCs)在宫腔粘连大鼠子宫内膜中的迁移情况,文中提取鉴定及PKH26标记hAMSCs,检测PKH26染色剂对hAMSCs生物学特性的影响;利用机械感染双重法建立大鼠宫腔粘连模型并经尾静脉移植PKH26标记的hAMSCs,荧光共聚焦显微镜下观察PKH26标记的hAMSCs移植后在大鼠子宫内膜中的分布情况。结果显示,PKH26染色剂对细胞的活性、周期、凋亡等无明显影响,PKH26标记的阳性细胞主要分布在大鼠受损的子宫内膜中。表明PKH26标记技术是一种安全有效的示踪方法,可用于hAMSCs移植在治疗宫腔粘连时的示踪研究。     

早孕小鼠子宫内膜钙网蛋白的表达规律

采用RT-PCR、间接免疫荧光组织化学、Western 印迹及原位杂交技术分别检测未孕(d0)和妊娠d1、d2、d3、d4、d5、d6、d7天小鼠子宫内膜中钙网蛋白(calreticulin, CRT)的表达规律, 探讨CRT在胚胎着床中的作用.结果显示CRT mRNA在妊娠小鼠子宫内膜中的表达明显高于未孕小鼠(P <0.05), 且随着妊娠天数的增加呈逐渐增强的趋势.间接免役荧光组织化学结果显示CRT表达于子宫内膜基质细胞、腺上皮以及腔上皮, 并在妊娠第4、5天基质细胞的胞浆中呈现高峰.实验结果提示, CRT在妊娠早期子宫内膜的持续表达, 可能通过调节整合素介导的细胞信号通路而调节胚胎滋养层细胞的黏附、侵袭, 参与胚胎着床.     

小鼠骨髓瘤中肿瘤干细胞的初步分析

探讨小鼠骨髓瘤(SP2/0细胞)中肿瘤干细胞存在与否。以克隆形成试验检测SP2/0细胞中具有形成克隆能力细胞的大体比例;采用BrdU标记滞留试验检测SP2/0细胞中含有DNA永生化链的细胞,即具有干细胞特性的细胞;检测SP2/0细胞中具有干细胞特性的SP细胞存在情况及其比例。结果显示,SP2/0细胞中有一部分细胞具有形成克隆的能力;SP2/0细胞中含有DNA永生化链的细胞;SP2/0细胞中存在SP细胞,其比例约为0.7%。而且SP2/0细胞中存在肿瘤干细胞。     

小鼠围着床期子宫内膜甲基化CpG结合域蛋白2的表达规律

为研究甲基化CpG结合域蛋白2（methyl-CpG binding domain protein 2,MBD2）在围植入期小鼠子宫内膜的表达规律,通过采用实时荧光定量PCR（Real-time fluorescence quantitative PCR,qPCR）、Western blot和免疫组化技术检测未孕小鼠（d0）和不同孕天小鼠子宫MBD2的表达情况。qPCR结果显示,d0至d7的小鼠子宫内膜组织均有MBD2 mRNA表达,在d5至d7高表达。MBD2蛋白在子宫内膜的表达规律与qPCR结果相符。MBD2蛋白在孕d1到d4中度表达于腔上皮、腺上皮和基质细胞,在d5至d7基质细胞表达增强,主要表达于蜕膜区。假孕小鼠子宫内膜中,MBD2在腔上皮、腺上皮和基质细胞中中度表达,d5至d7基质细胞表达明显减弱。动物模型中,宫角注射MBD2基因反义寡聚脱氧核苷酸,可抑制MBD2的表达,降低人工诱导蜕膜化反应和蜕膜化标志物PRL的表达。MBD2在早孕小鼠子宫内膜的表达模式提示其可能参与了蜕膜化过程。     

多潜能乳腺干细胞的发现

小鼠乳腺由多种不同类型的上皮细胞构成。多潜能干细胞位于乳腺发育的顶端,是乳腺中所有分化细胞类型的来源。然而,这群多潜能乳腺干细胞此前尚未通过特定的标记基因得到鉴定,其存在性也备受争议。借助乳腺干细胞体外培养体系,从Wnt信号通路入手,发现了蛋白C受体基因Procr。在乳腺中,Procr作为一个新的Wnt信号通路的靶基因,能够标记多潜能乳腺干细胞。Procr标记乳腺基底细胞中的一个亚群,这个亚群的细胞低表达基底细胞普遍表达的角蛋白,表现出上皮–间充质转化的特性。Procr阳性的细胞在移植实验中表现出最高的乳腺重建率,体内追踪Procr阳性细胞的后代,发现Procr阳性细胞能够在发育过程中分化形成乳腺上皮的所有细胞类型。多潜能乳腺干细胞的发现结束了乳腺中多潜能干细胞存在性的争议,对乳腺癌的诊断及靶向治疗具有重大意义。     

Identification of a putative intestinal stem cell and early lineage marker; musashi-1

There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day post-irradiation samples of BDF1 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / - ) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stem cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.     

肝细胞生长因子对人羊水干细胞迁移和黏附能力的影响

目的分离培养及鉴定羊水干细胞（hAFSC）,并研究肝细胞生长因子（HGF）对羊水干细胞迁移、黏附能力的影响。方法使用细胞贴壁法分离培养羊水干细胞,细胞免疫荧光及westernblot鉴定羊水干细胞,Transwell小室分析HGF对羊水干细胞迁移的作用。明胶贴壁法分析HGF对羊水干细胞黏附能力的作用。两组之间数据的比较采用独立样本t检验。结果分离的羊水干细胞均表达特异性标记物Oct-4、c-kit、SSEA-4、CD105。HGF在体外对hAFSC的迁移有趋化作用,对照组和HGF组每个视野的迁移细胞数分别为38±2.5和80±3.2。对黏附能力有促进作用,对照组和HGF组每个视野的黏附细胞数分别为19±1.5和50±2.7,差异均有统计学意义（P〈0.01）。结论 HGF可趋化羊水干细胞的迁移,增强羊水干细胞的黏附能力。     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

Function of RNA-binding protein Musashi-1 in stem cells

Musashi is an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system. The first member of the Musashi family was identified in Drosophila. This protein plays an essential role in regulating the asymmetric cell division of ectodermal precursor cells known as sensory organ precursor cells through the translational regulation of target mRNA. In the CNS of Drosophila larvae, however, Musashi is expressed in proliferating neuroblasts and likely has a different function. Its probable mammalian homologue, Musashi-1, is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). Mammalian Musashi-1 augments Notch signaling through the translational repression of its target mRNA, m-Numb, thereby contributing to the self-renewal of NSCs. In addition to its functions in NSCs, the role of mammalian Musashi-1 protein in epithelial stem cells, including intestinal and mammary gland stem cells, is attracting increasing interest.     

Isolation and characterization of human mammary stem cells

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.     

Sertoli细胞饲养层对小鼠精原干细胞增殖的影响

摘要 目的 以小鼠睾丸支持细胞(Sertoli)为饲养层,小鼠胚胎成纤维细胞(STO) 饲养层做对照,研究它对小鼠精原干细胞增殖的影响。方法 用无血清StemPro-34 SFM培养基培养2～5日龄小鼠精原干细胞,分别用相差显微镜观察,免疫组化法研究Sertoli饲养层对精原干细胞生物学行为的影响。结果 发现精原干细胞在Sertoli及STO两种饲养层上的一周内的生物学行为非常相似,但培养1周后,Sertoli细胞作饲养层的培养体系中保留的精原干细胞要比对照组明显增多,约有30%的精原干细胞能存活下来并能维持存活到60d以上。结论 Sertoli细胞作饲养层明显促进精原干细胞的更新增殖。