高增殖潜能集落形成细胞的分类,富集与调控

高增殖潜能集落形成细胞（ＨＰＰ－ＣＦＣ）是存在于小鼠和人骨髓及入胎肝、脐血中的一类较为原始的造血祖细胞。它在体外培养中可产生直径大于０．５ｍｍ，５００００细胞／集落的巨大集落；对５－氟尿嘧啶有相对耐受性；对多种造血生长因子的刺激有反应性。     

p21基因启动子甲基化对血管平滑肌细胞向泡沫细胞转化的影响

目的观察氧化低密度脂蛋白(ox-LDL)对血管平滑肌细胞(VSMC)p21基因启动子甲基化及对VSMC向泡沫细胞转化的影响。方法采用组织贴块法培养人脐血管平滑肌细胞,给予不同浓度氧化低密度脂蛋白ox-LDL(0,10mg/L,20mg/L,40mg/L)孵育24h,甲基化特异PCR(MS-PCR)检测p21基因启动子区CpG岛甲基化程度,MTT比色法测定VSMC增殖活性,油红O染色镜下观察计数泡沫细胞比率。结果 ox-LDL呈浓度依赖性促进p21启动子甲基化,同时平滑细胞增殖活性增高,向泡沫细胞转化增加。结论 ox-LDL可以通过p21基因甲基化促进VSMC向泡沫细胞转化,p21基因甲基化可能在动脉粥样硬化发生中起到一定作用。     

多种恶性肿瘤p21基因启动子及编码区点突变的检测

目的 探讨ｐ２１基因点突变是否与恶性肿瘤的发生发展过程有关,特别是与那些没有ｐ５３基因突变的恶性肿瘤发病的关系。方法 结合ＳＳＣＰ分析及ＤＮＡ序列测定技术,检测３４６例９种不同组织学类型的恶性肿瘤ｐ２１基因的启动子和主要的编码区。结果 ３４６例恶性肿瘤的ｐ２１基因启动子未检测到突变,但在Ｐ５３蛋白结合位点不游及下游处存在多态性变异。仅１例膀胱癌有一杂合性４４ｂｐ缺失突变。此外,在编码区还发现了１个     

野生型p53基因诱导肝癌细胞凋亡及p21（WAF1／CIP …

目的：研究野生型ｐ５３基因对人肝癌细胞系细胞凋亡的作用。方法：通过脂质体转染方法将野生型ｐ５３基因（ｗｔ－ｐ５３）导入ｐ５３缺陷的ＨＣＣ－９２０４细胞系中，并获稳定表达。结果：转染ｗｔ－ｐ５３后细胞生长缓慢，Ｇ１期细胞数量由转基因前的４８．５％增加到７８．０％，并有较多细胞逐渐死亡。电镜观察和ＤＮＡ分析证实细胞死亡方式主要是细胞凋亡。免疫组化结果显示细胞转染ｗｔ－ｐ５３后，ｐ２１ＷＡＦ１／ＣＩＰ１     

银屑病患者皮损p16INK4a基因CpG甲基化位点和频度

目的研究皮损表皮p16INK4a甲基化阳性的银屑病患者基因启动子区CpG甲基化的位点和频度。方法采集50例银屑病患者皮损处皮肤,分离表皮,提取DNA。采用甲基化特异性PCR和测序法检测p16INK4a基因启动子区CpG甲基化的位点和频度。结果p16INK4a基因启动子甲基化阳性的患者,约50%CpG发生甲基化,均出现于特定位点。结论p16INK4a甲基化阳性银屑病患者p16INK4a基因启动子并非完全发生甲基化。     

p21和p27基因多态性与肿瘤的相关性

做为细胞周期依赖性蛋白激酶抑制剂，p21和p27被认为是侯选的抑癌基因，在细胞周期的调控中发挥重要作用。p21和p27基因的突变或多态性可能与人类多种肿瘤的发生发展有关。研究该二基因的多态性可能有助于判断肿瘤的发病风险、临床特征以及预后。     

人胎盘CD133+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133+细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析,证明人胎盘存在早期造血干/祖细胞(HSPC). 方法 采用机械法制备人胎盘组织(PT)单细胞悬液,用Histopaque-1007分离出单个核细胞(MNC),经磁式分选(MACS)富集CD133+细胞,培养28 d后观察HPP-CFC集落形成能力,用流式细胞仪(FCM)对分选的细胞组份和HPP-CFC进行表型分析,实验全程用脐带血(UCB)作平行比较分析. 结果 培养28 d后,PT-CD133+与UCB-CD133+细胞组份分别扩增了266和362倍,前者低于后者(P<0.01);PT-CD133+与UCB-CD133+细胞中HPP-CFC分别为(32.4±11.2)/5×103、(17.7±5.7)/5×103,前者形成的HPP-CFC数量明显高于后者(P<0.01);PT.CD133+、UCB-CD133+细胞培养至28 d时,除UCB-CD133+组的CD133+CD34-亚群比例无明显改变外,CD133+CD34+、CD133-CD34+和CD133+CD34-(PT-CD133+组)亚型均比培养前减少. 结论 人胎盘组织CD133+细胞中存在HPP-CFC,说明胎盘CD133+细胞群中存在早期HSPC.     

肺鳞癌及癌前病变PCNA、p16、p21、p53表达及细胞凋亡观察

目的 :观察肺癌癌变过程中细胞增殖与凋亡活性的改变 ,分析它们在肺癌发生发展过程中的意义并探讨其调控基因的作用。方法 :收集 86例支气管黏膜活检标本 ,采用免疫酶标法对各组病例进行PCNA、p5 3、p2 1、p16蛋白表达检测 ,用 3′ OH末端DNA原位标记技术进行凋亡细胞检测。结果 :在正常与鳞化组 ,PCNA只在极个别细胞中表达 ,p2 1、p5 3未见表达 ,p16阳性表达率分别为 10 0 %、93 3% ,凋亡细胞阳性率为 (2 9± 5 6 ) %、(2 4± 4 7) %。轻、中度非典型增生组 ,PCNA也只在少数细胞中表达 ,p2 1、p5 3没有表达或极少数细胞表达 ,p16表达率分别为 91 7%、83 3% ,凋亡细胞阳性率分别为 (2 2 0± 4 3) %和 (18 5± 3 7) %。在重度非典型增生与高分化鳞癌组织中PCNA阳性率显著增加 ,而凋亡细胞阳性率却明显减少 ,p2 1、p5 3表达增加 ,p16显著下降。结论 :支气管黏膜癌变是一个由量变到质变的过程 ,而中到重度非典型增生是这一过程中的关键性阶段 ,不仅增殖活性增加 ,而且凋亡活性下降 ,它们的调控基因p16、p2 1、p5 3也发生了显著的变化 ,可能起着重要的调节作用。     

外源性野生型p53基因在两个肺腺癌细胞系中表达及对p16和p21基因的调控

目的 探讨p53基因在肺癌细胞周期中的作用机制，以及裸鼠体内基因治疗的作用。方法 用以腺病毒为载体的野生型p53基因pAdCMV-p53(Ad-p53)感染高转移肺腺癌95D细胞系和高浸润肺腺癌L-18系，对感染前后各细胞系的细胞生长曲线、p53、p16和p21基因的表达以及调亡进行分析。此外，用Ad-p53对两细胞系进行了裸鼠体内感染实验。结果 体外实验中，导入p53基因细胞系的生长均得到抑制，并且最终都出现凋亡，感染后的细胞中p53和p21基因的mRNA表达量明显增高，p16基因的mRNA表达量则无明显变化。p53基因治疗后的裸鼠，其中95D细胞系的肿瘤全部消失；L-18细胞系的腹腔注射组肿瘤全部消失，而皮下注射组无明显变化。结论 p53基因是一个有效的肿瘤抑制基因，其诱导细胞凋亡的途径与p16基因不同。腺病毒介导的野生型p53基因可以有效地控制肺癌细胞的发展和转移。     

p21WAF1／CIPI基因导入对p53突变人肺癌细胞系生长特性的影响

目的 探讨含突变ｐ５３基因癌细胞系ｐ２１基因的抗肿瘤作用。方法 构建人ｐ２１表达重组子,导入ｐ５３基因突变的人肺癌ＰＧ细胞每当增产同表达ｐ２１的细胞系;然后观察其形态太生长特性的改变;ＣＤＫ４,ＰＣＮＡ水平;对基因毒性物的反应性。结果 在含ｐ５３基因突变的ＰＧ细胞内导入的性ｐ２１基因,其高表达ｐ２１约是ＰＧ细胞的６倍,转谬为薄等异型性降低;生长速度下降（５０％）,叠落生长不明显;在０．５％血甭条件     

p73基因在卵巢上皮性肿瘤中的表达及甲基化研究

目的 探讨卵巢上皮性肿瘤中p73蛋白的表达和基因启动子的甲基化情况,并观察其与临床病理学特征的关系.方法 制备包括68例卵巢癌、37例卵巢交界性肿瘤和21例卵巢良性肿瘤的组织芯片,用免疫组织化学EnVision法检测上述组织中p73蛋白表达情况,用亚硫酸氧盐修饰后测序法检测13例新鲜卵巢癌组织及5例新鲜卵巢交界性肿瘤组织的p73基因启动子甲基化情况.结果 92.6％ (63/68)的卵巢癌表达p73,p73蛋白总体表达率均值为32％(p73表达率指p73阳性细胞数所占的百分比),其中浆液性癌( 26/26)的表达率均值为40％,高于其他组织类型的癌(P=0.006).按照卵巢癌发病模式区分,Ⅱ型卵巢癌p73表达率均值(40％)高于Ⅰ型卵巢癌(24％),P=0.010.卵巢癌中p73的表达与临床分期及组织学分级无相关性(均P＞0.05).卵巢交界性肿瘤组(30/37)和良性肿瘤组(12/21)p73的总体表达率均值分别为16％和15％,该两组肿瘤中浆液性肿瘤表达率均值均高于黏液性肿瘤(P-0.003,P=0.026).卵巢癌组的p73阳性表达率均值明显高于交界性肿瘤组和良性肿瘤组(均P ＜0.05),交界性肿瘤组与良性肿瘤组比较差异无统计学意义(P＞0.05).浆液性肿瘤( 49/53)中,卵巢癌组(26/26) p73阳性表达率均值明显高于交界性肿瘤组(12/14)和良性肿瘤组(11/13;P =0.024和P=0.002),而卵巢交界性肿瘤组和良性肿瘤组比较差异无统计学意义(P=0.428).黏液性肿瘤(15/27)中,卵巢癌组(6/7)p73阳性表达率均值高于良性肿瘤组( 1/8;p=0.032),而卵巢癌组与卵巢交界性肿瘤组(8/12)、交界性肿瘤组与良性肿瘤组比较,差异均无统计学意义(P=0.234和P=0.201).p73启动子的甲基化结果显示,13例卵巢癌有8例发生甲基化,但每例样本甲基化频率有所不同,总体甲基化频率均值为8.0％.5例交界性肿瘤有2例发生甲基化,总体甲基化频率均值为9.0％,两组比较差异无统计学意义(P＞0.05).卵巢癌组p73甲基化额率与组织类型、发病模式、组织学分级及临床分期均无相关性(均P＞0.05).结论 卵巢上皮性肿瘤多数表达p73,卵巢癌p73的表达率均值明显高于交界性肿瘤和良性肿瘤,浆液性肿瘤高于其他组织类型;p73蛋白表达率与p73基因甲基化程度不存在简单线性相关关系.     

原发胃癌中p16和p15基因表达及甲基化异常

为检测胃癌组织中抑癌基因p16,p15及其启动子区甲基化状态和P16、P15蛋白表达情况。选择p16、p15基因及启动子区域，用PCR－SSCP、MSP（甲基化特异的PCR）和测序法对100例胃癌患者的癌组织、癌旁正常组织和5例正常组织进行检测，同时用免疫组化法检测了癌组织和正常对照组织的P16和P15的表达。结果发现癌组织p16和p15基因启动子区甲基化率显著高于癌旁正常组织和正常对照；胃癌组织中，71％的病例P16表达阴性，54％的病例具有p16基因启动子区的高甲基化，无突变和纯合缺失检出；11％的病例P15表达阴性，9％的病例具有p15基因启动子区的高甲基化，p15异常与低分化胃癌有关，p15基因内含子1和外显子1内各发现1例DNA序列改变；癌组织中p16和p15基因启动子区甲基化与其蛋白表达密切相关。结果显示p16基因启动子区域高甲基化是胃癌中p16基因失活的关键因素之一，并在胃癌的发生发展中发挥重要作用；p15基因启动子区域高甲基化在胃癌中起一定作用。     

Characterization and expression of H-21 region gene products on bone marrow-derived macrophages

Antigen-presenting macrophages (M phi) were derived from day 7 cultures of bone marrow stem cells using L cell conditioned medium. The adherent bone marrow-derived macrophages (BMM phi) were 100% esterase-positive, 95% positive for C3 receptors, 93% positive for Fc receptors, and 95% actively phagocytic. Indirect immunofluorescence using anti-Ia monoclonal antibodies resulted in 60% Ia-positive BMM phi on day 7 of stem cell culture. BMM phi could stimulate mixed lymphocyte reaction (MLR) proliferation across an I-A subregion difference, but not across I-J subregion differences. This contrasted with splenic M phi which stimulated MLR proliferation across both an I-A and I-J subregion difference. The apparent lack of I-J subregion determinants on BMM phi correlated with their ability to function as antigen-presenting cells. In these experiments, BMM phi effectively reconstituted the trinitrophenyl-specific IgM plaque-forming cell (PFC) response of B cells but not the primary burro red blood cell (BRBC)-specific IgM-PFC response of M phi-depleted spleen cells. When BMM phi were added to BRBC-primed T and B cells, they reconstituted the secondary IgG PFC response to levels obtained using splenic M phi. These experiments relate the differential expression of H-21 region determinants on antigen-presenting cells with their functional capacity.     

The role of bone marrow-derived cells in fibrosis

There is a growing realization that bone marrow-derived cells (BMDCs) are a potential therapy for many diseases including ischemic heart disease, arterial stenosis and osteogenesis imperfecta. On the other hand, the fact that BMDCs may also contribute to fibrosis in many solid organs as well as to fibrosis surrounding tumours suggests that BMDCs are also involved in disease progression. This review focuses on the contribution of bone marrow cells to organ and tumour fibrosis, noting the utility of BMDCs as a potential new portal through which to direct anti-tumour therapies. Conversely, bone marrow cell therapy has been claimed to reduce fibrosis in some organs, highlighting a seemingly beneficial as opposed to a detrimental effect of BMDCs on organ fibrosis.     

Electrospun nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem cells

Interactions between bone marrow-derived hematopoietic stem cells (BM-HSCs) and their local microenvironment are an integral part of signaling control of BM-HSCs migration, proliferation and differentiation. We hypothesized that both substrate topographical and biochemical cues promote BM-HSCs adhesive behaviors, which are crucial for BM-HSCs' homing, self-renewal and lineage commitment within their microenvironment. We employed electrospinning technique to fabricate nanofiber scaffolds (NFS) with poly(dl-lactide-co-glycolide) blended with collagen I. NFS was further coated with E-selectin, a critical adhesive biomolecule. Capture efficiency study showed that blended NFS, after coated with E-selectin, significantly increased cell capture percentage from 23.40% to 67.41% within 30 min and from 29.44% to 70.19% within 60 min of incubation at room temperature. This study highlights the potential of using a biomimetic scaffold to design a site-specific niche-like unit for facilitating proliferation or differentiation functions of BM-HSCs.     

小鼠骨髓成熟与不成熟树突状细胞中RelB基因的表达

目的:探讨体外分离培养的小鼠骨髓来源的成熟与未成熟DC中核转录因子RelB(avianreticuloendotheliosisviral(v-rel)oncogenerelatedB)基因的表达。方法:无菌从C57BL/6小鼠股骨和胫骨中取出骨髓细胞,利用rmGM-CSF和rmIL-4联合诱导骨髓前体细胞产生未成熟的DC,未成熟的DC在培养结束前18h经LPS刺激获得成熟的DC,用流式细胞术分析它们的表型,用RT-PCR和免疫荧光染色法检测成熟与未成熟DC中,RelBmRNA和其蛋白的表达。结果:流式细胞术分析显示未成熟的DC中MHC-Ⅱ类分子和共刺激分子(CD86和CD40)呈低水平表达;而成熟的DC则呈高水平表达。RT-PCR和免疫荧光染色法检测结果均显示,RelB基因在未成熟的DC中呈低水平表达;而在成熟的DC中呈高水平表达,两者比较具有统计学意义(P<0.01)。结论:RelB基因的表达与小鼠骨髓来源的DC的成熟状态密切相关。抑制DC中RelB基因的表达,有可能诱导产生具有耐受原性的未成熟的DC。     

Identification of keratocyte-like cells differentiated from circulating bone marrow-derived cells in the mouse cornea

Bone marrow (BM)-derived stem cells have the potential to differentiate into multiple lineages of tissue resident cells. BM-derived cells have been detected in the mouse cornea, but most of these cells were found to be CD45⁺ or CD11b⁺ immunocompetent cells. Although some BM-derived cells in the cornea were negative for these cell surface markers, it has remained unclear whether cells of BM origin can differentiate into corneal resident cells. To address this issue, we subjected wild-type mice that had been exposed to a lethal dose of radiation to intravenous injection with BM cells from green fluorescent protein (GFP) transgenic mice. Two months after cell transplantation, fluorescence microscopy revealed the presence of numerous GFP⁺ cells throughout the cornea, with intense GFP fluorescence being apparent around the limbal region. Immunohistofluorescence analysis of corneal cross-sections revealed that most of the BM-derived GFP⁺ cells expressed CD45 or CD11b, although a few GFP⁺ cells were negative for these markers. Immunostaining of individual cells isolated from the corneal stroma, however, showed that a small proportion (~1 %) of GFP⁺ BM-derived cells expressed the keratocyte-specific proteoglycan keratocan. Our results suggest that BM-derived cells introduced intravenously are able to differentiate into resident cells of the corneal stroma.