碱性成纤维细胞生长因子对体外培养人牙周膜细胞的作用

背景：碱性成纤维细胞生长因子是具有多功能的细胞生长因子,对来源于中胚层及神经外胚层的细胞有明显的促进增殖作用。 目的：观察碱性成纤维细胞生长因子对体外培养人牙周膜细胞的作用。 方法：将第5代人牙周膜细胞,以1×108 L-1的浓度分别接种到96孔板,随机分成4组,分别加入含0,1,10,100 μg/L的碱性成纤维细胞生长因子、体积分数为15%胎牛血清的α-MEM培养基进行培养。在第1,3,5,7天测定细胞的增殖情况,在第1,7天检测碱性磷酸酶活性。 结果与结论：4组之间人牙周膜细胞增殖情况的差异有显著性意义(F=6.586,P=0.024),随着碱性成纤维细胞生长因子质量浓度的增大,吸光度值均增大,其中100 μg/L碱性成纤维细胞生长因子组的吸光度值均大于其他组(P < 0.05);碱性成纤维细胞生长因子各组的碱性磷酸酶活性均低于对照组(P=0.000),浓度越大,活性越低(P < 0.05)。结果显示碱性成纤维细胞生长因子在1-100 μg/L范围内质量浓度越高,促进人牙周膜细胞增殖和抑制碱性磷酸酶活性的作用越强。     

前列腺特异的转录因子NKX3.1上调 Dicer1 基因的表达

目的: 研究前列腺特异转录因子NKX3.1对 Dicer1 基因表达的影响。方法: 转染pcDNA3.1-NKX3.1至前列腺癌PC3细胞,通过G418筛选,有限稀释法克隆培养获得NKX3.1稳定转染的PC3细胞-PC3(+);采用基因芯片检测NKX3.1对前列腺癌PC3细胞基因组表达谱的影响。进一步应用RT-PCR、Western blotting进行验证。为进一步检测 Dicer1 表达增高、促进microRNA的成熟作用,构建了microRNA let-7a-1靶序列-报道基因融合质粒,转染PC3细胞和PC3(+)细胞,通过报道基因检测成熟microRNA let-7a-1对其靶序列的作用。结果: 基因组芯片显示,NKX3.1稳定表达的PC3(+)细胞中, Dicer1 表达明显高于PC3细胞。RT-PCR、Western blotting结果验证PC3(+)细胞中Dicer1 mRNA和蛋白质的表达水平均高于PC3细胞。microRNA let-7a-1靶序列-报道基因融合质粒,转染PC3细胞和PC3(+)细胞后,报告基因检测,PC3(+)细胞中荧光素酶活性明显低于PC3细胞。结论: NKX3.1可上调前列腺癌PC3细胞中Dicer1的表达,促进microRNA的成熟及作用。     

表皮干细胞体外培养体系的初步研究

目的探讨以成纤维细胞Ⅰ型胶原网格模型体外培养表皮干细胞的可行性。方法酶消化法获取细胞悬液，Ⅳ型胶原快速贴壁法分选表皮干细胞，将体外培养的表皮干细胞和成纤维细胞接种于0．01％的戊二醛交联的Ⅰ型胶原上，气-液界面培养2周，免疫组化法、流式细胞仪和细胞周期来鉴定贴壁细胞，共聚焦显微镜和扫描电镜观察细胞材料。结果贴壁细胞CK19和P63阳性表达，β1整合（CD29）表达率为98．57％，CD71表达率为9．05％，94．99％细胞处于静息期，以上均表明该细胞是表皮干细胞，并在Ⅰ型胶原上呈鱼鳞状排列。结论表皮干细胞在Ⅰ型胶原上生长良好，可以生成全层组织工程皮肤。     

氨基葡萄糖胶囊干预膝骨关节炎患者外周血单个核细胞中软骨代谢相关基因的表达

背景:目前关于氨基葡萄糖的研究多集中于对膝骨关节炎的治疗作用,但关于其对膝骨关节炎患者外周血中软骨代谢相关基因影响的研究有限。目的:观察氨基葡萄糖胶囊对膝骨关节炎的治疗效果及对软骨代谢相关基因表达的影响。方法:选取2017年3月至2019年2月郑州大学附属郑州中心医院收治的90例膝骨关节炎患者,另选取同期医院体检中心收入的40例健康受试者,检测并对比健康受试者与膝骨关节炎患者治疗前外周血单个核细胞中软骨寡聚基质蛋白、Ⅱ型胶原纤维α1、聚糖蛋白、特异性组织抑制物3基因表达水平。采用随机数表法将膝骨关节炎患者分为常规组和研究组,分别给予双氯芬酸钠缓释片、双氯芬酸钠缓释片+氨基葡萄糖胶囊治疗12周,对比治疗前后2组外周血单个核细胞中各基因表达水平、Lequesne指数,统计治疗期间2组不良反应发生情况。结果与结论:①与健康受试者比较,膝骨关节炎患者外周血单个核细胞中软骨寡聚基质蛋白、特异性组织抑制物3 mRNA相对表达量升高(P<0.05),Ⅱ型胶原纤维α1、聚糖蛋白mRNA相对表达量降低(P<0.05);②治疗前研究组和常规组外周血单个核细胞中各基因表达水平比较,差异均无显著性意义(P>0.05);治疗后2组外周血单个核细胞中软骨寡聚基质蛋白、特异性组织抑制物3 mRNA相对表达量均低于治疗前,且研究组低于常规组(P<0.05);治疗后2组外周血单个核细胞中Ⅱ型胶原纤维α1、聚糖蛋白mRNA相对表达量均高于治疗前,且研究组高于常规组(P<0.05);③治疗前研究组和常规组Lequesne指数比较,差异均无显著性意义(P>0.05);治疗后2组Lequesne指数均低于治疗前,且研究组低于常规组(P<0.05);④研究组和常规组不良反应发生率比较,差异均无显著性意义(P>0.05);⑤结果表明,氨基葡萄糖胶囊可有效改善膝骨关节炎患者临床症状且安全可靠,可能通过抑制软骨寡聚基质蛋白、特异性组织抑制物3基因表达,促进Ⅱ型胶原纤维α1、聚糖蛋白基因表达实现的。     

体外大鼠肝卵圆细胞的长期培养方法

背景：肝卵圆细胞是目前公认的成体肝干/祖细胞,但其体外长期培养时会不可避免地丢失干细胞的活性。目的：探索大鼠肝卵圆细胞体外长期培养的方法。方法：构建2-乙酰胺基芴/部分肝切除肝再生大鼠模型,通过酶消化和Percoll梯度离心分离纯化大鼠异质卵圆细胞并进行免疫染色鉴定,用含表皮生长因子、白血病抑制因子的培养基体外长期培养,后撤去表皮生长因子、白血病抑制因子,通过形态学的观察和分子标志物的检测判断其能否保持干/祖细胞活性。结果与结论：采用含表皮生长因子、白血病抑制因子的培养基体外培养卵圆细胞4个月后,大鼠异质卵圆细胞仍能表达肝细胞标志物ALB、胆管上皮细胞标志物CK-19,经不含表皮生长因子、白血病抑制因子的培养液继续培养后,卵圆细胞胎肝标志AFP表达量迅速下降。该研究结果表明大鼠肝卵圆细胞在表皮生长因子、白血病抑制因子等培养条件下可长期增殖并保持干细胞活性。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

酸性成纤维细胞因子基因体外转染肌卫星细胞

背景：研究报道外源性酸性成纤维细胞生长因子既可调节肌卫星细胞增殖和分化,又具有预防运动终板退变及肌萎缩的作用。 目的：通过酸性成纤维细胞因子基因转染大鼠骨骼肌卫星细胞,检测目的基因转染肌卫星细胞效果及基因表达情况,探讨建立有效预防运动终板退变及肌萎缩种子细胞可行性。 方法：取Wistar成年大鼠后肢肌肉,差速贴壁培养法分离纯化肌卫星细胞,观察细胞生长特性并做免疫组织化学鉴定;取第2代细胞,LipofectamineTM2000 Reagent转染试剂介导,将重组真核表达质粒pEGFP-N1-aFGF转染肌卫星细胞为实验组;阴性对照组转染空载质粒pEGFP-N1;空白对照组仅加入转染试剂。转染后24-72 h和传代后分别用倒置荧光显微镜观察细胞绿色荧光蛋白表达情况,计算转染效率。转染细胞行Western Blot检测酸性成纤维细胞因子表达。提取转染后72 h 细胞总RNA, 实时荧光定量PCR检测酸性成纤维细胞因子基因mRNA表达。 结果与结论：分离纯化细胞经免疫组织化学鉴定为肌卫星细胞。荧光显微镜观察到细胞转染6 h后即有绿色荧光发出,荧光强度和表达细胞总数在72 h达到高峰,传代后仍可观察到绿色荧光蛋白表达。实时荧光定量PCR证实目的基因mRNA表达水平远远高于对照组,Western Blot检测实验组有大量酸性成纤维细胞因子产生。提示酸性成纤维细胞基因转染肌卫星细胞可表达基因产物,有望作为基因工程种子细胞预防失神经支配后运动终板退变及肌萎缩。     

小鼠抗人前B细胞白血病同源盒基因3多克隆抗体的制备与鉴定

目的制备前B细胞白血病同源盒基因3(PBX3)多克隆抗体。方法采用反转录PCR扩增PBX3基因区序列,并重组入原核表达载体pGEX-4T-1,重组载体转化BL21大肠杆菌,异丙基-β-D硫代半乳糖苷(IPTG)诱导GST-PBX3融合蛋白的表达。SDS-PAGE纯化融合蛋白,并用GST-PBX3融合蛋白免疫BALB/c小鼠,收集经过免疫的小鼠血清即抗PBX3的多克隆抗血清,采用间接ELISA检测其效价,通过免疫细胞化学技术及Western blot法鉴定其特异性。结果成功构建pGEX-4T-1/PBX3原核表达载体,转化BL21菌株后可高效表达GST-PBX3融合蛋白,且以不可溶包涵体形式存在。SDS-PAGE纯化蛋白并免疫小鼠产生的抗PBX3血清可特异识别细胞外源过表达的PBX3蛋白,同时可检测到细胞外源性PBX3蛋白的细胞内定位情况。结论获得效价和特异性良好的PBX3多克隆抗体。     

E1A激活基因阻遏子蛋白过表达可抑制人血管平滑肌细胞的迁移

背景：成熟动脉中膜血管平滑肌细胞(vascular smooth muscle cells, VSMCs)分化稳态的丧失是血管老化的一个重要原因。 目的：观察E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)抑制人血管平滑肌细胞迁移的分子机制。 方法：应用已建立的稳定转染CREG过表达细胞hVSMCs-CREG和CREG表达沉默的hVSMCs-siCREG细胞株通过刮伤实验和Transwell小室细胞移行实验检测细胞迁移能力,通过Western blot检测转染前后细胞中CREG蛋白、基质金属蛋白酶和JNK表达及活化情况,应用JNK和基质金属蛋白酶9抑制剂阻断研究分析上述信号分子表达变化在细胞迁移中的作用。 结果与结论：Western blot结果证实,CREG蛋白表达在hVSMCs-CREG组中增加(P < 0.05),而在hVSMCs-siCREG组中减少(P < 0.05)。刮伤实验和Transwell实验分析结果证实hVSMCs-CREG组细胞较正常对照组细胞迁移能力受抑。相反,hVSMCs-siCREG组细胞迁移能力显著增加(P< 0.05)。Western blot和明胶酶谱分析证实hVSMCs-siCREG组细胞中MMP9活性明显增加(P < 0.05),同时JNK蛋白活化。进一步应用JNK抑制剂阻断研究证实,CREG蛋白表达抑制引起的血管平滑肌细胞迁移能力增加与细胞中基质金属蛋白酶9活性均受到剂量依赖性抑制。结果证实,CREG蛋白表达可抑制JNK-基质金属蛋白酶9信号通路的活化,以此抑制体外培养的人血管平滑肌细胞迁移。     

小鼠骨髓间充质干细胞向胰岛素分泌细胞诱导分化过程中调控PDX-1基因表达miRNAs的鉴定

目的实现小鼠骨髓间充质干细胞(BMSCs)向胰岛素分泌细胞(IPCs)的诱导分化并对分化过程中可能调控胰十二指肠同源异型盒基因-1(PDX-1)基因表达miRNAs进行鉴定。方法首先分离培养BMSCs,应用conophylline和尼克酰胺将其诱导分化为IPCs,采用双硫腙(DTZ)染色和免疫荧光检测胰岛素的表达。然后采用靶基因预测软件miRanda和Target Scan对调控PDX-1基因表达miRNAs进行预测并通过双荧光素酶报告基因系统鉴定。实时定量聚合酶链反应(Real-time PCR)检测诱导分化过程中miRNAs及PDX-1的表达。结果诱导分化后的细胞双硫腙染色呈猩红色,免疫荧光化学显示有胰岛素表达。生物信息学方法预测得到4个可能调控PDX-1表达的miRNAs,通过双荧光素酶报告基因系统检测发现其中的miR-149和miR-346能结合到PDX-1 mRNA的3’UTR并有效抑制其表达。Real-time PCR检测结果表明,miR-149和miR-346的表达水平与PDX-1表达呈负相关。结论 miR-149和miR-346能负性调控IPCs诱导分化过程中PDX-1的表达。     

槟榔碱对2型糖尿病大鼠胰腺β细胞PDX 1 mRNA表达的影响

目的：探讨槟榔碱对2型糖尿病大鼠β细胞分泌功能及胰腺十二指肠同源盒因子 1（pancreas duodenum homeobox 1,PDX 1）mRNA表达的影响。方法：采用高果糖饲料饲养Wistar大鼠12周制备2型糖尿病大鼠模型,40只实验动物随机分为5组：对照组、模型组、模型+1 mg/kg槟榔碱组、模型+5 mg/kg槟榔碱组、模型+10 mg/kg槟榔碱组。药物注射4周后股动脉取血检测空腹血糖、三酰甘油、总胆固醇、高密度脂蛋白、低密度脂蛋白、血清胰岛素,处死大鼠取胰腺组织,石蜡切片HE染色观察组织形态学变化,RT PCR检测β细胞内PDX 1及胰岛素mRNA的表达水平。 结果：（1）高糖作用引起Wistar大鼠胰腺β细胞PDX 1 及胰岛素mRNA 的表达水平显著下降（P＜0.01）;（2）1 mg/kg和5 mg/kg槟榔碱处理能显著上调高糖喂养Wistar大鼠胰腺β细胞PDX 1与胰岛素mRNA表达水平（P＜0.05,P＜0.01）。结论：槟榔碱可以通过上调PDX 1和胰岛素基因表达,改善高糖环境下的β细胞胰岛素合成和分泌功能的损伤。     

Expression survey of genes critical for tooth development in the human embryonic tooth germ.

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.     

胎儿牙齿硬组织发育的临床应用解剖

目的：探讨国人胎儿牙齿硬组织发育状况。方法：对正常胎龄10-38周的胎儿上、下颌骨作连续切片,观察乳、恒牙胚及其相关结构发生规律。结果：牙体硬组织形成于14-20周,牙齿的增殖、钙化、萌出同时进行,牙釉质、牙本质厚度随胎龄增长而增加,各乳磨牙牙尖总是近颊尖最先发牛,恒牙板总是位于乳牙胚舌侧。结论：国人牙体硬组织发育成熟较低,牙釉质、牙本质厚度与胎龄呈正相关。     

Expression of podoplanin in the mouse tooth germ and apical bud cells

This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud.     

麦胚系统真核表达小鼠伯氏疟原虫TIP样蛋白的实验研究

目的体外真核表达小鼠伯氏疟原虫TIP样蛋白（PbTIP）胞外域重组蛋白。方法合成PbTIP胞外域DNA片段,克隆入真核表达载体pEU-E01-His-TEV—MCS,采用CFS真核无细胞蛋白表达系统（麦胚系统）,在体外表达可溶性PbTIP胞外域重组蛋白,SDS-PAGE凝胶电泳和Western-blot检测并鉴定目的蛋白表达情况。结果获得了可溶性表达的PbTIP胞外域重组蛋白,分子量约70000Mr,与预期相符,目的蛋白浓度约为0．1mg／ml。结论采用麦胚系统在体外获得了较高浓度的可溶性PbTIP胞外域重组蛋白,不但为后续的PbTIP胞外域功能研究打下了良好的基础,也为众多疟原虫未知蛋白的功能研究提供了新的方法。     

In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n‐ethyl‐n‐nitrosourea (ENU), 6‐mercaptopurine (6‐MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration‐dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration‐related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6‐MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real‐time PCR results showed that the mRNA level expression of p53 increased and bcl‐2 decreased significantly with the increasing ENU, 6‐MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6‐MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6‐MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99–107, 2017. © 2017 Wiley Periodicals, Inc.     

人原生殖细胞的分离与培养

目的:探讨人原生殖细胞(PGC)的分离取材时机与体外培养方法,为人类胚胎生殖细胞(EG)的建系研究奠定基础。方法:取不同孕龄的人胚,分离人PGC,在不同培养体系中,观察其增殖与分化情况。结果:孕8、9周较孕7周人胚用酶机械法分离PGC后,原代克隆形成率高,以小鼠胚胎成纤维细胞或STO细胞作为饲养层,并且在培养液中加入hLIF、hbFGF\,hSCF,能较好地维持人PGC的增殖并保持未分化。结论:孕8、9周龄人胚为分离PGC的适宜材料,酶机械法分离PGC简单有效,饲养层细胞与生长因子为人PGC细胞体外培养所必需。     

Detection of Bromo-Deoxyuridine-and Proliferating Cell Nuclear Antigen-Immunoreactivities in Tooth Germ

The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU-and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ.