Osteogenic differentiation of bone mesenchymal stem cells regulated by osteoblasts under EMF exposure in a co-culture system

This study examined the osteogenic effect of electromagnetic fields （EMF） under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells （BMSCs） and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit （CCK-8）. For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase （ALP） staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure （2 h/day） could obviously promote prolifera- tion of BMSCs and osteoblasts, while long-term EMF （8 h/day） could promote osteogenic differen- tiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when ceils were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differ- entiation of BMSCs.     

Investigation of molecular mechanism of CNS symptoms induced by DDVP poisoning

Objective To Screen differentially expressed genes related to dichlorphos(DDVP) poisoning from rat‘s hippocampus using oligonucleotide microarray technology in order to elucidate the mechanism of DDVP poisoning. Methods We composed probes of 40 genes of our interest. The probes were retrotranscribed on plata glass and oligonucleotide microarray was formed. 0.5 ml DDVP was given to the rats in experimental group and 0.Sml-pumping brine was given to the rats in control group by hypodermic injection, twenty minutes after convulsion, all hippocampus were collected for total RNA extraction, cDNAs were marked with Cy3 and Cy5 for control group and experiment group respectively, and hybridized with loligonucleotide microarray. Hybridization signals were collected and analyzed following scanning by laser co-focal scanner. Results There were 8 differentially expressed genes identified. Conclusion Many genes expressing changed by DDVP poisoning could be analyzed in a time period by using oligonucleotide microarray, which provides a powerful method for further studies on the molecular mechanism of DDVP poisoning.     

Modulation of Gene Expression Networks underlying Realgar-Induced Differentiation of Acute Promyelocytic Leukemia Cells

Objective: To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. Methods: The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. Results: The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. Conclusion: The ubiquitin-proteasome degradation pathway may play an important role in the degradation of PML/RAR α fusion protein and the differentiation of NB4 cells.     

Effect of kidney-reinforcing,blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells from chronic aplastic anemia patients

OBJECTIVE:To explore the effect of kidney-reinforcing,blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells(MSCs) from patients with chronic aplastic anemia(CAA).METHODS:We used threeTraditional Chinese Medicine recipes,namely a kidney-reinforcing recipe(KRR),blood-activating and stasis-removing recipe(BASRR),and kidney-reinforcing,blood-activating and stasis-removing recipe(KRBASRR),and a normal saline control to prepare herbal medicine serum in Sprague Dawley rats.Thirty CAA patients were enrolled in the experimental group,including 17 kidney-Yang deficient patients and 13 kidney-Yin deficient patients.Ten healthy individuals were included in the control group.MSCs were isolated from bone marrow samples,and the cell density was observed to measure their proliferation ability by microscopy on days 2,7,and 14 after isolation.In addition,the expression of adhesion molecules of bone marrow MSCs(CD106,CD49d,CD31 and CD44) were detected by flow cytometry after 48 h of treatment with the four different herbal medicine serums.RESULTS:The proliferation of MSCs from kidney-Yang deficient and kidney-Yin deficient patients was weaker than that of MSCs from the control group.The expression of all adhesion molecules of bone marrow MSCs from CAA patients was obviously lower than that in the control group(P< 0.01).The expression of CD49d and CD31 in MSCs from patients with a kidney-Yin deficiency was lower than in those with a kidney-yang deficiency(P< 0.05 and P<0.01,respectively).For kidney-Yang deficient patients,CD31 expression in the KRBASRR group was significantly higher than that in the BASRR group(P<0.01),while CD44 in the KRBASRR group was significantly higher than that in both KRR and BASRR groups(P<0.01).For kidney-Yin deficient patients,CD106 and CD49d expression in the KRBASRR group was obviously higher than that in the KRR group(P<0.05),while CD31 and CD44 expression in the KRBASRR group was significantly higher than that in both KRR and BASRR groups(P< 0.05 and P<0.01,respectively).CONCLUSION:The bone marrow microenvironment in CAA patients is abnormal.The effect of KRBASRR may be better than that of KRR and BASRR for kidney-Yang deficient and kidney-Yin deficient patients by improving the expression levels of MSC adhesion molecules.     

DIFFERENTIAL EXPRESSION OF GENES IN OMENTAL FAT OF NORMAL WEIGHT AND OBESE SUBJECTS AND OBESE DIABETIC PATIENTS USING eDNA MICROARRAY

Objective To identify genes differentially expressed in omental fat of normal weight subjects, obese subjects and obese diabetic patients. Methods Using a high-density cDNA microarray, gene expression profile of omental fat from normal weigh subjects, obese subjects and obese diabetic patients were compared Results Totally, 119 and 257 genes were up-regulated in obese subjects and obese diabetic patients respectively, while 46 and 58 genes were down-regulated. A total of 77 genes, including PDK4 , which switched from carbohydrate to fatty acids as the primary source of fuel, were up-regulated in both obese and obese diabetic patients, while 8 genes, including key enzymes in lipid synthesis, such as HMG-CoA synthase, fatty acid synthase and stearoyl-CoA desaturase, were down-regulated in both groups. Tyrosine-3-monooxygenase/tryptophan 5-monooxygenase activation protein θ（ YWHAZ） , a negative regulator for insulin signal transduction, was up-regulated only in obese diabetic patient, but not in normal-glycemic obese subjects. Conclusion The study demonstrated that decrease of lipogenesis along with increase of fatty acids oxidation of adipose tissue could be a common cause of insulin resistance in obesity and type 2 diabetes, while block of insulin signal transduction may trigger the transition from obesity to diabetes. Further exploration of these genes will be useful in the understanding of the pathogenesis of obesity and diabetes.     

Effect of Tiantai No.1(天泰1号)on Gene Expression Profiles in Hippocampus of Alzheimer's Disease Rats by Bioinformatic Analysis

Objective:To study the effect of Tiantai No.1(天泰1号) on gene expression profile in hippocampus of Alzheimer's disease(AD) rat,molecular genetic target points of the effect of this drug were defined,its molecular genetic pharmacodynamic mechanism of anti-AD was further explored at molecular gene level,and a scientific basis was provided for its clinical availability and promotion.Methods:Thirty male SpragueDawley rats were divided into three groups with 10 rats per group:sham-operation group,model group and Tiantai No.1 group.Sterile surgical procedure was applied,the model group with bilateral hippocampal injection of Aβ_(1-40) was established,and normal saline was used instead of Aβ_(1-40)in the sham-operation group.One week after the models was made,rats were administered by gastric lavage once every day for three consecutive weeks.The rats of the sham-operation group and the model group were daily fed with purified water by lavage;the rats of the Tiantai No.1 group treated group were administered with Tiantai No.1 by lavage.Total RNAs of hippocampus tissues were extracted with Trizol,the changes of hippocampus gene expression profiles in the above three groups were analyzed by using Affymetrix rat whole genome expression profile microarray.Results:Microarray analysis showed that,compared with the sham-operation group,the hippocampus of the model group had 50 up-regulated genes with significant difference(fold change 2),and 21 down-regulated genes with significant difference(fold change 0.5);compared with the hippocampus of the model group,the hippocampus of the Tiantai No.1 group was found to have 5 up-regulated genes with significant difference(fold change 2) and 20down-regulated genes with significant difference(fold change 0.5).The functions of differentially expressed genes of the groups were involved in nervous system's development,neuronic differentiation and function-regulation,cellular growth and differentiation and apoptosis,synaptic occurrence and plasticity,inflammation and immune response,ion channels/transporters,cellular signal transduction,cellular material/energy metabolism and so on.Conclusion:Tiantai No.1 can regulate hippocampal function,and further regulate the brain function of animals in multiple gene target points by a number of ways.     

Difference of Gene Expression Profiles between Barrett＇s Esophagus and Cardia Intestinal Metaplasia by Gene Chip

The difference of gene expression profile changes in Barrett＇s esophagus （BE） and cardia intestinal metaplasia （CIM） epithelium was studied and the novel associated genes were screened in the early stage by cDNA microarray. The cDNA retro-transcribed from equal quantity mRNA from BE and CIM epithelial tissues were labeled with Cy3 and Cy5 fluorescence as probes. The mixed probe was hybridized with three pieces BiostarH-40s double dot human whole gene chip. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 3.0 software. It was found a total of 141 genes were screened out that exhibited differentially expression more than 2 times in all three chips. It was identified that in gene expression profiles of BE, 74 genes were up-regulated and 67 down-regulated as compared with CIM. The comparison between the difference of gene expression profile changes in BE and CIM epithelia revealed that there existed the difference between BE and CIM at gene level. 141 genes with the expression more than two time were probably related to the occurrence and development of BE and the promotion or progress in adenocarcinoma.     

Biological Characteristics of Human Bone Marrow Mesenchymal Stem Cell Cultured in Vitro

Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed, hMSCs were isolated from bone marrow and purified by density gradient eentrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow eytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90%. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs。     

体外培养大鼠骨髓间充质干细胞成骨基因表达谱的检测

目的:探讨体外培养大鼠骨髓间充质干细胞(MSC)成骨相关基因的表达谱,明确体外培养大鼠骨髓MSC的成骨潜能。方法:体外培养SD大鼠骨髓MSC,取第3代细胞提取总核糖核酸(RNA),制备杂交探针,并运用SuperArray公司提供的大鼠Oligo GEArray骨再生基因芯片(每张芯片可测113种基因)进行杂交,化学发光检测,通过X线胶片或台式扫描获得化学发光的芯片图像,使用网上提供的综合型GEArry表达分析配套软件(GEArryExpression Analysis Suite)进行完整的芯片数据分析。结果:检测体外培养大鼠骨髓MSC的113种成骨相关的基因,其中有31种基因表达强度较低,45种基因呈中等强度表达,37种基因表达强度较高。结论:体外培养大鼠骨髓MSC具有部分成骨细胞基因表型特征,但成熟成骨细胞的特征基因表达量较低,提示大鼠MSC是具有较强成骨细胞分化潜能的成纤维样细胞。     

糖皮质激素治疗三氯乙烯药疹样皮炎患者外周血淋巴细胞基因表达谱研究

目的探讨三氯乙烯药疹样皮炎在糖皮质激素治疗前后的基因表达谱变化。方法从1例三氯乙烯药疹样皮炎患者糖皮质激素处理前后的外周血中分离淋巴细胞，提取总KNA，合成cKNA并分别用Cy3和Cy5标记，与Agilent Human 1B寡核苷酸基因芯片杂交，研究基因表达谱变化。结果在检测的20173个基因中，筛选出共同差异表达基因1016个，其中上调618个，下调398个，和免疫与防御相关基因52个。结论和免疫与防御相关的52个基因可能为三氯乙烯药疹样皮炎的疾病相关基因。     

醛固酮瘤中醛固酮合成相关酶与相应调节基因表达的变化

目的 比较醛固酮瘤(APA)与正常肾上腺组织的基因表达谱,探讨醛固酮合成过程的差异与可能的调节异常。 方法 以术后病理学检查确诊为APA的组织标本（APA组,n=10）及正常肾上腺组织（对照组,n=7）作为研究对象。提取两种组织的总RNA,合成生物素标记的探针cRNA,与载有一组靶基因的基因表达谱芯片杂交;通过扫描荧光强度,计算机软件分析,得到两种组织的表达差异基因;对异常表达靶基因进行Real-time PCR验证。 结果 与对照组相比,APA组有97个基因表达上调,168个基因表达下调;醛固酮合成路径中,仅CYP11B2表达显著上调;生理性调节醛固酮合成的因子中,CYB5A、CYP17A1、DUSP1、HMGCR表达下调,RENBP和NR1H2表达上调;皮质醇合成关键酶CYP17A1表达被抑制。 结论 在APA的醛固酮合成路径相关酶与调节因子中,CYP11B2可能是关键合成酶;多数生理性调节因子被抑制,提示存在肿瘤性调节因素。     

基因芯片技术筛选2型糖尿病伴远端对称性多神经病变患者外周血单个核细胞差异表达基因

目的：应用基因芯片技术筛选2型糖尿病伴远端对称性多神经病变患者外周血单个核细胞（PBMC）与不伴远端对称性多神经病变糖尿病患者以及正常个体相比的差异表达基因.方法：采用包含5 075个功能已知人类基因的cDNA芯片检测2名2型糖尿病并发远端对称性多神经病变患者（DSPN组）,2名2型糖尿病不伴远端对称性多神经病变患者（DM组）以及2名年龄和性别匹配的正常个体（C组）PBMC基因表达谱,分析差异表达基因,并选择其中有差异表达的部分基因进行实时定量RT-PCR验证.结果：基因芯片技术筛选出22条差异表达基因,涉及细胞代谢与信号转导基因,原癌与抑癌基因,DNA合成和修复基因,离子通道与运输蛋白基因,DNA结合、转录和转录因子基因,细胞骨架组成基因等.其中上调基因4条,下调基因18条.实时定量RT-PCR测定下调基因中的AK1和FBXO7,与基因芯片技术结果一致.结论：2型糖尿病伴DSPN患者PBMC基因表达谱存在差异;DSPN可能涉及细胞代谢,信号转导和DNA合成等多个方面.     

基因芯片技术筛选结直肠癌腹膜转移相关基因

目的筛选和鉴定结直肠癌腹膜种植转移相关基因。方法收集手术切除3例伴有腹膜种植转移的结直肠腺癌病人原发病灶和正常黏膜的新鲜组织标本,提取总RNA,经逆转录合成cDNA后经体外扩增合成aRNA,Cy3荧光分子标记后和人类全基因组表达谱芯片杂交,采用经验贝叶斯方法(P≤0.05)筛选出差异表达基因,并用RT-PCR实验对部分差异表达基因进行验证。结果满足(P≤0.05)的基因共有105个。其中和正常组织相比,在肿瘤组织表达上调的基因有42个,表达下调的基因有63个。通过生物信息学分析,在表达上调基因中选择3条(S100P;PRDX1;SLPI)基因进RT-PCR行验证,结果与芯片结果完全相符。结论基因芯片技术通过筛选结直肠癌腹膜转移过程中发挥关键作用的基因,为寻找结直肠癌腹膜转移的分子标志物提供依据和参考。     

人参皂甙对K562细胞作用的基因表达谱研究

目的 运用基因芯片技术研究人参总皂甙(TSPG)作用后K562细胞基因表达谱的变化.方法 200 μg/ml TSPG作用于K562细胞3 d后,提取总RNA,合成cRNA并分别用cy3和cy5标记,与AgilentHuman 1B寡核苷酸基因芯片杂交,研究基因表达谱的变化.结果 TSPG刺激K562细胞后共有362个基因表达发生变化,与对照组K562细胞比较,表达上调的基因有20个,主要有代谢相关基因,信号转导相关基因,细胞受体相关基因等;表达下调的有342个,包括免疫防御相关基因,DNA结合与转录因子,代谢相关基因,细胞周期相关基因等.RT-PCR技术验证了FOSL1、E2F2、CCNE2和ODZ1四个基因表达的变化.结论 TSPG刺激K562细胞后,影响了细胞内一系列基因的表达.这些基因可能与TSPG的抗肿瘤机制有关.     

miR-30a-5p在人骨髓间充质干细胞向成骨细胞分化过程中的生物学功能及验证

目的：探讨并验证重组miRNA-30a-5p体外转染对人骨髓间充质干细胞（MSCs）向成骨细胞分化的生物学作用。方法：人工重组合成从人骨髓MSCs定向成骨分化差异性表达miRNA基因芯片结果中筛选成骨性表达量显著增高的miRNA-30a-5p。从人体骨髓中分离培养MSCs,成骨诱导分化过程中于体外转染重组miRNA-30a-5p,并通过茜素红S法染色检测钙盐沉积、碱性磷酸酶（ALP）钙钴法染色及ALP比色法定量检测成骨细胞ALP活性,对比观察研究重组miRNA-30a-5p在人骨髓MSCs定向成骨诱导分化过程中的生物学功能。结果：成功分离培养人骨髓MSCs并诱导其成骨定向分化。经体外向MSCs转染miRNA-30a-5p并诱导其成骨定向分化,转染效率为（37.32±2.43）%。分别于诱导培养第14、21天行ALP染色观察、ALP活性测定、茜素红钙盐结节染色检测各组细胞成骨活性,结果显示miRNA-30a-5p mimics转染组MSCs成骨分化特性显著性增强（P〈0.05）。结论：miRNA-30a-5p在MSCs定向成骨分化的过程中具有一定的促进增强作用,被转染的MSCs向成骨细胞定向分化表达有所增加,为进一步阐明人骨髓MSCs定向成骨分化的分子生化机制,细胞移植修复治疗骨缺损奠定了理论基础。     

DHPG诱导的大鼠海马脑片癫癎样放电模型基因表达谱分析

目的 研究代谢型谷氨酸受体-Ⅰ（mGluR-Ⅰ）激动剂D-对羟基苯甘氨酸（DHPG）诱导的大鼠离体海马脑片癫癎样放电模型的基因表达谱。方法 以含50 μmol DHPG的人工脑脊液持续灌流大鼠离体海马脑片,诱导建立癫癎样放电模型（DHPG组,n=3）。采用cDNA微阵列分析技术获得DHPG组的基因表达谱,与对照组（n=3）比较筛选出差异表达基因,并进行富集功能分类。结果 与对照组比较,DHPG组样本模型分别筛选出的上调基因206个,下调基因489个;其中差异表达达1.5倍的上调基因67个,下调基因86个;2.0倍以上的上调基因6个,0.5倍以下的下调基因25个。富集功能分类结果显示,差异表达基因功能涉及蛋白结合（19个）、分子功能、钙离子结合和核苷酸结合等多方面。结论 癫癎样放电及mGluR-Ⅰ激动剂的作用均涉及多种基因,是一复杂的调控过程。实验为进一步研究提供了依据。     

一种用于基因表达谱分析的基因芯片方法

目的：通过研究SLE患者外周血基因表达谱的改变，建立一种新型的基因芯片技术用于分子发病机理的研究。方法：提取总RNA，逆转录合成单链cDNA、双链cDNA，体外转录合成生物标记的cRNA与基因芯片进行杂交，通过抗体的检测标记荧光染料Cy3，基因芯片扫描仪进行图像扫描。结果：该方法有较高的重复性和稳定性，SLE患者与正常对照组相比较，鉴定出94个基因存在表达差异。结论：该方法能在较少起始标本量的情况下，有效地进行SLE致病基因的筛选，更好的理解SLE发生的分子机理，并且可在其它疾病研究中推广应用。