The role of microRNAs during the genesis of medulloblastomas induced by the hedgehog pathway

Constitutive hedgehog (Hh) signaling is associated with the genesis of medulloblastomas (MB). The objective of this study is to identify special microRNAs (miRNAs) regulated by the Hh pathway, and to clarify the role of miRNAs during the genesis of MB induced by sustained Hh activation. In the primary screening, we used stemloop RT-PCR to test the expression of 90 different miRNAs in the wildtype (WT) and Ptc-/- MEF cell lines. In the secondary screening, the miRNAs screened from the first screening were validated in the Sufu-/- MEF cell lines. We then verified the expression of miRNAs both in the normal cerebellar tissues and the MB induced by activated Hh pathway, and examined the expression of the other 21 miRNA members of the miR-154 cluster in the MB and normal cerebellum. In the first screening, 13 miRNAs showed significant differential expression in WT and Ptc-/- MEF cell lines, while 10 of them had significant difference in the Sufu-/- MEF cell line. Compared to the normal mouse cerebellum, only 2 miRNAs in 15 miRNAs were differentially expressed between the MB and normal cerebellar tissues. Among 21 members of the miR-154 cluster, 6 miRNAs were downregulated in the MB. Our study demonstrated that miR-154 may be regulated by the Hh pathway, and the activation of the Hh pathway led to the downregulation of the miR-154 cluster, resulting in the genesis of MB.     

Kinetics of the phenotype and function of murine peritoneal macrophages following acute inflammation

This study was undertaken to have a better understand for the process and the underlying mechanisms to limitmacrophage activation and population of activated macrophages.A comprehensive kinetics of cytokineproduction was performed in murine peritoneal macrophages recovered from Balb/c mice at various timeduring the course of an intraperitoneal injection with thioglycollate (TG).The expression of cell surfacemolecules such as MHC-Ⅰ,MHC-Ⅱ,B7-1 and B7-2 of these macrophages were also determined by flowcytometry.The present findings of our research suggested that the population of activated macrophages and theactivation of macrophages (including cytokines production and expression of cell surface functional molecules)were strictly controlled during inflammation process.This is one of the important mechanisms to retain the hosthomeostasis.Cellular & Molecular Immunology.2004;1(1):57-62.     

Effect of Glycoprotein ⅢaT1565C Mutation on FAK Phosphorylation

Objective To study the effect of Glycoprotein Ⅲ aT1565C mutation on the phosphorylation of focal adhesion kinase (FAK). Methods The recombinants of pcDNA3.1( + ) Ⅱb and pcDNA3.1( + )Ⅲa or pcDNA3.1( + )ⅢaT1565C were transfected into CHO cells by Lipofectamine 2000. Cell lines expressing wild-type and mutational GP Ⅱ b/Ⅲ a were screened by G418. The constructed CHO cell lines were examed through flow cytometry (FCM) to detect the expression of CD41 and CD61. lmmunoprecipitation, Western blot and FCM were employed to detect the tyrosine and serine 910 phosphorylation of FAK in CHO cells stimulated by fibrinogen (Fbg). Results CD41 and CD61 were highly expressed in both CHO cell lines detected by FCM, which was 97.19% and 99.71%,respectively. The tyrosine phosphorylation of FAK was detected in CHO cells expressing wild-type and mutational GP Ⅱb/Ⅲa stimulated by Fbg for 90min, which was 16.24% and 20.44%, respectively. There was no serine 910 phosphorylation of FAK observed in both CHO cell lines stimulated by Fbg for 90min. However, serine 910 phosphorylation of FAK in the two cell lines was increased after 48h of stimulation to 34.89% and 73.84%, respectively.Conclusions CHO cell lines stably expressing wild-type GP Ⅱ b/Ⅲ a and mutational GPⅡb/ⅢaT1565C were constructed successfully. GPⅢaT1565C mutation could enhance the serine 910 and tyrosine phosphorylation of FAK. Increased phosphorylation of FAK may enhance GPⅡb/Ⅲ a-mediated outside-in signal transduction.     

RGD-FasL induces apoptosis of pituitary adenoma cells

This study was to investigate the cytotoxic effects on pituitary adenoma cell lines GH3/MMQ/AtT20 induced by RGD-FasL and the underlying mechanism. Fas/DcR3 mRNAs were detected by RT-PCR and their surface expressions were measured by flow cytometry. Cytotoxicity exerted by RGD-FasL on tumor cells was measured with MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. The cell cycle and apoptosis was assessed by flow cytometry with PI staining. The expressions of caspase8/9/3, Bcl-2, RANKL and JNK2 were detected by Western blotting. Approximately 13.7% of GH3 cells, 25.5% of MMQ cells, 22.2% of AtT20 cells express Fas, while 23.9% of GH3 cells, 24.1% of MMQ cells, 4.6% of AtT20 cells express DcR3. The cytotoxic effects of FasL/RGD-FasL on tumor cells were all taken in a dose-dependent manner. Cell lines MMQ/AtT20 showed the same sensitivity to RGD-FasL as to FasL, while cell line GH3 was less sensitive to RGD-FasL. The cell cycle analysis indicated that RGD-FasL could inhibit cells in G0/G1 phase and G2/M phase. In MMQ and AtT20 cells treated with RGD-FasL, the AI was not significantly different from that treated with FasL, while in GH3 cells treated with RGD-FasL, the AI was lower than that treated with FasL. The expressions of caspase-8/9/3, RANKL and JNK2 were increased while that of Bcl-2 was decreased after treatment with RGD-FasL, suggesting that RGD-FasL induces apoptosis through caspase activation. We concluded that RGD-FasL could possibly be considered as a novel therapeutical candidate for the treatment of pituitary adenomas. Cellular ＆ Molecular Immunology.     

Zinc-deficient diet aggravates ventilation-induced lung injury in rats

We investigated the effects of zinc deficiency on acute lung injury(ALI)induced by mechanical ventilation.Male Sprague-Dawley rats were fed with a zinc-deficient or zinc-proficient diet for 4 weeks,and then received mechanical ventilation at normal frequency and pressure for 30 min.Total protein,cell count,the number of polymorphonuclear neutrophil(PMN)in the bronchoalveolar lavage(BAL),and vascular endothelial growth factor(VEGF)expression in the lung were determined.Activation of nuclear factor-κB(NF-κB)was detected by examining the phosphorylation of NF-κB(pNF-κB p65)and the expression of inhibitor of NF-κB(pI-kBα).Compared to the controls,total cell count and the number of PMNs were significantly increased to 160% and 140%,respectively,in zinc-deficient rats treated with ventilation.Activation of NF-κB was significantly increased and VEGF was also increased to three-folds.Zinc deficiency aggravated the inflammatory response in rats and was associated with the overexpression of VEGF in response to mechanical ventilation.Zinc supplementation may be beneficial to zinc-deficient patients during mechanical ventilation.     

构建肿瘤坏死因子α刺激基因6慢病毒表达及干扰载体及对人瘢痕疙瘩成纤维细胞增殖与凋亡的影响

BACKGROUND: Current research has shown that tumor necrosis factor α stimulated gene 6 (TSG-6) has anti-inflammatory effect, and the scar formation can be inhibited by local injection of TSG-6 protein at the early stage of trauma. However, the mechanism of this effect is still unclear. OBJECTIVE:To construct the lentiviral expression vector and shRNA vector for human TSG-6, with stable overexpression, transfection and interference, and to explore the effect of TSG-6 on proliferation and apoptosis of keloid fibroblast cell lines.  METHODS:Human keloid fibroblast cells were isolated from the keloid’s tissue by enzyme digestion and identified by immunocytochemistry assay. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed and transfected into human keloid fibroblast, exclusively. Expression levels of TSG-6 mRNA and protein were detected by RT-PCR and western blot assay. MTT assay and flow cytometry were used to estimate the cell proliferation and apoptosis in each group after transfection. In addition, expression of Bcl-2, p53 and active-caspase-3 were detected by western blot assay in each group. RESULTS AND CONCLUSION: (1) Human keloid fibroblasts were separated successfully. Under the light microscope, cells were spindle. Immunohistochemical staining for vimentin was performed in the fifth passage of cells, with the positive rate of 100%. Cells were negative for cytokeratin. (2) Recombinant lentiviral vectors and stably transfected cell lines were successfully established. TSG-6 gene expression was altered apparently. Compared with the control group, cell proliferation was delayed and apoptotic rate was noticeably increased in TSG-6 gene overexpression group. Cell proliferation increased and apoptotic rate decreased in the TSG-6 gene intervention group (P < 0.05). (3) Western blot assay results demonstrated that Bcl-2 expression reduced, P53 and Active-caspase-3 expression significantly increased in the TSG-6 gene overexpression group (P < 0.05). (4) These finding showed that TSG-6 could inhibit proliferation and induce apoptosis in keloid fibroblasts. Its mechanism may be associated with the down-regulation of Bcl-2 protein expression, up-regulation of P53 protein expression and increased Caspase-3 activity. 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     

Induction of tumor cell apoptosis via Fas/DR5

The apoptosis inducing effects on tumor cell lines MGC803, BEL7402 and HL60 by Fas ligand and anti-human DR5 monoclonal antibodies （anti-DR5 mAb） and the underlying mechanism was studied. Fas/DR5 mRNA was detected by RT-PCR. Cytotoxicity exerted by FasL/anti-DR5 mAb on tumor cell lines was measured by MTT assay and the induced apoptosis was determined by agarose gel electrophoresis. Flow cytometry was employed to analyze the mode of cell death. The mRNA expression of DR5 in MGC803 and BEL7402 cells after giving anti-DR5 mAb was up-regulated compared with control group, while it was down-regulated in HL60 cells in the same condition. The mRNA expression of Fas in HL60 was higher after giving FasL compared with control group, while it was lower in MGC803 and BEL7402. MGC803 and BEL7402 were sensitive to anti-DR5 mAb but partially to FasL, and HL60 was sensitive to FasL but less sensitive to anti-DR5 mAb. Apoptosis induced by Fas ligand and anti-DR5 mAb vary among tumor cell lines. The underlying mechanism may be relevant to Fas/DR5 mRNA expression, which was presented as the release of caspase-8 and Bcl-2.     

气虚血瘀型Corti器毛细胞损伤动物模型的建立

BACKGROUND: Traditional Chinese medicine holds therapeutic potential for deafness and tinnitus, but there is a lack of in-depth research about the underlying mechanism, so establishing an animal model maybe helpful to explore the mechanism. OBJECTIVE: To establish hair cell damage model in the Corti organ with Qi deficiency and blood stasis in guinea pigs, and to assess its effects. METHODS: Twelve adult guinea pigs were randomly divided into normal control and model groups by random number table method. Guinea pigs in the model group were firstly treated with the modified relieving stagnated Qi and abnormal starvation for 15 days to model Qi deficiency, and then stimulated hair cell damage with blood stasis by photochemical induction. The controls received no intervention. Subsequently, the detection of serum D-xylose content and DPOAE test were performed, and the cochlea morphology was observed under light microscope. RESULTS AND CONCLUSION: (1) The serum D-xylose content in the model group was significantly lower than that in the normal control group (P < 0.01). (2) DPOAE test revealed that the amplitudes of 1 560 and 3 125 Hz in the model group were lower than those in the normal control group and before modeling (P < 0.01). (3) In the model group, hemangiectasis and microthrombosis appeared in the spiral ligament, microvascular basement membrane and modiolus capillaries, and stria vascularis presented edema and degeneration; degeneration, necrosis or loss of hair cells were visible in the Corti organ. (4) To conclude, the model of hair cell damage in the guinea pig with Qi deficiency and blood stasis is established successfully, which will provide a research tool for figuring out the mechanism underlying the traditional Chinese medicine for treating deafness and tinnitus, especially sudden hearing loss. 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

A metastasis map of human cancer cell lines

Here we introduce an in vivo barcoding strategy that is capable of determining the metastatic potential of human cancer cell lines in mouse xenografts at scale.We validated the robustness,scalability and reproducibility of the method and applied it to 500 cell lines1,2 spanning 21 types of solid tumour.We created a first-generation metastasis map(MetMap)that reveals organ-specific patterns of metastasis,enabling these patterns to be associated with clinical and genomic features.We demonstrate the utility of MetMap by investigating the molecular basis of breast cancers capable of metastasizing to the brain-a principal cause of death in patients with this type of cancer.     

Study on the Establishment of a Rat Model for Transfusion-related Acute Lung Injury with Coronary Heart Disease and its Oxygen Balance

Objective: To establish a rat model of transfusion-related acute lung injury(TRALI) with coronary heart disease(CHD), and to analyze the safety of blood transfusion through oxygen balance. Methods: Forty-five 10-day-old male Wistar rats were purchased, and 35 of them were fed with high-fat diet to establish coronary heart disease rat models, and then 20 of them were selected to establish rat models of transfusion-related acute lung injury with coronary heart disease(model group, 10 rats),positiv...     

中国遗传性耳聋大家系永生细胞系的建立与保存

目的建立我国遗传性耳聋大家系患者永久淋巴母细胞株以研究核基因结构对线粒体DNA突变的修饰效应.方法采用EB病毒转化外周血淋巴细胞同时加环孢霉素A法,建立该大家系永生细胞系,其中患者14例,配偶及正常同胞18例:男性17例,女性15例.结果建株成功率达到90%以上,对已建立的永生细胞株经复苏和冻存的成功率为100%.细胞染色体制备及G显带核型分析正常.结论通过建立永生细胞系保存这一具有重要研究价值的家系遗传资源,为在细胞和分子水平上进一步开展遗传性耳聋的基础研究提供了宝贵的资料.     

CpG基序联合EB病毒建立永生化B淋巴母细胞系

目的 体外建立慢性乙型肝炎患者永生化的B淋巴母细胞系(LCLs).方法 用EB病毒感染自慢性乙型肝炎患者外周血中分离的单个核细胞(PBMC),加入CpG DNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性.光学显微镜下观察LCLs的形态特征,利用流式细胞术分析LCLs膜表面分子CD19和CD23的表达水平.结果 46例患者PBMC经EBV感染4周后,42例转化成永生化B淋巴母细胞系,成功率为91.3%.转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养.结论 CpG免疫调节基序联合EBV感染人PBMC,提高转化效率,转化后的LCL保持了成熟B淋巴细胞的生物学特性,可作为体外研究HBV特异性免疫应答的刺激细胞和靶细胞.     

男性性腺发育不全患者外周血淋巴细胞永生细胞系的建立

目的建立男性性腺发育不全患者外周血永生细胞系以保存性腺发育不全患者特有的基因组资源,为进一步探讨性腺发育不全的发生机理提供材料.方法收集40例男性性腺发育不全患者的外周血样品,采用EB病毒转化技术,把患者外周血B淋巴细胞转化成永生淋巴母细胞系;用遗传学方法检测其建系前后的遗传稳定性.结果建系成功40例,所有建成的细胞系冻存后复苏成功率100%,核型和DNA分析表明建系前后遗传是稳定的.结论性腺发育不全患者永生细胞系的建立,为进一步研究性腺发育不全导致的不育症分子水平发病机理提供随时可取的实验材料.     

罗伯逊易位型21三体永生淋巴细胞株的建立

目的建立并鉴定13／21易位与21／21罗伯逊易位21三体永生淋巴细胞株,为21三体罗伯逊易位的遗传学研究提供实验材料。方法筛查获得易位型21三体患者并收集外周血,培养B95-8细胞制备EB病毒感染液,采用EB病毒转化法获得永生淋巴细胞株,对第10、15、20代细胞进行G显带染色体分析。结果筛检出1例罕见的13／21易位与4例121／21罗伯逊易位型21三体患儿并成功建立永生淋巴细胞株,传代至第10、15、20代细胞的核型无显著差异。结论明病毒转化法可用于易位型21三体永生淋巴细胞株的建立,早期转化的细胞可为该病的研究提供实验基础。     

中华民族永生细胞库的建立

目的 为保存中国不同民族基因组,完整建立中国各民族永生细胞库,供永久性研究需要。方法 按照严格的采样标准和“知情同意”原则,采集不同民族群体外周血样,利用EB病毒转化B淋巴细胞为永生细胞的技术,建立中国各民族永生细胞库。结果 已建立了47个民族70个群体（含民族支系）的3982株永生细胞株,并保存了7210份DNA样本。建立了较为成熟和稳定的利用EB病毒转化B淋巴细胞为永生细胞的技术。结论 这是目前规模最大的较为完整的中国国家级中国各民族永生细胞库,可供永久性研究需要。已向国内多家人类基因组相关单位提供了细胞株和 DNA,进行相关研究。     

A human-human hybridoma producing cytotoxic antibody to HLA-B15, cross-reacting with B17, B5, B35 and B18

Mononuclear blood cells from a multiparous woman were transformed with Epstein Barr virus, and a cell line (Tr2D8) producing anti-HLA antibody was obtained. This cell line was immortalized by hybridization to the human fusion partners KR4 and KR12. While the EBV line died after 7 months, the hybridomas have remained stable for 13 months. The EBV line supernatant (40 micrograms IgM/ml) lysed peripheral blood mononuclear cells (PBMC) bearing B15, B17, B5 and B35. Consistent lysis of B18 bearing cells was only observed with lymphoblastoid cell lines. The supernatant from the Tr2D8 (EBV line X KR4) hybridoma (2.7 micrograms IgM/ml) only lysed B15 bearing PBMC. At a concentration of 13.5 micrograms IgM/ml, the hybridoma antibody lysed lymphoblastoid cell lines bearing B15, B17, B5, B35 and B18.     

HFRS患者汉滩病毒核蛋白特异性T细胞克隆及其靶细胞的建立

目的　建立肾综合征出血热 (HFRS)患者汉滩病毒 (HTNV)核衣壳蛋白 (NP)特异性CTL克隆 ,为HTNVNPT细胞表位鉴定及HFRS患者T细胞免疫功能研究奠定基础。方法　采用Fi coll密度梯度离心法分离HFRS患者外周血单个核细胞 (PBMC) ,用灭活HTNV和IL 2体外刺激 ,有限稀释法建立T细胞克隆 ,流式细胞术鉴定克隆表型。并用EB病毒 (EBV)转化B淋巴细胞 ,建立B淋巴母细胞样细胞系 (B LCL) ,以含HTNVS基因的重组痘苗病毒感染B LCL作靶细胞 ,以CTL克隆作效应细胞进行细胞毒杀伤试验 ,测定T细胞克隆的抗原特异性。结果　T细胞克隆能特异性识别表达NP的B LCL。在 5名患者中 ,3名有较高的杀伤率 ,并自 2名患者建立了 5株HTNV特异性CTL克隆 ,其表型为CD8 均大于 6 0 %。结论　成功建立了HFRS患者HTNVNP特异性CTL克隆及其靶细胞。NP是HFRS患者HTNV特异性CTL应答的主要靶抗原之一。     

Evolution of clonality and invasive behavior of Epstein-Barr virus immortalized lymphoblastoid cell lines in SCID mice brains.

BACKGROUND: Recently established Epstein-Barr virus immortalized lymphoblastoid cell lines express polyclonal immunoglobulins, are diploid, and grow into invasive tumors when injected intracerebrally into mice with severe combined immunodeficiency (SCID). It is unclear whether clonal selection of neurotropic cell lines occurs during long-term growth in the brain and the effect of this selection on brain invasiveness. EXPERIMENTAL DESIGN: Epstein-Barr immortalized lymphoblastoid cell lines from a normal Epstein-Barr negative donor were serially passaged seven times intracerebrally within groups of SCID/SCID CB 17 mice. Each cell line was injected into five or more animals during each passage. Clonality of the rescued cell lines, genotype, and brain invasiveness were examined. RESULTS: All mice developed extensive intracerebral lymphoproliferative disease within 10-18 days after injection. Intracerebral, subarachnoid, intraventricular, and perivascular lymphoid lesions were noted. Infiltrates were similar in all animals studied regardless of the passage number. Clonal B cell populations were detectable in lesions after the first passage by Southern blot hybridization using JH probe. Immunohistochemically, polyclonal tumors were seen initially, but after the fourth passage, monoclonal cytoplasmic immunoglobulin was predominantly expressed by all tumors. Minor bands seen in the early passages disappeared subsequently. Random chromosomal abnormalities appeared in the rescued cell lines after the third passage; however, after the sixth passage, the abnormalities became more consistent. Clonability in agarose was very low initially in both cell lines and increased significantly after the sixth passage. CONCLUSIONS: These experiments demonstrate that within the immunoprivileged conditions of the SCID mouse brain, the evolution of Epstein-Barr immortalized lymphocytes from polyclonal to oligo- and monoclonal cell lines with chromosomal abnormalities occurs very early. This evolution is not paralleled by increased invasiveness in vivo.     

免疫缺陷、着丝粒不稳定和面部异常综合征家系永生化淋巴细胞系的建立

目的 探讨建立免疫缺陷、着丝粒不稳定和面部异常(ICF)综合征家系永生化淋巴细胞系的方法,为保存这类罕见病家系完整的基因组进行长期研究提供材料.方法 应用EB病毒转染ICF综合征家系外周血淋巴细胞,光学显微镜下观察B95-8细胞系及永生化淋巴细胞系形态特征,流式细胞仪检测ICF综合征家系B细胞表达水平,用台盼蓝检测冻存...     

Method for generation of human B lymphoblastoid cell lines using Epstein-Barr virus

A detailed methodology is described for the successful immortalization of B lymphocytes to give lymphoblastoid cell lines using Epstein-Barr (EB) virus. A virus stock is prepared from B95-8 or Akata cell line, which spontaneously release high titers of virus, and the immortalizing titer of the preparation determined. B lymphocytes from fresh or cryopreserved samples are separated from the whole cell population, infected with EB virus and cultured for 4 weeks for the establishment of a lymphoblastoid cell line. The presence of EB virus encoded proteins in the immortalized cell line can be detected by indirect immunofluorescent staining.