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Apoptosis and its pathway in X gene-transfected HepG_2 cells

World Journal of Gastroenterology 2005年第28期11卷 4326-4331页

作者： Na Lin Hong-Ying Chen Dan Li Sheng-Jun Zhang Zhi-Xin Cheng Xiao-Zhong Wang Department of Gastroenterology Affiliated Union Hospital Fujian Medical University Fuzhou 350001 Fujian Province China

AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells *** groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159）（P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000）（P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.

关键词： HBx transfect HepG2 Apoptosis Fas FasL Bax Bcl-xL c-mvc

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Human β-NGF gene transferred to cat corneal endothelial cells

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International Journal of Ophthalmology(English edition) 2016年第7期9卷 937-942页

作者： Wen-Juan Luo Min Liu Gui-Qiu Zhao Chuan-Fu Wang Li-Ting Hu Xiang-Ping Liu Department of Ophthalmology the Affiliated Hospital of Medical College Qingdao University Central Laboratory of the Affiliated Hospital of Medical College Qingdao University

AIM： To transfect the cat corneal endothelial cells （CECs） with recombinant human β-nerve growth factor gene adeno-associated virus （AAV-β-NGF） and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS： The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco＇s modified Eagle＇s medium （DMEM） to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following： normal CEC control group, CEC-AAV control group and recombinant CEC- AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method （FCM）; cell morphology was observed under inverted phase contrast microscope. RESULTS： The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group （P〉0.05）; there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group （P〈0.05）. MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group （P〉0.05）. FCM result show

关键词： nerve growth factor corneal endothelialcell transfect proliferation

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Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

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Neural Regeneration Research 2017年第3期12卷 433-439页

作者： Tao Chen Yan Yu Liu-jiu Tang Li Kong Cheng-hong Zhang Hai-ying Chu Liang-wei Yin Hai-ying Ma Department of Neurosurgery the First Affiliated Hospital of Dalian Medical University Department of Histology and Embryology Dalian Medical University Department of Oncology Dalian Central Hospital

Cytoskeletal proteins are involved in neuronal ***-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal ***,the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains *** examine this,we established a rat model of traumatic brain injury by controlled cortical *** 72 hours after injury,2 × 10~7 cells/m L neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells（3 m L） were injected into the injured *** 1–3 weeks after transplantation,expression of neurofilament 200,microtubule-associated protein 2,actin,calmodulin,and beta-catenin were remarkably increased in the injury *** findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival,growth,and differentiation in the injury *** underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.

关键词： nerve regeneration brain-derived neurotrophic factor neural stem cells transfect differentiation traumatic brain injury cytoskeleton neurofilament microtubule-associated proteins calmodulin Wnt/β-catenin neural regeneration

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乙型肝炎病毒DNA经精细胞垂直传播的实验研究

乙型肝炎病毒DNA经精细胞垂直传播的实验研究

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作者：高慧婕青岛大学

学位级别：硕士

目的研究乙型肝炎病毒(hepatitis B virus,HBV)DNA在HBsAg、HBeAg阳性患者睾丸和附睾组织中的检出及组织细胞定位,观察体外培养的鼠精子细胞俘获HBV DNA的能力及HBV DNA在精细胞中的定位,采用精子载体法建立HBV DNA垂直传递的小鼠模型... 详细信息

目的研究乙型肝炎病毒(hepatitis B virus,HBV)DNA在HBsAg、HBeAg阳性患者睾丸和附睾组织中的检出及组织细胞定位,观察体外培养的鼠精子细胞俘获HBV DNA的能力及HBV DNA在精细胞中的定位,采用精子载体法建立HBV DNA垂直传递的小鼠模型,探讨HBV垂直传播的机制。方法 ①取22例HBsAg、HBeAg阳性患者的睾丸和附睾组织,以18例HBsAg、HBeAg阴性者睾丸和附睾组织作为阴性对照,应用巢式聚合酶链反应(nest-PCR)技术和荧光原位杂交(FISH)技术检测并定位睾丸和附睾组织中的HBV DNA;②体外培养小鼠及大鼠精细胞,阳离子转染剂介导HBV DNA转染精细胞。应用聚合酶链反应(PCR)技术检测鼠精细胞中HBV DNA的存在;制作精子涂片,FISH观察HBV DNA在精细胞中的定位;③采用精子载体法建立HBV DNA垂直传递小鼠模型,应用PCR技术和半定量逆转录聚合酶链反应(RT-PCR)技术检测HBV DNA在新生代小鼠中的表达情况。结果 ①22例乙肝患者睾丸和附睾组织中,18例组织检出HBV DNA,阳性率为81.8%,对照组未检测到HBV DNA,两组差别有高度统计学意义(P<0.001);荧光原位杂交检测到HBV DNA主要集中在大部分乙肝患者睾丸组织生精小管的生精上皮和基底部;②鼠精细胞原位杂交结果显示,荧光显微镜下精子样本中部分精子带有黄绿色阳性杂交信号,普通光镜下DAB显色部分精子带有黄褐色阳性杂交信号,主要在精子头部的中后部区域两侧和顶部,对照组无信号存在或只呈现本底水平染色。在高倍镜下,随机选取视野,统计携带HBV DNA的精子数目,荧光原位杂交显示阳性率为28.2%(P<0.001),DAB显色后显示阳性率为31%(P<0.001);③应用精子载体法获得31只新生代小鼠:11只小鼠检出HBV DNA,检出率为35.4%(11/31),其中又有3只检出HBV RNA。结论 ①乙肝患者睾丸和附睾组织中存在HBV DNA,定位于各级生精细胞内,为HBV的垂直传播提供了形态学依据;②精子对HBV DNA具有主动俘获性,可以携带HBV DNA;③应用精子载体法建立HBV DNA经精细胞垂直传递的小鼠模型,说明HBV DNA可经精细胞传递给下一代新生小鼠,并在新生代小鼠中表达复制。

关键词： Hepatitis B virus DNA Vertical Transmission transfect Sperm

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