B7-2基因工程抗体的制备及体内外抗B淋巴瘤作用研究

 构建B7-2人-鼠嵌合抗体基因表达质粒pIRES/ch3C8,经脂质体法转染真核表达细胞株CHO制备B7-2人-鼠嵌合抗体（命名为ch3C8）。该抗体能够识别人恶性B淋巴瘤细胞株Raji表面的B7-2分子并诱导其凋亡。ch3C8结构中来源于人Ig 的Fc段可介导高效的ADCC及CDC效应。经ch3C8结合的Raji细胞接种于BALB/c裸鼠后的致瘤性消失。该抗体在 B7-2相关肿瘤的生物治疗中具有潜在的应用价值。     

TIMP-1对肝癌细胞BEL-7402的增殖抑制

构建含人组织金属蛋白酶抑制TIMP1基因的表达载体，以磷酸钙沉淀法转染人肝癌细胞系^1BEL－7402，研究过表达TIMP1对肝癌细胞生长的影响，本研究首先从正常流产胎儿组织中提取总RNA，通过RT－PCR扩增获得TIMP1基因，并克隆到pcDNA6表达载体中，经酶切鉴定，序列测定结果正确后，转染入肝癌细胞系BEL－7402，采用MTT（四甲偶氮唑盐微量酶反应比色法）、细胞生长曲线等方法，经数据统计分析发现：TIMP1过表达能够抑制肝癌细胞BEL－7402的增殖。     

基于PiggyBac转座系统的低溢漏诱导型Tet-On过表达体系的建立及应用

为避免诱导基因稳定表达的Tet-On诱导表达系统溢漏表达,实现简便且高效的外源基因稳定诱导表达, 本研究拟在Tet-On调控的转录水平基础上,将基于稳定配体Shield-1的不稳定结构域FK506结合蛋白引入目的基因的N端,从蛋白水平控制其本底表达水平.为验证该系统的效果,本研究以荧光蛋白TdTomato为报告基因,经流式分析结果证明优化后的体系较原体系的溢漏表达在蛋白水平上降低7倍左右.将该系统应用于基于小鼠胚胎干细胞的体外牙向分化模型,在诱导因子Dox和稳定配体Shield-1的协同作用下,诱导表达牙齿发育相关转录因子Hand2提高了牙向分化诱导的完成度.     

应用SCID鼠筛选肝癌转移性亚克隆及M-H7402亚细胞系的建立

本研究应用人肝癌细胞SCID鼠转移模型，筛选和建立肝癌转移细胞系，旨在为肝癌转移研究提供实验材料．采用人肝癌细胞H7402，腋后背部皮下接种SCID鼠，接种后66天时，在荷瘤鼠赢弱时引颈处死，取其肺组织，进行原代细胞培养和传代培养，获得肝癌细胞H7402的亚细胞克隆，命名为M—H7402．然后，对M—H7402细胞进行了生物学鉴定，检测了细胞形态、染色体、细胞动力学、细胞周期、甲胎蛋白表达、癌基因表达和转移相关基因表达等指标．结果显示，M—H7402细胞的生长、增殖和形态等与亲本细胞H7402十分相近，其染色体形态仍为人类核型，众数维持在72～80之间，占75．0％．RT—PCR检测结果显示，M—H7402细胞甲胎蛋白表达为阳性．Western blot检测结果显示，与亲本细胞H—7402相比，癌基因C—myc表达水平较高，转移抑制基因nm23的表达水平明显下调．从肺组织中获得的肝癌转移细胞系M—H7402，在体外可连续传代培养，细胞形态不变．应用SCID鼠筛选获得的亚细胞克隆，与亲本细胞形成配对关系，可为肝癌细胞转移的研究提供新的实验材料。     

Inhibition of PKB/Akt activity involved in apigenininduced apoptosis in human gastric carcinoma cells

Apigenin is a flavonoid widely distributed in fruits and vegetables. It possesses growth inhibitory properties against numerous cancer cell lines. However,the molecular mechanism(s) by which api-genin elicits its effects have not been fully elucidated. Here we studied whether apigenin inhibits growth and induces apoptosis in human gastric carcinoma cells. We showed that the flavonoid inhibited growth of the cells and caused apoptosis,as evidenced by DNA Ladder,cleavage of pro-caspase-3 in a time-dependent manner. Induction of apoptosis was dependent on inhibition of the PKB/Akt activity. We found that while apigenin had no effect on the expression of Akt and Bad,it inhibited specific phosphorylation of the two proteins that are associated with pro-survival mechanisms. We propose that this important flavonoid induces apoptosis in gastric cancer cells by inhibiting Akt activity. Since Akt is often activated in cancers,our findings may have clinical implications.     

Human CMTM2/CKLFSF2 enhances the ligand-induced transactivation of the androgen receptor

CKLF (chemokine-like factor)-like MARVEL (MAL and related proteins for vesicle trafficking and membrane link domain) transmembrane domain containing (CMTM) is a novel gene family. One member of this family, CMTM2, also named chemokine-like factor superfamily 2 (CKLFSF2), is expressed highly in the testis and moderately in the prostate, marrow and peripheral blood cells. However, the function of human CMTM2 remains unknown. Here, we found that CMTM2 was upregulated in 5α-dihydrotestosterone (DHT)-treated LNCaP cells. We investigated the relationship between CMTM2 and the androgen receptor. Our results showed that CMTM2 enhanced DHT-mediated androgen receptor (AR) transactivation and the expression of prostate specific antigen (PSA). We also observed that CMTM2 enhanced the AR protein level, which was reversed by silencing endogenous CMTM2 expression, which suggested that CMTM2 might play an important role in maintaining the AR protein level. We also found that CMTM2 suppressed Akt activation. A previous study showed that Akt could phosphorylate AR at Ser210 and Ser790 and lead to AR ubiquitylation and degradation as well as suppression of AR activity. Taken together, suppressing Akt activation and increasing the AR protein level might be one of the mechanisms for the CMTM2-mediated enhancement of AR transactivation. Supported by National High Technology Research and Development Program of China (Grant Nos. 2006AA02A305 and 2002BA711A01) and National Natural Science Foundation of China (Grant Nos. 30271203 and 30671907)     

五味子中残留农药对卵巢颗粒细胞的毒性研究

探讨五味子中残留农药对卵巢颗粒细胞的毒性作用.MTT法测定农药提取物作用于卵巢颗粒细胞24、48、72 h后的细胞增殖情况;Western blot法检测农药提取物对卵巢颗粒细胞MAPK信号通路的ERK1/2及p-ERK1/2蛋白表达水平的影响.MTT结果显示农药提取物能够显著促进卵巢颗粒细胞增殖,48 h增殖率最高(P0.01),Western blot结果显示提取物能够促进ERK1/2、p-ERK1/2蛋白表达,与阴性对照组相比差异极显著(P0.01).因此农药提取物作用于体外培养的卵巢颗粒细胞能够显著促进细胞增殖,通过促进MAPK信号通路的ERK1/2、p-ERK1/2蛋白表达,对颗粒细胞产生毒性作用.     

人肿瘤细胞株中细小病毒H—1的DNA扩增与非结构蛋白NS—1基因的表达

研究三株人癌细胞和两株对照细胞对细小病毒Ｈ－１杀伤作用敏感性的分子机制．表明了在感染复数ｍｏｉ（ｍｕｌｔｉｐｉｃｉｔｙｏｆｉｎｆｅｃｔｉｏｎ）为５ｐｆｕ（ｐｌａｑｕｅｆｏｒｍｉｎｇｕｎｉｔ）／细胞的情况下，作为Ｈ—１病毒复制受纳细胞的人肝癌细胞株ＯＧＹ－７７０３和人胃癌细胞株ＳＧＣ—７９０１，能够支持病毒ＤＮＡ扩增和非结构蛋白ＮＳ—１基因的表达，这和作为阳性对照的由ＳＶ４０转化的新生儿肾细胞株ＮＢ—Ｋ一样，但对Ｈ—１病毒感染有抗性的人肾癌细胞株ＯＵＲ—１０和它的对照人胎肾细胞株ＨｕＫ—１，并不支持病毒ＤＮＡ扩增和ＮＳ—１蛋白的表达．本文结果指出，细小病毒Ｈ—１的杀伤作用与细胞中的病毒ＤＮＡ扩增及ＮＳ—１基因表达的程度相关．     

石松属植物化学成分抗炎活性的筛选

为研究2种石松属植物化学成分的抗炎活性,筛选具有较强抗炎活性的化合物,用LPS诱导RAW264.7细胞建立了体外炎症模型,采用MTT法检测了化合物对小鼠巨噬细胞株RAW264.7细胞增殖的影响.在安全浓度范围内(细胞存活率大于80%)给予药物干预,用Griess法检测了培养上清液中一氧化氮(NO)水平,以各化合物抑制NO释放的能力为筛选指标,评价了化合物的抗炎活性.结果表明:乙酰基二氢石松生物碱(16)、对香豆酸甲酯(21)、豆甾烷-3-酮-21-酸(22)具有较好的NO抑制率,并呈一定剂量依赖关系,提示它们具有一定的抗炎活性.     

HCAP1基因对Raji细胞凋亡及凋亡相关蛋白Bax/Bcl - 2的作用

目的：研究外源HCAP1基因产物对Burkitt淋巴瘤细胞系Raji细胞凋亡及凋亡相关蛋白Bax／Bcl-2的作用。方法：用脂质体介导的基因转移方法，把外源HCAP1基因转染到Raji细胞中，用流式细胞术和Hochest 33258荧光染色检测细胞凋亡；用Western blot检测外源HCAP1基因对凋亡相关蛋白Bax和Bcl-2蛋白表达的调节。结果：外源HCAPl基因导入Raji细胞24、48和96h后，细胞凋亡率增加。Western blot显示Bax／Bel-2蛋白表达比值明显增高。结论：转染外源性HCAP1基因可诱导Raji细胞凋亡，使Bax／Bcl-2蛋白表达比例上调可能是HCAP1诱导凋亡的机制之一。     

PinX1基因真核表达载体的构建及其对乳腺癌MCF-7细胞增殖的抑制作用

探讨了PinX1 基因在乳腺癌MCF-7 细胞生长和细胞周期中的作用, 初步探讨了该基因用于乳腺癌临床治疗的可行性. 采用RT-PCR 技术从293-T 细胞中扩增PinX1 基因, 将其克隆入真核表达载体pEGFP-C1 中, 再将重组质粒转染MCF-7 细胞. 通过real-time PCR 检测PinX1 基因的mRNA 表达, 用MTT 法检测转染前后细胞生长曲线的变化, 用流式细胞仪检测转染目的基因后细胞生长周期的改变. 检测结果表明, PinX1 基因已经在转染后MCF-7 细胞的细胞核内稳定表达, 乳腺癌细胞生长明显减缓(P <0.05), 增殖变慢(P <0.05), 细胞生长阻滞于G0/G1 期, 说明PinX1 基因可抑制乳腺癌MCF-7 细胞的生长和增殖.     

Cloning of aminopeptidase N promoter and its activity in hematopoietic cell and different tumor cell lines

Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expression in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may provide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radiotherapy.     

人PSF真核表达载体构建及其在CHO细胞中的表达

目的是构建人PSF基因真核表达载体pEGFP-N1-PSF,并在检测其在CHO细胞株中的表达情况。应用DNA重组技术和PCR方法从人宫颈癌Hela细胞中扩增PSF基因,插入pEGFP-N1真核表达载体,构建重组质粒pEGFP-N1-PSF并测序鉴定。将pEGFP-N1-PSF瞬时转染CHO细胞,通过Western blot和RT-PCR方法检测PSF的表达,荧光显微镜下观察绿色荧光蛋白表达。结果 CHO细胞转染pEGFP-N1-PSF真核表达载体后,RT-PCR和Western blot实验发现,在RNA和蛋白水平有PSF的表达,在荧光显微镜下可以观察到融合蛋白EGFP/PSF的表达。成功构建pEGFP-N1-PSF真核表达载体,证实其在CHO细胞中可以表达。     

RNA干扰阔口游仆虫γ-微管蛋白基因表达的研究

根据阔口游仆虫微管蛋白基因序列设计1对引物,扩增出692bp的γ-微管蛋白基因片段,将之重组到L4440载体中,重组质粒转化到RNAase缺陷型E.coli HT115菌株中,并用IPTG诱导双链RNA的表达,将经诱导后的重组菌喂食游仆虫,诱导出RNAi表型,具体表现为虫体变小、变圆,约两周后游仆虫全部死亡.电镜观察表明,经诱导后的游仆虫部分纤毛杆横切面显示结构为9+0,部分纤毛杆膨胀变形并开始瓦解,原高度有序的微管排列结构消失.进一步证实喂食法可有效地产生RNAi,γ-微管蛋白基因沉默后游仆虫无法维持正常形态并死亡,观察到纤毛杆的超微结构发生明显的变化,由此推测γ-微管蛋白的表达和细胞调控在游仆虫细胞微管骨架的装配及其细胞生命活动中起重要作用.     

过表达小鼠ERA蛋白对细胞系的影响

为了研究鼠ERA蛋白对细胞生长状态的影响,构建了可调控诱导表达载体pEGSH-mera,并与受体表达载体pERV3稳定共转染鼠成纤维细胞L-929。应用PonA进行诱导表达后,Western Blot检测其表达水平发现,细胞中鼠ERA蛋白明显增多;用MTT法测定其生长曲线发现,细胞生长加快;应用流式细胞仪检测其细胞周期发现,G2/M期细胞数量明显减少。所以,鼠ERA蛋白在细胞周期中发挥重要作用。     

局部晚期宫颈癌新辅助化疗后Bax蛋白作为治疗有效性的指标

探讨Bax蛋白与宫颈癌新辅助化疗疗效之间的关系,用免疫组化方法检测23例局部晚期宫颈癌组织新辅助化疗前后Bax蛋白的表达。结果化疗后2例为临床完全缓解,17例为临床部分缓解,4例患者对新辅助化疗无反应,总有效率为82．61％。NACT前有反应者和无反应者宫颈癌组织的Bax表达无统计学差异（P〉0．05）。对化疗有反应者NACT后BaxWS值有显著性差异（P〈0．05）;无反应者NACT后BaxWS值无统计学差异（P〉0．05）。不同病理类型和临床分期患者NACT前后Bax变化值无显著性差异（P〉0．05）;不同病理分级患者NACT前后Bax变化值有统计学差异（P〈0．05）。说明Bax与化疗疗效有显著相关性。NACT前后Bax变化值与临床分期和病理类型无关,而与病理分级有显著相关性。Bax有可能作为判断化疗疗效的指标。     

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study,we examined the expression of inducible nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) by immunohistochemical staining in 76 tissue sections collected from bepa-tocellular carcinoma(HCC) patients undergoing hepatectomy.Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody.We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC.Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody.The iNOS stain intensity of the liv-er tissue close to the tumor edge was stronger than that of HCC tissue,and the strongest was the hepatocytes colser to the tumor tissue.However,iNOS expression in 10 normal hepatic samples was undetectable.VEGF positive expression ratio was 84.8% in iNOS positive expression cases,and the ratio was 35.3% in negative cases.There was significant correlation(P=0.000) between iNOS and VEGF expression.Moreover,iNOS expression was significantly associated with bcl-2 and MVD,but without p53 expression.DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC.PI(Proliferating Index) and SPF(S-phase fraction)in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group.The present findings suggested that iNOS expression was significantly associated with angiogenesis,bcl-2 and cell proliferation of HCC.     

反义人端粒酶催化亚单位基因转染对人结肠癌细胞恶性表型的逆转作用

利用基因重组技术,人端粒酶催化亚单位基因反向插入真核表达载体pcDNA3.0,获得重组体pcDRTRT,通过脂质体法导入人结肠癌细胞株SW-111C,获得稳定转染细胞系,即反义细胞.该细胞易脱落,出现明显生长抑制现象;失去叠落生长能力;流式细胞仪(FCM)证实导入反义hTRT后,G0/1期细胞增加,G2M和S期细胞减少,增殖指数(PI)降低;且不能在软琼脂中形成集落.说明反义hTRT基因体外导入结肠癌细胞株SW-111C可以明显降低端粒酶活性,抑制结肠癌细胞的增殖且能使其恶性表型发生逆转.     

X连锁凋亡抑制蛋白对肺癌细胞生长和细胞周期的影响

为了研究靶向X连锁凋亡抑制蛋白的发夹状RNA对肺癌细胞中抗肿瘤作用,构建XIAP基因的shRNA表达载体,并设计阴性对照(psiRNA-Con)质粒,分别转染A549细胞;实时定量PCR和免疫印记法分别检测XIAP的mR-NA和蛋白的表达;四甲基偶氮唑盐试验(MTT)检测A549细胞的增殖;流式细胞术检测细胞周期分布。实验结果表明:转染psiRNA-XIAP组细胞XIAP mRNA和蛋白水平均低于正常对照组,生长慢于正常组(t=16.82和t=12.13,均P<0.01),G2/M期细胞所占比例增高(t=3.78,P<0.05),并出现亚G1峰。而阴性对照质粒psiRNA-Con未能下调XIAP的表达水平,对A549细胞增殖和细胞周期亦无明显影响。结论:针对XIAP的RNA干扰质粒特异性地抑制了其RNA和蛋白水平,使肺癌细胞A549生长减慢,诱导凋亡,并使其发生G2/M期阻滞,有望发展为新的抗肿瘤药物。     

氧化应激促进重组腺相关病毒2型体外转导分析

为揭示氧化应激在重组腺相关病毒2型(rAAV2)转导中的作用,以过氧化氢(H₂O₂)和铁过载(Fe-NTA)氧化应激为模型,在293T,LO-2,Hela,A549细胞中,从转基因表达量、阳性细胞数、基因组数和病毒衣壳分布等方面研究氧化应激对rAAV2转导的影响.实验结果表明:H₂O₂和Fe-NTA能促进rAAV2转导,转基因表达量、阳性细胞数、基因组数与对照组相比的差异具有显著的统计学意义;细胞转导12 h后,氧化应激组核周围衣壳分布数目显著比对照组多,氧化应激能够促使rAAV2长时间聚集在细胞核周围;当氧化应激产生的活性氧(ROS)被N-乙酰-L-半胱氨酸(NAC)消除,则氧化应激促rAAV2转导作用消失,说明氧化应激通过ROS发挥促进rAAV2转导作用.