KAI1基因转染的胰腺癌细胞系的建立

目的转染pCMV—KAI1重组质粒入人胰腺癌细胞株MiaPacaⅡ和Panc1，观察KAⅡ基因的表达，为进行KAI1基因在胰腺癌中抗转移作用的有关研究提供实验用靶细胞，并为今后利用KAI1基因治疗胰腺癌提供实验和理论依据。方法采用Western blot方法从7种胰腺癌细胞中筛选出无KAI1表达的MiaPacaⅡ和Panc1细胞株，通过脂质体转染法将pCMV—KAI1重组质粒转染到MiaPacaⅡ和Panc1中，经G418筛选4周，获得单克隆细胞系，应用Western blot、免疫荧光及免疫细胞化学方法检测转染细胞系KAI1蛋白的表达。结果获得了稳定表达KAI1基因的MiaPacaⅡ和Panc1胰腺癌细胞克隆；经Western blot分析显示转染后的胰腺癌细胞株MiaPacaⅡ与Panc1有明确的分子质量为29100KAI1蛋白表达，而未转染细胞无KAI1蛋白表达；免疫荧光显示，转染的胰腺癌细胞质中可见明显的荧光，而无转染的母细胞无荧光；免疫细胞化学检测显示，无转染的细胞呈阴性反应，转染的细胞呈中到强度的阳性反应。结论pCMV—KAI1重组质粒经转染人人胰腺癌细胞株MiaPacaⅡ和Panc1后可表达KAI1蛋白，从而建立了KAIl蛋白表达的MiaPacaⅡ和Panc1细胞系。     

HBx 基因转染对人肝癌细胞系 HepG2细胞增殖的影响

目的探讨HBx基因转染对人肝癌细胞系Hep G2细胞增殖的影响。方法用脂质体转染法将HBx真核表达载体pc DNA3/HBx瞬时转入Hep G2细胞(转染HBx细胞组);以转染空载体细胞组和未转染Hep G2细胞组作为对照。以流式细胞仪及荧光显微镜观察各组肝癌细胞增殖情况。结果转染HBx细胞组的Hep G2肝癌细胞生长较其他两组密集。流式细胞仪检测显示转染HBx细胞组的Hep G2细胞较多处于增殖期(G2期细胞占5.68%)。荧光显微镜图像显示转染HBx细胞组处于增殖期的Hep G2细胞比率为47%,转染空载体细胞组为28%,未转染Hep G2细胞组为25%;转染HBx细胞组Hep G2细胞处于增殖期的比率与其他两组比较有统计学差异(P均<0.05)。结论 HBx基因转染能促进人肝癌细胞系Hep G2细胞增殖。     

KAI1基因抑制胰腺癌细胞转移机制的探讨

目的　将pCMV KAI1重组质粒转染人高转移胰腺癌细胞株MiaPaCaⅡ中 ,观察转染前后KAI1基因对胰腺癌细胞侵袭转移能力和运动能力的影响 ,从基因水平探讨KAI1基因抗胰腺癌转移机制。方法　通过脂质体转染法成功将重组质粒转染到MiaPaCaⅡ中 ,利用Transwell小室测定细胞穿膜侵袭运动能力 ,采用SDS 聚丙烯酰胺凝胶电泳检测明胶酶活性。结果　pCMV KAI1重组质粒转染后 ,显微镜下细胞计数示转染后胰腺癌细胞穿透基底膜的数量较转染前明显减少 (P <0 0 5 ) ,细胞运动能力明显减弱。聚丙烯酰胺凝胶电泳显示负染带宽度比转染前变窄 ,亮度减弱 ,降解基质膜和细胞外基质的能力和侵袭转移能力下降。结论　KAI1基因控制胰腺癌细胞转移可能是通过抑制癌细胞侵袭及运动功能来实现的。     

单纯疱疹病毒Ⅰ型胸苷激酶基因转染人肺腺癌细胞A549的体内外实验研究

单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ１－ＴＫ）基因是一个药敏基因,转导该基因的细胞对非毒性抗病毒药更昔洛韦（ＧＣＶ）或阿昔洛韦（ＡＣＶ）敏感,能被其杀灭。通过探讨该基因转染肺癌细胞后对ＧＣＶ或ＡＣＶ的敏感性,为其进入临床治疗肺癌积累实验资料。本文用电穿孔法将含胸苷激酶（ＴＫ）基因的逆转录病毒载体导入人肺腺癌细胞Ａ５４９,在体内外观察转基因细胞对ＧＣＶ的敏感性。材料与方法合ＴＫ基因的逆转录病毒表达载体ＰＬＸＳＮ－ＴＫ参照文献［１］构建,Ａ５４９细胞购自上海细胞所。新霉素（Ｇ４１８）、四甲基偶氮唑盐（Ｍ…     

蚊抗药性相关糜蛋白酶基因稳定转染细胞系的建立与表达鉴定

目的 建立抗药性相关糜蛋白酶基因稳定转染细胞系。方法 采用PCR方法，以淡色库蚊抗药性品系CD-NA文库为模板，扩增出抗药性品系高表达的糜蛋白酶基因编码区；经T／A克隆测序鉴定后，亚克隆到真核表达载体piEl-3，构建重组昆虫细胞表达载体piEl-3／chy，经酶切和PCR鉴定后，与带有筛选标记的piEl-neo质粒共转染蚊细胞C6／36，通过G418选择培养，建立转染细胞系；用RT-PCR、Westernblotting鉴定糜蛋白酶基因的转录表达。结果 成功构建了真核表达载体piE1-3／chy，证明糜蛋白酶基因已转入C6／36细胞，建立了稳定转染细胞系，表达产物分子质量单位约30ku，与理论值相符。结论 稳定转染细胞系的建立和基因表达为进一步研究糜蛋白酶基因与抗药性的关系奠定了基础。     

体内心肌基因转染技术的研究进展

心力衰竭是各种心血管疾病的终末阶段,至今死亡率仍居高不下。近年来心力衰竭的基因治疗取得了很大的进步,有望成为心力衰竭新的治疗途径。然而,潜在治疗靶基因的选择、向大动物模型的过渡以及提高病毒转染技术的效率仍是目前难以克服的困难。本文仅就目前体内心肌基因转染技术的进展作一综述。     

蚊抗药性相关糜蛋白酶基因稳定转染细胞系的建立与表达鉴定

目的建立抗药性相关糜蛋白酶基因稳定转染细胞系。方法采用PCR方法,以淡色库蚊抗药性品系cD NA文库为模板,扩增出抗药性品系高表达的糜蛋白酶基因编码区;经T/A克隆测序鉴定后,亚克隆到真核表达载体pIE13,构建重组昆虫细胞表达载体pIE13/chy,经酶切和PCR鉴定后,与带有筛选标记的pIE1neo质粒共转染蚊细胞C6/36,通过G418选择培养,建立转染细胞系;用RT PCR、Westernblotting鉴定糜蛋白酶基因的转录表达。结果成功构建了真核表达载体pIE13/chy,证明糜蛋白酶基因已转入C6/36细胞,建立了稳定转染细胞系,表达产物分子质量单位约30ku,与理论值相符。结论稳定转染细胞系的建立和基因表达为进一步研究糜蛋白酶基因与抗药性的关系奠定了基础。     

鼠源性血管抑素基因转染对人肝癌细胞体内致瘤力的影响及新血管抑制作用

目的 研究血管抑素血管他丁（angiostatin）基因转染人肝癌细胞系HCC7721,对肿瘤细胞体外生长、周期分布、形态以及体内致瘤力的影响。探讨血管抑素的作用机制。方法 应用定向克隆技术构建鼠源性血管抑素血cDNA基因真核表达载体pcDNA3.1( )-angio,酶切鉴定和测序。采用脂质体基因转染技术将真核表达载体导入人肝癌细胞系HCC7721,新霉素G418筛选抗性克隆,设置转染空载体细胞为阴性对照和未转染细胞为空白对照。通过RNA点杂交和流式细胞荧光免疫检测血管抑素的表达,测定细胞生长曲线,计算细胞倍增时间,流式细胞仪检测细胞周期分布。建立动物模型,观察转染前后肿瘤细胞致瘤力的改变。免疫组化检测肿瘤组织微血管密度（MVD）和血管抑素体内表达。结果 成功构建带有碱基序列正确的血管抑素基因片段的重组真核表达载体pcDNA3.1（ ）-angio。分别转染脂质体/pcDNA3.1( ）-angio和脂质体/pcDNA3.1( ）的实验组,HCC7721肝癌细胞作阴性对照组经G418筛选,30d后均得到稳定的抗性细胞克隆,空白对照组在筛选1周后全部死亡。实验组HCC7721细胞在体内和体外均表达目的蛋白,而对照组细胞中无表达。与对照组相比,虽然实验细胞在体外的生长速度和细胞周期分布未发生明显改变。但在体内的致瘤力显著降低,瘤体增长缓慢,平均抑瘤率达47%。肿瘤MVD计数显示,实验组肝癌细胞形成的瘤体组织中MVD明显低于阴性对照组。结论 血管抑素不直接影响肝癌细胞HCC7721在体外的生物学特笥,但可强烈抑制体内肿瘤的形成,其机制可能是通过抑制肿瘤的新血管生成,间接发挥抑制肿瘤作用。     

Efficient gene delivery to articular cartilage using electroporation

Abstract

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

Efficient gene delivery to articular cartilage using electroporation

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

促甲状腺激素受体胞内环基因突变与乳头状甲状腺癌发病的相关性研究

目的探讨乳头状甲状腺癌的发病与促甲状腺激素受体(TSHR)基因突变的相关性。方法采用巢氏-多聚酶联反应-单链构象多态性分析(NEST-PCR-SSCP)和DNA测序方法,对65例乳头状甲状腺癌和44例正常甲状腺组织TSHR3个胞内环基因进行检测。结果NEST-PCR-SSCP检测乳头状甲状腺癌促甲状腺激素受体(TSHR)3个胞内环未发现明显带型异常;DNA测序后,检测的第3胞内环2例对照组织和3例甲状腺癌组织TSHR2000位点碱基均由C→T,使得所编码的601位氨基酸由组氨酸(CAT)→酪氨酸(TAT)(His→Tyr),余胞内环未发现基因突变。结论乳头状甲状腺癌TSHR3个胞内环未发现基因突变,提示乳头状甲状腺癌发病与TSHR胞内环基因突变无关;5例标本601位氨基酸均为酪氨酸,考虑中国人TSHR基因可能与国外人群存在差异,TSHR2000位点碱基存在基因多态性。     

Insight to drug delivery aspects for colorectal cancer

Colorectal cancer(CRC) is the third most common cancer diagnosed worldwide in human beings. Surgery, chemotherapy, radiotherapy and targeted therapiesare the conventional four approaches which are currently used for the treatment of CRC. The site specific delivery of chemotherapeutics to their site of action would increase effectiveness with reducing side effects. Targeted oral drug delivery systems based on polysaccharides are being investigated to target and deliver chemotherapeutic and chemopreventive agents directly to colon and rectum. Site-specific drug delivery to colon increases its concentration at the target site, and thus requires a lower dose and hence abridged side effects. Some novel therapies are also briefly discussed in article such as receptor(epidermal growth factor receptor, folate receptor, wheat germ agglutinin, VEGF receptor, hyaluronic acid receptor) based targeting therapy; colon targeted proapoptotic anticancer drug delivery system, gene therapy. Even though good treatment options are available for CRC, the ultimate therapeutic approach is to avert the incidence of CRC. It was also found that CRCs could be prevented by diet and nutrition such as calcium, vitamin D, curcumin, quercetin and fish oil supplements. Immunotherapy and vaccination are used nowadays which are showing better results against CRC.     

血清促甲状腺激素与甲状腺癌的关系

越来越多的证据显示,血清促甲状腺激素(TSH)是预测结节性甲状腺肿患者发生甲状腺癌的独立危险因素.即使血清TSH水平在正常范围内,结节性甲状腺肿患者发生甲状腺癌的风险也随着血清TSH水平的升高而逐渐增加.高水平的TSH与甲状腺癌的高发生率以及晚期甲状腺癌关系密切.血清TSH抑制治疗能降低肿瘤进展高危患者的复发率和死亡率.血清TSH在甲状腺癌的发展过程中起着非常重要的作用.     

基因芯片技术在肺癌研究中的应用进展

基因芯片技术是一项新的生物学技术,该技术具有高通量、大规模、高灵敏性等特点,在肺癌及其他恶性肿瘤研究中具有广泛的用途.本文就基因芯片技术在肺癌的早期诊断、耐药基因检测和转移相关基因检测中的应用情况作一综述.     

结节大小与甲状腺癌风险的临床研究

目的 分析手术切除的甲状腺结节大小与甲状腺癌患病风险之间的关系.方法 回顾性分析2001年至2011年于本院进行甲状腺切除手术的5 440例甲状腺结节患者,病历记录的甲状腺结节共7 642个,有病理结果的结节共6 907个,按照结节直径＜1.0cm、≥1.0cm且＜2.0 cm、≥2.0 cm且＜3.0 cm、≥3.0 cm且＜4.0 cm、≥4.0 cm分为5组,对不同直径分组甲状腺结节中甲状腺癌发生率进行分析比较.结果 (1)5 440例甲状腺结节患者中,评价的结节共6 907个,28.94％(1 999/6 907)的结节为甲状腺癌,其中为甲状腺乳头状癌的结节共1 947个(占所有恶性肿瘤的97.39％).(2)结节直径＜1.0 cm、≥1.0 cm且＜2.0 cm和≥2.0 cm这3组中,甲状腺癌发生率分别为51.6％、26.8％和11.2％ (P＜0.01).而在结节直径≥2.0 cm且＜3.0 cm、≥3,0cm且＜4.0 cm和≥4.0cm这3组中,甲状腺癌的发生率分别为12.1％、10.0％和9.5％ (P=0.213 2).(3)组织病理学为甲状腺癌的结节中,甲状腺乳头状癌所占比例呈随结节直径增加呈下降的趋势(＜1.0 cm:99.4％,≥1.0 cm且＜2.0 cm:98.4％,≥2.0cm且＜3.0 cm:90.3％,≥3.0cm且＜4.0 cm:89.5％,≥4.0 cm:68.4％),而滤泡细胞癌的比例逐渐增加(＜1.0 cm:0.5％,≥1.0cm且＜2.0 cm:1.3％,≥2.0 cm且＜3.0 cm:6.3％,≥3.0cm且＜4.0 cm:5.3％,≥4.0:21.1％).结论 甲状腺乳头状癌的发病风险随结节直径的增加而呈降低趋势,滤泡细胞癌与其他少见甲状腺癌的发病风险在较大直径的结节中增加.     

自身免疫性甲状腺疾病基因治疗的新进展

近年来,自身免疫性甲状腺疾病(AITD)的基因治疗在一些动物实验中取得成功,如通过干预Na /I-同向转运蛋白基因、甲状腺球蛋白基因、促甲状腺激素受体基因、白细胞介素-4基因等转基因治疗Graves病,通过干预白细胞介素-10基因、Fas配体基因、肿瘤坏死因子相关凋亡诱导配体基因等转基因治疗自身免疫性甲状腺炎。此外,细胞毒性T淋巴细胞相关抗原4、T细胞受体、X染色体等基因异常也与AITD发病有关,其基因治疗有待开展。基因治疗有望成为治疗AITD的一种新途径。     

甲状腺过氧化物酶基因c.2268dupT突变与甲状腺结节的相关性研究

目的 探讨先天性甲状腺功能减退症(先天性甲减)患者及其家系成员发生甲状腺结节与突变位点的关系.方法 58例先天性甲减患者入选,提取外周血白细胞基因组DNA,根据甲状腺功能及超声检查结果选择靶基因.PCR扩增甲状腺过氧化物酶(TPO)、双氧化酶2(DOUX2)、双氧化酶成熟因子2(DOUXA2)、促甲状腺激素受体(TSHR)、钠/碘协同转运体(NIS)基因的所有外显子,对纯化PCR产物进行测序.发现突变后对其家系成员进行筛查.患者及家系成员行甲状腺功能和99mTc甲状腺扫描或超声检查,然后分析5个基因的突变位点与甲状腺结节的关系.结果 16例先证者为复合杂合子或纯合子,其中10例为TPO基因突变,含c.2268dupT突变者6例.37例家系成员为杂合突变,其中TPO基因突变25例.TPO基因c.2268dupT突变先证者及杂合携带者共20例,其中12例并发甲状腺结节.与其他所有突变位点组相比,c.2268dupT突变组的甲状腺结节患病率较高,差异有统计学意义(x2=13.545,P ＜0.01).结论 TPO基因c.2268dupT突变为高频突变,该突变携带者发生甲状腺结节危险性较高.     

Dermatomyositis associated with thyroid cancer

We describe herein dermatomyositis (DM) associated with thyroid cancer in a 54-year-old woman. She was resistant to corticosteroids at first, but removal of the coexisting cancer resulted in improvement of DM. Reports on the association of DM with thyroid cancer are very few. However, recently, increasing incidence of thyroid cancer is pointed out. It is thought that increasing incidence reflects increased detection of subclinical disease due to increased diagnostic scrutiny, not an increase in the true occurrence of thyroid cancer. Thus, DM associated with thyroid cancer may be more frequent than we generally expected. We recommend that thyroid studies should be included in cancer investigations, particularly in DM cases resistant to corticosteroids.