HSV-TK自杀基因对涎腺多形性腺瘤细胞治疗的实验研究

目的研究单纯疱疹病毒胸苷激酶基因(Herpes simplex virus thymidine kinase，HSV-TK)／丙氧鸟苷(ganciclovir，GCV)系统对人涎腺多形性腺瘤体外基因治疗的作用。方法采用腺病毒包装重组脂质体介导的含有HSV-TK全长的cDNA真核表达质粒PDC312-HSVTK转染人涎腺多形性腺瘤细胞，通过RT-PCR检测TK基因在转染细胞中的表达；用MTT法检测HSV-TK／GCV系统对多形性腺瘤细胞的杀伤作用和旁观者效应：采用倒置显微镜、组织学染色等观察HSV-TK／GCV系统作用细胞后的形态学改变。结果HSV-TK基因转染24小时后，肿瘤细胞开始出现空泡性变；转染48小时后，RT-PCR从转染的细胞中成功扩增出1150bp的特异性TK基因片段。转染36小时后加入不同浓度的GCV治疗，细胞出现核固缩，胞浆裂解，随之细胞死亡、脱壁的现象。细胞存活率随HSV-TK／GCV作用时间延长而逐渐下降，当加入10^-4mol／LGCV治疗7天时，HSV-TK／GCV系统的杀伤作用最强，细胞存活率为10．3％。当TK阳性的细胞占细胞总数50％时，其旁观者效应最明显，细胞存活率为31．7％。结论HSV-TK／GCV系统对涎腺多形性腺瘤细胞具有明显的杀伤作用和旁观者效应。     

TK基因重组质粒的构建及在ACC-2细胞中的表达

目的：克隆胸腺嘧啶核苷激酶（TK）基因,构建质粒表达载体pIRES—TK,并利用该载体转染ACC-2细胞,建立了稳定表达TK基因的ACC-2细胞克隆。方法：通过PCR从逆转录病毒重组体pLNSX—TK中扩增出TK基因全长序列,将扩增产物定向插入到克隆载体pMD18-T中进行序列测定,测序正确后将其亚克隆到质粒表达载体pIRES中,构建重组表达载体pIRES—TK,用电穿孔法以该质粒转染ACC-2细胞,经G418筛选获得稳定表达TK基因的ACC-2细胞株,提取该细胞株的总RNA,用RT—PCR检测TK基因的表达。结果：PCR扩增出1128 bp大小的片段,测序结果与GeneBank报道的TK序列基本一致;阳性重组质粒pIRES—TK经XhoI和Mull双酶切后获得2692 Bb和1128 bp的片段;RT-PCR从转染细胞的总RNA中扩增出361 bp的预期片段。结论：成功扩增了TK基因的DNA片段;成功构建了质粒表达载体pIRES—TK;建立了稳定表达TK基因的ACC～2细胞株,为TK／GCV自杀基因系统在腺样囊性癌基因治疗中的应用奠定良好的基础。     

全反式维甲酸提高HSV-TK/GCV系统治疗舌鳞癌旁观者效应的研究

目的：探讨全反式维甲酸（all-trans retinoic acid,ATRA)对单纯疱疹病毒胸苷酸激酶（herpes simplex virus thymidine kinase,HSV-TK)／丙氧鸟苷（ganciclovir,GCV)系统治疗舌鳞癌旁观者效应的增强作用及机制。方法：应用免疫细胞化学、流式细胞仪等技术，对ATRA诱导舌鳞癌细胞系（Tca8113)细胞间隙连接蛋白基因（Cx43的表达进行分析；采用析因分析法分析ATRA对HSV－TK／GCV系统旁观者效应的影响。结果：舌鳞癌细胞经10^-7mol/L-10^-5mol/L ATRA处理后Cx43蛋白的表达明显提高；阳性细胞计数率由处理前的5．17％上升至30．53％（10^-5mol/L ATRA)，经t检验处理组与非处理组间差异有显著性（P＜0．01）；舌鳞癌细胞经ATRA处理后增强了HSV－TK／GCV系统的旁观者效应，两者间存在交互作用（P＜0．05）。结论：ATRA可显著提高HSV－TK／GCV系统的旁观者效应，该作用可能通过诱导Cx43蛋白的表达而实现。     

转染BMPⅡ型突变受体的Tca8113细胞的建立及意义

目的：建立稳定表达BMPⅡ型突变受体的Tca8113细胞，以便进一步从信号转导水平探讨、认识BMPs对口腔上皮恶变的作用机理；方法：用FuGENE6Transfection Reagent Kit真核转染试剂盒将携带BMPsⅡ型突变受体cDNA的真核表达载体转染Tca8113细胞；结果：得到抗G-418的阳性细胞克隆转染细胞，neo基因原位杂交阳性；结论：建立了稳定表达骨形成蛋白（BMPs）Ⅱ型突变受体的Tca8113细胞株。     

稳定表达cbfal的人牙乳头细胞模型的建立和鉴定

目的：建立能稳定表达核心结合因子a1(cbfal)的人牙乳头细胞模型。方法：体外培养人牙乳头细胞，脂质体法转染重组质粒，G418筛选抗性克隆，并从基因(原位杂交和RT-PCR)和蛋白水平(免疫组化和western blot)进行鉴定。结果：共获得三个G418抗性克隆，其中PC-3克隆能够稳定表达cbfal基因和蛋白。结论：成功建立了稳定表达核心结合因子a1的人牙乳头细胞模型PC-3。     

1稳定表达cbfa1的人牙乳头细胞模型的建立和鉴定

目的 :建立能稳定表达核心结合因子a1(cbfa1)的人牙乳头细胞模型。方法 :体外培养人牙乳头细胞 ,脂质体法转染重组质粒 ,G418筛选抗性克隆 ,并从基因 (原位杂交和RT -PCR)和蛋白水平 (免疫组化和westernblot)进行鉴定。结果 :共获得三个G418抗性克隆 ,其中PC -3克隆能够稳定表达cbfa1基因和蛋白。结论 :成功建立了稳定表达核心结合因子a1的人牙乳头细胞模型PC -3。     

腺病毒介导的HSV-TK/GCV系统治疗腺样囊性癌的体外实验研究

目的　体外观察腺病毒载体介导的单纯疱疹病毒胸苷激酶基因及羟基无环鸟苷系统 (ADV -HSV -Tk/GCV)对人腺样囊性癌细胞 (ACC -2及ACC -M )的杀伤作用。方法　分别将人腺样囊性癌细胞株 (ACC -2及ACC -M )用腺病毒构建的绿色荧光蛋白按 1∶10 ,1∶2 0 ,1∶3 0转染 ,在 12～ 3 6h内用荧光显微镜观察细胞的转染率。MTT法观察ADV -HSV -TK在不同感染复数 (5～ 60 )以及不同浓度GCV处理时对ACC -2及ACC -M的细胞杀伤作用以及“旁观者效应”。结果　体外研究发现 ,ACC -2及ACC -M均能被腺病毒 -绿色荧光蛋白转染。最小感染复数为 10～ 2 0 ,转染率与感染复数呈正相关。在感染复数为 5 ,GCV浓度为 2 5 μg/ml时 ,能够发挥细胞杀伤效应且出现剂量依赖性。MTT法检测发现经ADV -HSV -TK处理后 ,当GCV浓度为 2 5 μg/ml或高于该浓度时ACC -2及ACC -M的细胞抑制率为 40 % -60 % ,而当GCV单独作用时则无此作用。转染ADV -HSV -TK的细胞与未转染细胞按不同比例混合提示“旁观者效应”的存在 ,且转染细胞的比例越高 ,被杀伤的细胞比例越大。结论　腺病毒载体介导的单纯疱疹病毒胸苷激酶基因及羟基无环鸟苷系统 (ADV -HSV -TK/GCV)在体外能够有效抑制腺样囊性癌细胞 ,然而 ,其细胞杀伤效应及“旁观者效应”仍有待提高     

Semaphorin3F基因转染舌鳞状细胞癌细胞的方法研究

目的：探讨阳离子脂质体介导转染舌癌细胞的可行性，筛选含有Semaphorin3F（SEMA3F）的舌鳞状细胞癌细胞，检测SEMA3F基因在舌癌细胞中的表达。方法：应用阳离子脂质体转染方法将SEMA3F导入Tca8113细胞中，G418筛选阳性克隆，用RT—PCR检测转染组细胞中SEMA3F基因的表达。结果：Tea8113细胞中SEMA3F基因表达下调。筛选14d后，含有SEMA3F的转染组细胞克隆形成，SEMA3F在转染组细胞中表达稳定。结论：阳离子脂质体介导转染的外源性基因SEMA3F在Tea8113细胞获得稳定表达，为进一步实验奠定了基础。     

脆性组氨酸三聚体基因对黏液表皮样癌细胞生长抑制作用的研究

目的探讨外源性脆性组氨酸三聚体（FHIT）基因的转入对黏液表皮样癌细胞增殖和生长的抑制作用。方法通过透射电镜、流式细胞仪测试、免疫组化染色及肿瘤细胞裸鼠移植实验,将转入外源性FHIT基因前后的黏液表皮样癌MEC- 1细胞的体外生物学活性和体内成瘤能力进行比较。结果外源性FHIT基因转入黏液表皮样癌MEC- 1细胞后,S期细胞显著减少,G1期细胞增多;细胞倍增时间从（21.03±0.41）h延长为（26.86±0.71）h;软琼脂克隆形成率显著降低;细胞发生退行性改变和出现凋亡早期表现。FHIT基因转染后细胞间质黏液增多,说明细胞向黏液细胞分化。结论外源性FHIT基因能够抑制黏液表皮样癌MEC- 1细胞的生长和MEC- 1细胞在体内的成瘤活性,而且可以提高MEC- 1癌细胞的分化程度。     

外胚间充质干细胞永生化的实验研究

目的：探讨端粒酶催化亚基(hTERT）在外肌间充质十细胞永生化中的作用。方法：质粒pClneo—hTERT转染外胚问充质干细胞，经G418筛选后获得抗性克降。免疫细胞化学法及图像分析检测hTERT的表达，累计生长曲线检测细胞增殖能力。结果：转染后获3个抗性克隆。转染第3代细胞hTERT染色呈强阳性，转染后第15代细胞hTERT活性较正常培养的第15代细胞明显增强、转染细胞增殖能力有不同程度的增加，其中克隆2越过衰老期，寿命延长。结论：外源性hTERT介入可重建外胚间充质干细胞的端粒酶活性，使细胞延长寿命，得以永生。     

CO oxidation on stepped Rh[n(1 1 1) × (1 1 1)] single crystal electrodes: a chronoamperometric study

The CO electro-oxidation reaction has been studied on Rh[n(1 1 1) × (1 1 1)]-type electrodes in 0.5 M H₂SO₄ using chronoamperometry. The transients recorded on Rh(1 1 1), Rh(5 5 4), Rh(5 5 3) and Rh(3 3 1) are characterized by a current plateau, visible directly after charging of the double layer, followed by a main oxidation feature, which consists of two peaks, a pre-peak and a main peak. The current density in the plateau region is nearly constant over time and, thus, is of (quasi) zeroth order in CO coverage. A plot of log(j_plateau) vs. E_final gives a linear relationship with a slope of ca. 45 mV dec^?1, suggesting a second electron transfer as the rate determining step. Analogously to platinum, the current density plateau was ascribed to a Langmuir–Hinshelwood type reaction between CO_ads and OH_ads with no effective freeing of sites for OH adsorption due to relaxation of the CO adlayer. The presence of two peaks, rather than one, in the main oxidation feature can be explained by assuming a low surface mobility of CO and high oxidizability of rhodium surfaces. Indeed, dual step chronoamperometry shows that the mobility of CO on rhodium surfaces in aqueous media is very low. Since rhodium surfaces are known to oxidize readily, the pre-peak and main peak can be ascribed to CO reacting with OH, which adsorbs fast at the steps and more slowly at terrace sites. Since the geometry of the steps is nearly the same on each surface, the pre-peak appears structure insensitive, while the main peak shifts considerably with the step density. Introducing randomly distributed crystalline defects by cycling the electrodes repeatedly up to the surface oxidation region prior to each potential step experiment, results in a negative shift of the main peak, while the position of the pre-peak remains fixed. From the data presented, we conclude that the reaction nucleates at the steps and grows in the direction of the terraces.     

Assessment of a gel-type chelating preparation containing 1-hydroxyethylidene-1, 1-bisphosphonate

AIM: To test an aqueous gel containing 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) regarding its interactions with sodium hypochlorite, its calcium binding capacity, and its potential in preventing the formation of a smear layer when used in conjunction with rotary root canal preparation. METHODOLOGY: The experimental aqueous gel consisted of (w/v) 2% alginate, 3% aerosil, 10% Tween 80 and 18% HEBP. Interactions of gel components with hypochlorite were assessed using iodometric titration and monochromatic ultraviolet spectrometry. Two commercial paste-type chelators containing ethylenediaminetetraacetic acid (EDTA) and peroxide (RC-Prep and Glyde) served as controls. Calcium-binding capacities were measured in mixtures with a Ca2+ standard solution buffered at pH 10 using a calcium-selective measuring chain. Finally, root canals of 16 extracted single-rooted premolars per group were instrumented using ProFile instruments dipped in the experimental gel, RC-Prep, or nothing. Additionally, canals were rinsed with 10 mL of a 1% NaOCl solution during/after preparation. Smear scores in instrumented teeth were monitored using scanning electron microscopy. RESULTS: None of the experimental gel components showed short-term interactions with hypochlorite, whilst EDTA, peroxide, RC-Prep and Glyde immediately reduced the hypochlorite in solution. The experimental gel chelated 30 mg Ca2+ g-1, compared with 16 mg Ca2+ g-1 and 11 mg Ca2+ g-1 chelated by RC-Prep and Glyde respectively. Smear scores obtained with the experimental gel were significantly (P<0.05) lower than with RC-Prep in coronal and middle root thirds, whilst no differences were observed in apical root thirds. CONCLUSIONS: Under the conditions of this study, an HEBP gel appeared advantageous over currently available products.     

IL-1α、IL-1β和TNF-α mRNA 在实验性鼠根尖周炎中的表达

目的　检测IL-1α、IL-1β和TNF-αmRNA在鼠根尖周炎组织中的表达,了解上述细胞因子与根尖周炎的关系。方法　16只SD大鼠第一、二磨牙开髓,分别于术后3d、7d、14d、28d取磨牙根尖周组织,提取总RNA,用RT-PCR方法测定IL-1α、IL-1β和TNF-αmRNA的表达,用看家基因β-Actin作内参照进行半定量分析。结果　术后7d鼠根尖周组织中即有IL-1α、TNF-α、IL-1βmRNA的表达,14d达高峰,28d有所下降,但IL-1α和TNF-α的表达量大于IL-1β表达量,相差非常显著（P＜0.01）。结论　IL-1α和TNF-α可能是导致鼠根尖周炎的主要细胞因子。