两种转染方法介导质粒转染人舌鳞癌细胞的实验研究

目的:比较新型阳离子聚合物交联化聚乙烯亚胺(polyethylenimine-ethyleneglycol dimethacrylate,PEI-EGDMA)与阳离子脂质体TRANSfection转染人舌鳞癌(Tca8113)细胞的转染效率和细胞毒性.方法:将合成的含有绿色荧光蛋白报告基因的靶向血管内皮生长因子RNA干扰质粒,分别通过PEI-EGDMA和TRANSfection转染入Tca8113细胞中,每组设4 种浓度.转染后24 h,荧光显微镜下观察转染效果,计算各组的转染效率.48 h后四甲基偶氮唑盐(MTT)法行细胞毒性检测.结果:PEI-EGDMA与重组质粒质量比(W∶ W)控制在1.5∶ 1时转染效率最高,约为72%,与其余浓度组相比存在显著性差异(P<0.05).TRANSfection与重组质粒质量比控制在2∶ 1时,转染效率最高,约为55%,与其余各浓度组有显著性差异(P<0.05).在各自最佳转染条件下,PEI-EGDMA的转染效率明显提高.MTT结果显示在实验设定的浓度范围内,各组细胞的生长无显著性差异(P>0.05),细胞毒性较小.结论:新型阳离子聚合物PEI-EGDMA与TRANSfection相比转染效率更高,细胞毒性小,有望成为人舌鳞癌细胞基因转染的有效载体.     

硝苯地平对人牙龈上皮细胞bcl-2基因表达的影响

目的:体外观察硝苯地平(nifedipine,NIF)对人牙龈上皮细胞(human gingival epithelial cells,HGECs)bcl-2基因转录水平的调节,探讨NIF诱导的药物性牙龈增生(drug-induced gingival overgrowth,DGO)与凋亡抑制基因bcl-2的相关性.方法:采用牙周手术切除的健康牙龈组织.用酶消化法分离培养HGECs;免疫组织化学方法对培养细胞进行细胞鉴定;实时定量PCR技术检测不同浓度NIF(1 μg/ml、2 μg/ml和3 μg/ml)刺激下HGECs中bcl-2 mRNA水平,以0 μg/ml NIF为空白对照.采用SPSS 11.0软件包对所得数据进行单因素方差分析.结果:酶消化法获得的HGECs在体外培养中生长状态良好;免疫组织化学显示,HGECs抗角蛋白染色阳性,抗波形蛋白染色阴性;NIF处理24h后的HGECs bcl-2 mRNA水平随NIF浓度的增高而上升,3 μg/ml浓度组与空白对照组有显著差异(P＜0.05);NIF处理48h后.2 μg/ml、3 μg/ml浓度组HGECs bcl-2 mRNA水平与空白对照组差异明显(P＜0.05).结论:NIF调节体外培养的HGECs中bcl-2基因转录的水平.     

人肿瘤坏死因子-α基因转染对舌癌细胞的抑制作用

目的：探讨人肿瘤坏死因子－α（hTNF-α)基因转染对舌癌细胞生长的影响。方法：制备和纯化含hTNF-α基因穿梭质粒，用DOSPER阳离子脂质体介导转染Tca8113舌癌细胞，对照组只给予等量的脂质体，不加入质粒；转染基因，24，48，72，92h后用ELISA法检测细胞培养上清液中hTNF-α的浓度，并用MTT法检测细胞的存活率。结果：基因转染24，48，72，96h后转染组与对照组细胞培养上清液中hTNF-α浓度之间的差异均有显著性（P＜0．05），转染组各个时期浓度均高于对照组；MTT法检测相应各时期转染组与对照组平均OD之间的差异均有显著性（P＜0．05），转染组各个时期平均OD值均低于对照组，转染细胞的存活率降低，生长受抑制。结论：阳离子脂质体介导的hTNF-α基因体外转染可在Tca8113舌癌细胞中高表达并抑制舌癌细胞的生长。     

舌鳞癌细胞热处理后HSP27的表达变化

目的 观察热疗后舌鳞癌Tscca细胞中热休克蛋白27 (heat shock protein 27,HSP27)的变化及其对凋亡的作用.方法 常规培养的Tscca细胞分6组,对照组不加热,其余5组43℃水浴法热处理40 min后分别常规培养2、4、8、12、24h,应用蛋白组学方法检测HSP27变化.运用空载质粒(pcDNA3)、pcDNA3-HSP27质粒转染Tscca细胞24h,免疫印迹分析靶细胞中的HSP27表达.43℃水浴处理未转染及转染组细胞0 min、40 min,再培养24 h后流式细胞仪检测细胞凋亡率.结果 加热后12 h内细胞内HSP27持续升高.HSP27质粒转染细胞中HSP27蛋白表达增强.0 min热处理,未转染组、pcDNA3转染组及HSP27质粒转染组细胞凋亡率无差异;40min热处理,未转染组与pcDNA3转染组凋亡率无差异,HSP27质粒转染组分别与未转染组及空载质粒转染组比较,均有差异(P＜0.05).结论 热处理后舌鳞癌Tscca细胞中HSP27含量升高.HSP27的变化与Tscca细胞凋亡的变化有关.     

超声介导微泡破裂法促进外源性基因在小鼠NIH3T3细胞中的表达

目的观察超声介导微泡破裂法对目的基因重组人骨形态发生蛋白- 2（hBMP- 2）转染靶细胞NIH3T3效率的影响。方法复苏NIH3T3细胞并传至3~4代,待细胞生长状态良好后接种至六孔培养板;将六孔板中细胞随机分为2组,分别采用质粒DNA和脂质体法（脂质体组）,质粒DNA、超声和微泡法（超声介导微泡破裂组）转染目的基因;转染24~48 h后在荧光倒置显微镜下对各组细胞分别计数以计算转染效率,并采用ELISA实验测定转染后hBMP- 2蛋白质量浓度;使用SPSS 11.5软件包分析实验结果。结果脂质体组的转染效率为（7.30±1.58）%,超声介导微泡破裂组为（11.77±3.16）%（P<0.05）;脂质体组转染后hBMP- 2蛋白质量浓度为（1 164.35±724.67）pg/mL,超声介导微泡破裂组为（2 932.70±656.27）pg/mL（P<0.05）。结论超声介导微泡破裂法可以明显提高外源性基因hBMP-2在体外NIH3T3细胞中的转染效率和蛋白质量浓度,可以为牙周再生基因治疗提供一种新型基因转移系统。     

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     

bFGF与米贝拉地尔对人牙周膜成纤维细胞增殖的调节

目的:研究成纤维细胞生长因子(bFGF,以下用B代表)与米贝拉地尔(Mibefradil,以下用M代表)这两种药物对人牙周膜成纤维细胞(HPLFs)增殖的调节作用。方法:B分10.0、20.0、30.0 μg/L 3种浓度(分别为B1、B2、B3),M分2.5、5.0、10.0 μmol/L 3种浓度(分别为M1、M2、M3),再按加药时间分为1 d组、2 d组和3 d组,用WST法检测两种药物单独使用和按先后不同顺序联合使用后HPLFs的增殖率。结果:两种药物单独使用可分别起到促进和阻滞细胞增殖的效果,但是B作用时间过长细胞表现为增殖受阻滞。其中,B3作用1 d组细胞增殖率最高(P<0.05),M2作用1 d组细胞增殖率最低(P<0.05)。M仅对加用B3的细胞有明显阻滞作用,与单独使用B3的细胞增殖率有显著性差异(P<0.05)。另外,只有B1不能改变细胞加用M后增殖受阻滞的现象。所有联合用药后的细胞增殖率均与正常细胞增殖率无统计学差异。结论:bFGF与Mibefradil单独使用有各自的最佳作用浓度和作用时间。除过度增殖状态外,HPLFs增殖状态很难受到阻滞,并且处于增殖受阻滞状态的细胞很容易恢复增殖状态,以维持HPLFs不断处于增殖平衡状态。     

牙骨质基质胍提取物促牙龈成纤维细胞、成骨细胞粘附研究

目的 :鉴定牙骨质基质胍提取物内牙骨质蛋白的生物活性。方法 :制取牙骨质基质胍提取物 ,检测人牙龈成纤维细胞、MC3T3 E1成骨细胞两种细胞在含牙骨质基质提取物浓度分别为 2 .5、5、10、2 0μg/ml的DMEM培养液中孵化 1h的贴壁率并以加牛血清蛋白 1mg/ml为空白对照组。检测两种细胞在含牙骨质基质提取物浓度 10 μg/ml的DMEM培养液中孵化 30、60、90、12 0min贴壁率。 结果 :不同浓度的牙骨质基质提取物均可促进牙龈成纤维细胞和成骨细胞的粘附 ,并呈浓度依赖性 ,尤以 10 μg/ml的牙骨质基质提取物的浓度为较佳浓度。细胞的贴壁率随牙骨质基质提取物作用时间的延长逐步升高 ,并以 90min为较理想的作用时间。结论 :人牙骨质基质胍提取物含有可促进牙龈成纤维细胞和成骨细胞粘附的牙骨质活性蛋白     

人颊癌BcaCD885细胞Fas表达水平与移植瘤形成和增殖的关系

目的 :研究Fas表达水平与人颊癌BcaCD885细胞移植瘤形成、增殖及凋亡的关系 ,探讨其相关机制。方法 :用脂质体将真核表达重组质粒pBK -Fas导入人颊癌BcaCD885细胞。转染 72h后 ,收集转染及未转染细胞 ,每组细胞分别接种 10只裸鼠。观察移植瘤形成及增殖状况 ,绘制肿瘤生长曲线。实验结束采集肿瘤标本 ,称重 ,计算肿瘤生长抑制率。同时以RT -PCR检测两组移植瘤细胞FasmRNA表达 ,流式细胞仪检测移植瘤细胞凋亡、增殖和Fas蛋白表达。结果 :Fas转染组移植瘤形成时间平均延长 3 .4d ,各观察点移植瘤体积均小于未转染组 ,二者差异有显著性 (P <0 .0 1)。RT -PCR和流式细胞术检测显示 ,Fas转染上调FasmRNA表达 ,提高Fas蛋白阳性表达率、阳性表达强度和细胞凋亡指数 ,但不影响肿瘤细胞增殖指数。结论 :Fas基因转染抑制移植瘤形成和增殖与提高Fas表达、增加肿瘤细胞凋亡有关。     

生存素基因诱导人黏液表皮样癌细胞凋亡的实验研究

目的 研究生存素(survivin)反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)对人黏液表皮样癌高转移细胞株Mc3细胞的生长抑制效应及可能的作用机制,探讨生存素基因作为黏液表皮样癌基因治疗靶点的可行性.方法 设计合成靶向生存素特异性ASODN,转染Mc3细胞.将转染的Mc3细胞分为4组:空白对照组、脂质体转染组、生存素正义寡核苷酸(sense oligodeoxynucleotide,SODN)转染组、生存素ASODN转染组.转染后24、48及72 h倒置显微镜观察Mc3细胞形态学变化,甲基噻唑基四唑(MTT)法观察转染前后对细胞增殖的影响,流式细胞仪技术检测细胞凋亡率,脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记法分析细胞凋亡指数,半定量反转录聚合酶链反应分别检测生存素mRNA的表达.结果 生存素ASODN转染组的Mc3细胞数量明显少于其他组,脱壁悬浮细胞明显增多,可见典型凋亡改变,并呈时间依赖性.生存素ASODN转染组Mc3细胞G1～G0期前出现明显的亚二倍体凋亡峰,Mc3细胞的凋亡率在转染后24、48及72 h分别为12.96%、14.43%及22.69%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN对Mc3细胞增殖抑制率分别为22.35%、39.04%、43.46%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN转染组在细胞转染后24、48及72 h Mc3的凋亡指数分别为11.038%、12.172%及18.900%,明显高于其他组(P＜0.01),并呈时间依赖性(P＜0.05).生存素ASODN转染组Mc3细胞生存素mRNA在24、48及72 h的相对表达量分别为0.739±0.008、0.668±0.007、0.500±0.006,相对抑制率分别为18.21%、26.06%、44.82%,明显低于其他组(P＜0.01),并呈时间依赖性(P＜0.01).结论 生存素ASODN可抑制人黏液表皮样癌高转移细胞株Mc3的生长增殖,诱导其凋亡,同时亦可抑制Mc3细胞生存素mRNA的表达,并呈时间依赖性.生存素可作为黏液表皮样癌基因治疗研究的一个靶点.     

Variation in periodontal referral in 2 regions in theUK

This study investigated the periodontal referral patterns of general dental practitioners (GDPs) in Northern Ireland (NI) and North West England (NWE). A questionnaire dealing with periodontal referral was sent to all 520 GDPs registered in NI and to 274 GDPs in NWE. A usable return was made by 355 (68%) in NI and 189 (70%) in NWE. The NI dentists made significantly more periodontal referrals (median 5, range 0-80) in the year preceding the survey than those in NWE (median 2, range 0-50), p<0.001. Distance was the only factor significantly related to the referral rate in both regions with those who practised more than 25 miles from a specialist referring significantly fewer patients in both regions. In NI, there was a trend towards increased periodontal referral by GDPs who had attended more postgraduate courses; however, in NWE, this was not the case. The GDPs in NWE were significantly less likely than those in NI to refer patients with medical conditions. It is concluded that there is considerable variation in periodontal referral both within and between the 2 regions studied. It is further concluded that in many cases, non-disease factors, such as the accessibility of the specialist service, have powerful effects on the decisions made by dentists and patients in these regions (NI and NWE) in relation to periodontal referral. Much of the variance in referral in North West England, as in Northern Ireland, remains unexplained.     

Changes in cyclooxygenase metabolities in experimental periodontitis in Macaca mulatta

The four principal metabolites of cyclooxygenase (CO) were examined during the progression of experimental periodontitis in the rhesus monkey Macaca mulatta. Thirty-two monkeys were divided in four disease-matched groups. Three groups were treated with flurbiprofen, a potent CO inhibitor, at either 0.027, 0.27 or 7.1 mg/kg/day delivered systemically by a subcutaneously-implanted osmotic mini-pump. We have previously described the findings indicating that flurbiprofen treatment significantly retarded clinical attachment loss (ALOSS), redness and radiographic bone loss (BLOSS). This investigation focuses on the changes in CO metabolites which occur during disease progression of ligature-induced periodontitis and on the dose-response relationship of flurbiprofen, as it relates to disease inhibition and the suppression of ARA metabolites within the crevicular fluid (CF). In untreated animals there was a statistically significant 3-fold increase in CF levels of prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) at 3 months, as compared to baseline, which positively correlated with increases in redness, bleeding, ALOSS and BLOSS. CF-PGE2 and TxB2 levels reached a 6-fold peak at 6 months and returned to baseline by 12 months. Flurbiprofen (Fb) prevented the 3-month rise in TxB2, but did not affect the increase in PGE2. At 6 months, Fb administration caused a dose-dependent inhibition of both PGE2 and TxB2. Probit analysis of the dose-response data revealed that the concentration of Fb which caused a 50% inhibition of CF-TxB2 level (the IC50 value for TxB2 synthesis) was approximately two logs lower than the IC50 value for PGE2 synthesis, i.e. TxA2-IC50 = 0.013 vs. PGE2-IC50 = 1.35 mg flurbiprofen/kg/d. The slopes of the PGE2 and TxB2 inhibition curves were identical, consistent with a similar mechanism or singular enzyme for the site of action of Fb inhibition of CO activity. However, the kinetics and sensitivity of Fb inhibition were significantly different for the CO activity responsible for TxB2 and PGE2 synthesis, perhaps due to different compartmentalization of CO within different cell types.     

云南省五岁儿童乳牙龋病调查分析

目的：了解云南省５岁儿童乳牙龋齿患病情况。方法：随机抽取云南省三个城市三个农村的２１３２名５岁常住儿童,采用第二次全国口腔健康流行病学调查标准调查龋齿患病情况。结果：云南省５岁儿童乳牙患龋率为７５．７５％,龋均为４．４４,充填率仅为６．５７％,仅占构成比的４．３９％。结论：云南省５岁儿童乳牙龋齿患病率高,充填率低。儿童龋病防治应注重两个方面：１．加强对家长和教师的幼儿口腔卫生保健知识及方法的宣传。     

Mast cells in masseter muscle in experimental bruxism in man

Three human subjects performed tooth grinding for 25 min, and after 20 h biopsies of the right and left masseter muscles were examined for their contents of mast cells. In comparison with specimens from a control group of three subjects, there was an increase of degranulating mast cells in muscles that had performed bruxism.     

Age-related changes in anthropometric measurements in the craniofacial regions and in height in Down's syndrome

This cross-sectional study analyzed age-related changes in normal and abnormal measurements of the head and face in three age categories in 115 Down's syndrome patients 1 to 36 years old. The frequency of normal measurements significantly surpassed that of abnormal ones in each category. Clinically, the key task was to ascertain differences between the youngest and oldest patients. In age group 1 (1 to 5 years), normal measurements in three of the six craniofacial regions were significantly more frequent than abnormal ones. In age group 2 (6 to 15 years) the percentage of normal measurements significantly increased, influenced by higher growth rates in the period of maturation, which coincided with this category. In age group 3 (16 to 36 years) the percentage of normal measurements significantly increased in the head and ear but decreased in the other regions, significantly in the orbits. The frequency of both optimal and severely abnormal measurements changed significantly from age group 1 to 3 in only five measurements each, with no consistency in the direction of results. Abnormal measurements qualified as stigmata and were recorded in 40% (10 of 25) in five regions: three in the face; two in each of the head, orbits, and ears; and one in the nose. Marked epicanthi covering the endocanthion decreased from 35.0% in age group 1 to 8.7% in group 3. In age group 1, the frequency of normal body height (20.7%) in both sexes was significantly less than subnormal (70.3%) but significantly decreased in age group 2. Mean height in group 3 was enough to rule out short stature as a stigmata of Down's syndrome. The study was limited by small numbers, particularly in the variations of normal and abnormal, but the trend toward normality after maturation suggests that reconstructive surgery should be delayed until this time.