阿霉素磁性介孔氧化硅纳米颗粒对口腔鳞状细胞癌细胞的体外实验研究

目的:探讨阿霉素磁性介孔氧化硅纳米颗粒(Doxorubicin-loading Mesoporous Magnetic Silia Nanoparticles,DOX@M-MSNs)对口腔鳞状细胞癌细胞的体外靶向杀伤作用。方法:采用甲基噻唑基四唑(MTT)法,流式细胞仪及激光共聚焦显微镜检测DOX@M-MSNs对CAL-27的体外靶向抗肿瘤效果。结果:单纯磁性介孔氧化硅纳米颗粒表现出非常好的生物稳定性以及生物相容性,尽管浓度为100 mg/L时,细胞存活率仍超过80%。当浓度值有所提升及作用时间延长后,相较于单纯阿霉素原料组,DOX@M-MSNs对于肿瘤细胞的增殖抑制作用明显增强,差异有统计学意义(P<0.05)。流式细胞仪检测结果显示DOX@M-MSNs促进肿瘤细胞的凋亡率高于单纯阿霉素,随着药物作用时间延长,其对肿瘤细胞的抑制效果明显增强,凋亡率增高。激光共聚焦显微镜检测结果显示通过外加磁场,磁性区域的DOX@M-MSN释放的DOX于肿瘤细胞的聚集优于非磁性区域。结论:DOX@M-MSNs纳米颗粒能够选择性进入肿瘤细胞内,从而使得阿霉素对肿瘤细胞的毒性有所增强,进而使得其杀伤肿瘤细胞的效果显著提高,外加磁场可以显著地提升输送DOX@M-MSNs进到肿瘤细胞内的效率,进而提高肿瘤抑制效果。     

平阳霉素-活性炭纳米微粒对口腔鳞癌细胞系Tca8113和BcaCD885细胞体外的杀伤作用

目的探讨平阳霉素-活性炭纳米微粒（PYM-CH-NP）对人舌鳞癌细胞系Tca8113和颊鳞癌细胞系Bca-CD885的药物敏感性。方法采用甲基噻唑基四唑（MTT）法检测不同剂量的PYM-CH-NP和平阳霉素（PYM）在7个时间点（1～7 d）对Tca8113和BcaCD885的杀伤效应,计算各时间点2种剂型的半数抑癌率浓度值（IC50）、抑癌率和药物抗肿瘤作用强度的相对值（RAA）,选择药物作用后第5天观察量效关系。结果PYM-CH-NP和PYM对Tca8113和BcaCD885均有较强的抑癌作用,其抗癌效应与药物浓度和作用时间相关。结论改型后的PYM-CH-NP保留了PYM的抗癌活性,且具有缓释性,作用于肿瘤时可以维持周围有效的浓度和作用时间,有效地杀伤肿瘤细胞。     

基于介孔硅的姜黄素⁃siRNA共递送系统构建及其对巨噬细胞M2型极化的影响

目的构建以介孔硅为基础的姜黄素(curcumin,CUR)?siRNA共递送系统,探讨其对巨噬细胞向M2型极化的促进作用。方法采用常规溶胶?凝胶法制备介孔硅纳米粒(mesoporous silica nanoparticles,MSN),在其内部孔道吸附小分子CUR,外层吸附阳离子高分子聚乙烯亚胺(polymeric polyethyleneimine,PEI),与带负电的siRNA结合,形成(CUR@MSN)PEI/siRNA纳米共递送系统;采用透射电镜观察纳米粒制备过程;以不同浓度的纳米粒处理巨噬细胞RAW264.7,采用MTT方法检测细胞活力变化;采用FAM荧光标记的siRNA制备纳米粒后转染细胞,分别采用激光共聚焦扫描显微镜和透射电镜观察纳米粒的细胞摄取;采用靶向GAPDH的siRNA制备纳米粒后转染RAW264.7细胞,观察对靶基因的沉默效率,对照组为未处理细胞组、仅递送CUR组、CUR与siRNA阴性对照(siNC)共递送组,采用实时定量PCR检测沉默效率;进一步采用miRNA?130a?3p反义序列(antisense oligonucleotide,ASO)制备纳米粒转染巨噬细胞RAW264.7,观察对巨噬细胞极化的影响,对照组为未处理细胞组、仅递送CUR组、CUR与miRNA阴性对照(miNC)共递送组,采用Western Blot检测巨噬细胞M2极化标志物精氨酸酶1(arginase 1,Arg?1)的表达。结果(CUR@MSN)PEI/siRNA纳米共递送系统可成功构建;纳米粒呈现剂量依赖性的细胞毒性,在10μg/mL以下浓度处理组细胞活力在90%以上;纳米粒可被细胞高效摄取,显著抑制靶基因GAPDH的表达,效率约80%(P<0.05 vs.其余各组);仅递送CUR或CUR与miNC共递送组细胞Arg?1的表达提升约3倍(P<0.05 vs.未处理细胞组),在共递送CUR和ASO后能发挥二者的协同作用,促进巨噬细胞Arg?1表达约8倍(P<0.05 vs.其余各组)。结论CUR?siRNA共递送系统可高效转染巨噬细胞实现协同诱导其M2极化的作用。     

介孔二氧化硅纳米颗粒负载顺铂及其对TSCCs杀伤作用的实验研究

目的：利用介孔二氧化硅纳米颗粒负载顺铂,并研究对人舌鳞状细胞癌细胞（TSCCs）的杀伤作用。方法：以表面活性剂CTAB为模板合成MCM-41介孔二氧化硅纳米颗粒（Mesoporous Silica Nanoparticle,MSN）,并负载顺铂,通过扫描电镜和红外光谱观察颗粒表面形貌和功能团组成,通过碘化丙啶（PI）染色法观察TSCCs的细胞凋亡。结果：MSN平均粒径约200 nm,细胞结果显示载药纳米颗粒组死亡细胞明显多于其它各组（P〈0.01）,且单纯纳米颗粒组死亡细胞低于空白对照组。结论：MSN负载顺铂后显著增强了对肿瘤细胞的杀伤能力,有望应用于临床肿瘤化疗提高药效或降低药物并发症。     

载阿霉素介孔二氧化硅纳米粒对人舌癌Tca-8113细胞增殖及凋亡的影响

目的:探讨载阿霉素介孔二氧化硅纳米粒(MSNs@DOX)对体外培养的人舌癌Tca-8113 细胞增殖及凋亡的作用。方法:透射电镜(TEM)和氮吸附法对介孔二氧化硅纳米粒(MSNs)进行表征;紫外分光光度计检测阿霉素(DOX)释放行为;MTT法检测细胞增殖抑制作用;Hochest33342染色、流式细胞术检测细胞凋亡和周期分布;Western blot检测P53、Bcl-2、Bax 和Caspase-3 蛋白表达。结果:MSNs形态规则,分布均一,平均粒径、介孔孔径、比表面积和孔容分别为约30 nm,19.59682 nm、193.4296 m²/g、0.947625 cm³/g;MSNs细胞相容性良好;与单独DOX相比,载药组的细胞增殖抑制率及凋亡率更显著,且呈一定MSNs浓度依赖性(P<0.05);细胞明显阻滞于G0/G1期; P53、Bax、Caspase-3 表达显著升高,Bcl-2表达显著降低。结论:该载药纳米传输系统有望用于舌癌治疗。     

载银锌介孔钙硅纳米粒子根管密封性能的研究

目的 评价载银锌介孔钙硅纳米粒子(silver and zinc incorporated mesoporous calcium-silicate nanoparticles,Ag-Zn-MCSNs)作为根管封闭剂的理化性能及根管密封性能.方法 测定MCSNs、Ag-MCSNs、Zn-MCSNs、Ag-Zn-MCSNs...     

介孔二氧化硅包覆金纳米双锥近红外光响应药物载体的构建及抗菌性能

目的　制备介孔二氧化硅包覆金纳米双锥的药物载体,研究其表征、生物相容性、载药性能、近红外光响应性能及抗菌性能。方法　运用晶种介导方法合成金纳米双锥,并在其表面包覆介孔二氧化硅,采用扫描电镜、透射电镜和紫外分光光度计对其进行表征,并通过MTT实验检测其生物相容性;观察其米诺环素负载率及近红外光照射对其药物累积释放率的影响,探讨其抗菌效果。结果　成功制备介孔二氧化硅包覆金纳米双锥药物载体,透射电镜下可见无序的孔隙结构,对细胞增殖无明显影响,药物负载率为76.5%,经近红外光照射后药物累积释放量上升41.7%。体外抗菌实验显示显著抑制细菌生长达90%。结论　介孔二氧化硅包覆金纳米双锥药物载体具有良好生物相容性、近红外光响应药物释放及抗菌作用,在牙周局部缓控释抗菌中有应用的潜能。     

人口腔鳞癌细胞系对淋巴靶向葫芦素BE聚乳酸纳米微粒的敏感性研究

目的:探讨人口腔鳞癌细胞系(Tca8113和BcaCD885)对颈淋巴结靶向性葫芦素BE聚乳酸纳米微粒(CuBE- PLA-NP)的敏感性。方法:用四唑盐显色法(MTT法)检测用不同剂量的CuBE-PLA-NP和CuBE在1~8 d内8个时间点对Tca8113和BcaCD885的抗癌活性,计算出2种药物在8个时间点对2种癌细胞的抑癌率和药物的半数抑癌率浓度。结果:CuBE-PLA-NP和CuBE对Tca8113和BcaCD885均有强的杀伤作用,其杀伤效应均呈时间依赖性和剂量依赖性。结论:经改型后的淋巴靶向CuBE-PLA-NP没有降低CuBE成份的抗癌活性,同时,由于CuBE-PLA-NP具有淋巴靶向性,更有利于杀灭口腔癌颈淋巴结转移灶内的癌细胞。     

载银锌介孔钙硅纳米粒子与聚己内酯复合材料的体外生物活性研究

目的 评价载银(Ag)或锌(Zn)的介孔钙硅纳米粒子(mesoporous calcium-silicate nanoparticles,MCSNs)和聚己内酯(poly-ε-caprolactone,PCL)的复合材料的理化性能、体外生物活性及促进成骨能力.方法 通过加热熔融法制备PCL-MCSNs、PCL-Zn-M...     

钛表面氨基杂化介孔硅基纳米形貌保护涂层的构建及成骨效果评价

目的 在钛纳米管(titanium nanotube,TNT)形貌表面原位沉积可降解的氨基杂化介孔硅(amino-hybrid mesoporous silica,AHMS),探讨其对纳米形貌的保护作用及成骨效应。方法 通过阳极氧化法和油水两相法依次制备TNT、TNT@AHMS作为实验组,以酸蚀钛作为对照组(Ti);通过改变硅源用量比探索合成参数(3∶1,1∶1,1∶3);扫描电镜观察其表面形貌、水接触角测定仪测定亲水性、X射线光电子能谱仪分析元素组成;利用纳米压痕检测及超声震荡仪体外观察TNT@AHMS机械强度形貌保持效果;体外模拟浸泡实验观察其降解行为;利用MC3T3-E1细胞系观察细胞在材料表面的黏附、增殖和分化能力;利用SD大鼠股骨植入模型和Micro-CT验证AHMS对TNT形貌的保护作用及骨结合效果。结果 TNT、TNT@AHMS形貌均制备成功,硅源用量比为1:3;扫描电镜可见钛纳米管间均匀覆盖AHMS涂层,介孔径约4 nm;AHMS掺入后材料表面为亲水性(12.78°),可检测到氨基基团(NH₂-)存在,并在体外12 h内即可降解完全,从而重新暴露T...     

Role of MDR1 gene polymorphisms in gingival overgrowth induced by cyclosporine in transplant patients

Background and Objective: The aim of the present study was to determine the association between genotypes of the MDR1 gene, encoding P‐glycoprotein, and gingival overgrowth in transplant patients treated with cyclosporine, and to evaluate the effect of periodontal treatment in these patients. Material and Methods: Fifty transplant patients receiving therapy with cyclosporine and suffering from gingival overgrowth were subjected to nonsurgical periodontal treatment and received oral hygiene instructions. Hyperplastic index, periodontal probing depths, bleeding and plaque scores were recorded at baseline and after 3 and 6 mo. Patients were dichotomized into two groups: those with a hyperplastic index of < 30% (minimal gingival overgrowth) and those with a hyperplastic index of ≥ 30% (clinically significant gingival overgrowth). MDR1 C3435T and G2677T polymorphisms were evaluated in all patients and in 100 controls. Results: At baseline, 32 patients (64%) had minimal gingival overgrowth and 18 patients (36%) had clinically significant gingival overgrowth. The mutated C3435T genotype was significantly more frequent in the second group (p < 0.019). The significant association between gingival overgrowth and the 3435TT genotype was confirmed by logistic regression analysis (p < 0.031). The differences in hyperplastic index, observed at baseline between patients with the TT genotype and those with the CC/CT genotype disappeared in the second and third evaluation. The mean monthly change of the square root of the gingival overgrowth scores for all patients, assessed using linear models, was significantly different from baseline (?0.17 points per month, p < 0.00001); and this was particularly evident in subjects with renal transplant (?1.62, p < 0.01). Conclusion: Aetiological periodontal and self‐performed maintenance therapy is effective in reducing gingival overgrowth, particularly in subjects with the 3435TT genotype and in patients with renal transplant.     

shRNA沉默LASP-1对舌鳞状细胞癌SCC9细胞增殖和迁移的影响

目的：探讨LASP?1在人舌鳞状细胞癌细胞中的表达,并应用shRNA沉默人舌鳞状细胞癌细胞SCC9中LASP?1基因,研究LASP?1对SCC9细胞增殖和迁移能力的影响。方法构建携带绿色荧光蛋白的LASP?1 shRNA质粒及阴性对照组质粒LASP?1 shNC,通过脂质体转染SCC9细胞,采用MTT法检测细胞增殖;RT?PCR法检测细胞LASP?1 mRNA表达;Western blot检测细胞LASP?1蛋白的表达;运用Transwell小室实验检测细胞迁移能力。结果 LASP?1在舌鳞状细胞癌细胞中高表达;实验组和阴性对照组分别转染相应质粒48 h后细胞均有绿色荧光蛋白表达,表明质粒转染成功;实验组SCC9细胞LASP?1 mRNA和LASP?1蛋白表达明显降低;与空白对照组相比,实验组细胞培养48 h和72 h细胞存活率分别降低了（51.23±1.47）%和（50.07±2.11）%;Transwell实验结果显示, LASP?1基因被沉默后细胞迁移能力明显下降,比对照组降低约43%。结论 LASP?1在舌鳞状细胞癌细胞中高表达,下调LASP?1基因表达可抑制SCC9细胞的增殖和迁移。     

体外诱导Tca8113细胞产生耐药性的分析

目的探讨Tca8113细胞长期接触卡铂以后耐药性表达情况。方法以Tca8113细胞系为研究对象,采用间歇性加药,逐步递增剂量,体外连续培养诱导其产生耐药性。用MTT法检测细胞耐药指数,LsAB免疫组化法检测耐药蛋白P糖蛋白- 170（Pgp- 170）、谷胱苷肽S转移酶- π（GST- π）、多药抗药性相关蛋白（MRP）和拓扑异构酶Ⅱ（TopoⅡ）的表达,RT- PCR检测多药耐药基因（MDR）、MRP和GST- π的表达。结果诱导后Tca8113/卡铂（CBP）对氨甲喋呤（MTX）、CBP、平阳霉素（PYM）、长春新碱（VCR）的抗药性明显升高,但对5- 氟尿嘧啶（5- FU）抗药性增加不明显。在免疫组化和RT- PCR中Pgp- 170、MRP和GST- π表达升高,TopoⅡ表达降低。结论Tca8113细胞系在卡铂长期刺激的环境中多种耐药相关蛋白表达上升,表明肿瘤细胞的耐药现象受到多种基因的调节。Tca8113/CBP可以作为一个肿瘤耐药研究的平台。     

硫代磷酸化反义寡核苷酸对耐放疗舌癌细胞MDR1基因和P-gp表达的抑制作用

目的：研究反义硫代磷酸化寡核苷酸（phosphorothioate antisense oligonuc leotide,PS—ASOs）抑制人舌癌细胞耐放疗株Tca 8113多药耐药基因1（multidrug resistance gene 1,MDR1）以及MDR1蛋白（P-gp）表达的作用。方法：人工合成互补于MDR1基因mRNA特定片断的硫代磷酸化正反义寡核苷酸;PS—ASOs浓度分别为0．1μmol／L,0．25μmol／L,0．5μmol／L,以正义寡核苷酸作为实验对照,未转染的耐放疗组为空白对照,以5μl／ml脂质体Lipofectamine为载体转染耐放疗的Tca8113细胞株;逆转录多聚酶链反应（RT-PCR）测定转染后MDR1 mRNA表达,流式细胞技术（FCM）测定细胞膜P—gp表达。结果：与正义组和空白对照组相比,转染PS—ASOs后12h,MDRI mRNA和P—gP表达降低,转染24h,各剂量组表达均降至最低,在转染48h后,基本恢复到转染前水平,而正义组和空白对照组在各时段及各剂量组均无统计学差异;双因素方差分析：PS—ASOs不同浓度（P=0．00）、不同作用时间仳0．00）对下调MDRI mRNA、P—gp表达差异显著,结论：PS—ASOs能降低MDR1基因以及蛋白的表达;PS—ASOs的作用具有浓度依赖性和时间依赖性。     

TGF-beta 1 alone and in combination with calcium hydroxide is synergistic to TGF-beta 1 production by osteoblasts in vitro

AIM: To examine the effects of calcium hydroxide (Ca(OH)2), transforming growth factor-beta (TGF-beta 1), and Ca(OH)2/TGF-beta 1 coadministration on TGF-beta 1 and interleukin-6 (IL-6) synthesis by early (subculture 1) and late (subculture 5) osteoblast cultures. METHODOLOGY: Early and late cultures were established using bone cells harvested from 21-day-old fetal rat calvaria. Cell cultures of both early and late osteoblasts were divided into four groups: group 1, control; group 2, cells challenged with Ca(OH)2; group 3, cells challenged with TGF-beta 1; and group 4, cells challenged with Ca(OH)2 and TGF-beta 1 in combination. TGF-beta 1 and IL-6 levels for all groups were determined using ELISA methodology. RESULTS: ANOVA and Tukey HS analyses revealed that osteoblasts of groups 3 and 4 significantly increased (P < 0.001) TGF-beta 1 synthesis in both early and late cultures of osteoblasts. IL-6 was not detected in any of the groups considered in this study. CONCLUSIONS: Exogenous TGF-beta 1 has an autocrine effect on cell cultures of osteoblasts. Administration of TGF-beta 1 alone or in combination with Ca(OH)2 increases the synthesis of TGF-beta 1 in osteoblast cultures. Ca(OH)2 and TGF-beta 1 are compatible when placed in a culture of osteoblasts. Ca(OH)2 provides a favourable environment for the anabolic effects of TGF-beta 1.     

Interleukin-1 stimulates proteoglycan and hyaluronic acid production by human gingival fibroblasts in vitro

The effect of recombinant interleukin 1β [IL-1β] on proteoglycan and hyaluronic acid synthesis by human gingival fibroblasts has been investigated. It was found to stimulate gingival fibroblast proliferation in a dose dependent fashion with the midpoint of this response being in the 10⁻¹¹ mol/L range. At a concentration of 10⁻¹¹ mol/L, IL-1β stimulated proteoglycan synthesis by 40 per cent. Although IL-1β can stimulate cell proliferation and prostaglandin synthesis, its effect on proteoglycan synthesis was independent of these parameters. The kinetics of proteoglycan degradation in the presence or absence of IL-1β was monitored by pulse chase experiments and were found not to differ between treated and untreated cultures. The molecular size and carbohydrate composition of the proteoglycans was not affected by IL-1β. Additional studies revealed the synthesis of hyaluronic acid was also stimulated by IL-1β. As for the proteoglycans, inhibition of cell proliferation did not affect the stimulatory effect of IL-1β. However, blockage of prostaglandin synthesis abolished the stimulatory effect of IL-1β on hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis was found to be related to elevated levels of the enzyme hyaluronate synthetase. Molecular size analysis of newly synthesized hyaluronic acid revealed that cells treated with IL-1β synthesized more large molecular mass hyaluronic acid. Taken together, these findings are considered to reflect the ability of gingival fibroblasts to respond to inflammatory mediators in a manner indicative of early tissue repair.     

Synergistic enhancement of collagenous protein synthesis by human gingival fibroblasts exposed to nifedipine and interleukin-1-beta in vitro

Abstract: Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; however, many clinicians suggest that poor oral hygiene may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis by gingival fibroblasts is synergistically enhanced when they are exposed to both nifedipine (N) and the pro-inflammatory cytokine, interleukin-1-beta, a cytokine expressed in inflamed gingiva. Human gingival fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group 1 was exposed to media containing 0, 5, 50, or 500 pg/ml IL-1-beta, or 10⁻⁷ M N for 7 days; Group 2 was exposed to those concentrations of IL-1-beta +10⁻⁷ M N. [³H]-proline was added to the medium for the final 24 h. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (d.p.m./10³ cells) were compared by factorial ANOVA and Scheffè comparisons. Collagenous protein synthesis was significantly reduced by 5 pg/ml IL-1-beta +10⁻⁷ M N and enhanced by 500 pg/ml IL-1-beta +10⁻⁷ M N as compared to N or IL-1-beta alone. Thus, patients may be more susceptible to gingival overgrowth coincident to nifedipine therapy as a result of the synergistic enhancement of connective tissue synthesis by these agents.     

Role of interleukin-1 and prostaglandin in in vitro bone resorption induced by Actinobacillus actinomycetemcomitans lipopolysaccharide

Lipopolysaccharide (Y4 LPS) isolated from Actinobacillus actinomycetemcomitans strain Y4 induced bone resorption in BALB/c mouse calvaria organ culture. The calcium release from LPS-low responsive C3H/HeJ mouse calvaria by Y4 LPS was very low. Indomethacin almost completely inhibited prostaglandin E₂ (PGE₂) production by Y4 LPS-stimulated BALB/c mouse calvaria, but did not suppress interleukin-1 (IL-1) release from the calvaria, and partially suppressed the bone resorption. Dexamethasone strongly inhibited the PGE₂ and IL-1 production by Y4 LPS-stimulated BALB/c mouse calvaria. as well as Y4 LPS-induced bone resorption. Dexamethasone inhibited expression of membrane IL-1 on osteoblastic cells stimulated with Y4 LPS, but indomethacin did not. Furthermore, anti-IL-1 serum partially suppressed the calcium release from Y4 LPS-stimulated BALB/c mouse calvaria. These results suggest that both PGE₂ and IL-1 participate in Y4 LPS-induced bone resorption in vitro .