肽段-量子点对鳞状细胞癌细胞成瘤及淋巴转移能力影响的动物实验

目的 观察肽段连接的最大发射波长为655 nm的荧光量子点(quantum dots,QD655)对人舌鳞状细胞癌(Tca8113)和昆明小鼠淋巴高转移鳞状细胞癌(U14)的体内生长、增殖、凋亡和淋巴转移能力的影响.方法 ①用QD655分别标记Tea8113细胞(TcaS113-QD655)和U14细胞(U14一QD655),将Tca8113-QD655和U14-QD655分别接种于裸鼠和昆明小鼠背部皮下,比较Tea8113-QD655和Tea8113、U14-QD655和U14的体内成瘤情况,并用流式细胞仪检测比较体内成瘤的Tea8113-QD655和TcaS113细胞、U14-QD655和U14细胞的增殖和凋亡情况;②将U14-QD655和U14分别接种于昆明小鼠颊黏膜下,建立颊癌颈淋巴转移模型,比较U14-QD655和U14颈淋巴转移能力的变化.结果 Tca8113-QD655组和Tea8113组肿瘤的平均重量分别为(1.25±0.14)、(1.30±0.16)g(P>0.05),肿瘤的平均体积分别为(2.81±0.68)、(2.69±0.63)cm3(P>0.05);U14-QD655组和U14组肿瘤的平均重量分别为(1.17±0.08)、(1.22±0.10)g(P>0.05),肿瘤的平均体积分别为(2.27±0.56)、(2.38±0.61)cm3(P>0.05).Tea8113-QD655和Tca8113体内成瘤细胞的平均增殖指数分别为(47.42±1.71)%、(48.33±1.52)%(P>0.05),平均凋亡指数分别为(12.38±0.75)%、(11.79±0.64)%(P>0.05);U14-QD655和U14体内成瘤细胞的平均增殖指数分别为(61.78±2.41)%、(60.9±2.26)%(P>0.05),平均凋亡指数分别为(13.65±0.91)%、(12.78±0.83)%(P>0.05).U14-QD655颊癌和U14颊癌的颈淋巴结转移率分别为41%(11/27)、43(13/30)(P>0.05).结论 用量子点标记Tea8113和U14后不影响体内肿瘤生长和淋巴转移能力.     

近红外量子点连接的精氨酸-甘氨酸-天冬氨酸肽段荧光探针对口腔鳞状细胞癌的活体可视化成像

目的 探索用精氨酸-甘氨酸-天冬氨酸（RGD）肽段连接的近红外量子点荧光探针对口腔鳞状细胞癌（OSCC）的原位可视化成像情况。方法 将含有RGD序列的肽段与发射波长为800 nm的近红外量子点（QD800）偶联,制备QD800-RGD荧光探针。将人颊鳞状细胞癌BcaCD885细胞植入裸鼠颊部皮下建立OSCC模型。用QD800-RGD探针和CD105单克隆抗体对OSCC冰冻切片行直接免疫荧光双重染色,用激光扫描共聚焦显微镜观察QD800-RGD探针与肿瘤新生血管内皮细胞表达的整合素αvβ3的结合情况。将QD800-RGD探针通过尾静脉注射入OSCC模型动物,在不同时间点通过活体成像观察QD800-RGD对OSCC活体原位可视化动态成像情况。在12 h后处死荷瘤鼠取出肿瘤,检测QD800-RGD在体内与肿瘤新生血管内皮细胞表达的整合素αvβ3的结合情况。结果 QD800-RGD探针在体内和体外均能与OSCC肿瘤新生血管内皮细胞表达的整合素αvβ3特异性靶向结合。静脉注射QD800-RGD探针后能对体内OSCC进行清楚地可视化成像,在注射QD800-RGD后0.5~6 h内肿瘤成像最完整,信噪比最高,9 h时肿瘤荧光强度显著减低,但在12 h时仍能看到明显的肿瘤成像。结论 以肿瘤新生血管内皮细胞表达的整合素αvβ3为靶点,利用QD800-RGD探针经静脉注射后能对OSCC进行清晰地可视化成像,在OSCC的诊断和个体化治疗等方面有巨大的发展前景。     

成人骨性下颌偏斜患者与正常人下颌骨及颞下颌关节单光子发射型CT/CT同机融合骨显像的比较研究

Objective To investigate mandible and temporomandibular joint (TMJ) in adults with and without mandible deviation using SPECT/CT fusion imaging. Methods SPECT/CT fusion imaging over bilateral mandible and TMJ was performed in 20 adult patients with mandibular deviation and 15 adult volunteers without mandibular deviation. Results Compared with the control group, the radioactive intensity of contralateral side was higher than that of the deviated side in patients with mandibular deviation. The biggest difference was found in the condyle process (P＜0. 01) and the mandibular angle (P＜0. 01). Conclusions Based on accurate anatomical localization, SPECT/CT fusion imaging was very sensitive in detecting functional alteration in TMJ.     

肽段连接的近红外荧光量子点对人颊鳞癌BcaCD885细胞侵袭和转移能力的影响

目的 研究肽段连接的近红外荧光量子点(QDs)对人颊鳞癌BcaCD885细胞的生长、侵袭、黏附和趋化运动能力的影响.方法:1)用表面连接穿膜肽段最大发射波长为800nm的近红外荧光量子点(QD800)标记BcaCD885细胞(BcaCD885/QD800),用流式细胞仪检测QD800对BeaCD885细胞的标记率,用激...     

唑来膦酸胶原膜对骨代谢细胞的影响初探

目的 通过研究唑来膦酸胶原膜对骨代谢细胞的影响,探讨该载药膜是否具有抑制局部骨吸收、促进局部骨形成的能力.方法 分别以双层胶原膜(Bio-Gide(R))和单层胶原膜(BME-10X(R))为载体,吸附不同浓度唑来膦酸(zoledronic acid,ZA),制备唑来膦酸胶原膜,分别命名为BG0、BG1、BG2、BG3组和BM0、BM1、BM2、BM3组(BG代表双层胶原膜,BM代表单层胶原膜,0、1、2、3分别代表唑来膦酸浓度为0、1×10-4、1×10-3、1×10-2 mol/L),设置无胶原膜组为空白对照组.通过离体细胞培养,评价唑来膦酸胶原膜对破骨细胞和成骨细胞的影响.结果 唑来膦酸胶原膜与破骨细胞体外共培养显示:培养第7大,BG1、BG2、BG3、BM1、BM2、BM3组骨吸收陷窝面积百分比分别为18.80%、14.75%、14.28%、20.51%、15.77%、15.12%,明显低于BG0(31.53%)和BM0(32.22%,P＜0.05),载药量越高,其抑制效果越强.唑来膦酸胶原膜与成骨细胞体外共培养显示:培养第7天,BG2组增殖指数(7.00)明显高于BG0(6.90);培养第4天,BG2、BG3、BM2、BM3组碱性磷酸酶表达(分别为154.67、154.33、155.33、152.00 U/g)明显高于BG0(129.33 U/g)和BM0(127.67 U/g,P＜0.05).结论 唑来膦酸胶原膜可同时具有抑制骨吸收和促进成骨细胞增殖的能力.

Abstract：

Objective To develop zoledronic acid (ZA)-loaded collagen membranes, and to study its effect on osteoclast and osteoblast so as to investigate whether ZA-loaded membranes can inhibit local bone resorption and promote bone formation. Methods ZA-loaded double-layer (Bio-Gide(R)) and singlelayer(BME-10X(R)) collagen membranes were prepared and divided into eight groups according to the concentrations of ZA in the membrane, namely Group BG0, BG1, BG2, BG3 and BM0, BM1, BM2, BM3 (BG refers to Bio-Gide(R), BM refers to BME-10X(R), 0, 1,2, 3 refer to the concentrations of ZA, 0, 1 ×10-4, 1 × 10-3, 1 × 10-2 mol/L respectively). Blank control group was set without using collagen membrane. The effects of ZA-loaded membranes on osteoclast and osteoblast were assessed using in vitro cell culture models. Results In vitro coculture of ZA-loaded membrane with osteoclast for seven days showed that the percentage of bone resorption area in BG1, BG2, BG3, BM1, BM2, BM3 were 18.80%,14.75%, 14.28%, 20.51%, 15.77%, 15.12% respectively, which were lower than that in BG0 (31.53%) and BM0(32.22%, P ＜0.05), and the higher ZA loading was, the stronger its inhibition to osteoclast was. In vitro coculture of ZA-loaded membrane with osteoblast for four days indicated that alkaline pbosphatase(ALP) activities in BG2 (154.67 U/g), BM2 (154.33 U/g), BG3 (155.33 U/g), BM3 (152.00 U/g) were higher than that in BG0(129.33 U/g) and BM0(127.67 U/g, P ＜ 0.05). What's more, results from seven-day coculture showed that proliferation index in BG2(7.00) was higher than that in BG0(6.90). Conclusions ZA-loaded collagen membrane can not only inhibit osteoclastic bone resorption but also improve proliferation of osteoblast.     

抑制视网膜母细胞瘤结合蛋白2基因表达促进人脂肪基质细胞成骨向分化的研究

目的 探讨视网膜母细胞瘤结合蛋白2(retinoblastoma binding protein 2,RBP-2)在人脂肪基质细胞(human adipose-derived stromal cell,hASC)成骨向分化中的作用.方法 设计靶向RBP-2小干扰RNA(small interfering RNA,siRNA)序列4对,将相应寡核苷酸构建到慢病毒载体pLL 3.7中,于293T细胞进行病毒包装用于RBP-2基因敲低.采用RBP-2敲低效果好的两个siRNA序列进行病毒包装,并以非沉默siRNA的pLL 3.7质粒包装的慢病毒为对照组,感染hASC,培养第14天定量检测碱性磷酸酶(alkaline phosphatase,ALP)活性并行ALP染色及茜素红矿化结节染色,检测hASC成骨向分化的差异.结果 成功构建RBP-2基因RNA干扰慢病毒载体并获得相应慢病毒.慢病毒感染hASC后,RBP-2 mRNA及蛋白表达均被明显抑制.RBP-2 siRNA-1组和RBP-2 siRNA-4组的敲低效果较好,以此两组感染hASC.成骨诱导培养第14天RBP-2 siRNA-1组和RBP-2 siRNA-4组ALP活性[(299.2±22.7)、(224.3±17.7)U/g]高于对照组[(129.9±12.9)U/g,P＜0.05],RBP-2 siRNA-1组和RBP-2 siRNA-4组茜素红及ALP染色强于对照组.结论 成功构建的RBP-2 RNA干扰慢病毒能显著抑制RBP-2表达,并促进hASC向成骨细胞分化,即RBP-2可以抑制hASC的成骨向分化.

Abstract：

Objective To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell(hASC).Methods According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. Results The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group[(299.2 ±22.7), (224.3± 17.7) U/g]was much stronger than that in the control group[( 129.9 ± 12.9) U/g, P ＜ 0. 05]and the same result was achieved for the ALP staining and alizarin red staining. Conclusions The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC.It indicated that RBP-2 can repress the osteogenic differentiation of hASC.     

台阶式垂直闭合曲内收上颌切牙的三维有限元分析

目的 研究台阶式垂直闭合曲在三维空间内对上颌切牙位置的控制作用.方法 选择一名正常 志愿者,对其上颌牙列和牙槽骨进行三维螺旋CT扫描,只对上颌右侧中、侧切牙及牙槽骨进行建模和数据计算,利用Ansys软件生成右侧弓丝-托槽-上颌切牙段及牙周支持组织的三维有限元模型,最后根据镜像对称原理建立弓丝-托槽-上颌切牙段及牙周支持组织的三维有限元模型.模拟台阶式垂直闭合曲在临床上的使用情况加力,分析上颌切牙的位移趋势以及牙周支持组织中的应力分布规律.结果 台阶式垂直闭合曲作用下,上颌中切牙舌向、唇向最大位移分别为5.29×10-2和0.71×10-2 mm;龈向、向最大位移分别为10.47×10-3和10.20×10-3 mm;近中、远中最大位移分别为10.26×10-3和1.63×10-3 mm;侧切牙舌向、唇向最大位移分别为3.31×10-2和0.41×10-2 mm;龈向、向最大位移分别为10.52×10-3 和5.10×10-3 mm;近中、远中最大位移分别为6.29×10-3 和4.64×10-3 mm;二者均表现为舌向、龈向的近似整体移动趋势.中切牙牙齿、牙周膜、牙槽骨的最大应力值分别为31.35、2.52、4.64 MPa;侧切牙牙齿、牙周膜、牙槽骨的最大应力值分别为19.59、1.28、4.12 MPa;二者的应力分布规律相似,牙周膜对应力起缓冲作用.结论 台阶式垂直闭合曲在上颌切牙内收阶段可控制其在三维方向上的位置,对抗"钟摆效应",对临床实践具有一定参考意义.

Abstract：

Objective To investigate the displacement and stress distribution of upper incisors in three-dimensional(3D) space controlled by step-shaped vertical closing loop. Methods The maxillary teeth and alveolar bone of a volunteer with normal occlusion were scanned with 3D spiral CT. Modeling and calculation were only carried out on right upper central incisor, lateral incisor and their alveolar bone in order to simplify the procedures. A 3D finite element model of archwire-brackets-upper incisors and periodontal tissues was developed using Ansys finite element package. Finally, a 3D finite element model of archwire-brackets-upper incisors and periodontal tissues was established based on mirror symmetry principle. The displacement of maxillary incisors and stress distribution in periodontal tissues were analyzed. ResultsWhen step-shaped vertical closing loop was simply drew back 1 mm, the maximum displacement of upper central incisor in labial and lingual direction were 5.29×10-2 and 0.71×10-2 mm; 10.47×10-3 and 10.20×10-3 mm in gingival and occlusal direction, 10.26×10-3 and 1.63×10-3 mm in medial and distal direction; the maximum displacement of upper lateral incisor in labial and lingual direction were 3.31×10-2 and 0.41×10-2 mm, 10.52×10-3 and 5.10×10-3 mm in gingival and occlusal direction, 6.29×10-3 and 4.64×10-3 mm in medial and distal direction, the displacement trend of them were moving lingually and gingivally similar to bodily movement. The stress peach of upper central incisor, periodontal ligament and alveolar bone were 31.35, 2.52 and 4.64 MPa, the stress peach of upper lateral incisor, periodontal ligament and alveolar bone were 19.59, 1.28 and 4.12 Mpa, the stress distribution of them were similar and the periodontal ligament buffered the stress imposed on the tooth. Conclusions The position of upper incisors in 3D space could be controlled by step-shaped vertical closing loop and the pendulum effect could be confronted.     

长期低剂量牙龈卟啉单胞菌感染对脂蛋白基因敲除小鼠动脉粥样硬化形成的影响

目的 评价牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)口腔内低剂量、长期、多次接种是否可影响载脂蛋白基因敲除小鼠(apolipoprotein E-knocked out,ApoE-/-)的动脉粥样硬化(atherosclerosis,AS)形成进程和病变程度.方法 20只C57BL/6背景小鼠,将其中13只种属为C57BL/6背景的ApoE-/-小鼠按随机数字表随机分成两组,实验组7只使用1013个/L的牙周致病菌Pg进行口腔内涂菌接种,持续15周共75次,对照组6只同法口腔内涂擦无菌肉汤培养基,比较两组小鼠体质量、血总胆固醇、三酰甘油及主动脉AS病损面积及病理组织变化的情况.另7只野生型C57BL/6小鼠取其主动脉组织作为正常对照.结果 实验组ApoE-/-小鼠主动脉AS病损平均面积为(98 363.68±12 043.00)μm2,显著大于未接种的小鼠对照组[平均病损面积(62 985.06±7419.64)μm2],P=0.035;两组体质量、血总胆固醇和三酰甘油差异均无统计学意义;与野生型C57BL/6小鼠正常主动脉相比,ApoE-/-小鼠主动脉内壁均有凸向管腔内的AS病损,且动脉壁变平,实验组较对照组AS斑块更明显,动脉管腔更狭窄.结论 Pg口腔内低剂量长期多次接种可以加速ApoE-/-小鼠AS的进展.

Abstract：

Objective To assess the effect of longterm and lower oral inoculation with Porphyromonas gingivalis (Pg) on the progression of atherosclerosis in apolipoprotein E-knocked out (ApoE -/-) mice. Methods Six-week-old male ApoE -/- mice were inoculated orally with 0. 1 ml live Pg(1013/L) or bouillon culture-medium quintic per week for 15 consecutive weeks, altogether 75 times of inoculations. The lesion area of atherosclerosis in the aortic tree was measured by en face quantification by red oil O staining method. The atherosclerotic lesion was examined by histopathology. The levels of total cholesterol and triglycerides were compared. Results At 22 weeks after inoculation, the mean atherosclerotic lesion area in inoculated mice was (98 363.68 ± 12 043.00) μm2 ,which was significantly greater than that in noninoculated mice, which was (62 985.06 ± 7419. 64) μm2 (P = 0. 035).Conclusions Longterm lower oral inoculation of Pg can accelerate the progression of atherosclerosis in apolipoprotein E-knocked out mice.     

成人骨性下颌偏斜患者与正常人下颌骨及颞下颌关节单光子发射型CT/CT同机融合骨显像的比较研究

目的 采用单光子发射型(single-photo Emission CT,SPECT)CT/CT同机融合骨显像技术对成人骨性下颌偏斜患者与正常人下颌骨及颞下颌关节的生长差异进行比较研究.方法 选取成人骨性下颌偏斜患者20例和正常成人志愿者15例,进行SPECT/CT同机融合骨扫描检查,以分析比较下颌偏斜患者与正常人两侧下颌骨及颞下颌关节骨血流和骨代谢的差异性.结果 成人骨性下颌偏斜患者下颌骨不同部位的骨血流和骨代谢存在特定性差异,正常人下颌骨不同部位的骨血流和骨代谢存在特定性差异;正常人下颌骨不同部位左右两侧放射性计数值比值均接近于1,对称性较好;与正常对照组相比,骨性下颌偏斜患者放射性强度均为对侧高于偏斜侧;髁状突差异最大(P＜0.01),其次为下颌角(P＜0.01),下颌升支中份差异最小(P＜0.05);不同部位两侧差异均有统计学意义.结论 SPECT/CT同机融合骨显像在精确解剖定位的基础上,能更加准确显示颞下颌关节的功能变化.

Abstract：

Objective To investigate mandible and temporomandibular joint (TMJ) in adults with and without mandible deviation using SPECT/CT fusion imaging. Methods SPECT/CT fusion imaging over bilateral mandible and TMJ was performed in 20 adult patients with mandibular deviation and 15 adult volunteers without mandibular deviation. Results Compared with the control group, the radioactive intensity of contralateral side was higher than that of the deviated side in patients with mandibular deviation. The biggest difference was found in the condyle process (P＜0. 01) and the mandibular angle (P＜0. 01). Conclusions Based on accurate anatomical localization, SPECT/CT fusion imaging was very sensitive in detecting functional alteration in TMJ.     

固定矫治对错(牙合)患者口腔健康相关生活质量的影响

Objective To assess oral health-related quality of life in patients with fixed appliances.Methods Orthodontic patients were asked to complete the scale of general conditions(Chinese version,questionnaire 1)and oral health impact profile(OHIP)-14(Chinese version,questionnaire 2).Baseline data were collected at first visit and thereafter.The subjects finished questionnaire 2 at the 1st week,4th week,12th week and 24th week,respectively,after the fixed appliance was bonded.Data were analyzed to evaluate the various sample groups with different personal information and clinical parameters.Results were collated and analyzed using software package SPSS version 15.0.Results The most common negative effect was physical pain[55/222(27.8%)]and psychological discomfort[40/222(18.0%)],mainly in the first month.The total scores at five time points were 3,10,7,5 and 4,respectively.No difference was found in quality of life in patients between sixth month with fixed appliance and without appliance(P＞0.05).Age and education status affected the quality of life(P＜0.001).Conclusions Fixed orthodontic appliance therapy affected patients' oral health-related quality of life during treatment.The quality of life in the first month of treatment was mostly compromised and was improved later.     

近红外荧光量子点表皮生长因子受体单克隆抗体探针对头颈部鳞状细胞癌活体可视化成像和体内分布的动物实验研究

目的探讨近红外荧光量子点（QDs）表皮生长因子受体（EGFR）单克隆抗体（mAb）探针对头颈部鳞状细胞癌的原位可视化成像和体内分布情况。方法将发射波长为800 nm的近红外荧光QDs与EGFR mAb连接,制备QD800-EGFR mAb探针。在体外将QD800-EGFR mAb与人颊鳞状细胞癌BcaCD885细胞共培养30 min,使用激光扫描共聚焦显微镜（LSCM）观察QD800-EGFR mAb对BcaCD885细胞的结合情况。将QD800-EGFR mAb通过尾静脉注射到裸鼠头颈部鳞状细胞癌模型,在不同时间点通过活体成像观察QD800-EGFR mAb对头颈部鳞状细胞癌的可视化成像情况和QD800-EGFR mAb在体内的分布。结果静脉注射QD800-EGFR mAb探针后能对裸鼠头颈部鳞状细胞癌进行清楚的可视化荧光成像,成像一直持续到24 h,但在30 min～6 h时间段内肿瘤成像最完整和荧光信噪比最高。对体内QD800-EGFR mAb在肿瘤和器官的分布检测证明：QD800在肝中分布最多,在肿瘤中的聚集随着时间的延长逐渐下降,心、脑、肠、肺、胃中均未见有QD800。结论QD800-EGFR mAb探针对头颈部癌能进行清楚的可视化个体成像检测,在非侵入可视化成像研究头颈部癌的发生发展和个体化治疗等方面有巨大的发展前景。     

两种粒径量子点在人舌鳞癌细胞中双色荧光成像的应用研究

目的 研究粒径655 nm和525nm的两种量子点(quantum dots,QDs)对人舌癌Tca8113细胞内2种热休克蛋白( heat shock protein,HSP)进行特异性荧光标记的成像效果和稳定性,为今后的连续动态观察提供实验依据.方法利用QD655nm和QD525nm,对人舌癌Tca8113细胞内HSP90蛋白和HSP70蛋白进行特异性双重荧光标记,激光连续照射2420391 ms,在共聚焦显微镜下同时观测人舌癌Tca8113细胞内的HSP90蛋白和HSP70蛋白表达及分布,用软件Leica Confocal Software测量量子点QD655nm和QD525nm的荧光信号强度变化.结果 激光共聚焦显微镜下可见人舌癌Tca8113细胞内HSP90蛋白和HSP70蛋白均有明显表达,分别表现为红色和绿色荧光,两种蛋白重叠处呈黄色荧光,在激光连续照射中,两种荧光有所衰减,其中QD655nm的荧光强度值下降相对较快、幅度相对较大.结论 量子点荧光标记技术能同时对人舌鳞癌细胞中HSP90蛋白和HSP70蛋白进行双重标记,而且QD655nm与QD525nm都具有较强的光稳定性,均可用于蛋白的长时间动态监测,其中QD525nm的稳定性更好.     

量子点对口腔鳞癌Tca8113活细胞中bcl-2、bax的荧光标记观察

目的:探讨量子点对口腔鳞癌(OSCC)活细胞bcl-2、bax蛋白进行观察研究。方法:量子点QD605,QD545通过间接免疫荧光法,在激光共聚焦显微镜下观测人舌癌Tca8113活细胞内bcl-2、bax蛋白与量子点孵育0.5 h、1.0h、1.5 h和2.0 h的荧光成像。结果:量子点对OSCC活细胞中的bcl-2、bax蛋白的观察研究显示:在0.5 h～2.0 h时,QD605和QD545与bcl-2、bax蛋白结合,荧光从分布于胞浆边缘逐渐至胞浆。结论:量子点能对活细胞内蛋白进行观察。     

量子点荧光探针检测人舌鳞状细胞癌荷瘤裸鼠模型早期下颌下淋巴结转移的研究

目的 利用量子点标记的特异性细胞角蛋白抗体QDs605-CK（AE1/AE3）免疫荧光探针检测人舌鳞状细胞癌荷瘤裸鼠早期下颌下淋巴结转移率及微转移率,并与传统的免疫组织化学（IHC）染色及苏木精-伊红（HE）染色方法进行比较,为舌鳞状细胞癌的早期诊断与治疗提供一种新的检测方法。方法 传代培养人舌鳞状细胞癌Tca8113 细胞,接种于18只裸鼠舌体内（不过中线）,建立人舌癌荷瘤裸鼠下颌下淋巴结转移模型。接种6周后,处死裸鼠,解剖下颌下淋巴结,将同一淋巴结分为两份。一份作石蜡包埋半连续切片,行HE染色和IHC检测;另一份即刻液氮冷冻,制作冰冻切片行QDs605-CK（AE1/AE3）荧光探针检测。分别计算3种方法检测出的淋巴结转移率和微转移率。结果 量子点标记的免疫荧光染色检测出裸鼠下颌下淋巴结转移率为66.7%,其中微转移率为38.9%;IHC染色检测的淋巴结转移率为61.1%,其中微转移率为33.3%;HE染色检测的淋巴结转移率为27.8%。经统计学分析,3种方法的差异有统计学意义（χ2=6.379,P<0.05）,量子点标记的免疫荧光染色和IHC检测都优于HE染色,但是量子点标记的免疫荧光染色和IHC染色间的差异无统计学意义（χ2=0.120,P>0.05）。结论 量子点标记的QDs605-CK（AE1/AE3）免疫荧光探针能准确定位于下颌下淋巴结转移的肿瘤细胞的细胞质内,发出红色荧光,其特异性强,分辨率高,背景清晰,能够用于淋巴结转移灶及微转移灶的检测。     

口腔鳞癌细胞中bcl-2、bax的量子点双标免疫荧光成像研究

目的:利用量子点对口腔鳞癌细胞内bcl-2、bax蛋白进行双标免疫荧光成像研究。方法:利用量子点QD605和QD545通过双标免疫荧光法,在激光共聚焦显微镜下对人舌癌Tca8113细胞内bcl-2、bax蛋白同时观察识别。结果:不同粒径量子点可同时对舌癌细胞bcl-2、bax蛋白特异性识别,在激光共聚焦显微镜下观察到舌癌细胞bcl-2和bax蛋白高表达。QD605标记的bcl-2蛋白表现为红色荧光,QD545标记的bax蛋白表现为绿色荧光,2种蛋白在细胞内同一位置重叠呈现黄色荧光。激光连续照射1 h,3种颜色的荧光均未明显衰减。结论:量子点能同时对细胞内的2种蛋白进行双标免疫荧光成像。     

骨髓间充质干细胞向牙髓组织迁移体内模型的构建

目的：构建一种骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMMSCs）向牙髓等口腔组织迁移分化的体内模型,为进一步研究BMMSCs迁移、归巢机制从而诱导其修复、再生口腔组织奠定基础。方法：将第1代绿色荧光蛋白（Green flurensence protein,GFP）转基因小鼠的BMMSCs与野生型C57BL6小鼠的全骨髓细胞按一定比例混合后,尾静脉注射入亚致死量照射24h后的16只同品系小鼠体内（GFP-BMMSCs：2×105/只;BMCs：2×106/只）,28d后随机选取6只嵌合小鼠,检测其股骨BMMSCs中GFP＋BMMSCs细胞的比例。对同一处理组剩余小鼠右下颌磨牙牙髓施加刺激,7d后行多聚甲醛心脏灌注,并采集标本,常规脱钙处理后以自体左侧磨牙作为对照,组织学观察比较牙髓中荧光细胞数量改变。结果：嵌合模型股骨BMMSCs中GFP＋的比例为（76.32±3.46）%,口腔刺激侧牙髓中荧光细胞数量明显多于同一个体对照侧（P〈0.05）。结论：以荧光细胞作为示踪标记的骨髓移植动物可作为有效的研究模型探索骨髓间充质干细胞在体向牙髓等口腔组织迁移分化机制。     

应用量子点荧光探针检测裸鼠舌癌组织中bcl-2表达

目的探讨应用量子点荧光探针检测裸鼠舌鳞癌移植瘤组织中bcl-2蛋白的可行性及其研究价值。方法 10只BALB/c-nu裸鼠皮下接种舌鳞癌Tca8113细胞悬液,待瘤体长至约1cm3时处死裸鼠,取瘤组织制成石蜡包埋组织切片和冰冻切片,经病理诊断证实为瘤组织后,应用量子点荧光探针通过间接免疫荧光法标记,在荧光显微镜下观察组织切片中的bcl-2蛋白,并与免疫组织化学检测的bcl-2蛋白进行比较。结果量子点荧光探针可分别与石蜡和冰冻组织切片中的bcl-2蛋白结合,在紫外荧光激发下发出特定荧光,主要分布于细胞浆,与免疫组织化学检测的bcl-2蛋白定位相同。结论量子点荧光探针可应用于裸鼠舌鳞癌移植瘤组织中检测特定蛋白。