• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    IN SITU IMAGING OF BREAST CANCER CELLS USING GREEN SEMICONDUCTOR QUANTUM DOTS

    2010-10-08 12:09:50XuHengyiZoraidaAguilarSuHuaipengBenjaminJonesJohnDixonXiongYonghuaWeiHuaAndrewWang

    Xu Hengyi,Zoraida P.Aguilar ,Su Huaipeng,Benjamin J.Jones,John.D.Dixon,Xiong Yonghua,Wei Hua,Andrew Y.Wang

    (1.State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang,330047,P.R.China;2.Ocean Nano Tech LLC,2143 Worth Lane,Springdale,AR 72764,USA)

    INTRODUCTION

    The breast cancer is the second most common type of the cancer,the fifth most common cause of cancer deaths in the United States and the world and the most common cause of cancer deaths in women.Knowing the accurate stage of this disease is critical for patient survival.Sufficient treatment during the early stages of the breast cancer can prevent the spread and the transfer of the cancer cells to other parts of the body.Thus,one of the most recent interests for the breast cancer cell detection is highly sensitive and specific immunocy-tochemical staining which can be used to directly take the cancer cell image as accurate detection.

    Highly sensitive and specific immunocy-tochemical,and molecular methods[1-2]can detect disseminated tumor cells(DTCs)in the bone marrow and circulating tumor cells(CTCs)in the peripheral blood at the single cell level in the presence of millions of normal cells.In the peripheral blood,CTCs are detectable months to years after complete removal of the primary tumors,thus indicating that the secells can circulate different metastatic sites[3].

    The National Institutes of Health(NIH)estimates the cost of cancer in 2008 is$228.1 billion.Solid tumors derived from epithelial tissues,such as breast,prostate,lung or colorectal carcinomas are the most frequent forms of the cancer.Of the various cancer types reported,the breast cancer is the second most common type of the cancer and the fifth most common cause of the cancer death in the United States and the world(WHO).There are 11.38 million people alive who has a history of the cancer that in cluds those who are cured and with active disease in 2006 in the USA[4].In 2004,the breast cancer caused 519 000 deaths worldwide(7% of cancer deaths;almost 1%of all deaths,WHO).

    The breast cancer is composed of distinct diseases with different outcomes.Usually,clinical and pathological factors are used to determine the prognosis of patients.Currently,most of the diagnostic predictions are a combination of prognostic factors to adapt adjuvant treatment based on the prognosis prediction,such as the Saint Gallen Guidelines[5],National Institute of Health Guidelines[6],and Nottingham Prognostic Index Guidelines[7]as well as the Adjuvant Online Decision-Making Tools[8].

    Knowing the accurate stage of the breast cancer is critical for patient survival.Sufficient treatment during the early stages of breast cancer can prevent the spread and transfer of the cancer cells to other parts of the body.But the cancer cells need to be detected at an early stagein order to start life-saving treatment.Owing to inaccurate prognosis predictions,however,a substantial proportion of breast cancer patients receive adjuvant systemic therapy without gaining any benefit[9].There are just several cancer cells per milliliter in the blood during the early stages making these cells difficult to find and differentiate from millions of white and red blood cells,thus sensitive and accurate detection methods are needed for the diagnosis of the early stages of the breast cancer.

    Nanoparticles can be used for accurate immunocy-tochemical imaging of breast cancer cells.Among the nano particles currently in use,quantum dot(QD)nano particles have received considerable attention due to their inherent advantages[10-11].QDs have unique size-dependent physical properties,such as broad absorption spectrum,precision small bandwidth emission wavelength,thus enhancing chemical and photochemical stabilities,the endocytosis,the cooperative binding activity,and multi-functionalities for the targeted delivery and imaging[12-14].QD immunocy-to chemical imaging of breast cancer cells provides ultra bright signals that persist the unlimited time for observations.

    This paper presents the development of early stage in situ imaging of breast cancer cells(SKBR3)using green QDs as fluorescent signal generator.This study chooses a HER2 over expressing cell line,SK-BR3,and uses the anti-HER2/neu antibody as the specific recognizing antibody.The HER2 gene(HER2/neu and ErbB2 gene)is amplified by 20%—30% at the early-stage of breast cancer which makes it overactive[15].HER2 passes through the cell membrane and sends signals from the cell outside to the inside.However,in the cancer,HER2 sends signals without being stimulated by mitogen promoting invasion,survival,and growth of new vessels of cells(angiogenesis)[16].HER2 over expression can also confer resistance to cancer drugs[17].Thus,the detection of breast cancer cells using HER2 over expression has clinical advantages.

    QDs based imaging of breast cancer cells involves anti-HER2/neu antibody for labeling with the over expressed HER2 on the surface of SKBR3 cells.The complete assay involves SK-BR3+ Ab-biotin+ streptavid in~QDs.The SK-BR3 cells are grown in the culture and exposed to the biotin labeled antibodies followed by the exposure to streptavidin labeled QDs that utilize the strong and stable biotin-streptavidin interaction.Fluorescent images of the complete assay for SK-BR3 cells are evaluated on a microscope with a UV light source.QDs with different wavelengths of the emission are tested to assess the different imaging effects of QDs at different sizes and to establish the possibility of using multicolor cancer cells.

    1 MATERIAL AND METHODS

    1.1 Synthesis and characterization of green QDs

    Green water soluble carboxyl fun ctionalized Cd Se/ZnScore/shell QDs(catalog# QSH 530;λemission 530 nm,molar extinction coefficient 1.5×105M/cm)are synthesized in the house at Ocean Nanotech,LLC,and Springdale,AR.After synthesis following standard protocols,UV-visible absorption spectra are acquired with a HP UV-visible absorption spectrophotometer and photoluminescence spectra are recorded on a PE spectr of luorometer. Transmission electron microscopy(TEM)samples are prepared by dropping a chloroform solution of Cd Se/Cd S QDs on an agar carbon-coated copper grid(400mesh)and the solvent is evaporated.TEM images are obtained at 100 K magnification with a Philips CM 20 TEM operating at 200 k V.The PLQYs of Cd Se/Cd SQDs are measured according to the literature.Rhodamine 6G in ethanol is chosen as the reference standard(PLQY 95%)[18-19].All optical measurements are performed at room temperature under ambient conditions.

    1.2 Surface functionali zation of QDs with strep-tavidin

    To prepare the functionalized QDs, the streptavidin is conjugated to QDs by using sulfo-N HSand EDC covalent coupling chemistry.The QDs(4μM)are activated by adding and incubating with sulfo-NHS(sulfo-N-hydrox ysuccinimide)at a molar ratio 1 000∶1 and EDC(1-ethyl-3,3-dimethy lamino pro pyl carbodiimide hydrochloride)at molar ratio 1 000∶1 for 5 min in boratebuffer,p H 7.4,after which 2 mg of the streptavidin is added,vortexed thoroughly,and reacted for 2 h at the room temperature.At the end of 2 h,the reaction is quenched by adding 5μL of quenching buffer(Cat.# QB,Ocean Nano Tech,USA)and mixed for more 10 min.The streptavidin~QD conjugates are stored at 4°C about 12 h and purified by ultra centrifugation using a Beckman ultracentrifuge(Beckman,USA).

    The surface functionalized streptavidin~ QD conjugates are evaluated with a horizontal submerged gel electrophoresis apparatus(Mini-Sub-Cell GT,Bio-Rad,USA)using a 1.5% (w/v)agarose gel in Tris-acetate-EDTA(TAE)buffer,p H 8.5.For each well,20μL of the streptavidin~ QD conjugates at 100 nM is mixed with 5μL of 5× TAE loading buffer(5× TAE,25% (v/v)glycerol,0.25% (w/v)orange-Gat p H 8.5)before being loaded on the gel.The gel is resolved at 100 V for 30 min(Power Pak Basic,Bio-Rad,USA)and the photo is taken with a 2 s exposure using a gel imaging system(Alpha Imager HP 2006,Alpha Innotech,USA).

    1.3 Cell ref reshment and subculture

    HER2-overexpressing human breast cancer cell line(SK-BR3)used in this research is originally obtained from the American Type Culture Collection(ATCC,Manassas,VA)and is donated by Dr.Lily Yang from Emory University.Liquid nitrogen frozen SK-BR3 cells are thawed in 37°C water bath before transferring into a flask containing RPMI-1640 medium (Invitrogen,Carlsbad,CA,USA)supplemented with 10%fetal bovine serum(FBS,Hy Clone,Logan,UT,USA)and 1% of streptomycin/penicillin antibiotics(Hy Clone,Logan,UT,USA).The flask is placed in a humidified atmosphere with 5%CO2 at 37°Cin a cell culture incubator(Sanyo,Japan).The media is replaced once every three days.The cells are cultured about 80%—90%confluence before the harvest.During the harvest,the cells are washed two times with DPBS(Hy Clone, Logan,UT, USA)followed by adding 1 m L of trypsin(Hy Clone,Logan,UT,USA)to detach the cells from the flask.The trypsin is neutralized by adding 5 mL of fresh RPMI-1640 medium to the flask and is centrifuged at 250×g for 5 min,where g is the gravitational acceleration.The pelleted cells are resuspended in fresh RPMI-1640 and then counted with a Z1 cell counter(Beckman,USA)after 105cells are inoculated on a cover placed on a 6-well cell culture plate.The cells are sub-cultured under the same conditions until the cells are on the glass cover to reach 80%—90%confluence.

    1.4 Antibody modif ication

    Anti-HER2/neu antibody is purchased from Ray Biotech,Inc.(Norcross,GA)and modified with biotin for binding with streptavidin~ QDs.The conjugation process is performed using the hydrazide method.Briefly,1 mg anti-HER2/neu antibody is dissolved in MESbuffer at 5 mg/mL and 25μL of biotin hydrazide solution(50 mM)is added in a centrifuge tube.After thorough mixing,12.5μL of EDC solution(100 mg/mL)is added and mixed thoroughly.The solution is incubated for 2 h at the room temperature.The conjugates are purified by using the dialysis.

    1.5 In situ cell imaging with QDs

    The SK-BR3 cells grown on the glass cover are washed with 1 mL DPBS three times.After washing,1 mL of DPBS containing 1 mg/mL of BSA(Bovine Serum Albumin)is added to each well,followed by 1μg of biotin labeled anti-HER2/neu antibody.The plate is placed on a vortex(VWR,USA)and gently mixed in a 37°C chamber for 30 min.The wells are washed with 1 mL blocking buffer(Ocean catalog#BBB)five times with 1 min shaking sufficiently to remove non-specifically attached antibodies.After washing and blocking,1 mL DPBS with 1 mg/mL BSAis added to each well to which 10 picomole of streptavidin-QD conjugate is added.The plate is vortexed and incubated under the previous condition for 30 min.The blocking and washing steps are repeated three times.The glass cover slide is taken out gently and placed on a microscope.

    QD labeled cells are observed on an Emscope fluorescence microscope(Emscope,USA)using 200×magnification.Thecells are observed using UV light and digital photographs are taken at various sections of the slides.

    2 RESULTSAND DISCUSSION

    Early stage detection of breast cancer cells is critical for patient survival.Sufficient treatment during the early stages of the breast cancer can lead to higher survival rate as well as prevent the spread of cancer cells to other parts of the body.The most common method for detecting cancer cells is immuno fluorescence assays and immuno histochemical staining.QDs as labels of antibodies for fluorescent signal generation have become a viable alternative to enzyme and fluorescent dyes[1-2,20-22]. In this project,we used only one kind of the antibody,anti-HER2/neu antibody labeled with biotin in combination with streptavidin-QD conjugates for signal generation.The in situ images of breast cancer cells are observed and recorded using a fluorescence microscope with a UV light source.

    2.1 Size,surface charge,and optical properties of QDs

    The physical properties of QDs are established after synthesized.As shown in Fig.1,the TEM image shows that the Cd Se/ZnScore/shell QDs are approximately 7 nm in diameter without the surface coating.This number is confirmed with a Zetatracker(Zetatrac Company,USA).After surface coating with a triblock polymer,the diameter increases to 14 nm with a zeta potential of 31 mV.

    Fig.1 TEM image of Cd Se/ZnScore/shell QDs

    After surfacecoa ting modification,the water soluble Cd Se/ZnS core/shell QDs exhibite the same optical properties,such as UV-vis absorption and fluorescence emission. The quantum yield> 50% indicates that the modification process involving a polymer coating for water soluble conversion has little effect on the brightness of QDs(see Fig.2).

    2.2 Surface functionalizat ion of QDs and gel electrophoresis analysis

    Fig.2 UV-vis absorption(left sloping curves)and fluorescence emission spectra (symmetric peaks at 530 nm)of QDs in water

    The conjugation of the streptavidin to the QDs is confirmed with agarosegel electrophoresis shown in Fig.3.The QDs without the streptavidin label are expected to run more rapidly toward the positive electrodebecause of their smaller size and high negative charges.The streptavidin-QDs are expected to run slower because of their larger size and reduced charges due to protein shielding. The results of the gel electrophoresis reveal that carboxyl group modified QDs(Ocean catalog# SHP)have greater migration distances which are in agreement with their strongly negative zeta potential and the smaller size.

    Fig.3 Gel electrophoresis data(100 V for 30 min)for streptavidin-QDs

    The polarities of electrodes are such that the top is negative and the bottom is positive.The bands from left to right correspond to carboxylated QDsλ emission 530 nm(Lane 1)and streptavidin-QDsλ emission 530 nm(Lane 2).The binding of streptavidin on the surface of the QDs leads to an increase in the net size,thus the migration on the gel during electrophoresis is slower compared with the free QDs.

    2.3 Breast cancer cell(SK-BR3)culture

    The breast cancer cells grow fast as shown by the change of the medium on the second day of the culture.The cells are multi-angular as a attachment result to the bottom of the culture flasks.On the fourth day,the cells already reach 90%confluence so they are harvested and inoculated on the glass cover placed on a six-well culture plate.Three days after the inoculation,the glass cover shows 80%—90%confluence exhibiting a natural multi-angular cell shape that has characteristic of cells in situ(see Fig.4).

    Fig.4 Digital photograph of SK-BR3 cells grown on glass cover

    The cells are multi-angular and irregular in the shape with the good light reflection(white light source at 200×magnification).

    2.4 In situ cancer cell imaging using green QDs

    The breast cancer cells in the complete assay consisting of SK-BR3+ 1°Ab-biotin+ streptavidin-QDs appeared as bright green colored irregular structures in situ under UV illuminated condition(see Fig.5).The brighter fluorescence indicates that most of QDs are attached to the cell surface because anti-HER2/neu is an antibody against the cancer cell surface protein.Even after 48 h of the storage in a humidified atmosphere,most of the cells still keep their shape and the bright green fluorescence.QD labeled fluorescent cells are stableat 4°Cin excess of 48h.The control using FITC(data not shown)shows that the color is lighter and diminished after several hours of the storage. Lower detection limits are achieved during biological assays using QDs because of their brighter signals compared with the organic dye.

    Initial studies indicate that there are less bright signals in the incomplete assays that do not have 1°Ab-biotin. Using the blocking buffer#BBB with shaking and incubation for 1 min that is repeated three times.It can eliminate the signals from the incomplete assay.Thus,before finalizing the in situ imaging of the breast cancer cells using green QDs, non-specific binding(NSB)on the incomplete as well as the complete assays are successfully eliminated using blocking buffer#BBB.

    Fig.5(a)shows that in a complete assay involving SK-BR3+1°Ab-biotin+streptavidin-QDs under white light and Fig.5(b)shows that under the UV light,QDs haveλemission 530 nm green on a microscope at 200×magnification.

    Fig.5 Digital photographs of microscope images of breast cancer cells(SK-BR3)

    The streptavidin-QDs targeted biotin molecules bound to antibodies are specific to HER2 proteins on the surface of SK-BR3 breast cancer cells.This results in images with bright fluorescent cells on the microscope when using a UV light source.Compared QD-cell imaging along side imaging with fluoresce in(FITC)dye QD imaging is 90%brighter.The use of QDs can significantly enhance the signals because of their high quantum yield and stability.Furthermore,the QDimaged cells remain bright even after 48 h of the storage indicating stability of the QDs for imaging cells.Thus,the studies show that QD-labeled antibodies have a promise for the sensitive and specific imaging,and the detection of cancer cells.

    3 CONCLUSION

    Instead of direct conjugation of the antibodies to QDs,it is possible to use streptavidin-biotin as a bridge for assays.This can beused for different kinds of imaging combined with commercially available antibodies.The biotin-Ab and the streptavidin-QD combination can be used as a tool for various assays,northern blotting,western blotting,cell labeling,and other applications.Ocean′s super blocking buffer#BBB of ocean eliminates the non-specific signals.Thus,the use of blocking buffer# BBB will eliminate non-specific signals that can mninimize false positive results.Future work will focus on the optimization of the various assay parameters,the elimination of nonspecific signals from biological fluids,quantification,as well as multi-cell/multi-color QD detection. All the parameters for QD-cell imaging analysis areoptimized for the diagnosis of the early stage breast cancer cell.

    [1] Aguilar Z P.Small-volume detection of plasmodium falciparum CSP gene using a 50μm-diameter cavity with self-contained electrochemistry[J].Analytical Chemistry,2006,78(4):1122-1129.

    [2] Aguilar Z P,Fritsch I.Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for cryptosporidium parvum hsp70 m RNA[J].Analytical Chemistry,2003,75(15):3890-3897.

    [3] Pantel K,Brakenh off R H,Brandt B.Detection,clinical relevance and specific biological properties of disseminating tumour cells[J].Nature Reviews Cancer,2008,8:329-340.

    [4] Horner M J,Ries L A G,Krapcho M,et al.SEER Cancer Statistics Review:1975-2006[M].Bethesda:National Cancer Institute,2009.

    [5] Goldhirsch A,Wood W C,Gelber R D,et al.Progress and promise:highlights of the international expert consensus on the primary therapy of early breast cancer 2007[J].Annals of Oncology,2007,18(7):1133-1144.

    [6] Eifel P,Axelson J A,Costa JP,et al.National institutes of health consensus development conference statement: adjuvant therapy for breast cancer,November 1-3,2000[J].Journal of the National Cancer Institute,2001,93(13):979-989.

    [7] Blamey R,Ellis I,Pinder R S,et al.Survival of invasive breast cancer according to the Nottingham Prognostic Index in cases diagnosed in 1990-1999[J].European Journal of Cancer,2007,43(10):1548-1555.

    [8] Ravdin P M,Siminoff L A,Davis G J,et al.Computer program to assist in making decisions about adjuvant therapy for women with early breast cancer[J].Journal of Clinical Oncology,2001,19(4):980-991.

    [9] Early Breast Cancer Trialists′Collaborative Group(EBC TCG).Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival:an overview of the randomised trials[J].Lancet,2005,365:1687-1717.

    [10]Yang L,Mao H,Wang Y A,et al.Singlechain epidermal growth factor receptor antibody conjugated nanoparticles for in vivo tumor targeting and imaging[J].Small,2009,5(2):235-243.

    [11]Xu Hengyi,Xu Feng,Xiong Yonghua,et al.Application of synthesized quantum dots for cell imaging[C]//Proceedings of the International Symposium on Photonics and Optoelectronics.Wuhan:IEEE Computer Society Press,2009:1-4.

    [12]Rosi N L,Mirkin C A.Nanostructures in biodiagnostics[J].Chemical Reviews,2005,105(4):1547-1562.

    [13]Medintz I L,Uyeda H T,Goldman E R,et al.Quantum dot bioconjugates for imaging,labelling and sensing[J].Nature Materials,2005,4(6):435-446.

    [14]Gill R,Patolsky F,Katz E,et al.Electrochemical control of the photocurrent direction in intercalated DNA/Cd S nano particle systems[J].Angewandte Chemie International Ed,2005,44(29):4554-4557.

    [15]Bange J,Zwick E,Ullrich A.Molecular targets for breast cancer therapy and prevention[J].Nature Medicine,2001,7:548-552.

    [16]Ménard S,Pupa S M,Campiglio M,et al.Biologic and therapeutic role of HER2 in cancer[J].Oncogene,2003,22:6570-6578.

    [17]Kute T,Lack C M,Willingham M,et al.Development of hercept in resistance in breast cancer cells[J].Cytometry,2004,57A:86-93.

    [18]Mao W Y,Guo J,Yang W,et al.Synthesis of high-quality near-infrared-emitting Cd TeS alloyed quantum dots via the hydrothermal method[J].Nan otechnology,2007,18:485611.

    [19]Bao H B,Gong Y Y,Zhen L,et al.Enhancement effect of illumination on the photoluminescence of water-soluble Cd Te nanocrystals:toward highly fluorescent Cd Te/Cd S core-shell structure[J].Chemistry Materials,2004,16:3853-3859.

    [20]Xu Hengyi,Aguilar Z P,Su Huaipeng,et al.Breast cancer cell imaging using semiconductor quantum dots[J].ECS Transactions,2009,25(11):69-77.

    [21]Dudak FC,Boyaci?I H,Jurkevica A,et al.Determination of viable Escherichia coli using antibody-coated paramagnetic beads with fluorescence detection[J].Analytical and Bioanalytical Chemistry,2009,393(3):949-956.

    [22]Bauemner A J,Humiston M C,Montagna R A,et al.Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification durst[J].Analytical Chemistry,2001,73(6):1176-1180.

    Transactions of Nanjing University of Aeronautics and Astronautics2010年2期

    Transactions of Nanjing University of Aeronautics and Astronautics的其它文章
    頁(yè)輪干式磨削Ti6Al4V合金
    亚洲欧美清纯卡通| 美女午夜性视频免费| 十分钟在线观看高清视频www| 一本一本久久a久久精品综合妖精| 亚洲伊人色综图| 亚洲熟女毛片儿| 不卡av一区二区三区| 少妇猛男粗大的猛烈进出视频| av一本久久久久| 狂野欧美激情性xxxx| 国产一区亚洲一区在线观看| 久久久国产精品麻豆| 国产精品麻豆人妻色哟哟久久| 99九九在线精品视频| 久久九九热精品免费| 青春草视频在线免费观看| 精品国产国语对白av| 亚洲精品国产一区二区精华液| 你懂的网址亚洲精品在线观看| 80岁老熟妇乱子伦牲交| 免费少妇av软件| 国产无遮挡羞羞视频在线观看| 免费日韩欧美在线观看| 午夜日韩欧美国产| 久9热在线精品视频| 国产精品九九99| 精品亚洲乱码少妇综合久久| 日韩av不卡免费在线播放| 美女视频免费永久观看网站| av国产精品久久久久影院| 国产精品亚洲av一区麻豆| 性色av乱码一区二区三区2| 国产97色在线日韩免费| 天天添夜夜摸| 考比视频在线观看| 欧美av亚洲av综合av国产av| 婷婷成人精品国产| 97精品久久久久久久久久精品| 国产日韩一区二区三区精品不卡| 99热网站在线观看| 精品熟女少妇八av免费久了| 十八禁人妻一区二区| 美女大奶头黄色视频| 满18在线观看网站| 黄片小视频在线播放| 无遮挡黄片免费观看| 久久国产精品影院| 中文字幕av电影在线播放| 久久ye,这里只有精品| 一级毛片电影观看| 啦啦啦在线观看免费高清www| 亚洲av成人精品一二三区| 久久九九热精品免费| 最新的欧美精品一区二区| 国产精品 国内视频| 久久99一区二区三区| 丝袜美腿诱惑在线| 性色av乱码一区二区三区2| 成人国产一区最新在线观看 | 国产精品99久久99久久久不卡| 久久久久国产一级毛片高清牌| 免费观看av网站的网址| 一本一本久久a久久精品综合妖精| 亚洲av在线观看美女高潮| 99热全是精品| 国产视频一区二区在线看| 久热爱精品视频在线9| 国产精品免费视频内射| 亚洲欧美中文字幕日韩二区| 女性被躁到高潮视频| 午夜91福利影院| 久久热在线av| 少妇人妻久久综合中文| 亚洲成色77777| 国产精品香港三级国产av潘金莲 | kizo精华| 日本五十路高清| 亚洲成人免费电影在线观看 | 啦啦啦 在线观看视频| 久久精品熟女亚洲av麻豆精品| 嫩草影视91久久| 在线观看www视频免费| 热99国产精品久久久久久7| av不卡在线播放| 国语对白做爰xxxⅹ性视频网站| 国产日韩一区二区三区精品不卡| 一边亲一边摸免费视频| 首页视频小说图片口味搜索 | 男女边吃奶边做爰视频| 欧美黄色淫秽网站| 国产成人av教育| 亚洲av成人精品一二三区| 日韩一卡2卡3卡4卡2021年| 国产成人91sexporn| 一本—道久久a久久精品蜜桃钙片| 国产成人av教育| 只有这里有精品99| 欧美激情高清一区二区三区| 91精品三级在线观看| 久久av网站| 亚洲av美国av| 午夜福利乱码中文字幕| 亚洲成人免费av在线播放| 亚洲精品一区蜜桃| 国产野战对白在线观看| 午夜免费成人在线视频| 免费黄频网站在线观看国产| 久久国产精品男人的天堂亚洲| h视频一区二区三区| 宅男免费午夜| 亚洲av综合色区一区| 悠悠久久av| 人人澡人人妻人| 一本—道久久a久久精品蜜桃钙片| 十分钟在线观看高清视频www| 人成视频在线观看免费观看| 亚洲国产欧美一区二区综合| 亚洲久久久国产精品| 日韩中文字幕欧美一区二区 | 亚洲欧美色中文字幕在线| 国产在线观看jvid| 丝袜喷水一区| 精品亚洲成国产av| 精品人妻1区二区| 欧美在线黄色| 亚洲男人天堂网一区| 亚洲,欧美精品.| 日韩视频在线欧美| 美女视频免费永久观看网站| 国产成人a∨麻豆精品| 国产成人av激情在线播放| 国产人伦9x9x在线观看| 精品欧美一区二区三区在线| 亚洲av男天堂| 又大又黄又爽视频免费| 久久九九热精品免费| 国产精品国产三级专区第一集| 久久久久久久久久久久大奶| 夫妻性生交免费视频一级片| 国产精品九九99| 成年人免费黄色播放视频| 久久av网站| 欧美老熟妇乱子伦牲交| 欧美黑人欧美精品刺激| 熟女av电影| 亚洲,欧美精品.| 欧美激情 高清一区二区三区| 少妇精品久久久久久久| av天堂久久9| 一级,二级,三级黄色视频| 亚洲成色77777| 在线天堂中文资源库| 人体艺术视频欧美日本| 国产成人精品无人区| 亚洲av日韩在线播放| 青草久久国产| 亚洲国产精品一区二区三区在线| 国产亚洲欧美在线一区二区| 一本一本久久a久久精品综合妖精| 国产亚洲欧美在线一区二区| 色94色欧美一区二区| 性色av乱码一区二区三区2| 90打野战视频偷拍视频| 久久精品人人爽人人爽视色| 你懂的网址亚洲精品在线观看| cao死你这个sao货| 只有这里有精品99| 波多野结衣一区麻豆| 波多野结衣一区麻豆| 桃花免费在线播放| 制服人妻中文乱码| 可以免费在线观看a视频的电影网站| 视频区图区小说| 欧美在线一区亚洲| 亚洲成色77777| 日韩熟女老妇一区二区性免费视频| 久久狼人影院| 91麻豆精品激情在线观看国产 | 香蕉国产在线看| av网站免费在线观看视频| 亚洲精品久久久久久婷婷小说| 国产极品粉嫩免费观看在线| 久久久久视频综合| 国产一区二区 视频在线| 久久国产亚洲av麻豆专区| 美国免费a级毛片| 99热全是精品| 国产亚洲av片在线观看秒播厂| 巨乳人妻的诱惑在线观看| 国产成人系列免费观看| 午夜福利在线免费观看网站| 老司机午夜十八禁免费视频| 精品一区二区三区四区五区乱码 | 国产无遮挡羞羞视频在线观看| www.999成人在线观看| 色精品久久人妻99蜜桃| 两人在一起打扑克的视频| 亚洲,一卡二卡三卡| 晚上一个人看的免费电影| 亚洲人成网站在线观看播放| 久久久亚洲精品成人影院| 亚洲国产中文字幕在线视频| 亚洲精品日本国产第一区| 国产精品麻豆人妻色哟哟久久| 国产午夜精品一二区理论片| 亚洲,欧美,日韩| 肉色欧美久久久久久久蜜桃| 永久免费av网站大全| 国产有黄有色有爽视频| 51午夜福利影视在线观看| 一级片免费观看大全| 最新的欧美精品一区二区| 69精品国产乱码久久久| 亚洲国产av新网站| 欧美日韩亚洲国产一区二区在线观看 | 欧美日韩亚洲高清精品| 久久久久久久国产电影| 久久久国产欧美日韩av| 极品少妇高潮喷水抽搐| 国产精品麻豆人妻色哟哟久久| 蜜桃在线观看..| 在线天堂中文资源库| 最近最新中文字幕大全免费视频 | 真人做人爱边吃奶动态| 侵犯人妻中文字幕一二三四区| 亚洲国产看品久久| 亚洲av美国av| 久久女婷五月综合色啪小说| 久久精品国产亚洲av高清一级| 亚洲五月婷婷丁香| 性少妇av在线| 免费av中文字幕在线| 国产成人a∨麻豆精品| 亚洲一区中文字幕在线| 欧美成人精品欧美一级黄| 日韩电影二区| 亚洲av电影在线进入| 18在线观看网站| 中文字幕人妻熟女乱码| www.熟女人妻精品国产| 精品久久久精品久久久| 欧美 日韩 精品 国产| 国产午夜精品一二区理论片| 国产精品av久久久久免费| 日韩,欧美,国产一区二区三区| 看免费成人av毛片| 午夜福利在线免费观看网站| 色94色欧美一区二区| 久久九九热精品免费| 成年人黄色毛片网站| 日本色播在线视频| 午夜av观看不卡| 国产男人的电影天堂91| 亚洲欧洲精品一区二区精品久久久| 日日摸夜夜添夜夜爱| 亚洲成人免费电影在线观看 | 国产精品偷伦视频观看了| 精品欧美一区二区三区在线| 黄色毛片三级朝国网站| 欧美成人午夜精品| 久久毛片免费看一区二区三区| av网站免费在线观看视频| 在线av久久热| 欧美中文综合在线视频| 久久久精品94久久精品| 午夜激情久久久久久久| 精品久久蜜臀av无| 国产日韩一区二区三区精品不卡| 91精品国产国语对白视频| 国产片特级美女逼逼视频| 黄色 视频免费看| 美女午夜性视频免费| 亚洲国产日韩一区二区| 天堂中文最新版在线下载| 国产精品成人在线| 日韩大片免费观看网站| 丰满迷人的少妇在线观看| 中文字幕亚洲精品专区| 精品少妇一区二区三区视频日本电影| 极品少妇高潮喷水抽搐| tube8黄色片| 亚洲av电影在线进入| 男女免费视频国产| 七月丁香在线播放| 黄色怎么调成土黄色| 曰老女人黄片| 亚洲av在线观看美女高潮| 日韩制服丝袜自拍偷拍| 男女无遮挡免费网站观看| 五月开心婷婷网| 欧美成狂野欧美在线观看| 国产高清videossex| 国产精品久久久久成人av| 新久久久久国产一级毛片| 看十八女毛片水多多多| 亚洲av成人精品一二三区| 亚洲av电影在线观看一区二区三区| 日本欧美国产在线视频| 好男人电影高清在线观看| 国产精品久久久人人做人人爽| 成年美女黄网站色视频大全免费| 99热国产这里只有精品6| 青青草视频在线视频观看| 少妇 在线观看| 精品国产一区二区三区四区第35| 成人亚洲精品一区在线观看| 亚洲一卡2卡3卡4卡5卡精品中文| 一级毛片电影观看| 欧美日韩av久久| 在线观看一区二区三区激情| 久久国产精品大桥未久av| 久久精品久久久久久噜噜老黄| 色视频在线一区二区三区| 国产精品一二三区在线看| 日本欧美视频一区| 欧美变态另类bdsm刘玥| 一级毛片黄色毛片免费观看视频| 交换朋友夫妻互换小说| 久9热在线精品视频| 精品久久久久久电影网| 多毛熟女@视频| 老司机深夜福利视频在线观看 | 亚洲第一青青草原| 麻豆av在线久日| 国产精品欧美亚洲77777| 老司机在亚洲福利影院| 日韩欧美一区视频在线观看| 久久久久久久久久久久大奶| 自拍欧美九色日韩亚洲蝌蚪91| 国产片特级美女逼逼视频| 欧美精品高潮呻吟av久久| 国产成人精品久久二区二区91| 男女边吃奶边做爰视频| 在线 av 中文字幕| 天天躁狠狠躁夜夜躁狠狠躁| 性高湖久久久久久久久免费观看| 欧美在线一区亚洲| 亚洲精品乱久久久久久| 欧美日韩国产mv在线观看视频| 国产高清视频在线播放一区 | 精品亚洲乱码少妇综合久久| 亚洲av在线观看美女高潮| 天天添夜夜摸| 极品人妻少妇av视频| 国语对白做爰xxxⅹ性视频网站| 亚洲国产欧美日韩在线播放| 午夜福利免费观看在线| 一区在线观看完整版| 久久国产精品人妻蜜桃| 国产精品国产三级国产专区5o| 久热这里只有精品99| 国产成人精品久久久久久| 可以免费在线观看a视频的电影网站| 9191精品国产免费久久| 丝袜喷水一区| 高清黄色对白视频在线免费看| 国产精品欧美亚洲77777| 麻豆乱淫一区二区| 在线精品无人区一区二区三| 国产一区二区三区av在线| 人人澡人人妻人| 久久精品久久久久久久性| 午夜福利,免费看| a级毛片在线看网站| 成人三级做爰电影| 超碰成人久久| tube8黄色片| 国产精品 国内视频| 欧美日韩亚洲国产一区二区在线观看 | 黑人巨大精品欧美一区二区蜜桃| 国产亚洲欧美在线一区二区| 少妇人妻久久综合中文| av不卡在线播放| 国产主播在线观看一区二区 | 热99国产精品久久久久久7| 男人添女人高潮全过程视频| 香蕉国产在线看| 午夜久久久在线观看| 热99国产精品久久久久久7| 亚洲国产欧美日韩在线播放| 国产欧美日韩精品亚洲av| cao死你这个sao货| 国产精品免费大片| 午夜91福利影院| 日本五十路高清| 91麻豆精品激情在线观看国产 | 啦啦啦在线免费观看视频4| www.自偷自拍.com| 久久精品国产亚洲av高清一级| 校园人妻丝袜中文字幕| 亚洲成人免费av在线播放| 亚洲av日韩在线播放| 一级毛片电影观看| 欧美日韩亚洲高清精品| 最近中文字幕2019免费版| 午夜激情久久久久久久| 久久国产精品影院| 成人免费观看视频高清| 成人国产av品久久久| 美女国产高潮福利片在线看| 欧美在线黄色| 国产成人精品久久久久久| 观看av在线不卡| 久久久久久久久免费视频了| 在现免费观看毛片| 欧美97在线视频| 国产有黄有色有爽视频| 狂野欧美激情性xxxx| a 毛片基地| 亚洲,欧美精品.| 欧美日韩视频精品一区| 午夜福利视频精品| 国产又爽黄色视频| 亚洲欧美一区二区三区黑人| 一级毛片电影观看| 另类精品久久| 国产免费现黄频在线看| 成人三级做爰电影| 一级a爱视频在线免费观看| 久久鲁丝午夜福利片| 亚洲精品日韩在线中文字幕| 亚洲国产中文字幕在线视频| 亚洲成人免费电影在线观看 | 黄色片一级片一级黄色片| 日本欧美国产在线视频| 欧美黄色片欧美黄色片| 亚洲av男天堂| 9热在线视频观看99| 男女床上黄色一级片免费看| 成人国产av品久久久| 亚洲精品在线美女| 亚洲精品一二三| 18禁黄网站禁片午夜丰满| 亚洲精品国产av成人精品| 午夜精品国产一区二区电影| 国产一区二区三区综合在线观看| 桃花免费在线播放| 午夜91福利影院| 黄网站色视频无遮挡免费观看| 性高湖久久久久久久久免费观看| 国产精品 欧美亚洲| 成年av动漫网址| 永久免费av网站大全| 黄色 视频免费看| 国产精品香港三级国产av潘金莲 | 天堂8中文在线网| 午夜福利视频在线观看免费| 久久精品成人免费网站| 王馨瑶露胸无遮挡在线观看| 欧美av亚洲av综合av国产av| 久久免费观看电影| www.熟女人妻精品国产| 欧美+亚洲+日韩+国产| 国产成人精品久久二区二区91| 黄色怎么调成土黄色| 国产在线观看jvid| 亚洲天堂av无毛| 欧美人与性动交α欧美软件| 波多野结衣av一区二区av| 色精品久久人妻99蜜桃| 在线观看免费高清a一片| 成年动漫av网址| 欧美精品一区二区免费开放| 91九色精品人成在线观看| 两个人免费观看高清视频| 久久九九热精品免费| 国语对白做爰xxxⅹ性视频网站| 国产不卡av网站在线观看| 国产欧美日韩综合在线一区二区| 久久精品亚洲熟妇少妇任你| 人妻一区二区av| 亚洲免费av在线视频| 亚洲精品第二区| 男女床上黄色一级片免费看| 十分钟在线观看高清视频www| 日韩一区二区三区影片| 美女脱内裤让男人舔精品视频| 男女国产视频网站| 国产深夜福利视频在线观看| 天天躁夜夜躁狠狠久久av| 午夜91福利影院| 亚洲,欧美精品.| 自拍欧美九色日韩亚洲蝌蚪91| 久久久久久久大尺度免费视频| 永久免费av网站大全| 中文字幕高清在线视频| 久热这里只有精品99| 女人精品久久久久毛片| 国产视频一区二区在线看| 亚洲人成77777在线视频| 两个人看的免费小视频| 亚洲成人免费av在线播放| 亚洲熟女精品中文字幕| 国产精品99久久99久久久不卡| 国产精品一区二区在线观看99| 青春草亚洲视频在线观看| 欧美在线黄色| 日韩制服骚丝袜av| 精品国产超薄肉色丝袜足j| 亚洲七黄色美女视频| 在线观看免费高清a一片| 黄片小视频在线播放| www.熟女人妻精品国产| 精品人妻一区二区三区麻豆| 一区在线观看完整版| 九色亚洲精品在线播放| 丝袜美腿诱惑在线| xxxhd国产人妻xxx| 精品人妻在线不人妻| 亚洲av男天堂| 国产高清视频在线播放一区 | 日本黄色日本黄色录像| 黑人猛操日本美女一级片| 午夜视频精品福利| 欧美精品一区二区免费开放| 亚洲中文字幕日韩| 丝袜人妻中文字幕| 午夜激情av网站| 在线看a的网站| 亚洲av片天天在线观看| 男女床上黄色一级片免费看| svipshipincom国产片| 无遮挡黄片免费观看| 婷婷色综合大香蕉| 国产精品一区二区精品视频观看| 免费女性裸体啪啪无遮挡网站| 亚洲精品av麻豆狂野| 亚洲第一青青草原| 亚洲国产中文字幕在线视频| 国产亚洲av片在线观看秒播厂| 麻豆国产av国片精品| 亚洲成人免费电影在线观看 | 人人澡人人妻人| 日本欧美视频一区| 亚洲欧美成人综合另类久久久| 国产黄色视频一区二区在线观看| 国产精品av久久久久免费| 91老司机精品| 国产日韩欧美在线精品| 亚洲免费av在线视频| 免费看av在线观看网站| 亚洲av日韩在线播放| 亚洲欧美一区二区三区久久| 国产一卡二卡三卡精品| 亚洲精品乱久久久久久| 亚洲 国产 在线| 99热国产这里只有精品6| 美女视频免费永久观看网站| 久久99精品国语久久久| 九色亚洲精品在线播放| 岛国毛片在线播放| 国产人伦9x9x在线观看| 成年动漫av网址| 又粗又硬又长又爽又黄的视频| 91成人精品电影| 国产97色在线日韩免费| 亚洲av在线观看美女高潮| 久久久精品国产亚洲av高清涩受| 亚洲精品中文字幕在线视频| 只有这里有精品99| 久久人人爽人人片av| 亚洲国产欧美一区二区综合| 男人爽女人下面视频在线观看| 国产有黄有色有爽视频| 婷婷色综合www| 国产男女超爽视频在线观看| 天堂8中文在线网| avwww免费| 日韩大片免费观看网站| 97在线人人人人妻| bbb黄色大片| 麻豆乱淫一区二区| 老鸭窝网址在线观看| 成年人午夜在线观看视频| 国产伦人伦偷精品视频| 无限看片的www在线观看| 中文字幕另类日韩欧美亚洲嫩草| 王馨瑶露胸无遮挡在线观看| 国产精品.久久久| 少妇猛男粗大的猛烈进出视频| 成人免费观看视频高清| 中文字幕高清在线视频| 视频区欧美日本亚洲| 色播在线永久视频| 看十八女毛片水多多多| 国产成人a∨麻豆精品| 亚洲av电影在线进入| 精品久久久精品久久久| 色网站视频免费| 一级毛片我不卡| 黄频高清免费视频| 五月天丁香电影| 欧美精品一区二区大全| 黄色视频在线播放观看不卡| 51午夜福利影视在线观看| 欧美人与性动交α欧美软件| 最近最新中文字幕大全免费视频 | 男女床上黄色一级片免费看| 精品少妇久久久久久888优播| 亚洲成av片中文字幕在线观看| 久久人人爽av亚洲精品天堂| 国产成人一区二区在线| 我的亚洲天堂| 国产欧美日韩精品亚洲av| xxxhd国产人妻xxx| 岛国毛片在线播放| 9191精品国产免费久久| 人体艺术视频欧美日本| 亚洲自偷自拍图片 自拍| 99国产精品一区二区蜜桃av | 王馨瑶露胸无遮挡在线观看| 午夜福利在线免费观看网站| 9191精品国产免费久久| 国产有黄有色有爽视频| 在线 av 中文字幕|