• <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 ?

    Multiple exosome RNA analysis methods for lung cancer diagnosis through integrated on-chip microfluidic system

    2022-07-11 03:39:34YunxingLuZhoduoTongZhenhuWuXioyuJinLinZhouShihuiQiuChunjieShenHoYinHongjuMo
    Chinese Chemical Letters 2022年6期

    Yunxing Lu,Zhoduo Tong,Zhenhu Wu,Xioyu Jin,Lin Zhou,Shihui Qiu,Chunjie Shen,Ho Yin,Hongju Mo,?

    a State Key Laboratory of Transducer Technology,Shanghai Institute of Microsystem and Information Technology,Chinese Academy of Sciences,Shanghai 200050,China

    b Center of Materials Science and Optoelectronics Engineering,University of Chinese Academy of Sciences,Beijing 100049,China

    Keywords:Exosome Integrated processing RNA analysis Microfluidics Droplet digital PCR

    ABSTRACT Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos (e.g.,proteins,mRNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR (ddPCR).Gradient dilution experiments show great linearity over a large concentration range with R2=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.

    Lung cancer is one of the malignant tumors among worldwide which resulted in maximum number of cancer death [1].Because of the inconspicuous symptom in early stages and frequentlyoccurred metastasis and recurrence,lung cancer would be diagnosed in middle and advanced stages [2,3].As an accurate and noninvasive method,liquid biopsy [4–7]offers a promising future for cancer diagnosis.

    Exosomes are one of the biotargets in liquid biopsy [8–10].Exosomes are a kind of nanovesicles (~30–200 nm in diameter)that play a critical role in intercellular communication and “cargo”transformation [11].Derived by eukaryotic cells into bioliquids,exosomes have several outstanding superiorities,like high abundance(~1010particles in 1 mL plasma),high stability in bioliquids and easy acquisition.Secreted by parental cells,especially tumor cells,exosomes can be the “trucks” of proteins and nucleic acids “cargos”[12–14].In addition,hereditary information of parental cells can be read out through analyzing the nucleic acid components.Exosomal nucleic acids [15]include mRNA,lncRNA,miRNA and some DNA.Nevertheless,on account of complicacy of sample preparation,developing convenient and effective methods for analyzing exosome nucleic acids is in urgent need.

    Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one of the RNA detection methods [16],where expression of target gene is detected by reading out the fluorescence intensity of whole reaction-tube [17,18],therefore,detection biases may occur and rare mutation may be ignored in background noise.Digital polymerase chain reaction (dPCR)is a new detection method that achieved single-molecule detection [19–21],which is free from standard curves and provides a far more accurate method to rare RNA detection [22,23].In our recent researches,dPCR has proven itself as a powerful tool against exosomal cancer-derived lncRNA [24]and SARS-CoV-2 RNA [25].

    In this study,we developed an integrated multiple fluorescence droplet digital PCR (ddPCR) microfluidic system for on-chip exosome isolation and RNA analysis (Fig.1),which is sensitive,unbiased,high-throughput and inexpensive.Utilizing the system,exosome samples were firstly on-chip isolated in the processing chamber,mingled with lysis buffer and form reaction mixture.After that,the mixture was transformed through the microtubule into the liquid entrance and converged with mineral oil at the standard droplet structure.During the exosome processing and droplets generation,no off-chip operation was involved,thus liquid contamination was avoided.~25 μL reaction mixture was formed and up to 100,000,50μm-diameter droplets were generated and contained into reaction chamber,which is much higher than commercial chips (30,000–50,000) and the dead volume is much lower (~5% compared to 30%).Beside,a polymethyl methacrylate(PMMA) packing structure was assembled for liquid stabilizing and maintain the holistic system.Dilution testing showed the great stability of our system (R2=0.9998),beside,the multiple mutation analysis of 2 genes in 4 cell lines were consistent with cell researches [8].These results indicate our system to be a promising solution for exosomal RNA analysis.

    Fig.1.Schematic diagram of the integrated exosome analysis system.

    To be specific,the microfluidic system is consisted of four main sections.The exosome on-chip processing chamber,droplet generation structure,the droplet reaction chamber and the PMMA packing structure.The exosome processing chamber and droplet generation structure were fabricated by polydimethylsiloxane (PDMS)and connected with a microtubule.The droplet reaction chamber was composed by two-layered glass with a 50μm gap where droplets were maintained for single-layered reaction.Additionally,the PMMA packing structure was fabricated by machine tool for liquid transformation.The processing chamber was a through-hole punched by puncture needle,7 mm in diameter,4 mm high,allowing the volume over 120 μL for high flux reaction.As a comparison,typical microfluidic chamber fabricated by pattern transformation,was usually less than 10 μL.Therefore,our system was capable to high volume reaction with a larger concentration range.

    For droplet generation structure,standard cross-shaped droplet generation structure was designed.During our experiments,exosome reaction mix prepared in the chamber was transferred through micro-tubule into liquid entrance,free from pipette,and eliminated the potential risk of contamination.After generated,droplets were flowed into the large-volume (up to 25 μL) doublelayered glass chamber for RT-ddPCR reaction.Aiming at sufficient liquid transformation,a PMMA packing structure was designed and manufactured to maintain and stabilize the injection pressure of reaction phase.The structure packed the microfluidic system around and covered the processing chamber,avoiding pressure from decreasing by divulging.

    The microfluidic system was fabricated according to standard process for prototyping of microfluidic systems in PDMS.The pattern mold of the exosome procession platform was manufactured by standard soft lithography.To be specific,the pattern portrayed by CAD software was firstly printed onto the chromium photo mask,followed by projecting to a silicon wafer photoetching.After that,deep reactive ion etching (deep RIE) techniques and sulfuric acid rinsing were utilized for structure etching.Subsequently,the silicon mold was treated with trichloro-(1H,1H,2H,2Hperfluorooctyl) silane (Sigma Aldrich,St.Louis,MO,USA) vapor overnight and ready for chip molding.

    For microfluidic system fabrication,the PDMS (Sylgard 184 resin),cross-linker,and Triton X-100 (Sigma-Aldrich,St.Louis,MO,USA) were mixed at the ratio of 10:1:0.05 (w/w/w) and degassed under the vacuum condition.The mixture was then poured onto the silicon mold to form the patterned microfluidic structure.Baked at 70°C for 50 min,the PDMS structure was carefully exfoliated and punched the inlet and outlet.Lastly,the microfluidic structure was bonded to a clean glass through air plasma treatment.Then microtubule inserted to connect the outlet of processing chamber and liquid entrance.The droplet reaction chamber was assembled by two pieces of glass slides with a 50μm thick double-sided adhesive (DSA).Between the micro channel and the cubage,an interval was designed (named “venting gap”) for air bubbles elimination caused by pressure fluctuations.PMMA packing structure was depictedviaSolidworksTMand manufactured through machining workshop,then polished and assembled by screw and nut set.For system operation,the microfluidic system was packed by the PMMA structure and the processing chamber was sealed,therefore stable gas pressure for droplet generation was guaranteed.

    Aiming at characterizing the scale distribution and concentration of exosomes,nanoparticle tracking analysis (NTA) (Particle Metrix,Meerbusch,Germany) was utilized under the standard protocols.To be specific,10 μL exosome samples were diluted in phosphate buffer saline (PBS) to reach the proper concentration(108–109particles/mL).During the detection,particles were radiated by laser and captured and tracked by a CCD camera.The measurements were conducted at 20±3°C.Then the size distribution and concentration were determined by the NTA 3.2 Analytical Software Suite and shown in Fig.2a.A unimodal peak around 120 nm could be observed,which was in accordance with published researches.

    Fig.2.Exosome characterization through NTA (a),TEM (b) and western blot (c),scale bar 100 nm.

    Fig.3.Four main parts of the microfluidic system,(a) schematic diagram of the whole system,(b) prototype of the system,scale bar 2 mm,(c–e) exosome isolation processing in the chamber,scale bar 1 mm,(f) microtubule-based liquid transformation,(g) micrograph of the venting gap,scale bar 100μm,(h–k) schematic diagram and feature of the venting gap for air bubbles elimination,scale bar 200μm.

    For transmission electron microscopy (TEM),exosomes were dripped onto a bronze parafilm by pipette and washed by PBS sequentially,then quiescence for 10 min to drain exosomes and added a drop of 2% paraformaldehyde,dyed in 2% uranyl acetate at room temperature for exosome counterstain,followed by embedded in 0.13% methyl cellulose and 0.4% uranyl acetate for 10 min,and then imaged by microscopic observation.Fig.2b showed the morphology of exosomes,and typical saucer shape could be observed.

    For western blotting,exosome sample was then treated with loading buffer at the ratio of 4:1 to obtain protein lysate.After being boiled at 95°C for 5 min,the product then cooled down and conducted with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Followed by PVDF transformation,the lysate was incubated with anti-CD9 at 1:5000 and bound with horseradish peroxidase (HRP) secondary antibody.At last,the immunoreactive band was visualized under imaging system.The result was shown in Fig.2c.

    The whole exosome processing could be observed owing to the transparent chip (Figs.3a and b).In the exosome isolation process,the immune magnetic beads were dispersed in the exosome samples as shown in Fig.3c.After isolation,a magnet was placed under the chamber and the beads were collected at the center of the bottom (Fig.3d) and the supernatant was excluded (Fig.3e).Then,the lysis buffer and reaction mix were added in order to form the final mix.

    Following that,the exosome chamber was sealed and the chip was packed with the PMMA structure.The PMMA structure was designed to stablize the high gap pressure in the droplet generation.After assembly,the reaction mix was successfully transformed into the liquid entrance in Fig.3f (replaced by blue ink for observation convenience).

    During the usual droplet generation,it is worthy to note that air bubbles would be generated from pressure fluctuation.Bubbles would expand and shrink during the thermal cycle process,and cause droplets fusion and affect the subsequent analysis.In our system,this problem could be solved.A double-layered reaction chamber was placed below the droplet structure and forming a venting gap shown in Fig.3g.When bubbles accidently generated,they would rise out from the gap due to the floatage difference between liquid and air (Fig.3h).Figs.3i–k showed the whole processing of air bubbles elimination.

    DdPCR is one kind of absolute quantification detection method.Different from qPCR,which measures through Ct value,ddPCR counts the number of positive droplets and total droplets and calculates the copy number by Poisson distribution theory.Thus,it is of importance to set threshold to distinguish positive droplets from negative droplets.

    Fig.4.Droplets distinguishing.(a) Micrograph of droplets after thermal cycle reaction (left) and negative control (right),inserts are representative positive and negative droplets,scale bar 100μm,(b) gray values of total droplets groups after data classification.

    Fig.5.System performance examination.(a) Micrograph of droplets generation,scale bar 100μm,(b) linear regression fitting of the dilution exosome samples,(c–h) micrographs of droplets with a series of dilution factors of sample exosomes,scale bar 100μm.

    Compared to qPCR,ddPCR is self-contrasting,where both positive and negative units are analyzed together and we can distinguish positive signals by calculating fluorescence intensity of droplets.Image pro plus 6.0 was utilized to measure the gray values of total droplets (including positive and negative droplets)and data were classified into two groups.Fig.4a left showed the droplets with reaction mixture after reaction and Fig.4a right revealed the negative control (blank mixture with RNase-free water) to further avoiding false positive result.Beside,the gray values of all droplets were classified into two significant difference groups in Fig.4b,which were positive group and negative group.The mean value of positive group was 77.70 while the negative group was 37.69.Therefore,we set 57.695 as the threshold for droplet distinguishing.

    Beside,we tested the linearity of copy number variation to concentration of exosome sample under gradient dilution.Linearity reflected the accuracy of the system,a better linearity means better consistency to different concentration of samples and lead to more accurate experimental results,andvice versa.

    Ultracentrifuged samples from H1299 cell line supernatant were tested.Firstly,the concentration of samples was measured by NTA.Then the samples were divided into 6 groups at different dilution ratios.Following that,these samples were parallelly tested under the same operation procedure and the copy numbers were calculated and summarized.

    Fig.5a showed the droplets generation process,Figs.5c–h represented the micrographs of droplets with the dilution factor at 0.5,0.2,0.1,0.05,0.01 and 0.001,respectively.Data was calculated and summarized in Fig.5b.Linear regression fitting showed linear relationship between copy number and dilution factor,withR2=0.9998,which showed great detection accuracy of our microfluidic system.Through NTA,the concentration of exosomes is measured as 3.9×107μL?1.10 μL sample was tested in the experiments and therefore,the limit of detection (LOD) is down to 3.9×105exosomes.

    Mutations onEGFRandKRASare well-known in specific types of non-small cell lung cancers (NSCLCs).Identification of different mutant subtypes would benefit the cancer therapy [26,27].Therefore,to inspect the detection and distinguish ability of our system,we employed four kinds of exosomes from cancer cell lines,including H446,H1299,H1975 and A549,for on-chip exosome multiple mutation analysis.EGFR L858RandKRASgene mutations were chosen to be the detection targets in our study.

    Through parallel experiments,Fig.6 exhibited the representative micro-photos of the results.Figs.6a–h wereEGFR L858Rmutant results for exosomes from H446,H1299,H1975 and A549,under carboxyfluorescein (FAM) and hexachlorofluorescein (HEX) fluorescence field,respectively,while Figs.6i–p represented theKRASmutation for same cell line exosomes.From contrast experiments it was observed that exosomes from H1975 carriedEGFR L858Rmutation,and the other three kinds of exosomes (from H446,H1299 and A549) were wild type.Beside,forKRASmutation analysis,exosomes from A549 cell lines exhibited wild type,while other three kinds of exosomes (from H446,H1299 and H1975) showed mutation type.Note that there were several mutation types forKRAS,thus in this set of experiments we concentrated on whether one kind of exosomes express mutation or not.It is proved from our researches that exosomes from various cell lines exhibited heterogeneity,and the mutation of exosomes was in accordance to their parental cells.These results revealed the potential utilities of exosomes for non-invasive diagnosis and detections.

    Fig.6.Parallel detections of four kinds of exosomes for multiple EGFR L858R and KRAS mutation analysis.(a-h) showed EGFR L858R mutant results for exosomes from H446,H1299,H1975 and A549,respectively,and (i-p) showed the KRAS mutation for same exosomes.Scale bar 200μm.

    In this research,we present an integrated microfluidic system for exosomal RNA analysis.Exosome on-chip isolation,lysis and droplet-based digital RT-PCR reaction were accomplished through three connected functional sections,where off-chip contaminations were avoided.After the PCR thermal cycles,exosomal RNA was reacted with reaction mixture.Positive droplets exhibited significant fluorescence signal from background signal.Beside,gradient dilution parallel experiments revealed good linearity over the large concentration range,withR2=0.9998.For exosome mutation analysis,two oncogenes (EGFRandKARS) were explored in four kinds of cell lines including H446,H1299,H1975,A549 and turned out the differentiation results which was in accordance with published cell researches.

    Additionally,large volume of one-stop exosome sample and reaction mixture were achieved in our system for high throughput exosome isolation and the final reaction droplets reached~100,000.Our microfluidic chip demonstrated advantages including low-cost,high convenience,no contamination risk and disposability perspectives.By adjusting reaction mixture,it is also suitable for other gene analysis.The easily-assembled PMMA structure also facilitated sample testing.In short,our microfluidic system is prospective to exosomal RNA sensitive detection and analysis in precision diagnosis era.

    Declaration of competing interest

    The authors declare that they do not have any known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

    Acknowledgments

    The authors are grateful for the support of grants from National Natural Science Foundation of China (Nos.61971410,and 62001458),and Shanghai Sailing Program (No.20YF1457100).

    18禁裸乳无遮挡免费网站照片| 黄色成人免费大全| 免费搜索国产男女视频| 欧美又色又爽又黄视频| 欧美日韩精品网址| 日本在线视频免费播放| 欧美3d第一页| 51午夜福利影视在线观看| 免费电影在线观看免费观看| 手机成人av网站| 久久午夜综合久久蜜桃| 中文字幕精品亚洲无线码一区| 中文字幕熟女人妻在线| 欧美又色又爽又黄视频| 亚洲精品一卡2卡三卡4卡5卡| 午夜成年电影在线免费观看| 亚洲七黄色美女视频| 日韩欧美三级三区| 999精品在线视频| 97超级碰碰碰精品色视频在线观看| 久久中文字幕人妻熟女| 精华霜和精华液先用哪个| 精品国产超薄肉色丝袜足j| 国产亚洲精品综合一区在线观看| 国产视频内射| 亚洲欧美日韩高清专用| 久久中文字幕人妻熟女| 日本精品一区二区三区蜜桃| 麻豆久久精品国产亚洲av| 一级毛片高清免费大全| 国产高清videossex| 高清毛片免费观看视频网站| 久久香蕉国产精品| 欧美又色又爽又黄视频| 99视频精品全部免费 在线 | 色综合婷婷激情| 两人在一起打扑克的视频| 狠狠狠狠99中文字幕| 亚洲av日韩精品久久久久久密| 国产成人系列免费观看| 日本免费一区二区三区高清不卡| netflix在线观看网站| 欧美一级毛片孕妇| 老鸭窝网址在线观看| 亚洲18禁久久av| 国产又色又爽无遮挡免费看| 999久久久精品免费观看国产| 色综合婷婷激情| www.精华液| 两个人看的免费小视频| 麻豆av在线久日| 琪琪午夜伦伦电影理论片6080| 人人妻人人澡欧美一区二区| 色哟哟哟哟哟哟| 中文字幕高清在线视频| 精品99又大又爽又粗少妇毛片 | 听说在线观看完整版免费高清| 人人妻人人澡欧美一区二区| 丁香欧美五月| 不卡一级毛片| 一本综合久久免费| www.熟女人妻精品国产| 麻豆国产av国片精品| 国产亚洲欧美在线一区二区| 国产精品久久久人人做人人爽| 国内少妇人妻偷人精品xxx网站 | 岛国视频午夜一区免费看| 搡老岳熟女国产| 日韩人妻高清精品专区| av天堂中文字幕网| 黑人操中国人逼视频| 在线播放国产精品三级| 狂野欧美白嫩少妇大欣赏| 观看美女的网站| 日韩av在线大香蕉| 国产高清视频在线观看网站| 久久欧美精品欧美久久欧美| 国产精品一区二区精品视频观看| 国产精品野战在线观看| 欧美一级毛片孕妇| 日本a在线网址| 九色成人免费人妻av| 少妇人妻一区二区三区视频| 成人三级黄色视频| 一进一出抽搐gif免费好疼| 手机成人av网站| 丁香六月欧美| 99国产精品99久久久久| 人妻久久中文字幕网| 国产成人欧美在线观看| 国产精华一区二区三区| 精品久久久久久久末码| 国产精品精品国产色婷婷| 在线永久观看黄色视频| www.自偷自拍.com| 国产午夜福利久久久久久| 老司机在亚洲福利影院| 超碰成人久久| 日韩大尺度精品在线看网址| 两性午夜刺激爽爽歪歪视频在线观看| 99精品在免费线老司机午夜| 欧美国产日韩亚洲一区| 免费在线观看亚洲国产| 操出白浆在线播放| 桃红色精品国产亚洲av| 午夜免费激情av| tocl精华| a在线观看视频网站| 性色av乱码一区二区三区2| a级毛片a级免费在线| 欧美黄色片欧美黄色片| 91麻豆精品激情在线观看国产| 亚洲自拍偷在线| 97人妻精品一区二区三区麻豆| 午夜福利高清视频| 男人舔女人的私密视频| 在线a可以看的网站| 免费无遮挡裸体视频| 搡老熟女国产l中国老女人| 色综合婷婷激情| 国产熟女xx| 国产午夜精品论理片| 免费一级毛片在线播放高清视频| 国产1区2区3区精品| 成人18禁在线播放| 国产激情久久老熟女| 国产日本99.免费观看| 可以在线观看的亚洲视频| 高清毛片免费观看视频网站| 高清毛片免费观看视频网站| 搡老妇女老女人老熟妇| 一二三四社区在线视频社区8| 国产精品一及| 久久精品夜夜夜夜夜久久蜜豆| 五月伊人婷婷丁香| 2021天堂中文幕一二区在线观| 老汉色∧v一级毛片| 99视频精品全部免费 在线 | 日日夜夜操网爽| 中出人妻视频一区二区| 桃色一区二区三区在线观看| 欧美日韩福利视频一区二区| 99热这里只有精品一区 | 国产亚洲精品综合一区在线观看| 亚洲成av人片在线播放无| 欧美一级a爱片免费观看看| 亚洲第一电影网av| 日韩欧美国产在线观看| 中出人妻视频一区二区| 国产精品亚洲av一区麻豆| 亚洲欧美激情综合另类| 午夜精品久久久久久毛片777| 欧美成狂野欧美在线观看| 18禁裸乳无遮挡免费网站照片| 一个人观看的视频www高清免费观看 | 亚洲专区字幕在线| 久久久久久久久中文| 天天一区二区日本电影三级| 亚洲国产日韩欧美精品在线观看 | 亚洲人成网站高清观看| netflix在线观看网站| 日韩大尺度精品在线看网址| 久久久久免费精品人妻一区二区| 成人精品一区二区免费| 免费一级毛片在线播放高清视频| 一区二区三区国产精品乱码| 蜜桃久久精品国产亚洲av| 夜夜夜夜夜久久久久| 夜夜夜夜夜久久久久| 国产69精品久久久久777片 | 婷婷精品国产亚洲av| 国产激情偷乱视频一区二区| 一本精品99久久精品77| 成人精品一区二区免费| 亚洲国产日韩欧美精品在线观看 | 91麻豆av在线| 国产单亲对白刺激| 99久久综合精品五月天人人| 久久精品国产99精品国产亚洲性色| 免费在线观看成人毛片| 九色国产91popny在线| 国产成人av激情在线播放| 亚洲国产欧美网| 国产黄片美女视频| 在线观看一区二区三区| 亚洲18禁久久av| 五月玫瑰六月丁香| 一二三四社区在线视频社区8| 亚洲av成人av| 国产精品爽爽va在线观看网站| 亚洲国产欧美人成| 嫩草影院精品99| 在线观看美女被高潮喷水网站 | 精品欧美国产一区二区三| 十八禁人妻一区二区| 非洲黑人性xxxx精品又粗又长| 老司机午夜十八禁免费视频| 亚洲国产中文字幕在线视频| 国产久久久一区二区三区| 搞女人的毛片| 成人国产一区最新在线观看| 欧美黄色片欧美黄色片| 亚洲国产精品成人综合色| 午夜激情福利司机影院| 国产精品一区二区免费欧美| 欧美激情久久久久久爽电影| 午夜影院日韩av| 九九久久精品国产亚洲av麻豆 | www.www免费av| 午夜激情欧美在线| 两个人看的免费小视频| 黄色日韩在线| 久久久久久九九精品二区国产| 久久婷婷人人爽人人干人人爱| 国产一区二区激情短视频| 男人的好看免费观看在线视频| 欧美色欧美亚洲另类二区| 欧美成人免费av一区二区三区| 精品国产乱子伦一区二区三区| 欧美乱色亚洲激情| 男人舔女人的私密视频| 久久这里只有精品中国| 最近视频中文字幕2019在线8| 母亲3免费完整高清在线观看| 两人在一起打扑克的视频| 精品久久久久久久久久久久久| 高潮久久久久久久久久久不卡| 在线国产一区二区在线| 一二三四在线观看免费中文在| 两个人的视频大全免费| av欧美777| 嫩草影院精品99| av视频在线观看入口| 操出白浆在线播放| 亚洲国产精品久久男人天堂| a级毛片在线看网站| 少妇裸体淫交视频免费看高清| 两性夫妻黄色片| 欧美日韩亚洲国产一区二区在线观看| 精品久久久久久久人妻蜜臀av| 国产精品98久久久久久宅男小说| 女人高潮潮喷娇喘18禁视频| 99在线人妻在线中文字幕| 久久性视频一级片| 成人18禁在线播放| 超碰成人久久| 国产一区二区在线观看日韩 | 日韩精品中文字幕看吧| 女人高潮潮喷娇喘18禁视频| 精品国产三级普通话版| 一进一出好大好爽视频| 欧美成人性av电影在线观看| 精品无人区乱码1区二区| 国产aⅴ精品一区二区三区波| 国产亚洲精品一区二区www| 午夜福利在线观看免费完整高清在 | www.熟女人妻精品国产| 搡老熟女国产l中国老女人| 精品国产亚洲在线| 男人舔女人下体高潮全视频| 亚洲无线在线观看| 国产精品美女特级片免费视频播放器 | 脱女人内裤的视频| 不卡一级毛片| 亚洲国产精品sss在线观看| 国产精品自产拍在线观看55亚洲| 午夜福利在线观看吧| 亚洲欧美日韩高清专用| 日本在线视频免费播放| 欧美绝顶高潮抽搐喷水| 1000部很黄的大片| 亚洲熟妇中文字幕五十中出| 久久精品91无色码中文字幕| 成人鲁丝片一二三区免费| www.熟女人妻精品国产| 国产成人av激情在线播放| 免费av不卡在线播放| 国产高清激情床上av| 精品国产乱码久久久久久男人| 国产欧美日韩一区二区精品| 欧美大码av| 女同久久另类99精品国产91| 成人三级黄色视频| 亚洲精品美女久久久久99蜜臀| 亚洲性夜色夜夜综合| 欧美黑人巨大hd| 亚洲精品乱码久久久v下载方式 | 最近最新中文字幕大全电影3| av天堂中文字幕网| 丁香欧美五月| 免费看美女性在线毛片视频| 午夜精品久久久久久毛片777| 99热这里只有是精品50| 一个人看的www免费观看视频| 美女大奶头视频| 天天添夜夜摸| 成年人黄色毛片网站| 日本三级黄在线观看| 国产成+人综合+亚洲专区| 好男人电影高清在线观看| 国产欧美日韩精品亚洲av| 男人和女人高潮做爰伦理| 国产精品一区二区免费欧美| 国产精品99久久久久久久久| 欧美日本亚洲视频在线播放| 亚洲无线在线观看| 别揉我奶头~嗯~啊~动态视频| 午夜久久久久精精品| 久久久久免费精品人妻一区二区| 国产高潮美女av| 成年女人永久免费观看视频| 成年人黄色毛片网站| 99热这里只有是精品50| 欧美日韩综合久久久久久 | 看黄色毛片网站| 又紧又爽又黄一区二区| 中文字幕人成人乱码亚洲影| 长腿黑丝高跟| 男插女下体视频免费在线播放| 成人亚洲精品av一区二区| 欧美乱码精品一区二区三区| 老熟妇仑乱视频hdxx| 天堂√8在线中文| 麻豆成人午夜福利视频| 国产综合懂色| 国产亚洲av高清不卡| 巨乳人妻的诱惑在线观看| 少妇裸体淫交视频免费看高清| 国产成人精品久久二区二区91| 91麻豆av在线| 久久久久国产精品人妻aⅴ院| 男人和女人高潮做爰伦理| 久久久久久国产a免费观看| 国产成人精品久久二区二区91| 日韩有码中文字幕| 久久久精品大字幕| 黄色日韩在线| 亚洲片人在线观看| 91在线精品国自产拍蜜月 | 一本一本综合久久| 麻豆一二三区av精品| 国产精品久久久久久久电影 | 午夜两性在线视频| www日本黄色视频网| 91在线精品国自产拍蜜月 | 久久性视频一级片| 啦啦啦观看免费观看视频高清| 两个人的视频大全免费| 波多野结衣高清作品| 免费观看的影片在线观看| 啦啦啦观看免费观看视频高清| 亚洲精品在线美女| 亚洲av成人av| 午夜免费激情av| 淫妇啪啪啪对白视频| 女同久久另类99精品国产91| 黄片小视频在线播放| 性色avwww在线观看| 日本一本二区三区精品| 国产亚洲精品久久久久久毛片| av天堂在线播放| 国产精品av久久久久免费| 99久国产av精品| 一个人看的www免费观看视频| 99re在线观看精品视频| 嫩草影院入口| 国产高清videossex| 久久精品91蜜桃| 国产高清视频在线观看网站| 青草久久国产| 国产精品电影一区二区三区| 一进一出抽搐gif免费好疼| 国产黄a三级三级三级人| 热99re8久久精品国产| 日韩国内少妇激情av| 啦啦啦免费观看视频1| 色精品久久人妻99蜜桃| 国产综合懂色| 日本免费一区二区三区高清不卡| АⅤ资源中文在线天堂| 黄片大片在线免费观看| svipshipincom国产片| 国产乱人伦免费视频| 欧美午夜高清在线| 国产精品久久视频播放| 级片在线观看| 一区二区三区高清视频在线| 琪琪午夜伦伦电影理论片6080| 三级毛片av免费| 欧美日韩瑟瑟在线播放| 免费电影在线观看免费观看| 一本久久中文字幕| 99国产精品99久久久久| 欧美又色又爽又黄视频| 成在线人永久免费视频| 色视频www国产| 曰老女人黄片| 宅男免费午夜| 久9热在线精品视频| 日日摸夜夜添夜夜添小说| 神马国产精品三级电影在线观看| 国内毛片毛片毛片毛片毛片| 99久久综合精品五月天人人| 亚洲国产精品合色在线| 九九久久精品国产亚洲av麻豆 | 夜夜夜夜夜久久久久| 搞女人的毛片| 热99在线观看视频| 美女cb高潮喷水在线观看 | 国产91精品成人一区二区三区| 真人一进一出gif抽搐免费| netflix在线观看网站| 亚洲自拍偷在线| 性色avwww在线观看| av黄色大香蕉| 三级国产精品欧美在线观看 | 日日夜夜操网爽| 国产免费av片在线观看野外av| 在线观看免费午夜福利视频| 人妻丰满熟妇av一区二区三区| 久久久久久人人人人人| 亚洲性夜色夜夜综合| 免费看日本二区| 亚洲成人免费电影在线观看| 19禁男女啪啪无遮挡网站| 99热6这里只有精品| 亚洲专区字幕在线| 国产免费av片在线观看野外av| 可以在线观看的亚洲视频| 少妇熟女aⅴ在线视频| 一进一出好大好爽视频| 中国美女看黄片| 色综合婷婷激情| 久久精品人妻少妇| 中文字幕人妻丝袜一区二区| 又紧又爽又黄一区二区| 三级男女做爰猛烈吃奶摸视频| 精品久久久久久久毛片微露脸| 一卡2卡三卡四卡精品乱码亚洲| av天堂中文字幕网| 床上黄色一级片| 老司机午夜福利在线观看视频| 国语自产精品视频在线第100页| 五月玫瑰六月丁香| 成人鲁丝片一二三区免费| 亚洲 欧美 日韩 在线 免费| 露出奶头的视频| 丰满人妻熟妇乱又伦精品不卡| 久久久久久人人人人人| 亚洲精品中文字幕一二三四区| 欧美国产日韩亚洲一区| 日本免费一区二区三区高清不卡| 欧美成人一区二区免费高清观看 | av欧美777| bbb黄色大片| 网址你懂的国产日韩在线| 全区人妻精品视频| av国产免费在线观看| 欧美成狂野欧美在线观看| 亚洲国产日韩欧美精品在线观看 | 久久国产乱子伦精品免费另类| 又大又爽又粗| 国产成人精品久久二区二区免费| 国产精品久久久av美女十八| 欧美乱妇无乱码| 欧美绝顶高潮抽搐喷水| 国产欧美日韩精品亚洲av| 色视频www国产| 国产亚洲精品久久久久久毛片| 免费大片18禁| 免费观看精品视频网站| 18禁黄网站禁片免费观看直播| 香蕉国产在线看| 成年版毛片免费区| 国产精品九九99| 丰满人妻熟妇乱又伦精品不卡| 精品人妻1区二区| 成人无遮挡网站| 搞女人的毛片| 丰满人妻一区二区三区视频av | 国产精品九九99| 午夜成年电影在线免费观看| 亚洲 国产 在线| 成人无遮挡网站| 国内精品一区二区在线观看| 老汉色av国产亚洲站长工具| 美女扒开内裤让男人捅视频| 亚洲专区中文字幕在线| 日韩高清综合在线| 亚洲欧美日韩东京热| 久久久久久久午夜电影| 午夜成年电影在线免费观看| www.自偷自拍.com| 国模一区二区三区四区视频 | 天堂动漫精品| 特大巨黑吊av在线直播| 两个人的视频大全免费| 母亲3免费完整高清在线观看| 99国产精品99久久久久| 88av欧美| 欧美日韩亚洲国产一区二区在线观看| 国产三级中文精品| 99在线视频只有这里精品首页| 黑人操中国人逼视频| 岛国视频午夜一区免费看| 久久草成人影院| 亚洲成人久久性| 91在线精品国自产拍蜜月 | 国产综合懂色| 亚洲成人精品中文字幕电影| 欧美乱妇无乱码| 亚洲国产高清在线一区二区三| 日日干狠狠操夜夜爽| 亚洲午夜理论影院| 亚洲熟妇中文字幕五十中出| 亚洲精品乱码久久久v下载方式 | 老汉色∧v一级毛片| a在线观看视频网站| 两性午夜刺激爽爽歪歪视频在线观看| 国产一区二区在线av高清观看| 午夜成年电影在线免费观看| 18禁黄网站禁片免费观看直播| 熟女人妻精品中文字幕| 国内精品一区二区在线观看| 国内毛片毛片毛片毛片毛片| 国产探花在线观看一区二区| 国产av在哪里看| 18美女黄网站色大片免费观看| 少妇的逼水好多| 99热只有精品国产| 欧美日韩亚洲国产一区二区在线观看| 大型黄色视频在线免费观看| 久久性视频一级片| 国产精品99久久99久久久不卡| 极品教师在线免费播放| 国产成人av激情在线播放| 草草在线视频免费看| 亚洲欧美精品综合久久99| 97碰自拍视频| 好男人在线观看高清免费视频| 白带黄色成豆腐渣| 一夜夜www| 搡老熟女国产l中国老女人| 久久精品综合一区二区三区| xxx96com| 久久亚洲精品不卡| www.精华液| 国产精品av视频在线免费观看| 欧美丝袜亚洲另类 | 变态另类成人亚洲欧美熟女| 搡老妇女老女人老熟妇| 国产午夜精品论理片| 久久久久久大精品| 少妇的逼水好多| 久久香蕉国产精品| 波多野结衣巨乳人妻| 99精品欧美一区二区三区四区| 中文字幕最新亚洲高清| 亚洲片人在线观看| 色综合欧美亚洲国产小说| 亚洲真实伦在线观看| 国产麻豆成人av免费视频| 伊人久久大香线蕉亚洲五| 99久久国产精品久久久| 97碰自拍视频| 成人av在线播放网站| 精品电影一区二区在线| 国产精华一区二区三区| 在线a可以看的网站| 成人无遮挡网站| 精品不卡国产一区二区三区| 欧美激情在线99| 国产伦一二天堂av在线观看| 综合色av麻豆| 欧美最黄视频在线播放免费| 久久久久久久久久黄片| 国产在线精品亚洲第一网站| 亚洲av美国av| 精品久久久久久久久久免费视频| 亚洲激情在线av| 99热这里只有是精品50| 精品乱码久久久久久99久播| 欧洲精品卡2卡3卡4卡5卡区| 99re在线观看精品视频| 美女扒开内裤让男人捅视频| av欧美777| 亚洲国产精品久久男人天堂| 国产主播在线观看一区二区| 亚洲精品乱码久久久v下载方式 | 天天躁狠狠躁夜夜躁狠狠躁| 亚洲欧美激情综合另类| 999久久久国产精品视频| 精品一区二区三区四区五区乱码| 午夜免费成人在线视频| 国产视频内射| 亚洲成人精品中文字幕电影| 欧美丝袜亚洲另类 | avwww免费| 国产久久久一区二区三区| 香蕉av资源在线| 天堂影院成人在线观看| 黄片小视频在线播放| 两个人看的免费小视频| 美女高潮喷水抽搐中文字幕| 国产1区2区3区精品| 国产亚洲欧美在线一区二区| 亚洲中文日韩欧美视频| 最近视频中文字幕2019在线8| 亚洲无线观看免费| 黄频高清免费视频| 色老头精品视频在线观看| 国产成人精品无人区| 欧美日韩精品网址| 中国美女看黄片| 我要搜黄色片|