• <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 ?

    Synthesis of DNP-modified GM3-based anticancer vaccine and evaluation of its immunological activities for cancer immunotherapy

    2021-03-14 02:32:04HanLinHaofeiHongLipengFengJieShiZhifangZhouZhimengWu
    Chinese Chemical Letters 2021年12期

    Han Lin,Haofei Hong,Lipeng Feng,Jie Shi,Zhifang Zhou,Zhimeng Wu

    Key Laboratory of Carbohydrate Chemistry &Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China

    Keywords:GM3 Cancer vaccine Anti-DNP antibodies Glycoengineering Tumor-associated carbohydrate antigen

    ABSTRACT Tumor-associated carbohydrate antigens (TACAs) are attractive targets for vaccine development.In this context,we described a strategy combining artificial TACA and glycoengineering for cancer vaccine development.A 2,4-ditrophenyl (DNP)-modified GM3 intermediate was synthesized chemoenzymatically and conjugated to keyhole limpet hemocyanin (KLH),and the resulting bioconjugate was tested for its potential as a vaccine candidate.Mice immunological studies revealed that the DNP-modified GM3 (GM3-NHDNP) analog elicited strong and rapid immune responses by recruiting anti-DNP antibodies to facilitate the targeted delivery of the vaccine construct to antigen processing cells (APCs).Moreover,the endogenously produced anti-DNP antibodies,together with the elicited antibodies against GM3-NHDNP,may synergistically promote tumor binding and cancer cell death when the cancer cell surfaces are glycoengineered to express the GM3-NHDNP antigen.

    Tumor-associated carbohydrate antigens (TACAs) that are abundantly expressed on the surface of cancer cells are attractive biological targets for the development of cancer vaccines or cancer immunotherapies [1,2].However,carbohydrate antigens are typically T cell-independent and poorly immunogenic,which impedes the success of TACA-based vaccines [3].In addition,as self-antigens,most TACAs are tolerated by the immune system,which further creates difficulties in vaccine design and development [4].Over the past few decades,many novel vaccine strategies have been developed to overcome these problems [5,6].Among them,the non-natural TACA strategy that aims to promote the immunogenicity and mitigate the immune-tolerance of vaccines shows great potential.For example,artificial TACA-based vaccines,including Tn [7],sTn [8,9],GM3 [10],TF [11],Globo H[12]and MUC1 analogs [13],successfully provoked robust immune responses against these carbohydrate antigens.However,one potential issue of these strategies is that the antibodies generated by non-natural TACAs had limited cross-reactivity with the natural glycans expressed on the surface of cancer cells.

    To address this dilemma,a novel immunotherapy strategy that combined the non-natural TACA strategy and the glycoengineering of cancer cells was developed [9].Previous work demonstrated that an unnatural NeuNPhAc-containing TACA-based vaccine elicited a strong immune response but could not recognize the natural TACAs present on the surface of cancer cells.However,after the cancer cells were glycoengineered with the corresponding precursorN-phenylacetyl-D-mannosamine to express NeuNPhAcmodified TACAs on their surfaces,the pre-elicited antibodies were able to recognize and kill the engineered cancer cells effectively[14].Nevertheless,a primary requisite for the success of the abovementioned immunotherapies is to provide an effective immunization that could generate a strong and antigen-specific humoral and cellular immunity by a non-natural TACAs-based cancer vaccine.

    Endogenous antibodies are naturally occurring antibodies that exist in human sera.Redirecting endogenous antibodies,such as anti-galactose-α-(1,3)-galactose (α-Gal),anti-2,4-ditrophenyl(DNP),and anti-rhamnose (Rha) antibodies,to target cancer cells can activate the immune system to selectively kill the cancer cells[15,16].Recent studies demonstrated that the metabolic incorporation of DNP or Rha into the cell-surface glycocalyx redirected and enhanced the immune response for targeting cancer cells [17–22].Moreover,these studies validated that endogenous antibodies were able to mediate the targeted delivery of TACAs-based vaccines to antigen-processing cells (APCs),resulting in an antibodydependent enhancement (ADE) of immune responses [23–26].In our previous research,we demonstrated that the incorporation of the Rha antigen into bovine serum albumin (BSA) as a carrier protein led to a significant improvement in the immunogenicity of sTn in the presence of anti-Rha antibodies [25].These results illustrated the potential collaborative role of endogenous antibodies in vaccine development.

    Fig.1.Design and structure of the GM3-NHDNP glycoconjugate vaccine construct.

    Ganglioside GM3 is a common TACA that is highly expressed by several types of malignancies,such as lung,brain and breast cancers,as well as melanomas [27].The expression of GM3 influences the development and proliferation of cancer cells and is positively correlated with tumor malignancy.GM3 is one of the 50 most investigated cancer antigens for the development of cancer immunotherapies and has been widely targeted in vaccine development [28,29].Based on previous work related to non-natural TACA-based cancer vaccines and cell-surface glycoengineering,we designed and synthesized a new 5?-N-DNP-modified GM3 antigen(GM3-NHDNP) for use in cancer immunotherapy (Fig.1).The incorporation of the DNP hapten into the structure of GM3 had multiple purposes.First,the artificial GM3-NHDNP antigen could elicit strong immune responses and overcome immune tolerance because of its unnatural structure.Second,the targeted delivery of the GM3-NHDNP antigen to APCs could be achieved in an ADE manner to further enhance the antigen-presenting process in the presence of endogenous anti-DNP antibodies.Finally,the surface of cancer cells could be glycoengineered to express the GM3-NHDNP antigen using established metabolic approaches.Thus,it was expected that the endogenous anti-DNP antibodies,together with the elicited endogenous anti-GM3-NHDNP antibodies,would synergistically promote tumor binding and phagocytosis after effective vaccinations.In this context,we communicate the chemoenzymatic synthesis of 5?-N-DNP-modified GM3 and the evaluation of the immune activity of the GM3-NHDNP glycoconjugate in the presence of anti-DNP antibodies as a potential carbohydrate-based cancer vaccine.

    The synthesis of the DNP-modified GM3 analog required a key intermediate:5?-N-DNP-substituted sialic acid containing an aminocaproic acid linker (8).The intermediate was prepared by a chemoenzymatic method,as shown in Scheme 1.First,aminocaproic acid was reacted with 1-fluoro-2,4-dinitrobenzene in the presence of sodium bicarbonate to afford intermediate 6 in 83%yield,after which 6 was coupled to D-mannosamine using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) as the condensation reagent in the presence of triethylamine,affording the DNPmodified mannosamine derivative (Man-NHDNP) 7,albeit in only 30% yield.Bacterial sialic acid aldolase typically has a broad substrate specificity [30],so it was able to recognize Man-NHDNP (7)as a substrate in our experiment.Therefore,Man-NHDNP (7) was enzymatically transformed into 5?-N-DNP-substituted sialic acid 8 in the presence of sodium pyruvate in excellent yield (91%).

    Scheme 1.a) 1-Fluoro-2,4-dinitrobenzene,acetone,2 mol/L Na2CO3,r.t.,1 h,83%;b) D-Mannosamine,EDCI,Et3N,MeOH,r.t.,18 h,30%;c) Sialic acid aldolase,MgCl2(20 mmol/L),Tris-HCl buffer (100 mmol/L,pH 7.5),sodium pyruvate,DTT,37 °C,24 h,91%.

    The synthesis of the DNP-modified GM3 is provided in Scheme 2.An efficient chemoenzymatic “1+2” approach was applied to assemble the trisaccharide because the glycosyltransferases required for this biochemical transformation could be easily obtained either by recombinant expression inEscherichia coli(E.coli) or commercially.Accordingly,the lactose acceptor 10 was prepared with an azidoethyl group as a linker at the reducing end following a reported procedure [31].The GM3-NHDNP analog 11a was assembled by two-step,one-pot enzymatic transformation.First,the sialic acid derivatives 8 were converted to the CMP-Neu5R derivatives 9a byNeisseria meningitidisCMP-sialic acid synthetase (NmCSS) according to a previously published procedure [32].Then,the lactose acceptor 10 and the recombinant enzymePasteurella multocida α-(2→3)-sialyltransferase (PmST1) were added into the reaction mixture,which was incubated for 12 h at 37 °C.After quenching the reaction and a brief purification using a Bio-P2 column,the azido group on the reducing end of the 5?-NDNP-substituted GM3 analog 11a was reduced to the corresponding aminoethyl group,affording GM3-NHDNP 12a in 77% yield.Following a similar procedure,the natural GM3-NHAc 12b was prepared,as well.All of the intermediates and final compounds were characterized by1H and13C NMR and HRMS.

    Scheme 2.a) MgCl2 (20 mmol/L),Tris-HCl buffer (100 mmol/L,pH 8.8),CTP,NmCSS,37 °C,5 h;b) Compound 10,PmST1,37 °C,12 h,77%;c) Ph3P,H2O:THF (1:1),60 °C,overnight;d) disuccinimidyl glutarate,DMF:PBS (4:1),5 h,r.t.;e) KLH/HSA,PBS,r.t.,3 days.

    The GM3 analogs were conjugated to Keyhole limpet hemocyanin (KLH) or human serum albumin (HSA)viathe bifunctional glutaryl ester method,which is an established conjugation method that was not expected to affect the immunological properties of the conjugates [33].Briefly,the GM3 analogs 12a or 12b were reacted with a large excess (15.0 equiv.) of disuccinimidyl glutarate(DSG) in a solution of DMF and PBS buffer (4:1) to afford the corresponding monoesters 13a or 13b,respectively.The monoesters were then mixed with KLH or HSA in phosphate buffer saline (PBS)to generate conjugates 1–4,which were purified by centrifugal filter devices (10 kDa for HSA conjugates and 30 kDa for KLH conjugates).The antigen loading percentages of the resulting glycoconjugates 1–4 were determined by the Svennerholm method (described in the Supporting information),which were calculated to be 3%,6%,6.1% and 4.9%,respectively.

    To evaluate the immunological activities of our glycoconjugate vaccine candidates,we created three groups of mice.Group 1 (control group),which consisted of five mice,were immunized with natural GM3-NHAc-KLH (3).The mice in the second group were immunized with GM3-NHDNP-KLH (1) only,while the mice in the third group were first pre-immunized with DNP-ovalbumin (OVA)to establish a high level of anti-DNP antibodies (Fig.S2 in Supporting information) and then immunized with conjugate 1.All of these conjugates were premixed with Freund’s complete adjuvant to activate the vaccine.Mouse blood samples were collected on days 0,21 and 28,and the specific antibodies were determined by enzyme-linked immunosorbent assays (ELISA).The ELISA results from the antisera of the three groups on Day 28 are summarized in Fig.2.As expected,the mice in group 1 produced the lowest number of GM3-specific antibodies (approx.9784,Fig.2A),while the mice in group 2 generated a significantly higher number of antibodies specifically against GM3-NHDNP (approx.164502,Fig.2B).Therefore,the specific antibody production increased nearly 17-fold compared to the mice in group 1.This result revealed that the artificial DNP-modified GM3 demonstrated a significantly improved immunogenicity compared to GM3,which was in consistent with a previously published report [34].For the mice in group 3 that were pre-immunized with the DNP-OVA conjugate,the highest production of GM3-NHDNP-specific antibodies was observed (approx.424294,Fig.2C),which was approximately 2.6-fold higher than the mice in group 2.This result indicated that the endogenous anti-DNP antibodies further promoted delivery of the vaccine to the APCs,which resulted in better internalization,antigenpresenting efficiency and specific antibody production.

    Fig.2.Immunological evaluation of GM3 analog-KLH vaccine construct:(A) GM3-NHAc-specific antibodies in group 1;(B) GM3-NHDNP-specific antibodies in group 2;(C) GM3-NHDNP-specific antibodies in group 3 pre-immunized with DNP-OVA.(D) The average titers of GM3-NHDNP-specific total antibodies in pooled antisera collected on Days 0,21 and 27 from mice of groups 2 and 3.Error bars represent the standard deviation (SD) of five parallel experiments.?P < 0.05.

    Antibody cross-reaction analysis revealed that antibodies elicited by the GM3-NHDNP conjugate recognized the natural GM3 structure.In addition,three conjugates produced IgG antibodies,but not IgM antibodies,as the major isotype,indicating that T cell immunity was involved.We also compared the kinetic immune response in the mice in groups 2 and 3 (Fig.2D).For the mice in group 2,the total antibodies in the pooled antisera collected on Days 21 and 28 were 146782 and 165056,respectively,compared to 324068 and 403431,respectively,in the mice of group 3.This result suggested that a much faster and stronger immune response was produced in the group 3 mice,and it demonstrated that the DNP-specific antibodies mediated a faster immune response compared to group 2,which can help to reduce injection frequency in the vaccine protocol.

    Fig.3.(A) FACS assays of the binding between B16F10 cancer cells treated with precursor and mouse sera.(B) CDC activities of antisera from each group were measured using the CCK-8 assay.Error bars represent the SD of three parallel experiments.?P < 0.05,???P < 0.001.

    After establishing a successful immunization strategy,we then investigated the capability of the elicited GM3-NHDNP-specific antibodies and DNP-specific antibodies to recognize cancer cells that express the GM3-NHDNP antigen,which is crucial for any glycoengineering-based vaccine immunotherapy.For this purpose,we chose B16F10,a mouse melanoma cell line that abundantly expresses GM3 [14],to carry out thein vitrocell surface glycoengineering.First,the B16F10 cells were treated with the precursor (Man4Ac-DNP,compound S6 in Supporting information) formulated in liposomes.Then,they were detected for GM3-NHDNP expression by flow cytometry using either the Day 0 sera from group 3 (containing DNP-specific antibodies after DNP-OVA immunization) or the Day 28 antisera of groups 2 and 3.The serum collected from the mice without any immunization was used as the blank,and the FITC-conjugated goat anti-mouse antibody was used as secondary antibody.As shown in Fig.3A,the cells treated with precursor had a noticeably higher fluorescein isothiocyanate (FITC)intensity than the untreated cells,which indicated that the cancer cells metabolized Man4Ac-DNP and successfully expressed artificial GM3-NHDNP on the cell surface after a series of carbohydrate biosynthetic enzymes and the antibodies elicited by GM3-NHDNPKLH vaccine could recognize them efficiently.Moreover,the anti-DNP antibodies elicited by the DNP-OVA conjugate recognized the precursor-treated cells.Therefore,both anti-DNP and anti-GM3-NHDNP antibodies recognized and bound to the glycoengineered cancer cells,suggesting that they have potential for mediating cancer cell phagocytosis by immune cells.

    We further analyzed the amount of interleukin-6 (IL-6)-representing Th2 cells and interferon gamma (IFN-γ)-representing Th1 cells present in the antisera of the immunized mice by ELISA.As shown in Fig.S4 (Supporting information),IFN-γand IL-6 were both provoked in groups 1,2 and 3,which indicated that T cell immune responses were involved.

    Finally,the potential antitumor activities of the antibodies elicited by the GM3-NHDNP vaccine were evaluated by antibodymediated,complement-dependent cytotoxicity (CDC) analysisin vitro.For the CDC measurements,mouse melanoma B16F10 cells were glycoengineered to express the GM3-NHDNP carbohydrate on the cell surface,after which they were incubated with the Day 28 sera from groups 2 and 3 or Day 0 sera from group 3 (containing anti-DNP antibodies) and diluted rabbit complement at 37 °C for 4 h,respectively.Cell lysis was conducted using a commercial cell counting kit-8 (CCK-8).As presented in Fig.3B,the antibodies generated by DNP-OVA or GM3-NHDNP-KLH in groups 2 and 3 were able to trigger a significant cytotoxicity in the cancer cells.Comparable cytotoxicity to group 2 was only observed in the sera containing anti-DNP antibodies.Notably,the antibodies from the mice in group 3 exhibited the highest cytotoxicities compared to both DNP-OVA and group 2.The most potent cytotoxicity was attributed to the fact that group 3 had the highest antibody production,and the enhanced immune-triggered phagocytosis might have been synergistically mediated by anti-DNP antibodies and anti-GM3-NHDNP antibodies,indicating that both antibodies played important roles in this CDC.

    In summary,we described herein the synthesis of a new DNPmodified GM3 antigen for the development of a carbohydratebased cancer vaccine.In vivoimmunological studies demonstrated that the GM3-NHDNP conjugate provoked robust and rapid immune responses in the presence of endogenous anti-DNP antibodies,which mediated a targeted delivery of the vaccine construct to the APCs.We validated that this strategy has the potential to utilize the metabolic glycoengineering of cancer cells for manifesting immunotherapy.In vitroCDC studies revealed that an enhanced cancer cell death was triggered by both pre-immunized anti-DNPand anti-GM3-NHDNP-specific antibodies.In future work,we aim to further optimize the structure of the GM3-NHDNP conjugate,including the linker and the position of DNP modification,to improve the glycoengineering efficiency of the surface of the cancer cells.Considering the abundance of anti-DNP antibodies in human sera and the success of non-natural TACAs-based cancer vaccine,this strategy could potentially be applied to the development of other TACAs-based vaccines.

    Declaration of competing interest

    The authors declare no competing financial interest.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (Nos.21907038,32000904),the Natural Science Foundation of Jiangsu Province (No.BK20200601,China),the National Postdoctoral Program for Innovative Talents of China(No.BX20200153),the Health and Family Planning Commission of Wuxi,China (No.Z202005),and the Social Development Key Project of Jiangsu Province (No.BE2019632,China).This research was partly supported by the 111 Project (No.111-2-06,China)and the National First-class Discipline Program of Food Science and Technology (No.JUFSTR20180101,China).We appreciate Prof.Hongzhi Cao from Shandong University for providing us the enzymesNeisseria meningitidisCMP-sialic acid synthetase (NmCSS)andPasteurella multocida α-(2→3)-sialyltransferase (PmST1)[32].

    Supplementary materials

    Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2021.04.034.

    国产蜜桃级精品一区二区三区| 午夜日韩欧美国产| 女生性感内裤真人,穿戴方法视频| 国产精品av久久久久免费| 精品福利观看| 久99久视频精品免费| 又紧又爽又黄一区二区| 91九色精品人成在线观看| 亚洲国产欧美一区二区综合| 欧美+亚洲+日韩+国产| 久久 成人 亚洲| 亚洲欧美激情综合另类| 国产色视频综合| 男女床上黄色一级片免费看| 嫩草影视91久久| 亚洲午夜精品一区,二区,三区| 国产日韩一区二区三区精品不卡| 久久精品亚洲av国产电影网| 99国产精品免费福利视频| 亚洲熟女毛片儿| 亚洲欧美一区二区三区黑人| 男女午夜视频在线观看| 老熟妇仑乱视频hdxx| 女性被躁到高潮视频| 精品久久久久久久久久免费视频 | 精品国产一区二区久久| 91麻豆精品激情在线观看国产 | 日本黄色视频三级网站网址| 成人亚洲精品av一区二区 | 最新美女视频免费是黄的| 黄色片一级片一级黄色片| 亚洲熟妇熟女久久| 99re在线观看精品视频| ponron亚洲| 国产乱人伦免费视频| 欧美人与性动交α欧美软件| 男男h啪啪无遮挡| 国产成人免费无遮挡视频| 成人精品一区二区免费| 看黄色毛片网站| 人人妻人人澡人人看| a在线观看视频网站| 日本欧美视频一区| 国产男靠女视频免费网站| 国产视频一区二区在线看| aaaaa片日本免费| 老熟妇乱子伦视频在线观看| xxxhd国产人妻xxx| 最新在线观看一区二区三区| 99国产综合亚洲精品| 少妇被粗大的猛进出69影院| 少妇粗大呻吟视频| 久久精品亚洲精品国产色婷小说| 夜夜夜夜夜久久久久| 琪琪午夜伦伦电影理论片6080| www日本在线高清视频| 在线国产一区二区在线| x7x7x7水蜜桃| 久久中文字幕人妻熟女| 国产成人精品在线电影| 国产精品久久久av美女十八| 99久久久亚洲精品蜜臀av| 免费久久久久久久精品成人欧美视频| 亚洲久久久国产精品| 久久人人精品亚洲av| 国产无遮挡羞羞视频在线观看| 又黄又粗又硬又大视频| 久久人妻av系列| 国产区一区二久久| 成人免费观看视频高清| 少妇被粗大的猛进出69影院| avwww免费| 日韩三级视频一区二区三区| 一个人免费在线观看的高清视频| 欧美精品一区二区免费开放| 多毛熟女@视频| 美女福利国产在线| 久久婷婷成人综合色麻豆| 一级a爱片免费观看的视频| 国产精品二区激情视频| 国产成人精品久久二区二区91| 国产欧美日韩综合在线一区二区| 男人的好看免费观看在线视频 | 免费高清在线观看日韩| 波多野结衣高清无吗| 涩涩av久久男人的天堂| 亚洲视频免费观看视频| 亚洲成av片中文字幕在线观看| 国产午夜精品久久久久久| 国产免费现黄频在线看| videosex国产| 一级片免费观看大全| av福利片在线| 国产熟女午夜一区二区三区| 岛国在线观看网站| 免费在线观看日本一区| 久久久久精品国产欧美久久久| 久久性视频一级片| 母亲3免费完整高清在线观看| 18禁美女被吸乳视频| 俄罗斯特黄特色一大片| 欧美黄色片欧美黄色片| 免费看a级黄色片| 日韩免费av在线播放| 亚洲中文字幕日韩| 亚洲欧美日韩另类电影网站| 国产精品野战在线观看 | 色老头精品视频在线观看| 亚洲午夜理论影院| 一级黄色大片毛片| 国产亚洲精品第一综合不卡| 伊人久久大香线蕉亚洲五| 无遮挡黄片免费观看| 精品国产美女av久久久久小说| 黑丝袜美女国产一区| 制服诱惑二区| 夫妻午夜视频| 丰满迷人的少妇在线观看| 91精品国产国语对白视频| 可以在线观看毛片的网站| 看黄色毛片网站| 亚洲一区二区三区色噜噜 | 99re在线观看精品视频| 久久人妻熟女aⅴ| 69精品国产乱码久久久| 欧美久久黑人一区二区| 黑人巨大精品欧美一区二区mp4| 女人高潮潮喷娇喘18禁视频| 美女高潮到喷水免费观看| 日韩人妻精品一区2区三区| 亚洲五月天丁香| 精品久久久久久电影网| 我的亚洲天堂| 成人永久免费在线观看视频| 在线观看66精品国产| 国产亚洲精品第一综合不卡| 亚洲av电影在线进入| 亚洲色图 男人天堂 中文字幕| 自拍欧美九色日韩亚洲蝌蚪91| 91大片在线观看| 亚洲中文日韩欧美视频| 成人av一区二区三区在线看| 午夜免费观看网址| 国产伦人伦偷精品视频| 国产麻豆69| 高潮久久久久久久久久久不卡| 日日爽夜夜爽网站| 亚洲一区二区三区色噜噜 | 欧美黑人欧美精品刺激| 一级,二级,三级黄色视频| 国产精品久久久久久人妻精品电影| 香蕉国产在线看| 久久久国产成人免费| 十分钟在线观看高清视频www| 欧美最黄视频在线播放免费 | 91精品国产国语对白视频| 亚洲欧美激情在线| 国产一卡二卡三卡精品| 一个人观看的视频www高清免费观看 | 国产精品偷伦视频观看了| 麻豆国产av国片精品| 国产av一区在线观看免费| 老司机亚洲免费影院| 亚洲伊人色综图| 国产一区二区三区综合在线观看| 在线观看午夜福利视频| 精品国产乱子伦一区二区三区| 变态另类成人亚洲欧美熟女 | 嫩草影视91久久| 国产一卡二卡三卡精品| 免费女性裸体啪啪无遮挡网站| a在线观看视频网站| 夜夜夜夜夜久久久久| 亚洲欧美一区二区三区久久| 天天影视国产精品| 两性午夜刺激爽爽歪歪视频在线观看 | 精品久久蜜臀av无| 精品国产国语对白av| 国产高清视频在线播放一区| 精品乱码久久久久久99久播| 91精品三级在线观看| 男女下面插进去视频免费观看| 丝袜人妻中文字幕| 免费少妇av软件| 欧美不卡视频在线免费观看 | 90打野战视频偷拍视频| 日本vs欧美在线观看视频| 亚洲午夜理论影院| 免费在线观看日本一区| 亚洲精品国产色婷婷电影| 两性夫妻黄色片| 99热国产这里只有精品6| 日日爽夜夜爽网站| 欧美乱色亚洲激情| 亚洲欧美日韩无卡精品| av有码第一页| 国产午夜精品久久久久久| 成人手机av| 欧美日韩亚洲国产一区二区在线观看| 在线视频色国产色| 久久久国产精品麻豆| 99riav亚洲国产免费| 精品久久久久久电影网| 国产精品野战在线观看 | 国产精品一区二区在线不卡| 亚洲人成电影免费在线| 午夜老司机福利片| 欧美乱码精品一区二区三区| 神马国产精品三级电影在线观看 | 免费在线观看影片大全网站| 久久亚洲精品不卡| 亚洲一区二区三区欧美精品| 国产精品一区二区免费欧美| 在线观看免费视频日本深夜| 桃红色精品国产亚洲av| 91九色精品人成在线观看| 国产主播在线观看一区二区| 又大又爽又粗| 欧洲精品卡2卡3卡4卡5卡区| 12—13女人毛片做爰片一| 国产成人系列免费观看| 嫩草影院精品99| 视频区图区小说| 热99国产精品久久久久久7| 日韩免费av在线播放| 日本黄色视频三级网站网址| 国产一区二区激情短视频| 波多野结衣一区麻豆| 欧美另类亚洲清纯唯美| 国产精品野战在线观看 | 国产精品自产拍在线观看55亚洲| 日本一区二区免费在线视频| 水蜜桃什么品种好| 亚洲久久久国产精品| 69精品国产乱码久久久| av福利片在线| 真人一进一出gif抽搐免费| 国产精品自产拍在线观看55亚洲| 大型av网站在线播放| 1024视频免费在线观看| 国产亚洲欧美在线一区二区| 亚洲狠狠婷婷综合久久图片| 欧美黑人欧美精品刺激| 久久中文字幕一级| 天天添夜夜摸| 19禁男女啪啪无遮挡网站| 天天添夜夜摸| 久久精品91无色码中文字幕| 久久精品成人免费网站| 亚洲熟妇中文字幕五十中出 | 免费观看人在逋| 亚洲国产欧美一区二区综合| 99精品久久久久人妻精品| 成熟少妇高潮喷水视频| 久久伊人香网站| 国产激情欧美一区二区| 亚洲色图综合在线观看| 日韩中文字幕欧美一区二区| 国产男靠女视频免费网站| 88av欧美| 精品福利永久在线观看| 亚洲国产欧美一区二区综合| 国产成年人精品一区二区 | 日本撒尿小便嘘嘘汇集6| 午夜成年电影在线免费观看| 国产成人av激情在线播放| 欧美精品啪啪一区二区三区| 欧美+亚洲+日韩+国产| 精品久久久久久,| 一区福利在线观看| 91成人精品电影| 午夜免费观看网址| 欧美黑人精品巨大| 欧美黄色片欧美黄色片| 久久久国产精品麻豆| 两性夫妻黄色片| 法律面前人人平等表现在哪些方面| 欧美日韩国产mv在线观看视频| 777久久人妻少妇嫩草av网站| 涩涩av久久男人的天堂| 亚洲精品美女久久久久99蜜臀| 久久中文字幕人妻熟女| 免费在线观看黄色视频的| 色尼玛亚洲综合影院| 亚洲自偷自拍图片 自拍| 午夜免费成人在线视频| 亚洲精品一二三| 中文字幕人妻丝袜制服| 性色av乱码一区二区三区2| a在线观看视频网站| 女人精品久久久久毛片| xxxhd国产人妻xxx| 老司机深夜福利视频在线观看| 亚洲 欧美 日韩 在线 免费| 亚洲精品一二三| 国产一区二区三区在线臀色熟女 | 久99久视频精品免费| 精品第一国产精品| 好看av亚洲va欧美ⅴa在| 亚洲色图av天堂| 午夜两性在线视频| 国产av一区二区精品久久| 国产三级黄色录像| av超薄肉色丝袜交足视频| 日韩人妻精品一区2区三区| 日韩大码丰满熟妇| 欧美丝袜亚洲另类 | 男人的好看免费观看在线视频 | 在线观看免费视频网站a站| 午夜免费观看网址| 国产蜜桃级精品一区二区三区| 亚洲精品美女久久av网站| 免费在线观看黄色视频的| 精品第一国产精品| 国产精品99久久99久久久不卡| 在线观看免费视频网站a站| 久久人妻福利社区极品人妻图片| 大型av网站在线播放| a级毛片在线看网站| 99久久国产精品久久久| 操美女的视频在线观看| 欧美一级毛片孕妇| 久久久久精品国产欧美久久久| 99久久综合精品五月天人人| 国产成人啪精品午夜网站| 中文字幕最新亚洲高清| 在线十欧美十亚洲十日本专区| 色老头精品视频在线观看| 欧美亚洲日本最大视频资源| 亚洲自拍偷在线| 亚洲中文av在线| 日韩高清综合在线| 国产亚洲欧美精品永久| 亚洲 欧美一区二区三区| 亚洲自偷自拍图片 自拍| 免费日韩欧美在线观看| 亚洲专区国产一区二区| 女人高潮潮喷娇喘18禁视频| 婷婷六月久久综合丁香| 日日干狠狠操夜夜爽| 欧美色视频一区免费| 老司机午夜福利在线观看视频| 国产精品一区二区免费欧美| 老司机亚洲免费影院| 国产亚洲av高清不卡| 久久精品国产综合久久久| 两性午夜刺激爽爽歪歪视频在线观看 | 亚洲免费av在线视频| 极品人妻少妇av视频| 久久久久精品国产欧美久久久| 妹子高潮喷水视频| 18禁国产床啪视频网站| 日韩有码中文字幕| 高潮久久久久久久久久久不卡| 成人特级黄色片久久久久久久| 久久久久久久午夜电影 | 亚洲色图综合在线观看| www日本在线高清视频| 国产日韩一区二区三区精品不卡| 成人黄色视频免费在线看| 制服人妻中文乱码| 两性午夜刺激爽爽歪歪视频在线观看 | 久久久国产欧美日韩av| 在线免费观看的www视频| 亚洲狠狠婷婷综合久久图片| 三级毛片av免费| 亚洲精品久久午夜乱码| 天堂俺去俺来也www色官网| 亚洲精品在线观看二区| 亚洲av电影在线进入| 99国产精品一区二区蜜桃av| 一进一出抽搐动态| 中文字幕精品免费在线观看视频| 国产成人系列免费观看| 大陆偷拍与自拍| 欧美中文日本在线观看视频| 欧美国产精品va在线观看不卡| www.自偷自拍.com| 黄色视频不卡| 搡老乐熟女国产| 国产99白浆流出| 成人免费观看视频高清| 欧美中文综合在线视频| 亚洲一区中文字幕在线| 啦啦啦免费观看视频1| 亚洲五月天丁香| 亚洲avbb在线观看| 免费在线观看影片大全网站| 免费高清在线观看日韩| 精品福利永久在线观看| 校园春色视频在线观看| 91老司机精品| 国产视频一区二区在线看| 大陆偷拍与自拍| 在线观看免费日韩欧美大片| 一级毛片高清免费大全| 国产视频一区二区在线看| a级片在线免费高清观看视频| 操出白浆在线播放| 色播在线永久视频| 亚洲第一av免费看| 成年人免费黄色播放视频| xxxhd国产人妻xxx| av免费在线观看网站| 男女床上黄色一级片免费看| 首页视频小说图片口味搜索| 一本大道久久a久久精品| 老熟妇仑乱视频hdxx| 99在线视频只有这里精品首页| 一级毛片高清免费大全| 亚洲精品美女久久av网站| 99在线视频只有这里精品首页| 久久久久久久久中文| 三上悠亚av全集在线观看| 亚洲人成77777在线视频| 日韩一卡2卡3卡4卡2021年| 韩国av一区二区三区四区| 在线观看一区二区三区激情| 天堂中文最新版在线下载| 色尼玛亚洲综合影院| 黑人操中国人逼视频| 在线观看免费视频日本深夜| 亚洲精品一二三| 久久久久久大精品| 女人精品久久久久毛片| 黑人操中国人逼视频| 久久香蕉精品热| 91字幕亚洲| 国产一区在线观看成人免费| 国产成人精品久久二区二区91| 国产精品偷伦视频观看了| 欧美另类亚洲清纯唯美| 老司机靠b影院| 午夜免费成人在线视频| 午夜福利免费观看在线| 午夜成年电影在线免费观看| 在线观看免费视频日本深夜| 久久热在线av| 成年女人毛片免费观看观看9| 9热在线视频观看99| 少妇被粗大的猛进出69影院| 超色免费av| 国产精品野战在线观看 | 久热爱精品视频在线9| 岛国视频午夜一区免费看| 久久草成人影院| 自线自在国产av| 亚洲色图 男人天堂 中文字幕| 欧美中文综合在线视频| 日本欧美视频一区| av片东京热男人的天堂| 国产深夜福利视频在线观看| 男人的好看免费观看在线视频 | 黄色怎么调成土黄色| 女生性感内裤真人,穿戴方法视频| 黄色丝袜av网址大全| 欧美黑人精品巨大| 精品国产乱子伦一区二区三区| 亚洲精品久久成人aⅴ小说| 精品国产一区二区久久| 欧美日韩亚洲高清精品| 香蕉丝袜av| 在线国产一区二区在线| av视频免费观看在线观看| 大陆偷拍与自拍| 成人精品一区二区免费| 亚洲中文av在线| 国产精品一区二区在线不卡| av视频免费观看在线观看| 美女 人体艺术 gogo| 天天影视国产精品| 性少妇av在线| 日韩中文字幕欧美一区二区| 美女高潮到喷水免费观看| 久久久久精品国产欧美久久久| 久久精品国产亚洲av香蕉五月| 精品无人区乱码1区二区| 午夜精品国产一区二区电影| 香蕉国产在线看| 丰满饥渴人妻一区二区三| 久久精品成人免费网站| 精品国产国语对白av| 五月开心婷婷网| av国产精品久久久久影院| 久久天堂一区二区三区四区| av福利片在线| 丝袜在线中文字幕| 一区在线观看完整版| 午夜影院日韩av| 99riav亚洲国产免费| 在线观看免费视频日本深夜| 久久天躁狠狠躁夜夜2o2o| 日韩大尺度精品在线看网址 | 成人国产一区最新在线观看| 一区二区三区精品91| 一级a爱视频在线免费观看| 日日干狠狠操夜夜爽| 国产乱人伦免费视频| 亚洲七黄色美女视频| 亚洲一区二区三区不卡视频| 午夜福利在线观看吧| 麻豆成人av在线观看| 高清黄色对白视频在线免费看| 国产视频一区二区在线看| 亚洲av成人av| 看免费av毛片| 性色av乱码一区二区三区2| 中文字幕精品免费在线观看视频| 久久中文字幕一级| 一本大道久久a久久精品| 亚洲人成电影观看| 日本黄色视频三级网站网址| 两人在一起打扑克的视频| av天堂在线播放| 性色av乱码一区二区三区2| 人人妻人人添人人爽欧美一区卜| 国产片内射在线| 欧美乱色亚洲激情| 国产高清激情床上av| 在线观看免费日韩欧美大片| 看黄色毛片网站| 国产三级在线视频| 中国美女看黄片| 美女福利国产在线| 亚洲国产精品999在线| 欧美在线一区亚洲| 91精品国产国语对白视频| 久久欧美精品欧美久久欧美| 久久亚洲真实| 欧美日韩国产mv在线观看视频| 欧美午夜高清在线| 国内久久婷婷六月综合欲色啪| 伊人久久大香线蕉亚洲五| 一夜夜www| 色婷婷久久久亚洲欧美| 宅男免费午夜| 操美女的视频在线观看| 国产精品1区2区在线观看.| 精品久久久久久久毛片微露脸| 法律面前人人平等表现在哪些方面| 纯流量卡能插随身wifi吗| 亚洲七黄色美女视频| 亚洲午夜理论影院| 国产精品九九99| 欧美日韩精品网址| 露出奶头的视频| 黄色毛片三级朝国网站| 国产一区二区三区综合在线观看| 91精品国产国语对白视频| 亚洲久久久国产精品| 精品久久久久久久久久免费视频 | 久久亚洲精品不卡| 亚洲国产中文字幕在线视频| 日韩欧美一区二区三区在线观看| 欧美中文综合在线视频| 日韩免费av在线播放| 69精品国产乱码久久久| av在线播放免费不卡| 精品久久久久久成人av| 精品国产美女av久久久久小说| 热re99久久国产66热| 啦啦啦 在线观看视频| 中文字幕高清在线视频| 成人亚洲精品一区在线观看| 91在线观看av| 亚洲七黄色美女视频| 又黄又爽又免费观看的视频| 亚洲av日韩精品久久久久久密| 亚洲av成人一区二区三| 十八禁网站免费在线| 99精品在免费线老司机午夜| 国产三级黄色录像| 好看av亚洲va欧美ⅴa在| 国产午夜精品久久久久久| 色尼玛亚洲综合影院| 美女高潮喷水抽搐中文字幕| 黄色怎么调成土黄色| 日本精品一区二区三区蜜桃| 最新美女视频免费是黄的| 18禁国产床啪视频网站| xxxhd国产人妻xxx| 极品教师在线免费播放| 自拍欧美九色日韩亚洲蝌蚪91| 久久人妻熟女aⅴ| 法律面前人人平等表现在哪些方面| 日韩欧美一区二区三区在线观看| 国产片内射在线| 中文亚洲av片在线观看爽| 欧美日韩视频精品一区| av免费在线观看网站| 午夜亚洲福利在线播放| 国产精品98久久久久久宅男小说| 亚洲av第一区精品v没综合| 国产精品1区2区在线观看.| 国产色视频综合| xxx96com| 日韩精品青青久久久久久| 欧美成人性av电影在线观看| 亚洲午夜精品一区,二区,三区| 中文字幕人妻丝袜制服| 水蜜桃什么品种好| 欧美黄色淫秽网站| 久热这里只有精品99| 成人手机av| av欧美777| 9色porny在线观看| 黄色 视频免费看| 亚洲成a人片在线一区二区| 国产不卡一卡二| 日本三级黄在线观看| 女人高潮潮喷娇喘18禁视频| 成年人黄色毛片网站| 日韩欧美国产一区二区入口| 精品欧美一区二区三区在线| 麻豆久久精品国产亚洲av |