• <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 ?

    Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

    2010-06-29 10:12:47ZhongZhouSiJieQunLiHaiZhiQiZhiJunHeWeiHuandYiNingLi

    Zhong-Zhou Si, Jie-Qun Li, Hai-Zhi Qi, Zhi-Jun He, Wei Hu and Yi-Ning Li

    Changsha, China

    Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

    Zhong-Zhou Si, Jie-Qun Li, Hai-Zhi Qi, Zhi-Jun He, Wei Hu and Yi-Ning Li

    Changsha, China

    BACKGROUND:Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirusmediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats.

    METHODS:Adenoviruses encoding hIL-10 orβ-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4℃in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups.

    RESULTS:Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-α, MIP-2, ICAM-1 mRNA, and NF-κB protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ.

    CONCLUSIONS:Donor pretreatment with Ad-hIL-10 downregulates the expression of proinflammatory cytokines TNF-α, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, bysuppressing NF-κB activation and subsequent expression of proinflammatory mediators.

    (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)

    adenovirus vector; interleukin 10; ischemia-reperfusion injury; gene transfer

    Introduction

    Ischemia-reperfusion injury (IRI), an antigenindependent component of harvesting insult, affects the outcome after transplantation and plays an important role in post-transplant complications, including primary nonfunction and acute and chronic rejection.[1,2]The development of new strategies to minimize hepatic IRI could have a major impact on the outcome after transplantation.

    Interleukin 10 (IL-10), a Th2-type cytokine, is known to modulate inflammatory responses by inhibiting the production of proinflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-8[3-5]and upregulating monocyte production of soluble TNF receptor and IL-1 receptor antagonist.[6]Administration of recombinant IL-10 (rIL-10) inhibits hepatic warm IRI by suppressing nuclear factorkappa B (NF-κB) activation and subsequent expression of proinflammatory mediators.[7]However, recombinant cytokines, including IL-10, are short-lived and have limited access to the tissue interstitiumin vivo.[8,9]This short half-life is likely to be an obstacle to the use of rIL-10 in transplantation.[10]Gene transfer techniques are attractive vehicles for prolonging the expression of shortlived proteins such as cytokines. Based on these data, we constructed a recombinant adenovirus vector, Ad-hIL-10, expressing the hIL-10 gene and evaluated its effects on preventing IRI in a rat model of liver transplantation.

    Methods

    Construction of recombinant adenoviral vectors

    The hIL-10 cDNA was cloned by PCR from pCDNA3.1-hIL-10, as described,[11]then cloned into the Kpn Ⅰand Hind Ⅲ sites of pShuttle-CMV. The plasmid was linearized by restriction digestion and co-transformed into BJ5183 cells with pAdEasy-1 to produce recombinant adenovirus plasmid. This plasmid was subsequently transfected into AD-293 cells to produce packaging recombinant adenovirus granules. After identification, the desired recombinant adenovirus was purified by density gradient ultracentrifugation and titrated. Packaging recombinant adenovirus granules (Ad-hIL-10 and Ad-lacZ) with high purity and titer (2×1010IU/ml) were generated after density gradient ultracentrifugation.

    Animals and surgical procedures

    Sprague-Dawley rats (10-12 weeks of age, weighing 250-300 g; Experimental Animal Center, Medical College, Central South University, Changsha, China) were used as donors and recipients. All animal experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care. A rat nonarterialized orthotopic liver transplantation mode was performed according to the techniques described by Kamada.[12]

    Experimental design

    The experiments were conducted in three groups of rats: an Ad-hIL-10 group, an Ad-lacZ control group, and a saline control group. One milliliter of Ad-hIL-10, Ad-lacZ (2×1010IU), or 0.9% saline was injected via the superior mesenteric vein into prospective donor animals after a simple laparotomy. Forty-eight hours later, the donor livers were harvested, and stored for 12 hours in lactated Ringer's solution at 4 ℃ prior to transplantation. Blood and liver tissue samples were obtained 24 hours after reperfusion (n=6).

    Histology

    Liver tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin. Histological severity of IRI in liver grafts was graded using the Suzuki classification, in which sinusoidal congestion, hepatocyte necrosis, and ballooning degeneration are graded from 0 to 4.[13]No necrosis, congestion, or centrilobular ballooning is given a score of 0, while severe congestion, ballooning degeneration, or >60% lobular necrosis is given a value of 4. All histological evaluations were done in a doubleblinded fashion.

    Expression of cytokine mRNA in liver tissue

    To study the expression of cytokine mRNA, we used competitive template reverse transcription-polymerase chain reaction (RT-PCR) as described.[14]Briefly, total RNA was extracted from frozen liver samples with an RNeasy mini kit (Qiagen, Chatsworth, CA, USA). Five micrograms of RNA was reverse transcribed with oligo(dT) primers and SuperScript reverse transcriptase (Invitrogen GIBCO, USA). The primer sequences for TNF-α, hIL-10, intercellular adhesion molecule-1 (ICAM-1), macrophage-inflammatory protein-2 (MIP-2) and β-actin were: β-actin sense, 5'-CTC TTC CAG CCT TCC TTC CT-3' and antisense, 5'-TAG AGC CAC CAA TCC ACA CA-3'; ICAM-1 sense, 5'-CTC TGC TCC TGG TCC TGG T-3' and antisense, 5'-CGT GAA TGT GAT CTC CTT GG-3'; MIP-2 sense, 5'-CCT CAA CGG AAG AAC CAA AG-3' and antisense, 5'-CAA GAC ACG AAA AGG CAT GA-3'; hIL-10 sense, 5'-TCT TGC AAA ACC AAA CCA CA-3' and antisense, 5'-TGA GGG TCT TCA GGT TCT CC-3'; TNF-α sense, 5'-TAT GGC TCA GGG TCC AAC TC-3' and antisense, 5'-TGG TCA CCA AAT CAG CGT TA-3'. Various cycles of PCR were performed at the annealing temperature that was optimized empirically for each primer pair: 26 cycles, 72 ℃ (hIL-10); 30 cycles, 72 ℃ (ICAM-1); 28 cycles, 72 ℃ (TNF-α); 34 cycles, 60 ℃ (MIP-2); and 28 cycles, 72 ℃ (α-actin). PCR products were analyzed in ethidium bromide-stained 2% agarose gels, and scanned with Kodak 1D Image Analysis software (version 2.0; Eastman Kodak, Rochester, NY, USA). All samples were normalized against the respective α-actin template cDNA ratio.

    Western blotting analysis

    Protein was extracted from tissue samples with protein lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100, pH 7.2). Protein (30 μg/sample) in SDS-loading buffer (50 mmol/L Tris, pH 7.6, 10% glycerol, 1% SDS) was subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The gel was then stained with Coomassie blue for protein loading. The membrane was blocked with 3% dry milk and 0.1% Tween 20 (USB, Cleveland, OH, USA) in PBS. Polyclonal rabbit anti-rat NF-κB Ab (Santa Cruz, CA, USA) was used. The membranes were incubated with antibodies and developed according to the enhanced chemiluminescence protocol (GE Healthcare, USA). Relative quantities of NF-κB proteins were determined by densitometry (Kodak Digital Science 1D Analysis Software, Rochester, NY, USA).

    Table 1. ALT and AST serum levels in rats after 24-hour reperfusion (mean±SD)

    Table 2. Histologic criteria (after Suzuki) for grading liver IRI after 24-hour reperfusion (mean±SD)

    Table 3. Intragraft expression of TNF-α mRNA, ICAM-1 mRNA, MIP-2 mRNA, hIL-10 mRNA, and NF-κB protein after 24-hour reperfusion (mean±SD)

    Fig. A-D: RT-PCR analysis of cytokine mRNAs in liver allograft after 24-hour reperfusion. E: Effects of Ad-hIL-10 on the expression of NF-κB detected by Western blotting analysis after 24-hour reperfusion.

    Statistical analysis

    All data were expressed as mean±SD. Comparisons between the groups were performed using Student'sttest or one-way analysis of variance (ANOVA) when necessary. The histological samples were subjected to the Kruskal-Wallis test to search for significant differences among the groups. SPSS 14.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. APvalue less than 0.05 was considered statistically significant.

    Results

    Ad-hIL-10 pretreatment improves hepatocyte function and ameliorates histologic signs of IRIThe serum levels of ALT and AST were significantly lower in the Ad-hIL-10 group than in the saline and Ad-lacZ control groups at 24 hours after reperfusion (Table 1). In correlation with the serum levels of ALT and AST, the saline and Ad-lacZ control groups revealed moderate to severe hepatocyte necrosis with prominent sinusoidal or vascular congestion (Table 2).

    Expression of cytokine mRNAs and NF-κB protein in the liver allografts

    The expression of hIL-10 mRNA was detected by RTPCR in Ad-hIL-10-treated grafts but not in control grafts treated with saline or Ad-lacZ. Successful transduction of Ad-hIL-10 to the hepatic graft was thus confirmed at themRNA level. RT-PCR revealed markedly decreased levels of TNF-α, ICAM-1, and MIP-2 mRNA in Ad-hIL-10-treated rats, compared to the saline and Ad-lacZ control groups (Table 3 and Fig. A-D). These findings imply that hIL-10 gene transfer leads to a decrease in the expression of proinflammatory cytokine levels in the allografts. Western blotting was used to evaluate the expression of NF-κB in hepatic grafts, and significantly decreased NF-κB was noted in the Ad-hIL-10 group compared to the two control groups (Fig. E).

    Discussion

    In this study, we constructed a recombinant adenovirus vector to express hIL-10, and hIL-10 mRNA expression was detected by RT-PCR in Ad-hIL-10-treated grafts but not in control grafts treated with saline or Ad-lacZ. Successful transduction of Ad-hIL-10 to the hepatic graft was thus confirmed at the mRNA level. Adenoviral gene transfer of hIL-10 downregulated the expression of NF-κB and mRNA of proinflammatory mediators (MIP-2, ICAM-1, and TNF-α), thus improving liver function and preserving hepatocyte integrity and architecture. The current study complemented recent studies showing that exogenous rIL-10 inhibits liver injury caused by warm IRI in mice,[7]and that a selective gene therapy approach provides evidence that hIL-10 protects the liver graft from IRI in a clinically relevant cold IRI model.

    In experimental animal models, two distinct phases in the development of organ injury induced by hepatic ischemia and reperfusion can be identified. During the initial phase, Kupffer cells are activated and release reactive oxygen species and proinflammatory cytokines, including TNF-α.[15,16]Although these products may directly injure liver parenchymal cells, organ dysfunction is minimal. The enhanced production of TNF-α, however, plays an important role in the initiation of a cascade of events that causes significant liver injury mediated by neutrophils. One of the main functions of TNF-α is the upregulation of adhesion molecules and neutrophil-attracting CXC chemokines.[17,18]The coordinated efforts of adhesion molecules such as ICAM-1, and CXC chemokines such as MIP-2, mediate the recruitment of neutrophils into the liver. Sequestered neutrophils release proteases and reactive oxygen intermediates, which directly damage hepatocytes and endothelial cells and also contribute to capillary plugging, causing hepatic hypoperfusion.[19,20]

    A common link between cytokines (TNF-α), chemokines (MIP-2), and adhesion molecules (ICAM-1) involved in the development of hepatic IRI is their transcriptional regulation. Each of these mediators is controlled, at least in part, by the transcription factor NF-κB.[21-24]The primary form of NF-κB consists of a heterodimer of NF-κB (p50) and RelA (p65), which in most cells is retained in the cytoplasm complexed with inhibitory proteins of the IκB family.[25]In response to inflammatory stimuli, including oxidant stress and proinflammatory cytokines such as TNF-α or IL-1, IκB proteins are phosphorylated, ubiquinated, and degraded in a process requiring the 26S proteasome.[23]Degradation of IκB proteins unmasks the nuclear localization sequence of NF-κB subunits and induces gene transcription. Recent studies found that attenuation of NF-κB activation and subsequent reduction in TNF-α production after sustained ischemia play important roles in the protective mechanism of ischemic preconditioning against hepatic IRI.[26,27]These data suggest a central role of NF-κB activation in the initiation of hepatic IRI, and inhibition of NF-κB activation could protect against hepatic IRI. Therapeutic strategies against proinflammatory mediators in liver grafts with NF-κB decoy oligodeoxynucleotides[28]or by adenoviral IκB[29]gene transfer have already proven effective in reducing IRI. In the present study, the data showed that IRI-induced liver NF-κB activation can be inhibited by IL-10. This is in accordance with a previous study showing that IL-10 suppresses nuclear translocation of NF-κB by preserving the expression of the inhibitory IκB protein, IκBa, in lung cells.[30]

    In conclusion, we have documented striking cytoprotection by Ad-hIL-10 against hepatic cold IRI. Downregulation of the proinflammatory cytokines TNF-α, MIP-2, and ICAM-1 mRNA may be associated with this protection. Given the central role of NF-κB activation in the initiation of hepatic IRI, hIL-10 protects against hepatic cold IRI, at least in part, by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.

    Acknowledgement

    The pCDNA3.1-hIL-10 plasmid was a kind gift from Jie-Xiong Tan, MD (State Key Laboratory of Genetics, Changsha, China).

    Funding:None.

    Ethical approval:All animal experiments were conducted in accordance with the guidelines approved by the Chinese Association of Laboratory Animal Care.

    Contributors:SZZ and LJQ proposed the study and wrote the first draft. LJQ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. QHZ is the guarantor.

    Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

    1 Sun HW, Shen F, Zhou YM. Influence of perfusion by gaseous oxygen persufflation on rat donor liver. Hepatobiliary Pancreat Dis Int 2006;5:195-198.

    2 Howard TK, Klintmalm GB, Cofer JB, Husberg BS, Goldstein RM, Gonwa TA. The influence of preservation injury on rejection in the hepatic transplant recipient. Transplantation 1990;49:103-107.

    3 de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 1991;174:1209-1220.

    4 Dagvadorj J, Naiki Y, Tumurkhuu G, Hassan F, Islam S, Koide N, et al. Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression. Innate Immun 2008;14:109-115.

    5 Tudor C, Marchese FP, Hitti E, Aubareda A, Rawlinson L, Gaestel M, et al. The p38 MAPK pathway inhibits tristetraprolin-directed decay of interleukin-10 and proinflammatory mediator mRNAs in murine macrophages. FEBS Lett 2009;583:1933-1938.

    6 Cassatella MA, Meda L, Bonora S, Ceska M, Constantin G. Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. Evidence for an autocrine role of tumor necrosis factor and IL-1 beta in mediating the production of IL-8 triggered by lipopolysaccharide. J Exp Med 1993;178:2207-2211.

    7 Yoshidome H, Kato A, Edwards MJ, Lentsch AB. Interleukin-10 suppresses hepatic ischemia/reperfusion injury in mice: implications of a central role for nuclear factor kappaB. Hepatology 1999;30:203-208.

    8 Wissing KM, Morelon E, Legendre C, De Pauw L, LeBeaut A, Grint P, et al. A pilot trial of recombinant human interleukin-10 in kidney transplant recipients receiving OKT3 induction therapy. Transplantation 1997;64:999-1006.

    9 Donckier V, Loi P, Closset J, Nagy N, Quertinmont E, Le Moine O, et al. Preconditioning of donors with interleukin-10 reduces hepatic ischemia-reperfusion injury after liver transplantation in pigs. Transplantation 2003;75:902-904.

    10 Blazar BR, Taylor PA, Smith S, Vallera DA. Interleukin-10 administration decreases survival in murine recipients of major histocompatibility complex disparate donor bone marrow grafts. Blood 1995;85:842-851.

    11 Li JQ, Qi HZ, He ZJ, Hu W, Si ZZ, Li YN, et al. Cytoprotective effects of human interleukin-10 gene transfer against necrosis and apoptosis induced by hepatic cold ischemia/reperfusion injury. J Surg Res 2009;157:e71-78.

    12 Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery 1983;93:64-69.

    13 Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation 1993;55:1265-1272.

    14 Ke B, Ritter T, Kato H, Zhai Y, Li J, Lehmann M, et al. Regulatory cells potentiate the efficacy of IL-4 gene transfer by up-regulating Th2-dependent expression of protective molecules in the infectious tolerance pathway in transplant recipients. J Immunol 2000;164:5739-5745.

    15 Matsuno T, Sasaki H, Nakagawa K, Ishido N, Matsuda H, Sadamori H, et al. Fas antigen expression and apoptosis in kidney allografts. Transplant Proc 1997;29:177-178.

    16 Jaeschke H, Bautista AP, Spolarics Z, Spitzer JJ. Superoxide generation by Kupffer cells and priming of neutrophils during reperfusion after hepatic ischemia. Free Radic Res Commun 1991;15:277-284.

    17 Colletti LM, Cortis A, Lukacs N, Kunkel SL, Green M, Strieter RM. Tumor necrosis factor up-regulates intercellular adhesion molecule 1, which is important in the neutrophildependent lung and liver injury associated with hepatic ischemia and reperfusion in the rat. Shock 1998;10:182-191.

    18 Kuboki S, Shin T, Huber N, Eismann T, Galloway E, Schuster R, et al. Hepatocyte signaling through CXC chemokine receptor-2 is detrimental to liver recovery after ischemia/ reperfusion in mice. Hepatology 2008;48:1213-1223.

    19 Jaeschke H, Smith CW. Mechanisms of neutrophil-induced parenchymal cell injury. J Leukoc Biol 1997;61:647-653.

    20 Llacuna L, Marí M, Lluis JM, García-Ruiz C, Fernández-Checa JC, Morales A. Reactive oxygen species mediate liver injury through parenchymal nuclear factor-kappaB inactivation in prolonged ischemia/reperfusion. Am J Pathol 2009;174:1776-1785.

    21 Collart MA, Baeuerle P, Vassalli P. Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B. Mol Cell Biol 1990;10:1498-1506.

    22 Stein B, Baldwin AS Jr. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and coopera-tivity between C/EBP and NF-kappa B. Mol Cell Biol 1993;13: 7191-7198.

    23 Widmer U, Manogue KR, Cerami A, Sherry B. Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines. J Immunol 1993;150:4996-5012.

    24 Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J 1995;9:899-909.

    25 Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225-260.

    26 Funaki H, Shimizu K, Harada S, Tsuyama H, Fushida S, Tani T, et al. Essential role for nuclear factor kappaB in ischemic preconditioning for ischemia-reperfusion injury of the mouse liver. Transplantation 2002;74:551-556.

    27 Ricciardi R, Shah SA, Wheeler SM, Quarfordt SH, Callery MP, Meyers WC, et al. Regulation of NFkappaB in hepatic ischemic preconditioning. J Am Coll Surg 2002;195:319-326.

    28 Xu MQ, Shuai XR, Yan ML, Zhang MM, Yan LN. Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft. World J Gastroenterol 2005;11:6960-6967.

    29 Takahashi Y, Ganster RW, Ishikawa T, Okuda T, Gambotto A, Shao L, et al. Protective role of NF-kappaB in liver cold ischemia/reperfusion injury: effects of IkappaB gene therapy. Transplant Proc 2001;33:602.

    30 Lentsch AB, Shanley TP, Sarma V, Ward PA. In vivo suppression of NF-kappa B and preservation ofikappa B alpha by interleukin-10 and interleukin-13. J Clin Invest 1997;100:2443-2448.

    August 10, 2009

    Accepted after revision February 12, 2010

    Author Affiliations: Department of Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha 410011, China (Si ZZ, Li JQ, Qi HZ, He ZJ, Hu W and Li YN)

    Hai-Zhi Qi, MD, Department of Organ Transplantation, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tel: 86-731-85295808; Fax: 86-731-85295808; Email: szz5858@medmail.com.cn)

    ? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.

    只有这里有精品99| 高清毛片免费观看视频网站| 91精品国产九色| 日韩视频在线欧美| 婷婷精品国产亚洲av| 国产一区二区三区av在线 | 亚洲成av人片在线播放无| 高清午夜精品一区二区三区 | 午夜精品一区二区三区免费看| 欧美最新免费一区二区三区| 国产成人91sexporn| 欧美在线一区亚洲| 桃色一区二区三区在线观看| 麻豆成人av视频| 精品人妻偷拍中文字幕| 国产精品,欧美在线| av视频在线观看入口| 亚洲成a人片在线一区二区| 免费一级毛片在线播放高清视频| 91久久精品电影网| 亚洲自偷自拍三级| 18禁裸乳无遮挡免费网站照片| 国产精品久久久久久久久免| 九草在线视频观看| av免费观看日本| 欧美一区二区亚洲| 人妻系列 视频| 国产精品野战在线观看| 悠悠久久av| 99热只有精品国产| 午夜老司机福利剧场| 亚洲人成网站在线播| 国产精品乱码一区二三区的特点| 亚洲av中文av极速乱| 又粗又硬又长又爽又黄的视频 | 成年版毛片免费区| www日本黄色视频网| 在线观看美女被高潮喷水网站| 免费在线观看成人毛片| 少妇的逼水好多| 国产女主播在线喷水免费视频网站 | 伦理电影大哥的女人| 91av网一区二区| 免费大片18禁| 色视频www国产| 国产精品人妻久久久久久| 亚洲精品日韩在线中文字幕 | 日本熟妇午夜| 97人妻精品一区二区三区麻豆| 免费看美女性在线毛片视频| 亚洲一区高清亚洲精品| 日日摸夜夜添夜夜添av毛片| 久久人人精品亚洲av| 我要看日韩黄色一级片| 久久人人爽人人片av| 久久久久久久久中文| 日韩一本色道免费dvd| 精品久久国产蜜桃| 人妻少妇偷人精品九色| 日韩一区二区视频免费看| av在线蜜桃| 国产黄a三级三级三级人| 久久久久网色| 啦啦啦观看免费观看视频高清| 国产极品精品免费视频能看的| 亚洲一级一片aⅴ在线观看| 中国美女看黄片| av黄色大香蕉| 麻豆久久精品国产亚洲av| 精品午夜福利在线看| 99久久精品国产国产毛片| 真实男女啪啪啪动态图| 国产一级毛片七仙女欲春2| 99热只有精品国产| 欧美色视频一区免费| 欧美潮喷喷水| 免费看a级黄色片| 一级二级三级毛片免费看| 麻豆av噜噜一区二区三区| 91狼人影院| 欧美日韩在线观看h| а√天堂www在线а√下载| 成人二区视频| www.av在线官网国产| 一区福利在线观看| 久久午夜福利片| 天堂影院成人在线观看| 国产精品久久久久久久电影| 亚洲内射少妇av| 三级毛片av免费| 在线国产一区二区在线| 村上凉子中文字幕在线| 亚洲七黄色美女视频| 中文字幕制服av| 久久久久久久久久久免费av| 国产精品人妻久久久影院| 在现免费观看毛片| 99久久精品一区二区三区| 国产乱人视频| 国产精品精品国产色婷婷| 国产单亲对白刺激| 国产精品国产高清国产av| 亚洲美女视频黄频| 亚洲精品国产av成人精品| 亚洲在线自拍视频| 日韩精品青青久久久久久| 美女xxoo啪啪120秒动态图| 成人鲁丝片一二三区免费| 欧美激情久久久久久爽电影| 国产蜜桃级精品一区二区三区| 午夜免费激情av| 青春草视频在线免费观看| 亚洲精品亚洲一区二区| 日韩成人av中文字幕在线观看| 成熟少妇高潮喷水视频| 成人亚洲欧美一区二区av| 国产精品免费一区二区三区在线| 亚洲人与动物交配视频| 两性午夜刺激爽爽歪歪视频在线观看| 国产私拍福利视频在线观看| 久久久久性生活片| 少妇熟女aⅴ在线视频| 久久亚洲精品不卡| 午夜福利视频1000在线观看| 午夜免费男女啪啪视频观看| 久久亚洲精品不卡| 亚洲欧美日韩高清专用| 国产在线男女| 热99在线观看视频| 1024手机看黄色片| 欧美日本视频| 国产高清激情床上av| 毛片女人毛片| 色5月婷婷丁香| 日韩精品有码人妻一区| 一本一本综合久久| 国产成人午夜福利电影在线观看| 亚洲美女视频黄频| 国产精品久久久久久精品电影小说 | 国产成人freesex在线| 黄色日韩在线| 国产精品久久久久久精品电影| 午夜福利视频1000在线观看| 熟妇人妻久久中文字幕3abv| 国产精品国产三级国产av玫瑰| 熟女人妻精品中文字幕| 亚洲无线观看免费| 日韩精品有码人妻一区| 能在线免费看毛片的网站| 麻豆乱淫一区二区| 国产大屁股一区二区在线视频| 久久精品国产亚洲av涩爱 | 99久久精品国产国产毛片| 女人被狂操c到高潮| 午夜视频国产福利| 精品一区二区三区人妻视频| 亚洲av男天堂| 日韩欧美精品免费久久| 久久草成人影院| 欧美日韩国产亚洲二区| 天天躁夜夜躁狠狠久久av| 久久精品影院6| 日韩亚洲欧美综合| 国产成人a区在线观看| 天天一区二区日本电影三级| 亚洲真实伦在线观看| 听说在线观看完整版免费高清| 午夜激情欧美在线| 国产成人午夜福利电影在线观看| 大香蕉久久网| 欧美又色又爽又黄视频| 久久综合国产亚洲精品| 一卡2卡三卡四卡精品乱码亚洲| 亚洲在久久综合| 欧美日韩在线观看h| 九九久久精品国产亚洲av麻豆| 三级男女做爰猛烈吃奶摸视频| 国产不卡一卡二| 免费大片18禁| 国产老妇伦熟女老妇高清| 国内久久婷婷六月综合欲色啪| 欧美+日韩+精品| 日韩精品青青久久久久久| 国产视频首页在线观看| 美女 人体艺术 gogo| 天堂av国产一区二区熟女人妻| 亚洲中文字幕一区二区三区有码在线看| 免费av毛片视频| 久久久久九九精品影院| 国产av麻豆久久久久久久| 亚洲国产精品sss在线观看| 91久久精品国产一区二区三区| 久久久久网色| 亚洲色图av天堂| 中文欧美无线码| 69人妻影院| 成人美女网站在线观看视频| 热99在线观看视频| 久久精品夜色国产| 白带黄色成豆腐渣| 麻豆国产97在线/欧美| 如何舔出高潮| 亚洲精品久久久久久婷婷小说 | 午夜老司机福利剧场| 久久久欧美国产精品| 国产高清激情床上av| 熟女人妻精品中文字幕| h日本视频在线播放| 99在线人妻在线中文字幕| 国产单亲对白刺激| 日本-黄色视频高清免费观看| 我要看日韩黄色一级片| 中文字幕人妻熟人妻熟丝袜美| 22中文网久久字幕| 免费人成视频x8x8入口观看| 亚洲丝袜综合中文字幕| 男女做爰动态图高潮gif福利片| 国产淫片久久久久久久久| 日韩国内少妇激情av| 国产成年人精品一区二区| 成年免费大片在线观看| 天堂中文最新版在线下载 | 看片在线看免费视频| 国产精品av视频在线免费观看| 亚洲成人久久爱视频| 国产精品国产高清国产av| 亚洲欧洲日产国产| 久久精品国产亚洲av天美| 亚洲精品自拍成人| 一本久久精品| 精品少妇黑人巨大在线播放 | 99在线视频只有这里精品首页| 亚洲最大成人中文| 亚洲精品粉嫩美女一区| 久久精品国产鲁丝片午夜精品| 日本五十路高清| 午夜福利视频1000在线观看| 九草在线视频观看| av在线天堂中文字幕| 干丝袜人妻中文字幕| 永久网站在线| 亚洲精品影视一区二区三区av| 日韩在线高清观看一区二区三区| 99在线人妻在线中文字幕| 蜜桃久久精品国产亚洲av| 国产精品久久久久久亚洲av鲁大| 在线免费十八禁| 亚洲av二区三区四区| 乱人视频在线观看| 日韩视频在线欧美| 日韩欧美一区二区三区在线观看| 国产精品久久久久久久久免| 久久热精品热| 一本一本综合久久| 免费av不卡在线播放| 嫩草影院精品99| 十八禁国产超污无遮挡网站| 国产精品久久久久久精品电影| 国产69精品久久久久777片| 亚洲第一区二区三区不卡| 亚洲av熟女| 97超碰精品成人国产| 亚洲av免费在线观看| 极品教师在线视频| 日韩av不卡免费在线播放| 在线观看午夜福利视频| 一个人看视频在线观看www免费| 亚洲欧洲日产国产| av在线老鸭窝| 国产成人91sexporn| 性欧美人与动物交配| 男的添女的下面高潮视频| 能在线免费看毛片的网站| 天天一区二区日本电影三级| 免费观看人在逋| 国产大屁股一区二区在线视频| 男的添女的下面高潮视频| 成人无遮挡网站| 精品人妻熟女av久视频| 最近最新中文字幕大全电影3| 欧美最新免费一区二区三区| 亚洲综合色惰| 亚洲成a人片在线一区二区| 国产老妇女一区| 国产精品爽爽va在线观看网站| 如何舔出高潮| 夜夜看夜夜爽夜夜摸| 国产成年人精品一区二区| 国产精品美女特级片免费视频播放器| 人人妻人人看人人澡| 3wmmmm亚洲av在线观看| 两个人视频免费观看高清| 精品久久久久久久久久免费视频| 亚洲成人精品中文字幕电影| 99久久无色码亚洲精品果冻| 一级毛片aaaaaa免费看小| 精品久久久久久久久亚洲| 久久久久九九精品影院| av在线亚洲专区| h日本视频在线播放| 亚洲欧美日韩无卡精品| 男女视频在线观看网站免费| 可以在线观看毛片的网站| 色噜噜av男人的天堂激情| 亚洲欧美中文字幕日韩二区| 亚洲在久久综合| 国产高清视频在线观看网站| 欧美3d第一页| 夜夜看夜夜爽夜夜摸| 国产黄色小视频在线观看| 99热这里只有是精品50| 神马国产精品三级电影在线观看| 天天躁日日操中文字幕| 国产精品电影一区二区三区| av免费观看日本| 1024手机看黄色片| 成人永久免费在线观看视频| 日本色播在线视频| 亚洲精品国产av成人精品| 国产精品野战在线观看| 国产精品无大码| 国模一区二区三区四区视频| av专区在线播放| 深夜a级毛片| 男人的好看免费观看在线视频| 国产在线男女| 男女下面进入的视频免费午夜| 精品欧美国产一区二区三| 亚洲成av人片在线播放无| 久久这里只有精品中国| 乱系列少妇在线播放| 免费看光身美女| 深夜精品福利| 免费看av在线观看网站| 国产黄片美女视频| a级毛片免费高清观看在线播放| 老司机福利观看| av在线蜜桃| av在线播放精品| 久久人妻av系列| 人人妻人人澡欧美一区二区| 岛国在线免费视频观看| 日本一本二区三区精品| 3wmmmm亚洲av在线观看| 久久人人爽人人片av| 成人特级黄色片久久久久久久| 一区二区三区四区激情视频 | 91av网一区二区| 麻豆国产av国片精品| 三级经典国产精品| 又粗又硬又长又爽又黄的视频 | 麻豆一二三区av精品| 国产 一区精品| 国产精品嫩草影院av在线观看| 久久久久免费精品人妻一区二区| 精品一区二区免费观看| 99热这里只有是精品在线观看| 欧美区成人在线视频| 乱人视频在线观看| 天堂影院成人在线观看| 99国产极品粉嫩在线观看| 一级毛片我不卡| 国产伦在线观看视频一区| 国产成年人精品一区二区| 成人二区视频| 我的老师免费观看完整版| 欧美激情在线99| 亚洲人成网站在线观看播放| 免费电影在线观看免费观看| 啦啦啦啦在线视频资源| 边亲边吃奶的免费视频| 欧美xxxx性猛交bbbb| 国产爱豆传媒在线观看| 久久精品夜色国产| 人人妻人人澡欧美一区二区| 蜜桃亚洲精品一区二区三区| 搡女人真爽免费视频火全软件| 亚洲美女搞黄在线观看| 久久99热这里只有精品18| 99久久人妻综合| 国产成人91sexporn| 一本精品99久久精品77| 亚洲一级一片aⅴ在线观看| 十八禁国产超污无遮挡网站| 九色成人免费人妻av| 久久这里有精品视频免费| 国产一区二区在线av高清观看| 成人欧美大片| 一级二级三级毛片免费看| 麻豆成人av视频| 校园人妻丝袜中文字幕| 亚洲色图av天堂| videossex国产| 国产精品久久久久久久电影| 亚洲国产色片| 插逼视频在线观看| 偷拍熟女少妇极品色| 成人美女网站在线观看视频| 国产精品无大码| 一本一本综合久久| 国产精品不卡视频一区二区| 99热网站在线观看| 一级av片app| 日韩av不卡免费在线播放| 插阴视频在线观看视频| 久久亚洲国产成人精品v| 国产精品人妻久久久影院| 国产精品久久电影中文字幕| 亚洲真实伦在线观看| 国产淫片久久久久久久久| 波野结衣二区三区在线| 免费观看人在逋| 日韩一区二区视频免费看| 久久久久久伊人网av| 国产精品久久久久久久久免| 亚洲精品乱码久久久久久按摩| 日韩高清综合在线| 看免费成人av毛片| 噜噜噜噜噜久久久久久91| 国产成人a区在线观看| 国产高清激情床上av| 干丝袜人妻中文字幕| 中文欧美无线码| 欧美极品一区二区三区四区| 国产精品免费一区二区三区在线| 亚洲丝袜综合中文字幕| 久久综合国产亚洲精品| h日本视频在线播放| 高清毛片免费观看视频网站| 国产爱豆传媒在线观看| 又爽又黄无遮挡网站| eeuss影院久久| 亚洲精品乱码久久久v下载方式| 色吧在线观看| 天堂av国产一区二区熟女人妻| 国产精品精品国产色婷婷| 少妇熟女aⅴ在线视频| 一区二区三区免费毛片| 人妻夜夜爽99麻豆av| 国产中年淑女户外野战色| 亚洲欧美精品专区久久| 99热这里只有精品一区| 爱豆传媒免费全集在线观看| a级一级毛片免费在线观看| 最近视频中文字幕2019在线8| 久久久久久久久久成人| 性插视频无遮挡在线免费观看| 欧美三级亚洲精品| 美女 人体艺术 gogo| 91av网一区二区| 在线播放国产精品三级| 长腿黑丝高跟| 亚洲人成网站在线观看播放| 亚洲欧美日韩卡通动漫| 日本-黄色视频高清免费观看| 亚洲天堂国产精品一区在线| 在线免费观看的www视频| 国产av麻豆久久久久久久| 偷拍熟女少妇极品色| 三级经典国产精品| 麻豆成人午夜福利视频| 91aial.com中文字幕在线观看| 精品久久久久久成人av| 亚洲熟妇中文字幕五十中出| 91狼人影院| 久久久精品欧美日韩精品| 联通29元200g的流量卡| 激情 狠狠 欧美| 婷婷亚洲欧美| 夜夜爽天天搞| 伦理电影大哥的女人| 18禁在线无遮挡免费观看视频| 精品一区二区三区人妻视频| 国产69精品久久久久777片| 久久这里只有精品中国| 伦精品一区二区三区| 日日摸夜夜添夜夜爱| av.在线天堂| 亚洲人与动物交配视频| 26uuu在线亚洲综合色| 又黄又爽又刺激的免费视频.| 精品久久国产蜜桃| 久久久久国产网址| 久久久色成人| 国产真实伦视频高清在线观看| 九九爱精品视频在线观看| av又黄又爽大尺度在线免费看 | 亚洲国产精品合色在线| 99久国产av精品国产电影| 国产亚洲5aaaaa淫片| 精品99又大又爽又粗少妇毛片| av又黄又爽大尺度在线免费看 | 一级黄色大片毛片| 最近2019中文字幕mv第一页| 日韩中字成人| 中文字幕久久专区| 国产黄色小视频在线观看| 亚洲中文字幕日韩| 国产视频内射| 91麻豆精品激情在线观看国产| a级一级毛片免费在线观看| 一区二区三区四区激情视频 | 国产69精品久久久久777片| 精品久久久久久久久亚洲| 国产日韩欧美在线精品| 日韩成人av中文字幕在线观看| 国产熟女欧美一区二区| 青春草国产在线视频 | 如何舔出高潮| 波多野结衣高清作品| 日日摸夜夜添夜夜添av毛片| 最后的刺客免费高清国语| 此物有八面人人有两片| 亚洲国产精品久久男人天堂| 一级毛片电影观看 | 91久久精品电影网| 成人亚洲精品av一区二区| 国产精品一区二区三区四区久久| 国产伦一二天堂av在线观看| 性色avwww在线观看| 午夜福利在线在线| 精品人妻一区二区三区麻豆| 精品久久国产蜜桃| 免费无遮挡裸体视频| 国产精品一区www在线观看| 免费观看a级毛片全部| 久久久精品大字幕| 国产黄色小视频在线观看| 国产极品精品免费视频能看的| 欧美丝袜亚洲另类| 美女国产视频在线观看| 日韩成人伦理影院| 欧美成人一区二区免费高清观看| 日韩精品青青久久久久久| 色尼玛亚洲综合影院| 亚洲精品国产成人久久av| 免费av不卡在线播放| 中文字幕精品亚洲无线码一区| 99久久精品热视频| kizo精华| 性色avwww在线观看| 只有这里有精品99| 国产高清三级在线| 嫩草影院新地址| 麻豆久久精品国产亚洲av| 特大巨黑吊av在线直播| 久久人人爽人人爽人人片va| 少妇高潮的动态图| 热99re8久久精品国产| www.色视频.com| 在线国产一区二区在线| 国产成人影院久久av| 2021天堂中文幕一二区在线观| 精品人妻一区二区三区麻豆| 欧美一区二区亚洲| 身体一侧抽搐| 精品日产1卡2卡| 国产av不卡久久| 亚洲色图av天堂| 99热6这里只有精品| 亚洲欧美成人综合另类久久久 | 最好的美女福利视频网| 老师上课跳d突然被开到最大视频| 我要看日韩黄色一级片| 国产精品一区二区三区四区免费观看| 六月丁香七月| 人妻夜夜爽99麻豆av| 在线免费观看不下载黄p国产| 国产三级在线视频| 成熟少妇高潮喷水视频| 久久婷婷人人爽人人干人人爱| 给我免费播放毛片高清在线观看| 寂寞人妻少妇视频99o| 国产片特级美女逼逼视频| 国产精品综合久久久久久久免费| 大型黄色视频在线免费观看| 久久这里只有精品中国| www.色视频.com| 又黄又爽又刺激的免费视频.| 草草在线视频免费看| 国产成人a区在线观看| 综合色av麻豆| 看免费成人av毛片| 插逼视频在线观看| 熟女人妻精品中文字幕| 亚洲精品久久久久久婷婷小说 | 色噜噜av男人的天堂激情| 日韩欧美国产在线观看| 免费不卡的大黄色大毛片视频在线观看 | 99riav亚洲国产免费| av在线蜜桃| 干丝袜人妻中文字幕| 精品国产三级普通话版| 国产 一区 欧美 日韩| 亚洲人成网站在线观看播放| 一夜夜www| 婷婷精品国产亚洲av| 在现免费观看毛片| 亚洲精品久久久久久婷婷小说 | 99久久精品国产国产毛片| 男女啪啪激烈高潮av片| 晚上一个人看的免费电影| 久久久久久伊人网av| 一级毛片aaaaaa免费看小| 噜噜噜噜噜久久久久久91| 欧美人与善性xxx| 美女内射精品一级片tv| 午夜福利在线观看免费完整高清在 | 国产精品永久免费网站| 日日摸夜夜添夜夜添av毛片| 高清日韩中文字幕在线| 国产精品电影一区二区三区| 午夜亚洲福利在线播放| 夫妻性生交免费视频一级片| 麻豆国产av国片精品|