• <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 ?

    Human induced pluripotent stem cell based in vitro models of the bloodbrain barrier: the future standard?

    2017-01-12 03:11:33WinfriedNeuhaus

    Human induced pluripotent stem cell based in vitro models of the bloodbrain barrier: the future standard?

    There is an urgent and tremendous need for human disease models in drug development in order to improve preclinical predictability. In the case of brain disorders drugs have to cross the blood-brain barrier (BBB) to enter the central nervous system (CNS). It was estimated that more than 95% of the drugs cannot cross the BBB. In the case of biopharmaceutics, it seems to be even more difficult for them to overcome the BBB and reach their target sites.e major tasks of the BBB are to maintain CNS homeostasis and prevent entrance of pathogens and toxins, but also to be a part of the brain’s waste disposal system. In a simplistic view, the BBB could be understood as a kind of bidirectional, active filter system. The tightening component of the BBB is the brain capillary endothelial cells (BCECs)which differ from peripheral endothelial cells by forming tight junctions sealing the intercellular gaps, possessing no fenestrae and exhibiting strongly reduced transcytosis(Avdeef et al., 2015).

    Changes of the BBB functionality have been reported for a myriad of diseases, chronic as well as acute ones such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), epilepsy, pain,brain tumor, stroke, and traumatic brain injury. More and more data suggest that alterations of the BBB functionality are not only disease’s symptoms, they are contributing to disease progressions and targeting the BBB can milden the adverse outcomes.is probably relates to the fact that the functionality of the BBB is strongly related to its microenvironment. It has been shown that cells from the CNS such as astrocytes, pericytes and neurons can modulate BBB functionality and vice versa.is collaboration of various cell types at the BBB is summarized in the term neurovascular unit (NVU) and it seems probable that disturbances in this communication involving a changed BBB might also effect the functionality of astrocytes (probably also of oligodendrocytes or microglia) and neuronal cells. Shear stress applied by the blood flow has to be considered as another factor regulating the BBB phenotype. All these facts underline that the need for proper, complex BBB models is enormous, because it is of essential interest not only to screen molecules for their BBB permeability, but also to understand the biology and the biology of the diseases involving the BBB.e idea could be to elucidate the specific changes during diseases and then either to consider or target these changes in the treatment strategies. Moreover,complex models might clarify not only the reasons why a drug cannot enter the CNS, they might also lead to the discovery of novel delivery routes which are upregulated during the diseases and could be utilized for drug delivery systems especially for biopharmaceutics. This knowledge then could be incorporated in simpler screening models being more feasible for drug development processes.

    To study the BBB in vitro, a huge array of different models has been developed and characterized. They are based on immortalized, tumor as well as primary brain endothelial cells from different species and cultivated as mono – or co-cultures together with mainly astrocytes or pericytes.ere are well established models based on porcine, bovine or rat primary brain endothelial cells reaching high values of the transendothelial electrical resistance(TEER) over 1,000 ?·cm2as a measure for high paracellular tightness similar to in vivo ranges. Also several cell lines– although exhibiting mainly lower paracellular tightness- have proven their value as screening tools to study drug permeability, signaling pathways or to develop disease models. However, reflecting the fact that high paracellular tightness is especially important for cellular polarity and correct localization of transporter proteins, current human models need to be essentially improved. Most human BBB in vitro models are based on immortalized BCECs such as hCMEC/D3, hBMEC, TY10 and BB19 and lack significant paracellular tightness. Use of primary human BCECs is critical because of their limited access via e.g. biopsy or autopsy and ethical issues. Biopsy obtained cells from e.g. surgery of epilepsy or tumor patients retain the risk of impurities with diseased cells. Interestingly, there are some commercial sources for human BCECs. However,most of the models applying primary human BCECs from these sources show low paracellular tightness challenging their advantages in relation to their cost. Some attempts to improve the barrier properties of human immortalized cell lines were somehow successful such as including shear stress in microfluidic or hollow-fiber models. Furthermore, cultivation on hydrogels simulating the soCNS tissue also enhanced localization of tight junction associated proteins. However, although these approaches confirmed the importance of mimicking the in vivo environment in order to come closer to a human in vivo like phenotype,finally the cell source still seems to be very critical.

    Recent developments of in vitro models of the BBB based on stem cells are very promising. Beginning with the first protocol from the group of Eric Shusta (together with first author Ethan Lippmann and Abraham Al-Ahmed) published in Nature Biotechnology in 2012, human induced pluripotent stem cells (hiPSCs) have been differentiated into BCECs exhibiting BBB marker expression as well as major BBB properties such as high paracellular tightness (TEER > 1,000 ?·cm2) or a distinct transport barrier(Lippmann et al., 2012). It was shown that these cells expressed endothelial markers von Willebrand factor (vWF),CD31 and Tie-2 as well as BBB markers such as amino acid transporter SLC1A1, glucose transporter SLC2A1 or efflux transporters ABCB1, ABCG2 and ABCCs. In addition, the functionality of ABC-transporters such as ABCB1 was confirmed. Especially, the expression of vWF seemed to be crucial as a marker for a mature endothelium(Lippmann et al., 2012; Appelt-Menzel et al., 2017). Until now, some protocol developments with regard to medium composition (e.g., addition of retinoic acid, usage of medium E6), timing and co-cultivations have been published yielding to maximum TEER values of > 6,000 ?·cm2and shortened differentiation durations to eight days (Hollmann et al., 2017). In the meantime, we and others were able to confirm the reproducibility of the protocols and reported the improved barrier properties yielding distinct tight junction formation shown by freeze fracture electron microscopy (Katt et al., 2016; Appelt-Menzel et al.,2017). A comprehensive overview about the development of hiPSCs-based in vitro BBB models was recently given in a review by Lauschke et al. (2017). Alternatively, BBB in vitro models based on stem cells derived from cord blood have been developed. While having the potential of forming also a distinct paracellular barrier, these models have not achieved such high TEER values as hiPSCs-based models did (~175 ?·cm2vs. 2,000–5,000 ?·cm2), but might be stable for a longer period (2–3 weeks). However, in general hiPSCs-based models seem to possess currently significant advantages with regard to their broader applicability. hiPSCs protocols are available to reprogram hiPSCs from several sources such as skin fibroblasts or cells isolated from the amniotic fluid or urine enabling non-invasive personalized collection. Moreover, there is a vast number of protocols to cultivate and propagate hiPSCs with very little ethical concerns and numerous hiPSC lines are available. For example, recent projects in the EU led to the installation of stem cell banks (StemBANCC,EBiSC) collecting and characterizing a significant number of hiPSC lines from patients.ese patient-derived hiPSCs can also be used to establish BBB disease in vitro models which might recapitulate the disease phenotype more closer. A remarkable example for this is the recently published work of Vatine et al. (2017) who formed a human BBB model based on hiPSCs from a patient with a mutation in the transporter MCT8. MCT8 is a thyroid hormone transporter, and mutations in MCT8 cause neuropsychomotor impairments. They showed that the hiPSCs-BBB model from patients with specific MCT8-mutations revealed a decreased transport of L-3,30,5-triiodothyronine (T3).Controls with isogenic hiPSCs-based BBB models with CRISPR/Cas9 induced MCT8 malfunction and its subsequent rescue confirmed their results.e authors hypothesized that lower CNS concentration of T3 due to restricted BBB permeation caused the observed neuronal dysfunction. Another recent example is the study from Lim et al.(2017) who generated a hiPSCs-BBB model from patients suffering from Huntington’s disease (HD). They demonstrated that HD-hiPSCs BCECs exhibited intrinsic abnormalities in barrier and angiogenesis properties and linked this to underlying pathways such as Wnt-signaling. In this context, we are currently involved in a project funded by the BMBF in Germany (“HiPSTAR”-project) to generate several Alzheimer’s disease hiPSCs-BBB models based on different mutations and to investigate relevant functional differences.

    Several data were obtained from hiPSCs-BCECs mono-cultures without considering cells from the microenvironment. Protocols are available to differentiate astrocytes, pericytes and neural stem cells from hiPSCs.us,isogenic models with several cell types of the NVU derived from the same hiPSC clone seem to be feasible. Canfield et al. (2017) showed a first perspective example of isogenic,multicellular human BBB models. This could become a very important aspect in the future when considering the already known, significant role of NVU cells for the BBB breakdown in animal in vitro disease models. In this regard, recently Yamamizu et al. (2017) demonstrated in a hiPSCs-BBB model the role of neural cells for BBB property induction via the Notch-signaling pathway.e general,pivotal role of astrocytes, pericytes and neural stem cells for the improvement of the paracellular tightness was already highlighted in studies from the group of Eric Shusta and Ethan Lippmann. Beyond that, co-cultivation of hiPSC-BCECs with astrocytes, pericytes and neural stem cells resulted in a significant change of the transcellular permeability of caffeine (Appelt-Menzel et al., 2017). In summary, these results indicate that - although the paracellular barrier is already very tight in hiPSC-BCEC mono-culture models - inclusion of further cells of the NVU might uncover the regulation of transport pathways and BBB properties which are possibly relevant for the translation of the data to the in vivo situation.

    Future developments might comprise novel protocols for BCEC differentiation without the currently necessary co-differentiation of BCECs with neural stem cells and the subsequent cell separation by different matrix proteins after cell reseeding.is would improve the stability and robustness of the protocols. In this context, Katt et al. (2016)already increased the amount of BCECs after hiPSC-differentiation to > 90%. Another point to be developed is the longevity of the models.e application of shear stress could improve the life-time of BBB models from days to several months. Longevity might pave the way to study chronic processes such as mild inflammation or nutritional changes. In this context, we are currently establishing models culturing hiPSC-BCECs in hollow-fiber devices(is project is funded by SET, a foundation to promote research leading to the replacement, reduction or refinement of animal studies). In addition to the intended longterm models, hiPSC-based BBB models of ischemic insults such as stroke and traumatic brain injury are currently under development within this project. A first study of an in vitro BBB ischemia model based on hiPSCs-BCECs was published in the journal Fluids and Barriers of the CNS in 2016. Major, remaining questions deal with the fact of species differences and how far the hiPSC-BBB models really reflect the human BBB or how artificial the found barrier properties are. For example, the expression of some barrier forming claudins were found in hiPSC-BBB models by us (claudin-4, Appelt-Menzel et al., 2017) and others (claudin-6, -8 and -9, Lim et al., 2017) which have not been found yet to be relevant in animal BBB models.A comprehensive analysis of the tight junction protein expression in human brain tissue as well as brain capillaries is still missing and highly needed in order to classify the data obtained with the hiPS-BCECs.e comparison of transporter proteins showed distinct differences of the expression of e.g. ABCB1 or ABCG2 in brain capillaries of rodents, marmosets and human. Also the total protein amount of claudin-5 was significantly altered in these samples indicating that the findings in hiPSC-BCECs could also be due to species differences. In this regard, data of the MCT8 deficiency model are an excellent example for the usage of hiPSCs-based BBB models to elucidate species differences (Vatine et al., 2017). Within their studies they found out that mice express a different set of transporters responsible for thyroid hormone delivery into the CNS in comparison to humans.

    With respect to the future, pre-differentiated hiPSC-BCECs could be applied in the construction of 3D models such as spheroids or organoids in order to support the development of a brain vasculature within these models. Especially current brain organoids lack a proper vascular system possessing no access points for intravenous drug administration making it difficult to study BBB permeability. Other future 3D models based on hiPSC-BCECs might use cultivation in or on plastic scaffolds or hydrogels with defined 3D structures and incorporated luminal channels for BCECs culture. Other basic questions still have to be answered in relation to, for example, the role of epigenetic or gender influences or how complex will a future hiPSC-BBB assay have to be for drug screening applications.

    In summary, hiPSCs-based BBB models are the first human BBB models with in vivo like paracellular barrier properties. These models possess an enormous potential for preclinical disease models especially to elucidate and reflect disease and species dependent differences.

    Winfried Neuhaus*

    AIT – Austrian Institute of Technology GmbH,Competence Center Health and Bioresources,Unit Molecular Diagnostics, Muthgasse 11, 1190 Vienna, Austria

    *Correspondence to:Winfried Neuhaus, winfried.neuhaus@ait.ac.at.

    orcid: 0000-0002-6552-7183 (Winfried Neuhaus)

    Accepted:2017-10-10

    How to cite this article:Neuhaus W (2017) Human induced pluripotent stem cell based in vitro models of the blood-brain barrier: the future standard?Neural Regen Res 12(10):1607-1609.

    Plagiarism check:Checked twice by ienticate.

    Peer review:Externally peer reviewed.

    Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under identical terms.

    Open peer review report:

    Reviewer: Colin Barnstable, Pennsylvania State University, USA.

    Appelt-Menzel A, Cubukova A, Gunther K, Edenhofer F, Piontek J, Krause G, Stuber T, Walles H, Neuhaus W, Metzger M (2017) Establishment of a human blood-brain barrier co-culture model mimicking the neurovascular unit using induced pluri- and multipotent stem cells. Stem Cell Reports 8:894-906.

    Avdeef A, Deli MA, Neuhaus W (2015) In Vitro Assays for Assessing BBB Permeability. In: Blood-brain Barrier in Drug Discovery, pp 188-237: John Wiley.

    Canfield SG, Stebbins MJ, Morales BS, Asai SW, Vatine GD, Svendsen CN,Palecek SP, Shusta EV (2017) An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells. J Neurochem 140:874-888.

    Hollmann EK, Bailey AK, Potharazu AV, Neely MD, Bowman AB, Lippmann ES (2017) Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells. Fluids Barriers CNS 14:9.

    Katt ME, Xu ZS, Gerecht S, Searson PC (2016) Human brain microvascular endothelial cells derived from the BC1 iPS cell line exhibit a blood-brain barrier phenotype. PLoS One 11:e0152105.

    Lauschke K, Frederiksen L, Hall VJ (2017) Paving the way toward complex blood-brain barrier models using pluripotent stem cells. Stem Cells Dev 26:857-874.

    Lim RG, Quan C, Reyes-Ortiz AM, Lutz SE, Kedaigle AJ, Gipson TA, Wu J,Vatine GD, Stocksdale J, Casale MS, Svendsen CN, Fraenkel E, Housman DE, Agalliu D,ompson LM (2017) Huntington’s disease iPSC-derived brain microvascular endothelial cells reveal WNT-mediated angiogenic and blood-brain barrier deficits. Cell Rep 19:1365-1377.

    Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A,Palecek SP, Shusta EV (2012) Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30:783-791.

    Vatine GD, Al-Ahmad A, Barriga BK, Svendsen S, Salim A, Garcia L, Garcia VJ, Ho R, Yucer N, Qian T, Lim RG, Wu J,ompson LM, Spivia WR,Chen Z, Van Eyk J, Palecek SP, Refetoff S, Shusta EV, Svendsen CN (2017)Modeling psychomotor retardation using iPSCs from MCT8-deficient patients indicates a prominent role for the blood-brain barrier. Cell Stem Cell 20:831-843.e5.

    Yamamizu K, Iwasaki M, Takakubo H, Sakamoto T, Ikuno T, Miyoshi M,Kondo T, Nakao Y, Nakagawa M, Inoue H, Yamashita JK (2017) In vitro modeling of blood-brain barrier with human iPSC-derived endothelial cells, pericytes, neurons, and astrocytes via Notch signaling. Stem Cell Reports 8:634-647.

    10.4103/1673-5374.217326

    国产高潮美女av| 男女视频在线观看网站免费| 长腿黑丝高跟| 国产精品亚洲一级av第二区| 久久精品aⅴ一区二区三区四区| 国产蜜桃级精品一区二区三区| 天堂动漫精品| 无遮挡黄片免费观看| 亚洲成人精品中文字幕电影| 日本五十路高清| 日韩欧美精品v在线| 久久99热这里只有精品18| 精品乱码久久久久久99久播| 日本与韩国留学比较| 一级a爱片免费观看的视频| 国产三级在线视频| 人人妻,人人澡人人爽秒播| 黄色 视频免费看| 亚洲专区国产一区二区| 久久精品亚洲精品国产色婷小说| 97超视频在线观看视频| 91av网一区二区| 91久久精品国产一区二区成人 | 精品久久蜜臀av无| 中出人妻视频一区二区| 性欧美人与动物交配| 在线播放国产精品三级| 啦啦啦韩国在线观看视频| 看免费av毛片| 久久精品国产综合久久久| 老司机深夜福利视频在线观看| 成人国产综合亚洲| 精品国产超薄肉色丝袜足j| 午夜视频精品福利| av中文乱码字幕在线| 久久亚洲精品不卡| 亚洲中文av在线| 国产伦人伦偷精品视频| 成人亚洲精品av一区二区| 特级一级黄色大片| 草草在线视频免费看| 亚洲aⅴ乱码一区二区在线播放| 精品久久蜜臀av无| 女同久久另类99精品国产91| 一二三四社区在线视频社区8| 两性夫妻黄色片| 观看免费一级毛片| 日韩免费av在线播放| 欧美午夜高清在线| 精品欧美国产一区二区三| 黑人欧美特级aaaaaa片| 国产精品综合久久久久久久免费| 噜噜噜噜噜久久久久久91| 在线免费观看不下载黄p国产 | 黄频高清免费视频| 国产激情久久老熟女| 国产精品一区二区三区四区免费观看 | 两个人的视频大全免费| 亚洲,欧美精品.| 桃红色精品国产亚洲av| 亚洲欧美日韩高清在线视频| 99国产极品粉嫩在线观看| 嫩草影院精品99| 日本撒尿小便嘘嘘汇集6| 色综合婷婷激情| 欧美+亚洲+日韩+国产| 成人特级av手机在线观看| 中文字幕精品亚洲无线码一区| 看片在线看免费视频| 男人舔奶头视频| 综合色av麻豆| 午夜福利成人在线免费观看| 亚洲中文字幕日韩| 日韩三级视频一区二区三区| 国产亚洲欧美98| 国产成+人综合+亚洲专区| 少妇人妻一区二区三区视频| 午夜福利在线观看吧| 中文在线观看免费www的网站| 欧美三级亚洲精品| 一个人免费在线观看的高清视频| 午夜福利成人在线免费观看| 亚洲中文字幕一区二区三区有码在线看 | 欧美日韩亚洲国产一区二区在线观看| 久久香蕉精品热| 免费看美女性在线毛片视频| 男人和女人高潮做爰伦理| 国内精品久久久久精免费| 1000部很黄的大片| 一夜夜www| 国产97色在线日韩免费| 在线观看66精品国产| 国产91精品成人一区二区三区| 欧美成狂野欧美在线观看| 国产欧美日韩精品一区二区| 成人国产一区最新在线观看| 久久中文字幕一级| svipshipincom国产片| 国产亚洲av高清不卡| 美女高潮喷水抽搐中文字幕| 18禁黄网站禁片午夜丰满| 亚洲午夜理论影院| 精品国产三级普通话版| 国产真实乱freesex| 一级毛片高清免费大全| 日韩欧美精品v在线| 99国产精品一区二区蜜桃av| 久久午夜综合久久蜜桃| 97碰自拍视频| 男女之事视频高清在线观看| 色尼玛亚洲综合影院| 午夜两性在线视频| 九九热线精品视视频播放| av片东京热男人的天堂| 曰老女人黄片| 99在线人妻在线中文字幕| 99久久国产精品久久久| 精品福利观看| 麻豆国产97在线/欧美| 国内精品久久久久久久电影| 一二三四在线观看免费中文在| 久久久久久久久久黄片| 美女高潮的动态| 色老头精品视频在线观看| 国产免费av片在线观看野外av| 久久性视频一级片| 欧美日韩国产亚洲二区| 一a级毛片在线观看| 国产黄片美女视频| 国产成年人精品一区二区| 亚洲人成电影免费在线| 国产精品国产高清国产av| 91麻豆精品激情在线观看国产| tocl精华| 两性午夜刺激爽爽歪歪视频在线观看| 一区福利在线观看| 精品熟女少妇八av免费久了| 国产午夜精品论理片| 少妇丰满av| 一本久久中文字幕| 亚洲专区字幕在线| 国产91精品成人一区二区三区| 成人av在线播放网站| 成人永久免费在线观看视频| 欧美黑人欧美精品刺激| 久久久久国产一级毛片高清牌| 变态另类成人亚洲欧美熟女| 综合色av麻豆| 亚洲天堂国产精品一区在线| 午夜福利18| 欧美成人一区二区免费高清观看 | 制服丝袜大香蕉在线| 亚洲国产精品久久男人天堂| 嫩草影视91久久| 日本a在线网址| 亚洲天堂国产精品一区在线| 18禁观看日本| 51午夜福利影视在线观看| 精品久久久久久久久久久久久| 亚洲成人久久性| 国内毛片毛片毛片毛片毛片| 黑人欧美特级aaaaaa片| 国产真人三级小视频在线观看| 亚洲五月婷婷丁香| 亚洲最大成人中文| 成年女人永久免费观看视频| 国产av一区在线观看免费| 免费在线观看影片大全网站| 狠狠狠狠99中文字幕| 最近最新中文字幕大全电影3| 久久久久久九九精品二区国产| 观看美女的网站| 中文字幕最新亚洲高清| 日韩人妻高清精品专区| 男女视频在线观看网站免费| 国产亚洲精品av在线| 久久精品夜夜夜夜夜久久蜜豆| 一个人看的www免费观看视频| 午夜免费成人在线视频| 亚洲国产精品成人综合色| 亚洲精品久久国产高清桃花| 一本精品99久久精品77| 欧美不卡视频在线免费观看| 一进一出好大好爽视频| 成人无遮挡网站| 日韩欧美在线二视频| 精品福利观看| 啦啦啦免费观看视频1| cao死你这个sao货| 一本精品99久久精品77| 精品一区二区三区视频在线观看免费| 老汉色av国产亚洲站长工具| 日韩有码中文字幕| 国产伦在线观看视频一区| 91av网一区二区| 欧美成人一区二区免费高清观看 | 曰老女人黄片| 一区福利在线观看| 最近最新中文字幕大全免费视频| 十八禁人妻一区二区| 波多野结衣巨乳人妻| avwww免费| 精品一区二区三区视频在线 | 丁香欧美五月| 日韩有码中文字幕| 亚洲精品一卡2卡三卡4卡5卡| 女生性感内裤真人,穿戴方法视频| 成人三级做爰电影| 日韩欧美国产一区二区入口| 两性夫妻黄色片| 熟妇人妻久久中文字幕3abv| 久久香蕉国产精品| 久久热在线av| 婷婷六月久久综合丁香| 热99re8久久精品国产| 亚洲欧美精品综合一区二区三区| 免费看美女性在线毛片视频| 麻豆一二三区av精品| 热99在线观看视频| 国产精品亚洲av一区麻豆| 亚洲色图av天堂| 搡老熟女国产l中国老女人| 黄色成人免费大全| 久久精品影院6| xxxwww97欧美| 亚洲va日本ⅴa欧美va伊人久久| 久久99热这里只有精品18| 精品一区二区三区视频在线 | 亚洲在线自拍视频| 两个人的视频大全免费| 在线观看美女被高潮喷水网站 | 色综合亚洲欧美另类图片| 国产亚洲精品久久久com| 国产探花在线观看一区二区| 日本五十路高清| 黄频高清免费视频| 天堂影院成人在线观看| 午夜福利在线观看免费完整高清在 | av中文乱码字幕在线| 舔av片在线| 99热6这里只有精品| 精品国内亚洲2022精品成人| 亚洲电影在线观看av| 欧美黑人欧美精品刺激| 久久性视频一级片| 精品一区二区三区av网在线观看| 日本黄色视频三级网站网址| 国产精品一区二区三区四区久久| 午夜精品久久久久久毛片777| 日本一本二区三区精品| 欧美三级亚洲精品| 亚洲精品国产精品久久久不卡| 久99久视频精品免费| netflix在线观看网站| 男人舔奶头视频| 亚洲黑人精品在线| 香蕉久久夜色| 淫秽高清视频在线观看| 国产精品野战在线观看| 中文字幕人妻丝袜一区二区| 色尼玛亚洲综合影院| 亚洲美女黄片视频| 亚洲天堂国产精品一区在线| 日韩欧美精品v在线| 欧美日韩福利视频一区二区| 国产极品精品免费视频能看的| 最近最新中文字幕大全免费视频| 香蕉久久夜色| 欧美日韩中文字幕国产精品一区二区三区| av黄色大香蕉| 久久精品aⅴ一区二区三区四区| 亚洲成a人片在线一区二区| 色哟哟哟哟哟哟| 男女视频在线观看网站免费| 色老头精品视频在线观看| 综合色av麻豆| 中文字幕高清在线视频| 九色成人免费人妻av| 日本在线视频免费播放| 无遮挡黄片免费观看| 国产美女午夜福利| 国产高清视频在线播放一区| 日本一二三区视频观看| 国产亚洲av高清不卡| av视频在线观看入口| 9191精品国产免费久久| 99精品欧美一区二区三区四区| 亚洲国产中文字幕在线视频| 99re在线观看精品视频| 精品久久蜜臀av无| 国产成人系列免费观看| 国产亚洲欧美98| 18禁观看日本| 久久九九热精品免费| 成人高潮视频无遮挡免费网站| 视频区欧美日本亚洲| 91老司机精品| 国产高清视频在线观看网站| 日本一本二区三区精品| 88av欧美| 淫妇啪啪啪对白视频| 草草在线视频免费看| 久久草成人影院| 久久久久亚洲av毛片大全| 99精品久久久久人妻精品| 全区人妻精品视频| 又大又爽又粗| 精品不卡国产一区二区三区| 免费看十八禁软件| 精品不卡国产一区二区三区| 日韩高清综合在线| 小说图片视频综合网站| 最近最新免费中文字幕在线| 国产精品98久久久久久宅男小说| 99国产精品一区二区蜜桃av| 婷婷亚洲欧美| 男女之事视频高清在线观看| 天天一区二区日本电影三级| 搡老妇女老女人老熟妇| 欧美乱色亚洲激情| 午夜亚洲福利在线播放| 一个人看视频在线观看www免费 | 欧美色欧美亚洲另类二区| 一区二区三区激情视频| 男人的好看免费观看在线视频| 精品福利观看| 欧美xxxx黑人xx丫x性爽| 国内精品一区二区在线观看| 精品免费久久久久久久清纯| 欧美日韩瑟瑟在线播放| 欧美成人性av电影在线观看| 成人高潮视频无遮挡免费网站| 偷拍熟女少妇极品色| 99久久99久久久精品蜜桃| 亚洲中文av在线| 啦啦啦韩国在线观看视频| 日本精品一区二区三区蜜桃| 啦啦啦观看免费观看视频高清| 精品乱码久久久久久99久播| xxx96com| 波多野结衣高清作品| 久久人妻av系列| 一进一出好大好爽视频| 久久人妻av系列| 久久精品国产清高在天天线| 亚洲天堂国产精品一区在线| 亚洲av美国av| 怎么达到女性高潮| 成年女人永久免费观看视频| 午夜日韩欧美国产| 免费看美女性在线毛片视频| 日韩国内少妇激情av| 欧美日韩乱码在线| 欧美激情久久久久久爽电影| 一个人观看的视频www高清免费观看 | 国产不卡一卡二| 精品午夜福利视频在线观看一区| 午夜影院日韩av| 最新在线观看一区二区三区| 午夜福利18| 在线免费观看不下载黄p国产 | 精品久久久久久成人av| 日本 av在线| 精品99又大又爽又粗少妇毛片 | 亚洲人成网站高清观看| 免费高清视频大片| 波多野结衣巨乳人妻| 久久久久久久午夜电影| 真人做人爱边吃奶动态| 亚洲性夜色夜夜综合| 中文资源天堂在线| 国产黄色小视频在线观看| 日韩国内少妇激情av| 一夜夜www| 少妇熟女aⅴ在线视频| 国产伦人伦偷精品视频| 老熟妇乱子伦视频在线观看| www.www免费av| 日韩中文字幕欧美一区二区| 久9热在线精品视频| 黄色 视频免费看| 后天国语完整版免费观看| 国产午夜福利久久久久久| 男人和女人高潮做爰伦理| 黄色 视频免费看| 在线观看日韩欧美| 国产av在哪里看| 首页视频小说图片口味搜索| 少妇丰满av| 欧美中文综合在线视频| 午夜免费观看网址| 亚洲欧美日韩高清在线视频| 亚洲av日韩精品久久久久久密| 看黄色毛片网站| 综合色av麻豆| 不卡av一区二区三区| 亚洲av熟女| 国产黄片美女视频| 国产亚洲精品久久久com| 老汉色∧v一级毛片| 国产淫片久久久久久久久 | 成人精品一区二区免费| 久久中文字幕人妻熟女| 国产精品99久久久久久久久| 欧美在线一区亚洲| 亚洲人成网站在线播放欧美日韩| 一本一本综合久久| 国产乱人视频| 成年免费大片在线观看| 男女下面进入的视频免费午夜| 亚洲国产精品久久男人天堂| 亚洲精品色激情综合| 精品国内亚洲2022精品成人| 亚洲性夜色夜夜综合| 免费看光身美女| 色播亚洲综合网| 在线观看免费视频日本深夜| 真人一进一出gif抽搐免费| 12—13女人毛片做爰片一| 热99re8久久精品国产| 亚洲乱码一区二区免费版| 欧美中文日本在线观看视频| 九九久久精品国产亚洲av麻豆 | 在线观看一区二区三区| 观看免费一级毛片| 久久久久久久午夜电影| 免费看光身美女| 嫩草影院入口| 999久久久精品免费观看国产| av女优亚洲男人天堂 | 欧洲精品卡2卡3卡4卡5卡区| 99精品欧美一区二区三区四区| 国产av麻豆久久久久久久| 舔av片在线| www日本在线高清视频| 久久天躁狠狠躁夜夜2o2o| 夜夜看夜夜爽夜夜摸| 亚洲人成电影免费在线| 村上凉子中文字幕在线| 欧美国产日韩亚洲一区| 免费高清视频大片| 亚洲一区二区三区不卡视频| 99久久无色码亚洲精品果冻| 国产一级毛片七仙女欲春2| 国产精品美女特级片免费视频播放器 | 日本一二三区视频观看| 嫩草影视91久久| 老司机午夜福利在线观看视频| 国产伦精品一区二区三区视频9 | 国产成+人综合+亚洲专区| 麻豆成人午夜福利视频| 婷婷精品国产亚洲av在线| 老司机午夜福利在线观看视频| 国产伦精品一区二区三区视频9 | 天天一区二区日本电影三级| 俺也久久电影网| 日韩免费av在线播放| 免费无遮挡裸体视频| 长腿黑丝高跟| 国产熟女xx| 91久久精品国产一区二区成人 | 欧美又色又爽又黄视频| 亚洲欧美精品综合久久99| 在线永久观看黄色视频| 亚洲欧美日韩高清在线视频| 亚洲狠狠婷婷综合久久图片| 又黄又爽又免费观看的视频| a在线观看视频网站| 日本免费一区二区三区高清不卡| xxxwww97欧美| 久久亚洲精品不卡| 亚洲欧美日韩高清在线视频| 国产精品自产拍在线观看55亚洲| 在线十欧美十亚洲十日本专区| www.www免费av| 18禁观看日本| 精品午夜福利视频在线观看一区| 国产精品,欧美在线| 国产激情偷乱视频一区二区| 少妇裸体淫交视频免费看高清| h日本视频在线播放| 欧美成狂野欧美在线观看| 久久久久亚洲av毛片大全| 亚洲avbb在线观看| 亚洲熟妇中文字幕五十中出| 国产亚洲精品一区二区www| a级毛片在线看网站| 日本黄大片高清| 欧美色欧美亚洲另类二区| 国产主播在线观看一区二区| 精品国产乱子伦一区二区三区| 国产精品自产拍在线观看55亚洲| 网址你懂的国产日韩在线| tocl精华| 精品久久久久久,| 国产高清videossex| 日韩三级视频一区二区三区| 免费看光身美女| 国产亚洲精品久久久com| 国产av在哪里看| 国产av一区在线观看免费| 成年女人永久免费观看视频| 国产毛片a区久久久久| 精品欧美国产一区二区三| 午夜福利18| 久久午夜综合久久蜜桃| 久久久精品欧美日韩精品| 黄色日韩在线| bbb黄色大片| 午夜福利欧美成人| 脱女人内裤的视频| 日本a在线网址| 久久精品91蜜桃| 国产成人aa在线观看| 国产真人三级小视频在线观看| 亚洲在线自拍视频| 国产伦人伦偷精品视频| 国产极品精品免费视频能看的| 色视频www国产| 久久天堂一区二区三区四区| 99久久99久久久精品蜜桃| 精品久久久久久久末码| 久久精品人妻少妇| 午夜成年电影在线免费观看| 国产精品日韩av在线免费观看| 国产精品一区二区三区四区免费观看 | 久久这里只有精品19| 久久精品aⅴ一区二区三区四区| 国产欧美日韩精品一区二区| 欧美zozozo另类| 日韩欧美 国产精品| 色av中文字幕| 久久香蕉国产精品| 国产黄片美女视频| 三级毛片av免费| 国产伦精品一区二区三区视频9 | 婷婷精品国产亚洲av| 女警被强在线播放| 国产伦精品一区二区三区视频9 | 亚洲色图av天堂| 女警被强在线播放| 国产免费av片在线观看野外av| 少妇的逼水好多| 久久亚洲真实| 国产三级中文精品| 国产人伦9x9x在线观看| 嫩草影院精品99| 中文亚洲av片在线观看爽| 最新在线观看一区二区三区| 99精品欧美一区二区三区四区| 超碰成人久久| 成人性生交大片免费视频hd| 男人舔女人下体高潮全视频| 99久久精品一区二区三区| 嫩草影院入口| av中文乱码字幕在线| 真实男女啪啪啪动态图| 一个人免费在线观看电影 | 欧美3d第一页| 十八禁网站免费在线| 天天一区二区日本电影三级| 国产真实乱freesex| 国产高清激情床上av| 亚洲av片天天在线观看| 这个男人来自地球电影免费观看| 免费看a级黄色片| 精品一区二区三区av网在线观看| 岛国在线免费视频观看| 午夜福利高清视频| 久久久久精品国产欧美久久久| 舔av片在线| 亚洲精品在线美女| 亚洲av电影不卡..在线观看| 成人特级av手机在线观看| 99视频精品全部免费 在线 | 91麻豆精品激情在线观看国产| 亚洲一区二区三区不卡视频| 欧美色视频一区免费| 国产主播在线观看一区二区| 91在线观看av| 久久久色成人| 亚洲美女视频黄频| 亚洲国产日韩欧美精品在线观看 | 午夜精品一区二区三区免费看| av视频在线观看入口| 成人国产一区最新在线观看| 国产综合懂色| 欧美成人性av电影在线观看| www.自偷自拍.com| 两个人看的免费小视频| 日本a在线网址| 又粗又爽又猛毛片免费看| 天堂影院成人在线观看| 一夜夜www| 大型黄色视频在线免费观看| 最新美女视频免费是黄的| 最近最新中文字幕大全免费视频| 91字幕亚洲| 中文字幕最新亚洲高清| 亚洲七黄色美女视频| 国产三级中文精品| 99热这里只有是精品50| 国产成人精品久久二区二区91| 大型黄色视频在线免费观看| 观看免费一级毛片| 欧美zozozo另类| 日本五十路高清| 免费在线观看日本一区| 麻豆av在线久日| 91av网一区二区| 人人妻人人看人人澡| 欧美成狂野欧美在线观看| 男女做爰动态图高潮gif福利片| 国产伦一二天堂av在线观看| 日韩有码中文字幕|