• <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 ?

    Percutaneous transhepatic portal catheterization guided by ultrasound technology for islet transplantation in rhesus monkey

    2012-07-07 15:48:28FengGaoShaoDongAiShengLiuWenBinZengandWeiWang

    Feng Gao, Shao-Dong Ai, Sheng Liu, Wen-Bin Zeng and Wei Wang

    Changsha, China

    Percutaneous transhepatic portal catheterization guided by ultrasound technology for islet transplantation in rhesus monkey

    Feng Gao, Shao-Dong Ai, Sheng Liu, Wen-Bin Zeng and Wei Wang

    Changsha, China

    BACKGROUND:Pig islet xenotransplantation has the potential to overcome the shortage of donated human islets for islet cell transplantation in type 1 diabetes. Testing in nonhuman primate models is necessary before clinical application in humans. Intraportal islet transplantation in monkeys is usually performed by surgical infusion during laparotomy or laparoscopy. In this paper, we describe a new method of percutaneous transhepatic portal catheterization (PTPC) as an alternative to current methods of islet transplantation in rhesus monkeys.

    METHODS:We performed ultrasound-guided PTPC in five adult rhesus monkeys weighing 7-8 kg, with portal vein catheterization confirmed by digital subtraction angiography. We monitored for complications in the thoracic and abdominal cavity. To evaluate the safety of ultrasound-guided PTPC, we recorded the changes in portal pressure throughout the microbead transplantation procedure.

    RESULTS:Ultrasound-guided PTPC and infusion of 16 000 microbeads/kg body weight into the portal vein was successful in all five monkeys. Differences in the hepatobiliary anatomy of rhesus monkeys compared to humans led to a higher initial complication rate. The first monkey died of abdominal hemorrhage 10 hours post-transplantation. The second suffered from a mild pneumothorax but recovered fully after taking only conservative measures. After gaining experience with the first two monkeys, we decreased both the hepatic puncture time and the number of puncture attempts required, with the remaining three monkeys experiencing no complications. Portal pressures initially increased proportionalto the number of transplanted microbeads but returned to preinfusion levels at 30 minutes post-transplantation. The changes in portal pressures occurring during the procedure were not significantly different.

    CONCLUSIONS:Ultrasound-guided PTPC is an effective, convenient, and minimally invasive method suitable for use in non-human primate models of islet cell transplantation provided that care is taken with hepatic puncture. Its advantages must be weighed against the risks of procedure-related complications.

    (Hepatobiliary Pancreat Dis Int 2012;11:154-159)

    ultrasound-guided; portal venous catheterization; islet xenotransplantation; rhesus monkey

    Introduction

    In recent years, the technologies related to the isolation and purification of islet cells and immunosuppressive therapies have been improved. These, along with international multi-center studies, suggest that pancreatic islet transplantation may play a greater role in the treatment of type 1 diabetes. Islet cell transplantation is one of the most effective treatments for type 1 diabetes.[1]Unfortunately, only a subset of patients have benefitted from islet transplantation due to the limited availability of donor islets. Pig islet xenotransplantation is one of the most promising means of alleviating this shortage.[2,3]It must be tested on nonhuman primates before large-scale clinical application.[4-6]The rhesus monkey is a valuable experimental model for diabetes research, including experimental pancreatic islet transplantation.[7,8]At present, intraportal islet transplantation in non-human primates is usually performed by cannulation of a mesenteric vein during laparotomy or laparoscopy.[9]In this paper, we describe a new method of intraportal microbead injection in rhesus monkeys that can serve as an alternative tocurrent methods of islet cell transplantation. This is a minimally invasive method involving ultrasound-guided percutaneous transhepatic portal catheterization (PTPC) for delivery of transplanted alginate microbeads into the portal vein. The hepatobiliary anatomy of the rhesus monkey is significantly different from that of the human being. In addition to a much smaller size, the rhesus monkey liver is composed of six relatively separate lobes (Fig. 1).[10]Alginate microbeads are similar in size to islets and show good biocompatibility. Here, we imitated islet transplantation using alginate microbeads instead of islets in order to evaluate the safety of ultrasound-guided PTPC. It proved to be a relatively easy, convenient, and feasible way to reach the portal bloodstream in the rhesus monkey. However, these advantages have to be balanced against a potentially higher rate of procedure-related complications in this non-human primate model.

    Methods

    Experimental animals

    Five rhesus monkeys 6-8 years old, each weighing 7-8 kg, were purchased from Gaoyao Kangda Laboratory Animal Science and Technology Co. Ltd. (Gaoyao, China). The protocols were approved by the Animal Ethics Committee of the Third Xiangya Hospital, Central South University (Changsha, China). Our protocols followed the guidelines regarding the care of laboratory animals issued by the Ministry of Science and Technology of China in 2006.[11]

    Preparation and counting of micro-capsules

    Fig. 1. Gross anatomy of the rhesus monkey hepatic portal vein, showing that it is composed of six relatively separate lobes.

    Alginate/poly-1-lysine/alginate microbeads were prepared as described previously.[12]Sodium alginate (from Sigma, USA) was dissolved in water to prepare the 1.5% (W/V) solution for injection. Then methylene blue was added for staining, and the mixture was stirred overnight, sterilized at high temperature, and stored at 4 ℃. An aqueous solution of 1.1% (W/V) calcium chloride was prepared. Then the filtered solution was sterilized at high temperature and stored at 4 ℃. The stained 1.5% (W/V) sodium alginate solution was loaded into a static microcapsule generator. The small droplets that formed were collected. The droplets were put into the 1.1% (W/V) calcium chloride suspension to produce microbeads. The solution was filtered with a 400-μm filter, and these microbeads were washed with phosphate-buffered saline (PBS). The morphologic features of the microbeads before transplantation were observed under an optical microscope.

    One drop of microcapsule suspension was dripped onto a counting plate along the rim and the number of microcapsules was determined under a low-power lens (×10). The number of capsules in four fields was calculated using the following formula: Number of microcapsules in 1 mL suspension=[(Number of microcapsules in four fields)/4]×10 000.

    PTPC and infusion of alginate microbeads

    The resulting microbeads were sized 50-300 μm in diameter: of the total beads, 26.1±6.0% were 50-150 μm, 35.5±5.7% were 151-250 μm, and 38.4±2.7% were 251-300 μm. Rhesus monkeys were slowly infused with microbeads suspended in 20 mL heparinized saline (200 U/mL) via the portal vein at 16 000 microbeads/kg. All procedures were carried out under general anesthesia induced by thiopentone sodium (15 mg/kg) and maintained with oxygen and 1%-2% halothane. Access to the portal vein was achieved using a percutaneous transhepatic technique to insert a lengthened 18G indwelling needle (1.6×170 mm; Wenzhou Lihua Corp., China) into the main portal vein under ultrasound guidance (color Doppler ultrasound; Acuson Corp., Issaquah, WA, USA) and digital subtraction angiography (DSA; Siemens, Forchheim, Germany).

    Each monkey was placed in the left lateral position. A 10-mL syringe was fixed on the lengthened 18G indwelling needle. The needle was inserted into the distal end of the right central lobe branch of the portal vein (equivalent to the right anterior branch of the human portal vein), and monitored by ultrasound via a rectal ultrasound probe placed in the intercostal space. If the syringe was pulled and blood flowed freely, a guidewire was inserted into the main portal vein along the needle. Then the internal metal needle was withdrawn while the external flexible catheter was inserted into the main portal vein. DSA was performed to show the location of the catheter and the structure of the portal vein both inside and outside the liver (Fig. 2).

    Fig. 2. DSA image of ultrasound-guided percutaneous transhepatic portal catheterization. The catheter was located properly in the portal vein.

    Infusion of the microbead suspension into the portal vein was carried out after PTPC in two separate injections. Each injection was performed at 2 mL/min to infuse a total of 10 mL microbead suspension. Each injection was followed by a 10-minute pause. Portal venous pressure (mmHg) was recorded before the two injections, immediately after each injection, and 30 minutes after the final injection using a pressure transducer (Medex, Hillard, OH, USA). The infusion route in the portal vein before and after microbead infusion was monitored by angiography (Siemens, Germany). After the operation, the intraparenchymal liver tract was sealed with gelfoam to prevent bleeding.[13]Each monkey was pressure-bandaged and remained anesthetized for 6-12 hours.

    Histological evaluation

    Histological analysis was performed at the end of the study, either 1 week after portal venous infusion or at autopsy, to determine whether the microbeads remained in the liver. Liver specimens were fixed in 10% formalin and processed in paraffin blocks for standard hematoxylin and eosin (HE) staining.

    Statistical analysis

    Data were expressed as mean±SEM. The statistical significance of differences between the groups was determined by repeated measures analysis of variance. AP<0.05 was considered statistically significant.

    Results

    Ultrasound-guided PTPC in rhesus monkeys

    Average total puncture time was 5 minutes per monkeyand the success rate of PTPC was 100%. Complications occurred after surgery in two monkeys. One experienced a mild pneumothorax, which resolved by itself, and the other suffered an intra-abdominal hemorrhage and died (Table). For animal 1 (animals are numbered in chronological order), difficulty was encountered in trying to distinguish the intrahepatic versus the extrahepatic segments of the portal vein only by intra-operative visualization of the liver. Because the technicians were unfamiliar with the hepatobiliary anatomy of the rhesus monkey, we mistakenly punctured the right branch of the portal vein, and the entirety of which is extrahepatic in the monkey. This caused bleeding at the puncture site, which was extrahepatic and at risk for persistent bleeding due to lack of the natural tamponade effect with intrahepatic portal vein puncture. The monkey died of blood loss and shock 10 hours after surgery. Due to the slow rate of bleeding, it was hard to monitor for postprocedural bleeding using ultrasound alone, even when this was performed immediately after surgery. In animal 2, since the diaphragm of the rhesus monkey is lower than that in humans, we failed to follow the respiratory rhythm and inadvertently inserted the needle when the diaphragm was not at its highest point as it would be during maximal expiration. The thoracic cavity was injured, causing pneumothorax. No specific therapeutic measure was taken for the pneumothorax and it healed by itself. We used the lessons from the first two operations to improve our technique. Total operation time was shortened greatly and the subsequent three monkeys experienced no complications.

    Table. PTPC in rhesus monkeys

    Changes in portal venous pressure at different times during PTPC and microbead injection

    Fig. 3. Effects of infused microbeads on portal venous pressure. Portal venous pressure was measured in recipient animals infused with microbeads via the portal vein at 16 000 microbeads/kg before the infusion procedure (pre), and after the first (1st) and second (2nd) injection of microbeads, and 30 minutes after the procedure (post). The data are presented as mean±SEM (n=5) (P>0.05).

    Fig. 4. HE-stained liver sections showing intact microbeads in the hepatic sinus (left) and the portal vessels (right) of all recipient rhesus monkeys.

    The microbeads were transplanted via the portal vein at 16 000 microbeads/kg body weight, and the portal venous pressure before, during, and after transplantation was measured (Fig. 3). The portal venous pressure was monitored in all animals both before and after microbead injections. The changes in the pressure were found to be related to the numbers of microbeads injected. Compared to the mean basal pressures recorded before the microbead injection (10.37±5.43 mmHg), the portal vein pressure increased slightly when about 50% of the microbeads had been transplanted, reaching a peak when all of the microbeads had been transplanted. This returned to the baseline by 30 minutes after completion of the procedure. The differences between the portal venous pressure measured before, during, and after the transplantation were not statistically significant.

    State of injected microbeads in hepatic vessels and hepatic sinuses of recipient animals

    HE-stained liver sections showed intact microbeads in the hepatic sinuses and hepatic vessels of all recipient monkeys after the intraportal injection (Fig. 4).

    Discussion

    Fig. 5. Ultrasonography of the right portal vein branch of the central lobe (red arrow), showing its 3.3-mm inner diameter.

    The development and application of medical imaging techniques have made it possible to perform minimally invasive punctures of the portal vein. Ultrasonographic technology has also accelerated progress in interventional medicine. Liver tissue is a good target for ultrasound imaging given that the internal and external structures can generally be visualized clearly. Selective transhepatic catheterization of the portal vein was first used as a diagnostic tool and as a means of acquiring access to bleeding esophageal varices.[14]This technique has a complication rate of <5%.[15]Percutaneous liver access for transhepatic cholangiography, biliary drainage procedures, and liver biopsy has become widely practiced, and has a proven safety record. However, portal vein puncture is difficult in rhesus monkeys because the animal is small, weighing around 6-8 kg or roughly one tenth the weight of an adult human. The anatomy of the rhesus monkey liver is significantly different from that of the human liver. It is clearly divided into six separate lobes, while the division of the lobes in humans is far more subtle, making it appear as one smooth organ. Moreover, the intercostal space in the rhesus monkey is narrow, the position of the diaphragm is low, the portal vein has a small caliber, and the common portal vein and its left and right branches are extrahepatic. The vessel suitable for puncture, the right portal vein branch of the central lobe, is short and small (inner diameter about 3 mm, length about 5 mm) (Fig. 5). It is therefore necessary to determine the safety and feasibility of ultrasound-guided PTPC in rhesus monkeys.

    In these experiments, we successfully established a rhesus monkey model of ultrasound-guided PTPC with a lengthened indwelling needle and a rectal ultrasound probe. The model has the following 5 major advantages. First, minimal invasiveness. In this model, the stress on the monkey is reduced, which makes for easier postoperative care and faster recovery. Second, single-step catheterization. After puncturing the vessel, the catheter is inserted directly with a guide wire as the needle is withdrawn. This reduces damage to the liver. After the conventional Selding puncture, the needle track mustbe expanded before the catheter can be inserted.[16]This is more complicated, requires more time, and increases both the damage to the surrounding liver tissue and the incidence of complications. Third, guidance by rectal ultrasound probe. The rectal ultrasound probe is so small that it can be easily placed into the intercostal space of rhesus monkeys, avoiding the ribs. The frequency is as high as 6-8 MHz, which is suitable for displaying the tiny structures of the portal vein. In addition, free guidance in narrow and small spaces is possible without much risk of collision between the puncture apparatus and the probe, making it possible to observe the needle’s path of insertion in a multi-angle, multi-directional manner.[17]Fourth, compatibility with percutaneous transhepatic portal vein catheterization. This method can be used with PTPC, the most common method in clinical pancreatic islet transplantation.[1,18-21]Fifth, the possibility of single-surgery treatment. Currently, an additional islet cell injection is usually required to maintain insulin independence. Our method makes multiple injections of additional islets in the same rhesus monkey possible without any need for new surgery.

    A few additional issues should be acknowledged: a) High-quality ultrasound guidance is required. b) The puncture must be performed in careful coordination with the monkeys’ respiratory rhythm. c) Large rhesus monkeys are preferred because their vessels are bigger and easier to puncture.

    In the above experiments, alginate microbeads, with good biocompatibility and similar sizes to islets, were used as islet substitutes for transplantation into the portal system. The safety of this method was assessed by monitoring the portal venous pressure. This rose as the number of transplanted alginate microbeads increased, but 30 minutes post-transplantation, the pressure returned to the pre-transplantation level. These variations in portal pressure are not necessarily indicative of danger. However, some clinical islet transplantations have involved much greater increases.[22]These changes in portal pressure are related to the volume of tissue injected and do not seem to be associated with the occurrence of complications or other undesirable outcomes.[23]All of the experimental animals survived except animal 1, which died of a hemorrhage, a peri-procedural complication unrelated to portal venous pressure changes. This animal’s death was secondary to operator unfamiliarity with the position and structure of the liver and portal vein. Despite the relatively small number of experimental animals, the success in the last three shows a promise for the feasibility and safety of ultrasound-guided PTPC. Our experience highlights the important differences in hepatobiliary anatomy between humans and monkeys, as well as the challenges in performing intricate procedures in animals only one tenth the size of humans. Our research could help some investigators and scientists avoid some of the pitfalls, such as those we encountered during our first 2 animals, which may pave the way for further research into xenotransplantation of pig islets into type 1 diabetes patients.

    In conclusion, sufficient knowledge of the model animal’s hepatobiliary anatomy, proper ultrasound guidance, skill, and careful pre-operative preparation and postoperative care are keys to establishing the rhesus monkey model of ultrasound-guided PTPC. Ultrasound-guided PTPC is a convenient, effective, and minimally invasive method suitable for use in nonhuman primate models of islet cell transplantation provided that care is taken with hepatic puncture. Its advantages must be weighed against the risks of procedure-related complications.

    Acknowledgement:We thank Rui-Zhen Li and Ping Zhou for their valuable suggestions for this work and their excellent technical assistance.

    Contributors:WW proposed the study. GF and ZWB wrote the first draft. GF, ASD and LS analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WW is the guarantor.

    Funding:The study was supported by grants from the Natural Science Foundation of Hunan Province (11JJ4078) and the National Natural Science Foundation of China (30900359 and 30900377).

    Ethical approval:Animal protocols were approved by the Animal Ethics Committee of the Third Xiangya Hospital, Central South University of China.

    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 Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006;355: 1318-1330.

    2 Igaz P. Xenotransplantation of islets of Langerhans: a possible therapy for diabetes mellitus? Wien Med Wochenschr 2001; 151:291-294.

    3 Cozzi E, Bosio E. Islet xenotransplantation: current status of preclinical studies in the pig-to-nonhuman primate model. Curr Opin Organ Transplant 2008;13:155-158.

    4 Sykes M, d'Apice A, Sandrin M; IXA Ethics Committee. Position paper of the Ethics Committee of the International Xenotransplantation Association. Xenotransplantation 2003; 10:194-203.

    5 Dufrane D, Gianello P. Pig islet xenotransplantation into nonhuman primate model. Transplantation 2008;86:753-760.

    6 Bottino R, Cooper DK. Islet xenotransplantation: the pig-tonon-human primate model. Xenotransplantation 2008;15:104-106.

    7 Hering BJ, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transpl Immunol 2009;21:81-86.

    8 Marigliano M, Casu A, Bertera S, Trucco M, Bottino R. Hemoglobin A1C Percentage in Nonhuman Primates: A Useful Tool to Monitor Diabetes before and after Porcine Pancreatic Islet Xenotransplantation. J Transplant 2011;2011: 965605.

    9 Hirshberg B, Montgomery S, Wysoki MG, Xu H, Tadaki D, Lee J, et al. Pancreatic islet transplantation using the nonhuman primate (rhesus) model predicts that the portal vein is superior to the celiac artery as the islet infusion site. Diabetes 2002;51:2135-2140.

    10 Chen Z, Wu D. Laboratory animal science. 1st Ed, Changsha: Hunan Science & Technology Press; 2006:91.

    11 The Ministry of Science and Technology of the People's Republic of China. Guidance suggestions for the care and use of laboratory animals. 2006-09-30. Available from: http://www. most.gov.cn/fggw/zfwj/zfwj2006/200609/t20060930_54389. htm.

    12 Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908-910.

    13 Villiger P, Ryan EA, Owen R, O'Kelly K, Oberholzer J, Al Saif F, et al. Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 2005;5:2992-2998.

    14 Viamonte M Jr, LePage J, Lunderquist A, Pereiras R, Russell E, Viamonte M, et al. Selective catheterization of the portal vein and its tributaries. Preliminary report. Radiology 1975;114: 457-460.

    15 Abdalla EK, Hicks ME, Vauthey JN. Portal vein embolization: rationale, technique and future prospects. Br J Surg 2001;88: 165-175.

    16 Lunderquist A, Vang J. Sclerosing injection of esophageal varices through transhepatic selective catheterization of the gastric coronary vein. A preliminary report. Acta Radiol Diagn (Stockh) 1974;15:546-550.

    17 Shi L, Tian F, Cai Z, et al. Ultrasonic guided percutaneous trans-hepatic biliary drainage (analysis of 2156 cases). J Surg Concepts Pract 2007;12:380-381.

    18 Gaglia JL, Shapiro AM, Weir GC. Islet transplantation: progress and challenge. Arch Med Res 2005;36:273-280.

    19 Robertson RP. Islet transplantation as a treatment for diabetes - a work in progress. N Engl J Med 2004;350:694-705.

    20 Korsgren O, Nilsson B, Berne C, Felldin M, Foss A, Kallen R, et al. Current status of clinical islet transplantation. Transplantation 2005;79:1289-1293.

    21 Owen RJ, Ryan EA, O'Kelly K, Lakey JR, McCarthy MC, Paty BW, et al. Percutaneous transhepatic pancreatic islet cell transplantation in type 1 diabetes mellitus: radiologic aspects. Radiology 2003;229:165-170.

    22 Casey JJ, Lakey JR, Ryan EA, Paty BW, Owen R, O'Kelly K, et al. Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 2002;74:913-915.

    23 Bucher P, Mathe Z, Bosco D, Becker C, Kessler L, Greget M, et al. Morbidity associated with intraportal islet transplantation. Transplant Proc 2004;36:1119-1120.

    July 13, 2011

    Accepted after revision December 16, 2011

    Author Affiliations: Cell Transplantation & Gene Therapy Institute (Gao F, Ai SD, Liu S and Wang W), and Department of Ultrasound (Gao F), Third Xiangya Hospital; School of Pharmaceutical Sciences (Zeng WB), Central South University, Changsha 410013, China

    Wei Wang, MD, PhD, Cell Transplantation & Gene Therapy Institute, Third Xiangya Hospital, Central South University, Changsha 410013, China (Tel: 86-731-88618411; Fax: 86-731-88852936; Email: wawe01cn@yahoo.com.cn)

    ? 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

    10.1016/S1499-3872(12)60141-6

    亚洲精品国产一区二区精华液| 欧美久久黑人一区二区| 永久免费av网站大全| 亚洲精品美女久久av网站| a级片在线免费高清观看视频| 国产日韩欧美亚洲二区| 在线观看www视频免费| 国产精品免费视频内射| 亚洲专区国产一区二区| 午夜福利影视在线免费观看| 青青草视频在线视频观看| 亚洲av电影在线观看一区二区三区| 91麻豆av在线| 自拍欧美九色日韩亚洲蝌蚪91| 18禁裸乳无遮挡动漫免费视频| 中文字幕人妻熟女乱码| 在线观看免费日韩欧美大片| av网站免费在线观看视频| 色网站视频免费| 大陆偷拍与自拍| 亚洲人成77777在线视频| 蜜桃在线观看..| 精品亚洲成国产av| 国产精品麻豆人妻色哟哟久久| 中文字幕制服av| 晚上一个人看的免费电影| 久久久久久人人人人人| 欧美亚洲日本最大视频资源| 亚洲精品一区蜜桃| 日本av手机在线免费观看| 免费av中文字幕在线| 久久久国产一区二区| av网站在线播放免费| 熟女少妇亚洲综合色aaa.| 一级毛片 在线播放| 精品国产一区二区久久| 99热国产这里只有精品6| 又大又黄又爽视频免费| 日本一区二区免费在线视频| 日本午夜av视频| 欧美日韩一级在线毛片| 精品国产一区二区久久| 操美女的视频在线观看| 777米奇影视久久| 婷婷丁香在线五月| 午夜久久久在线观看| 日韩中文字幕视频在线看片| 日韩中文字幕视频在线看片| 少妇的丰满在线观看| 精品久久蜜臀av无| 久久久国产一区二区| 欧美另类一区| 中国国产av一级| 国产精品一区二区免费欧美 | 一本一本久久a久久精品综合妖精| 久久天堂一区二区三区四区| 亚洲男人天堂网一区| 精品人妻熟女毛片av久久网站| 99国产精品免费福利视频| 99国产精品免费福利视频| 午夜福利乱码中文字幕| 丝袜在线中文字幕| 老司机深夜福利视频在线观看 | 一区二区三区四区激情视频| 黄色毛片三级朝国网站| 男男h啪啪无遮挡| 午夜日韩欧美国产| av电影中文网址| 国产精品一区二区精品视频观看| 麻豆乱淫一区二区| 悠悠久久av| www.自偷自拍.com| 亚洲成人免费av在线播放| 国产精品麻豆人妻色哟哟久久| 人妻 亚洲 视频| 少妇被粗大的猛进出69影院| 在线精品无人区一区二区三| 成人免费观看视频高清| 日本a在线网址| 男女下面插进去视频免费观看| 久久精品亚洲av国产电影网| 操出白浆在线播放| 色婷婷av一区二区三区视频| 午夜激情久久久久久久| 久久ye,这里只有精品| 啦啦啦在线免费观看视频4| 9191精品国产免费久久| 夜夜骑夜夜射夜夜干| 97精品久久久久久久久久精品| 国产精品久久久久久精品古装| 在线亚洲精品国产二区图片欧美| 亚洲伊人色综图| 国产xxxxx性猛交| 欧美黑人精品巨大| 午夜福利一区二区在线看| 91精品三级在线观看| 国产av精品麻豆| 欧美精品一区二区大全| 制服诱惑二区| 国产欧美日韩综合在线一区二区| 久久久久久亚洲精品国产蜜桃av| 777米奇影视久久| 亚洲精品久久午夜乱码| 国产免费一区二区三区四区乱码| 欧美性长视频在线观看| 亚洲欧美日韩另类电影网站| 欧美日韩av久久| 精品久久蜜臀av无| 女人精品久久久久毛片| 国产精品 国内视频| 成人手机av| 国产精品一国产av| 国产高清不卡午夜福利| 久久中文字幕一级| 亚洲视频免费观看视频| 午夜福利一区二区在线看| 深夜精品福利| 成人亚洲欧美一区二区av| 国产福利在线免费观看视频| 国产女主播在线喷水免费视频网站| 一级片免费观看大全| 高清av免费在线| 久久久精品94久久精品| 中文字幕最新亚洲高清| 男男h啪啪无遮挡| 国产精品久久久久久精品电影小说| 九色亚洲精品在线播放| 国产精品99久久99久久久不卡| 国产视频一区二区在线看| 黑人巨大精品欧美一区二区蜜桃| 日韩电影二区| 在线精品无人区一区二区三| 老司机影院毛片| 亚洲黑人精品在线| 国产精品一国产av| 亚洲国产欧美在线一区| 搡老乐熟女国产| 国产免费一区二区三区四区乱码| 丝袜脚勾引网站| 蜜桃国产av成人99| 精品亚洲成a人片在线观看| av欧美777| 99re6热这里在线精品视频| 精品国产乱码久久久久久男人| 大型av网站在线播放| 只有这里有精品99| 午夜老司机福利片| 国产男人的电影天堂91| 黄色怎么调成土黄色| 国语对白做爰xxxⅹ性视频网站| 亚洲精品成人av观看孕妇| 三上悠亚av全集在线观看| 国产真人三级小视频在线观看| 亚洲人成网站在线观看播放| 美女高潮到喷水免费观看| 日本a在线网址| 国产精品国产三级专区第一集| 亚洲av国产av综合av卡| 亚洲av成人精品一二三区| 亚洲欧洲精品一区二区精品久久久| 大片免费播放器 马上看| 一级a爱视频在线免费观看| 黑人巨大精品欧美一区二区蜜桃| 精品亚洲成a人片在线观看| 人妻一区二区av| 性色av乱码一区二区三区2| 久久久亚洲精品成人影院| 国产一区二区在线观看av| 精品国产一区二区久久| 久久影院123| 丰满少妇做爰视频| 丰满饥渴人妻一区二区三| 丝瓜视频免费看黄片| 国产又爽黄色视频| 看免费成人av毛片| 久久久久久久久久久久大奶| 亚洲av成人精品一二三区| 国产激情久久老熟女| 高清不卡的av网站| 一级a爱视频在线免费观看| av线在线观看网站| 亚洲,欧美,日韩| 亚洲七黄色美女视频| 国产极品粉嫩免费观看在线| 夫妻午夜视频| 国语对白做爰xxxⅹ性视频网站| 亚洲精品中文字幕在线视频| 巨乳人妻的诱惑在线观看| 老汉色av国产亚洲站长工具| 99国产精品一区二区蜜桃av | 亚洲av国产av综合av卡| 在线av久久热| 人人妻人人添人人爽欧美一区卜| 国产老妇伦熟女老妇高清| www.熟女人妻精品国产| 国产成人欧美在线观看 | 韩国高清视频一区二区三区| 国产片特级美女逼逼视频| 99久久综合免费| 久久人人爽av亚洲精品天堂| 熟女少妇亚洲综合色aaa.| 中文字幕人妻熟女乱码| 如日韩欧美国产精品一区二区三区| 亚洲精品日韩在线中文字幕| 国产女主播在线喷水免费视频网站| 日韩熟女老妇一区二区性免费视频| 亚洲人成网站在线观看播放| 日韩 欧美 亚洲 中文字幕| 熟女av电影| 国产精品 国内视频| 成人国语在线视频| 国产免费视频播放在线视频| 亚洲欧美激情在线| 美女中出高潮动态图| 成人免费观看视频高清| 91国产中文字幕| 一边亲一边摸免费视频| 91精品国产国语对白视频| 亚洲专区国产一区二区| 美女中出高潮动态图| 国产三级黄色录像| 欧美日韩视频精品一区| 男女高潮啪啪啪动态图| 亚洲午夜精品一区,二区,三区| 亚洲欧美色中文字幕在线| 亚洲图色成人| 成人免费观看视频高清| 欧美大码av| 亚洲一区二区三区欧美精品| 精品人妻1区二区| 精品福利观看| 国产精品一区二区免费欧美 | 久久久久久久精品精品| 高清欧美精品videossex| 亚洲国产欧美日韩在线播放| 欧美变态另类bdsm刘玥| 51午夜福利影视在线观看| 国产国语露脸激情在线看| 欧美精品av麻豆av| 午夜91福利影院| 亚洲精品国产区一区二| 日韩欧美一区视频在线观看| 狠狠婷婷综合久久久久久88av| 亚洲人成电影免费在线| 欧美少妇被猛烈插入视频| 午夜日韩欧美国产| 晚上一个人看的免费电影| 亚洲成av片中文字幕在线观看| 麻豆国产av国片精品| 久久久久精品国产欧美久久久 | 日本一区二区免费在线视频| 国产精品欧美亚洲77777| 九色亚洲精品在线播放| 人妻 亚洲 视频| 男的添女的下面高潮视频| 日韩一卡2卡3卡4卡2021年| 午夜福利视频精品| 久久久久久人人人人人| 看免费成人av毛片| 欧美日韩成人在线一区二区| 欧美另类一区| 久久久久网色| 午夜影院在线不卡| 欧美日韩综合久久久久久| 少妇人妻 视频| 在线观看免费高清a一片| 少妇粗大呻吟视频| 国产一区二区激情短视频 | 日本色播在线视频| 制服人妻中文乱码| 亚洲欧美日韩另类电影网站| 少妇被粗大的猛进出69影院| 欧美日韩视频高清一区二区三区二| 亚洲天堂av无毛| 亚洲欧美日韩高清在线视频 | 一区二区av电影网| 大片免费播放器 马上看| 午夜免费成人在线视频| 国产熟女欧美一区二区| 日韩大片免费观看网站| 不卡av一区二区三区| 午夜影院在线不卡| 国产成人精品久久久久久| 一本色道久久久久久精品综合| 美国免费a级毛片| 精品人妻1区二区| 90打野战视频偷拍视频| 女警被强在线播放| 看免费av毛片| 三上悠亚av全集在线观看| 女人久久www免费人成看片| 亚洲国产av新网站| 中文字幕高清在线视频| 丁香六月天网| 永久免费av网站大全| 国产激情久久老熟女| 精品少妇一区二区三区视频日本电影| 人妻 亚洲 视频| 亚洲一区二区三区欧美精品| 中文字幕最新亚洲高清| 婷婷成人精品国产| 看十八女毛片水多多多| 少妇人妻 视频| 午夜日韩欧美国产| 精品亚洲成国产av| 男女下面插进去视频免费观看| 亚洲国产中文字幕在线视频| 9热在线视频观看99| 日韩精品免费视频一区二区三区| 一级,二级,三级黄色视频| 久久热在线av| 老汉色∧v一级毛片| 欧美人与善性xxx| 亚洲色图 男人天堂 中文字幕| 久久综合国产亚洲精品| 婷婷成人精品国产| 看十八女毛片水多多多| 成年女人毛片免费观看观看9 | 日日夜夜操网爽| 亚洲精品国产av蜜桃| 老司机影院毛片| 成人国产av品久久久| 久久鲁丝午夜福利片| 国产精品秋霞免费鲁丝片| 欧美国产精品va在线观看不卡| 无遮挡黄片免费观看| 亚洲成色77777| 老司机午夜十八禁免费视频| 国产免费一区二区三区四区乱码| 国产亚洲av片在线观看秒播厂| 大话2 男鬼变身卡| 精品一区在线观看国产| 国产精品久久久久久精品古装| 岛国毛片在线播放| 亚洲伊人久久精品综合| 色婷婷久久久亚洲欧美| 欧美xxⅹ黑人| 婷婷丁香在线五月| 一级黄片播放器| av有码第一页| 最新在线观看一区二区三区 | 国产免费又黄又爽又色| 电影成人av| 亚洲 欧美一区二区三区| www.自偷自拍.com| 免费女性裸体啪啪无遮挡网站| 大码成人一级视频| 国产精品一区二区精品视频观看| 国产91精品成人一区二区三区 | 女人高潮潮喷娇喘18禁视频| 最近最新中文字幕大全免费视频 | h视频一区二区三区| 丝袜人妻中文字幕| 久久国产精品人妻蜜桃| 国产成人欧美| 国产亚洲精品久久久久5区| 欧美中文综合在线视频| 亚洲五月色婷婷综合| 国产在线视频一区二区| 操美女的视频在线观看| 一边摸一边抽搐一进一出视频| kizo精华| 国产精品成人在线| 咕卡用的链子| 另类亚洲欧美激情| 久久99精品国语久久久| 大片免费播放器 马上看| 青春草视频在线免费观看| 午夜av观看不卡| 我要看黄色一级片免费的| 午夜免费男女啪啪视频观看| 欧美日韩福利视频一区二区| 国产免费一区二区三区四区乱码| 成年动漫av网址| 日韩伦理黄色片| a级毛片黄视频| 亚洲国产精品999| 亚洲成国产人片在线观看| 97精品久久久久久久久久精品| 成年人免费黄色播放视频| 操美女的视频在线观看| 久久久久国产精品人妻一区二区| 啦啦啦 在线观看视频| 一本色道久久久久久精品综合| 黄网站色视频无遮挡免费观看| 天天添夜夜摸| 亚洲伊人久久精品综合| 亚洲中文日韩欧美视频| 男女无遮挡免费网站观看| 中文欧美无线码| 欧美人与性动交α欧美软件| videos熟女内射| 人人妻人人澡人人爽人人夜夜| www.精华液| 男人爽女人下面视频在线观看| 青春草视频在线免费观看| 观看av在线不卡| 热re99久久国产66热| 亚洲少妇的诱惑av| 欧美日韩成人在线一区二区| 精品卡一卡二卡四卡免费| 蜜桃在线观看..| 日韩一卡2卡3卡4卡2021年| 久久国产精品影院| 成人亚洲精品一区在线观看| 亚洲国产中文字幕在线视频| 每晚都被弄得嗷嗷叫到高潮| 久久人妻熟女aⅴ| 亚洲情色 制服丝袜| 自线自在国产av| 精品免费久久久久久久清纯 | 亚洲精品国产色婷婷电影| 日本五十路高清| 亚洲精品日韩在线中文字幕| 丰满少妇做爰视频| 18禁国产床啪视频网站| xxxhd国产人妻xxx| 国产精品香港三级国产av潘金莲 | 亚洲精品成人av观看孕妇| 精品国产一区二区三区久久久樱花| √禁漫天堂资源中文www| 久久人人97超碰香蕉20202| a级毛片在线看网站| 91精品国产国语对白视频| 在线观看免费视频网站a站| 视频区图区小说| 久久久久国产精品人妻一区二区| 国产成人精品在线电影| 亚洲欧洲精品一区二区精品久久久| 精品亚洲成a人片在线观看| 丝袜人妻中文字幕| 2021少妇久久久久久久久久久| 亚洲,欧美精品.| 日韩大片免费观看网站| 一区二区三区激情视频| 美女脱内裤让男人舔精品视频| 搡老乐熟女国产| 免费黄频网站在线观看国产| 波多野结衣一区麻豆| 欧美日韩精品网址| 国产高清不卡午夜福利| 久久影院123| 精品亚洲成国产av| 丰满饥渴人妻一区二区三| 一本综合久久免费| 亚洲成人免费av在线播放| 夫妻性生交免费视频一级片| 99久久精品国产亚洲精品| 99久久综合免费| 精品人妻一区二区三区麻豆| 亚洲成色77777| 两人在一起打扑克的视频| 亚洲欧美清纯卡通| 三上悠亚av全集在线观看| 黄色一级大片看看| 18禁黄网站禁片午夜丰满| 欧美xxⅹ黑人| 国产成人影院久久av| 久久午夜综合久久蜜桃| 欧美日韩av久久| 久久国产精品男人的天堂亚洲| 在线看a的网站| 午夜福利视频在线观看免费| 国产成人一区二区在线| 午夜av观看不卡| xxxhd国产人妻xxx| 涩涩av久久男人的天堂| 操出白浆在线播放| 99热全是精品| 国产欧美亚洲国产| 国产精品三级大全| 久久精品国产a三级三级三级| 汤姆久久久久久久影院中文字幕| 一本久久精品| 波野结衣二区三区在线| 亚洲国产精品一区三区| 欧美在线黄色| 国产成人91sexporn| 丝袜在线中文字幕| 母亲3免费完整高清在线观看| 一本大道久久a久久精品| 久久鲁丝午夜福利片| 亚洲欧洲精品一区二区精品久久久| 天天影视国产精品| 极品人妻少妇av视频| 成年av动漫网址| 欧美日韩视频精品一区| 国产精品免费视频内射| 免费观看人在逋| 国产成人免费无遮挡视频| 男女床上黄色一级片免费看| 国产激情久久老熟女| 日本欧美视频一区| 亚洲av成人精品一二三区| 日韩一区二区三区影片| 日韩人妻精品一区2区三区| 超碰成人久久| 中文字幕人妻丝袜制服| 亚洲精品av麻豆狂野| 精品人妻熟女毛片av久久网站| 中文字幕最新亚洲高清| 一区福利在线观看| 日韩大片免费观看网站| 黄色 视频免费看| 只有这里有精品99| 少妇 在线观看| 久久99一区二区三区| 婷婷色综合www| 国产有黄有色有爽视频| 欧美日韩国产mv在线观看视频| 国产精品一区二区在线观看99| 日日爽夜夜爽网站| 老汉色av国产亚洲站长工具| 欧美国产精品一级二级三级| 男女无遮挡免费网站观看| 亚洲成国产人片在线观看| 涩涩av久久男人的天堂| 欧美日韩福利视频一区二区| 国产无遮挡羞羞视频在线观看| 视频在线观看一区二区三区| 水蜜桃什么品种好| 男的添女的下面高潮视频| a级毛片黄视频| 交换朋友夫妻互换小说| 中文乱码字字幕精品一区二区三区| 男女之事视频高清在线观看 | 一级黄片播放器| 日韩av不卡免费在线播放| www日本在线高清视频| 亚洲九九香蕉| 又黄又粗又硬又大视频| 久久人妻福利社区极品人妻图片 | 国产精品.久久久| av国产精品久久久久影院| 99re6热这里在线精品视频| 黄片播放在线免费| 亚洲男人天堂网一区| 搡老乐熟女国产| 亚洲成人国产一区在线观看 | 考比视频在线观看| 国产男女内射视频| 精品人妻在线不人妻| 国产亚洲午夜精品一区二区久久| 男人添女人高潮全过程视频| 首页视频小说图片口味搜索 | 精品国产一区二区久久| 亚洲图色成人| 桃花免费在线播放| av线在线观看网站| 少妇人妻 视频| 十八禁高潮呻吟视频| 女人精品久久久久毛片| 亚洲伊人色综图| 国产成人免费观看mmmm| tube8黄色片| 国产男人的电影天堂91| 久久精品熟女亚洲av麻豆精品| 2018国产大陆天天弄谢| 亚洲精品久久成人aⅴ小说| 亚洲国产中文字幕在线视频| 午夜影院在线不卡| 欧美人与性动交α欧美精品济南到| 高清欧美精品videossex| 成年人免费黄色播放视频| www.熟女人妻精品国产| 日韩制服丝袜自拍偷拍| 只有这里有精品99| 欧美国产精品va在线观看不卡| 十八禁网站网址无遮挡| 黑人猛操日本美女一级片| 精品亚洲成a人片在线观看| 嫁个100分男人电影在线观看 | 精品视频人人做人人爽| 欧美日韩福利视频一区二区| 国产精品久久久久久精品电影小说| 看免费成人av毛片| 夫妻午夜视频| videos熟女内射| 别揉我奶头~嗯~啊~动态视频 | 色视频在线一区二区三区| 久久精品熟女亚洲av麻豆精品| 99热国产这里只有精品6| 最近手机中文字幕大全| av线在线观看网站| 国产精品成人在线| 亚洲一区中文字幕在线| 国产精品.久久久| 久久综合国产亚洲精品| 亚洲精品美女久久av网站| 美女福利国产在线| 久久国产精品大桥未久av| 精品国产国语对白av| 精品免费久久久久久久清纯 | 丰满饥渴人妻一区二区三| 成人黄色视频免费在线看| 黄网站色视频无遮挡免费观看| 国产精品一国产av| 好男人电影高清在线观看| 性少妇av在线| tube8黄色片| 国产成人啪精品午夜网站| 亚洲视频免费观看视频| 亚洲 欧美一区二区三区| 99国产综合亚洲精品| 欧美性长视频在线观看| 母亲3免费完整高清在线观看| 国产日韩欧美视频二区| av福利片在线| 国产亚洲av片在线观看秒播厂| 777米奇影视久久| 久热这里只有精品99| 纵有疾风起免费观看全集完整版| 亚洲自偷自拍图片 自拍| 久久99一区二区三区| 建设人人有责人人尽责人人享有的|