• <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 ?

    Autosomal recessive 333 base pair interleukin 10 receptor alpha subunit deletion in very early-onset inflammatory bowel disease

    2021-12-06 08:54:40JiaJiaLvWenSuXiaoYanChenYiYuXuXuChunDiXuXingDengJieBinHuangXinQiongWangYuanXiao
    World Journal of Gastroenterology 2021年44期
    關(guān)鍵詞:實踐證明放養(yǎng)密度青蝦

    Jia-Jia Lv, Wen Su, Xiao-Yan Chen, Yi Yu, Xu Xu, Chun-Di Xu, Xing Deng, Jie-Bin Huang, Xin-Qiong Wang,Yuan Xiao

    Abstract

    Key Words: Interleukin 10 receptor alpha subunit mutation; Very early-onset inflammatory bowel disease; Whole-genome sequencing; Immunodeficiency; Crohn’s disease; Wholeexon sequencing

    INTRODUCTION

    Inflammatory bowel disease (IBD) in children < 6 years of age is known as very earlyonset IBD (VEO-IBD)[1 ] and represents a specific disease course with a distinct phenotype that can be more severe and refractory than classic IBD[2 -3 ]. Recent studies suggested that patients with VEO-IBD, particularly those with symptoms such as perianal disease soon after birth, suffer from failed treatment, indicating a monogenic type of disease[4 -6 ].

    By utilizing next-generation sequencing (NGS), many genetic disorders associated with epithelial defects or immunodeficiencies have been found in patients[7 -9 ].Notably, interleukin 10 receptor alpha subunit (IL10 RA) dysfunction is the most common cause of the disease in East Asians, particularly in the Chinese, Japanese, and Korean populations[10 -12 ].

    According to our previous retrospective study, increased serum ferritin levels in VEO-IBD patients are indicative of monogenic disease, and very high serum levels of IL-10 suggest that patients with VEO-IBD are more likely to have IL10RAmutations[13 ].

    We report four cases clinically diagnosed with VEO-Crohn's disease with high serum IL-10 levels, indicating IL10RAdysfunction. However, neither results of targeted gene panel sequencing (TGPS) nor those of whole-exome sequencing (WES)in the probands were conclusive. Whole-genome sequencing (WGS) was performed in two patients, and a novel 333 -bp deletion in IL10RAwas identified. The results of trio-WES for the other two patients were subsequently reanalyzed, and the same novel 333 -bp deletion was found.

    MATERIALS AND METHODS

    Patients

    Four patients with VEO-IBD, including two boys and two girls, were enrolled in our study. The medical history and clinical characteristics of the patients are summarized in Table 1 . All patients were of Chinese Han ethnicity and were born to parents who were non-consanguineous, presented disease at the age of less than 1 year (range: 11 d to 8 mo), and experienced severe diarrhea with fistulas in the perianal region; blood samples were collected from three healthy volunteers.

    Written informed consent was obtained from the parents of the four patients who participated in the study. This study was approved by the Institutional Review Board of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (No. 2019 -15 ).

    養(yǎng)殖生產(chǎn)實踐證明,青蝦與小龍蝦混養(yǎng)是可行的。在飼養(yǎng)期間要注意以下的問題:①要合理控制青蝦與小龍蝦的放養(yǎng)密度,密度過大時小龍蝦可能攝食青蝦,青蝦也可以攝食小龍蝦。②混養(yǎng)池塘水體的溶解氧應(yīng)當(dāng)保持在5.0mg/L以上,最低也不能低于3.0mg/L。③要保證充足的飼料供應(yīng),以防青蝦與小龍蝦之間,或青蝦內(nèi)部間,或小龍蝦內(nèi)部間相互殘殺。④青蝦與小龍蝦一旦達到商品規(guī)格,即捕撈出售。

    Whole-genome sequencing

    Sample preparation and WGS were carried out by Beijing Berry Genomics Co., Ltd.(Beijing, China). The quality of the isolated genomic DNA was verified using the following two methods: (1 ) DNA degradation and contamination were monitored by electrophoresis on 1 % agarose gels; and (2 ) DNA concentration was measured using the Qubit DNA Assay Kit and Qubit 2 .0 Fluorometer (Life Technologies, Carlsbad, CA,United States).

    A total of 1 μg DNA per sample was used as the input material for DNA library preparation. The DNA sequencing library was generated using the CLEANNGS DNA kit following the manufacturer’s recommendations, and indexing codes were added to each sample. Briefly, genomic DNA samples were enzymatically disrupted to a size of 350 bp. The DNA fragments were then end-polished, a-tailed, and ligated with a fulllength adapter for Illumina sequencing, followed by further polymerase chain reaction(PCR) amplification. After the PCR products were purified (AMPure XP system,Beckman, Brea, CA, United States), libraries were analyzed to determine theirsize distribution using an Agilent 2100 Bioanalyzer (Santa Clara, CA, United States) and quantified by qPCR.

    Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the NovaSeq 5000 /6000 S4 Reagent Kit (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. After cluster generation, the DNA libraries were sequenced on an Illumina NovaSeq 6000 platform, and 150 -bp pairedend reads were generated.

    The pathogenicity of all mutations was further evaluated according to the American College of Medical Genetics and Genomics guidelines.

    Isolation and stimulation of peripheral blood mononuclear cells

    Peripheral blood mononuclear cells (PBMCs) were isolated according to a previous study, with minor modifications[14 ]. Briefly, blood was drawn from patient D and healthy controls by standard venipuncture in our pediatric ward and collected into a tube containing ethylenediamine tetraacetic acid. Blood (4 mL) was diluted 1 :1 with sterile RPMI 1640 medium (Hyclone, Logan, UT, United States) at room temperature(RT) and carefully dropped into a 15 -mL tube (Corning, Inc., Corning, NY, United States) containing 4 mL Ficoll-Paque Plus (GE Healthcare, Little Chalfont, United Kingdom). Notably, diluted blood was present on the surface of the Ficoll gradient.The 15 -mL tube was centrifuged at 800 × g at RT for 20 min (brake off), after which the buffy coat was carefully aspirated and transferred to another sterile 15 -mL tube. After washing the cells with 5 mL RPMI 1640 medium three times by centrifugation at 400 ×g for 15 min at RT, most of the supernatant, as much as possible, was pipetted off.

    Cells were aspirated with complete RPMI 1640 (10 % fetal bovine serum and 1 %penicillin-streptomycin) and cultured in 6 -well plates at a density of 2 × 106 cells/well.Four groups of PBMCs from patients or healthy controls were established as follows:Unstimulated phosphate buffered saline (PBS), lipopolysaccharide (LPS) (100 ng/mL),LPS (100 ng/mL) + IL-10 (20 ng/mL), and IL-10 (20 ng/mL)[15 ]. Cells were cultured in the indicated milieu for 12 h at 37 °C. Proteins in PBS-, LPS-, and IL-10 -stimulated PBMCs were collected in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors for western blot analysis. The supernatants of the PBS-,LPS-, and LPS + IL-10 -stimulated PBMCs were collected to determine tumor necrosis factor-α (TNF-α) level.

    Table 1 Clinical and laboratory characteristics of patients

    Western blot analysis

    Western blotting was performed as described previously[16 ]. Polyvinylidene fluoride membranes were blotted with monoclonal antibodies against phos-signal transducer and activator of transcription 3 (STAT3 ) (Tyr705 ), phos-STAT3 (Ser727 ), STAT3 (Cell Signaling Technology, Danvers, MA, United States), and glyceraldehyde-3 -phosphate dehydrogenase (Servicebio, Wuhan, China). Horseradish peroxidase-conjugated antimouse and anti-rabbit (Cell Signaling Technology) secondary antibodies were detected using a chemiluinescent substrate (Millipore, Billerica, MA, United States). Images were captured using an automatic chemiluminescence image analysis system (Tanon,Shanghai, China).

    Enzyme-linked immunosorbent assay

    The supernatant of PBMCs after stimulation with PBS, LPS or LPS + IL-10 was collected. IL-10 and TNF-α levels were determined using sandwich ELISA kits(DAKEWE, Shenzhen, China) according to the manufacturer’s instructions.

    Statistical analysis

    Continuous variables are presented as the mean ± SEM, and the unpaired two-tailed Student’st-test or analysis of variance was used to compare the differences between groups as appropriate. Bonferroni correction was used for pairwise comparisons(GraphPad Prism v.5 .0 software; GraphPad, Inc., La Jolla, CA, United States). Statistical significance was set atP< 0 .05 .

    RESULTS

    Clinical characteristics of four patients with VEO-IBD

    All four patients had severe diarrhea (> 6 times/d) and hematochezia during the first year of life. Patients C and D suffered from the disease during the newborn period. In addition to gastrointestinal symptoms, all cases exhibited extraintestinal manifestations, such as perianal abscesses, skin tags (Figure 1 A), rectoperineal fistula, failure to thrive, recurrent otitis, urinary tract or respiratory infection, folliculitis, and even sepsis (Table 1 ). Elevated numbers of peripheral white blood cells and platelets and decreased hemoglobin and albumin levels were found in each patient. Remarkably,immune-related investigations showed that all patients had high serum levels of IL-10 (Table 1 ). All patients underwent colonoscopy and intestinal biopsy under general anesthesia, revealing erosive lesions (Figure 1 B), and were diagnosed with Crohn’s disease.

    Identification of a novel compound heterozygous mutation in IL1 0 RA

    Before admission, all patients underwent TGPS or trio-WES. Two heterozygous pathogenic variants ofIL10 RA were detected in three patients (patient A: c.301 C>T,p.R101 W; patients B and C: c.537 G>A, p.T179 T) (Figure 1 C and Supplementary Figures 1 and 2 ). Mutation of c.537 G>A occurred at the exon-intron boundary of exon 4 , which is a variant hotspot and disrupts RNA splicing (Figure 2 A). No pathogenic or likely pathogenic variants were found in patient D.

    AsIL10RAmutation causes infantile IBD in an autosomal recessive manner and serum levels of IL-10 were very high in the four infantile patients with IBD, which is a valuable clinical indicator for identifying infantile IBD as a monogenic disease as we demonstrated previously[13 ], we suspected that mutations had been overlooked in WES owing to the techniques’ limitations. After performing WGS in patients A and B,the breakpoints of the novel deletion were identified by manual review and correction.The deletion was located at chr11 :117857030 upstream of exon 1 and chr11 :117857362 in intron 1 , which contains the 5 ′-untranslated region (UTR), all of exon 1 , and part of intron 1 in IL10 RA (Figure 1 D and Figure 2 B). PCR revealed a paternally-inherited 333 -bp deletion in addition to the point mutations mentioned above (Figure 1 E). The deletion and point mutations were inherited from both parents and eventually constituted compound heterozygotes in patients B (Figure 1 F) and A (Supplementary Figure 1 ).

    After identifying the 333 -bp deletion in the gene in patients A and B, we reanalyzed the trio-WES data for patients C and D, specifically in the region from 117857030 and 117857362 on chromosome 11 and detected the same deletion. Patient C was a compound heterozygous carrier for c.537 G>A, p.T179 T (maternal), and the 333 -bp deletion (paternal) (Supplementary Figure 2 ), and patient D was homozygous for the 333 -bp deletion.

    Histological and functional analysis of patient DIL1 0 RA deletion

    Histological findings in a specimen from the colon obtained during colonoscopy revealed oval-shaped intramural abscesses in the submucosa (Figure 3 A). Figure 3 B shows a higher magnification of inset 1 in Figure 3 A, depicting intramural microabscesses.

    To determine whether the novel 333 -bp deletion in IL10 RA caused IL-10 R dysfunction and subsequently inhibited TNF-α production, supernatants of cultured PBMCs were collected and used to determine TNF-α levels. In healthy controls, LPS stimulation caused a remarkable increase in TNF-α production, whereas addition of IL-10 significantly decreased its abundance. Although LPS led to increased TNF-α production in patient D, this phenomenon was not reversed by the addition of IL-10 ,as observed in the healthy controls (Figure 3 C).

    To clarify the exact mechanisms involved, PBMCs were isolated from patient D because the patient was homozygous for theIL10RAdeletion. PBMCs were stimulated with LPS in the presence or absence of IL-10 . The results of western blot analysis showed that in PBMCs from healthy controls, both LPS and IL-10 stimulation caused an increase in the phosphorylation of STAT3 at Tyr705 but not at Ser727 (Figure 3 D).In PBMCs from patient D, LPS stimulation also induced increased phosphorylation of STAT3 at Tyr705 but not at Ser727 . However, IL-10 stimulation failed to significantly increase phosphorylation of STAT3 at Tyr705 in PBMCs of patient D compared with that in PBS-stimulated PBMCs. No significant differences in STAT3 phosphorylation at Ser727 were observed among the PBS-, LPS-, and IL-10 -stimulated PBMCs from patient D (Figure 3 D).

    Figure 1 Identification of a novel 333 -bp deletion spanning interleukin 10 receptor alpha subunit exon1 . A: Perianal skin tag; B: Endoscopic image of ulcerations; C: Sanger DNA sequencing verified a compound heterozygous variant (c.537 G>A) inherited from the mother in patient B; D: Whole-genome sequencing (WGS) data showing sequencing read pairs at breakpoints chr:117857030 and chr:117857362 of interleukin 10 receptor alpha subunit (IL10 RA); E:Polymerase chain reaction validated the heterozygous deletion of 333 bp spanning exon1 inherited from the father; F: WGS revealed compound heterozygous variants of IL1 0 RA in patient B with very early-onset inflammatory bowel disease. bp: Base pair; HD: Healthy donor.

    DISCUSSION

    VEO-IBD is challenging to diagnose and treat because the patients are critically ill and exhibit numerous potential mono-genic defects. Approximately 56 Mendelian genetic defects that can lead to IBD-like colitis have been identified, some of which show almost 100 % penetrance, such as defects in IL-10 , IL10 RA, IL10 RB, FoxP3 , and XIAP[1 ].NGS has led to breakthroughs in the diagnosis of genetic diseases, including monogenic VEO-IBD. According to a recent single-center study performed by Crowleyet al[17 ], 7 .8 % of VEO-IBD (141 patients) and 13 .8 % of infantile-onset IBD (29 patients)cases had rare variations associated with monogenic genes. This prevalence was lower than that reported in Chinese or European studies, which was approximately 31 .9 %-45 .2 %[12 ,18 ]. In our center, we found that 60 .3 % of patients with infant-onset IBD had monogenic disease, with mutations inIL10RAidentified as the most common defect[13 ].

    Figure 2 Schematic diagram of disrupted RNA splicing and 333 -bp deletion in interleukin 10 receptor alpha subunit. A: Schematic diagram of disrupted splicing caused by c.537 G>A in interleukin 10 receptor alpha subunit (IL10 RA) between the boundary of exon4 and intron4 ; B: Schematic diagram of IL1 0 RA 333 -bp deletion. bp: Base pair.

    We evaluated four patients who had Crohn’s disease from early infancy and exhibited failure to thrive, severe perianal disease, and resistance to medication. These characteristics indicate the presence of underlying genetic conditions[7 ]. Although the patients underwent NGS in a local hospital, neither TGPS nor WES revealed conclusive results. However, all patients showed very high serum IL-10 levels, and three patients had a disease-causing heterozygous mutation inIL10RA. According to our previous research, serum IL-10 levels > 33 .05 pg/mL in patients with VEO-IBD strongly indicates the presence of IL10 RA dysfunction[13 ]. Thus, we predicted that additionalIL10RAmutations were missed during TGPS or WES. We did not detect the 333 -bp deletion in IL10RAin two patients until WGS was performed. We then analyzed the same deletion in the other two patients by reanalyzing the trio-WES data.Finally, all patients were precisely diagnosed with VEO-IBD owing to compound heterozygous mutations inIL10RAin three patients and homozygous deletion involvingIL10RAin one patient.

    NGS, including TGPS and WES, is a powerful tool for identifying Mendelian genetic diseases in patients with VEO-IBD. The position paper on VEO-IBD by NASPGHAN/ESPGHAN suggests that NGS combined with the patient clinical history represents a vital component of the diagnostic approach[1 ]. A previous multicenter study showed that molecular diagnosis was achieved in 32 % of patients with VEO-IBD when NGS was employed[18 ]. However, clinical NGS applications have limitations such as short read lengths, relatively high error rates, and incomplete coverage. Non-coding, yet potentially functional regions, and approximately 5 % of exons are poorly covered in WES[19 ]. It is difficult to detect variants involving extensive deletions/insertions or short tandem repeats[20 ]. Charbit-Henrion et al[18 ]reported three WES-negative cases harboring large deletions inLRBAandNCF1.Compared to WES, WGS can detect all single-nucleotide variants, small indels, large indels, and copy number variants. In our cases, the 333 -bp IL10RAdeletion contained a 5 ′-UTR, exon 1 , and part of intron 1 . This large deletion was easy to overlook when WES was used because of its technical limitations and insufficient bioinformatics analysis. After detecting this deletion, we requested re-analysis of the WES data for patients C and D, specifically for the 333 -bp region spanning IL10 RA exon 1 . As expected, these two patients harbored the deletion. These results indicate that WES can detect the 333 -bp deletion, which was easily overlooked in bioinformatics analysis because of algorithm defects and insufficient experience. WGS compensates for the limitations of WES. Our results indicated that WGS should be performed in VEO-IBD cases with negative WES results, particularly in those with infantile-onset IBD and treatment failure.

    Figure 3 Histopathochemistry and functional results of homozygous interleukin 10 receptor alpha subunit mutation. A: Histological findings in a colonic specimen obtained during colonoscopy showing oval-shaped intramural abscesses; B: Higher magnification of inset A; C: Determination of TNF-α levels in the supernatant of cultured PBMCs from patient D after stimulation with PBS, LPS, or LPS + IL-10 in vitro; D: Western blot results of PBMCs isolated from patient D after stimulation with LPS or IL-10 . Phosphorylation of STAT3 at Tyr705 and Ser727 and total STAT3 protein were detected (n = 3 ). aP < 0 .05 , bP < 0 .01 , and cP <0 .001 were considered as statistically different.

    A comprehensive range of defects may be associated with VEO-IBD. It is difficult to differentiate every patient based on these defects and their underlying genetic disorders. A small number of patients show distinct phenotypes associated with specific functions, such as IL-10 /IL-10 R defects, IPEX, CGD, and XIAP[1 ]. IL10RAmutation-induced VEO-IBD shows specific characteristics such as refractory pancolitis, perianal defects, fistulas, and growth failure, which occur during the neonatal period. An assay that can detect the lack of IL-10 inhibition in LPS can confirm receptor mutations[1 ]. Nevertheless, this assay is complicated and not routinely available in most hospitals in China. In a retrospective study, we found that the assay was useful for diagnosing IL10 RA defects when the serum level of IL-10 was > 33 .05 pg/mL. The assay sensitivity was very close to 100 %, and the specificity was approximately 84 %[13 ]. Elevated serum levels of IL-10 in patients with VEO-IBD indicated that even such high level of IL-10 could not inhibit TNF-α release and alleviate inflammation. Thus, serum IL-10 level may be a substitute for determining IL-10 inhibition in LPS functional testing. Therefore, when classic symptoms and laboratory results indicate that patients may have IL-10 /IL-10 R dysfunction but WES is inconclusive,IL-10 /IL10 RA/IL-10RBmust be investigated and analyzed. It is recommended to use WGS or specific PCR to detect whether a large deletion involving these genes has occurred.

    Interestingly, TNF-α production in LPS-stimulated PBMCs was not as robust in patient D as in control subjects; in our study, patient D was administered with anti-TNF-α antibody (Infliximab) before blood collection, which may have led to relatively low TNF-α production in the supernatant of LPS-stimulated PBMCs compared to that in healthy controls. Another possible reason is that increased TNF-α in the blood of patient D led to activation of the TNF-α receptor, resulting in JNK signaling-dependent inhibition of Bcl-2 expression, which acts as a major anti-apoptosis protein[21 ].The lack of a comprehensive functional test forIL10RAmutation is the major limitation of our study, although we conducted western blotting to determine the possible mechanism ofIL10 RA mutation-induced dysfunction of IL10 RA signaling. Further studies are needed to explore other potential differences in IL10 RA signaling.

    CONCLUSION

    Using WGS, we identified a novel 333 -bp deletion in IL10RAthat contributed to four cases of clinically diagnosed VEO-IBD with inconclusiveIL10RAmutations. Most importantly, we confirmed that typical clinical manifestations and increased serum levels of IL-10 strongly indicate the existence of IL-10 R dysfunction even when WES results are negative.

    ARTICLE HIGHLIGHTS

    Research results

    Results of WGS revealed a novel 333 -bp deletion encompassing exon 1 of IL10RAin patients A and B, which was also found in patients C and D after reanalyzing their WES data. Patient D was homozygous for the 333 -bp deletion. All four patients showed elevated serum IL-10 levels. In vitro, IL-10 -stimulated PBMCs from patient D failed to induce STAT3 phosphorylation at Tyr705 and minimally suppressed TNF-α production induced by LPS. Phosphorylation at Ser727 in PBMCs was not affected by LPS or LPS + IL-10 in both healthy subjects and patient D.

    Research conclusions

    Genome-wide uniformity of coverage of WGS identified a novel 333 -bp deletion inIL10RAin four patients with VEO-IBD, whereas the results of initially performed WES were inconclusive. WGS, which was more informative than WES, is the most important comprehensive second-tier genomic test for monogenic diseases in the clinic.

    Research perspectives

    We will customize a multiplex ligation-dependent amplification probe of the 333 -bp deletion inIL10 RA to help diagnose IL10RAmutation-related monogenic diseases.

    ACKNOWLEDGEMENTS

    The authors would like to thank the members of the Department of Pediatrics and Pediatric Laboratory.

    猜你喜歡
    實踐證明放養(yǎng)密度青蝦
    北方高寒地區(qū)淡水青蝦孵化育苗試驗
    青蝦有價無市,塘口價賣到看120元/斤!這個小品種為何沒有得到重視?
    高等數(shù)學(xué)解題中概率論方法的實踐分析
    放養(yǎng)小龍蝦蝦苗注意事項
    青蝦池塘種植蘆葦?shù)驹囼灴偨Y(jié)
    越冬青蝦放養(yǎng)管理三要點
    黃顙魚工廠化育苗技術(shù)
    論素質(zhì)測評(一)
    川西竹林地生態(tài)養(yǎng)雞放養(yǎng)密度篩選的研究
    春放魚種的適宜密度
    亚洲国产精品合色在线| 性色av乱码一区二区三区2| 麻豆av在线久日| 可以免费在线观看a视频的电影网站| 丁香六月欧美| 999久久久国产精品视频| 久久精品国产清高在天天线| 国产精华一区二区三区| 国内精品久久久久精免费| 三级男女做爰猛烈吃奶摸视频| 亚洲成人中文字幕在线播放| 欧美zozozo另类| 欧美中文日本在线观看视频| 女人爽到高潮嗷嗷叫在线视频| 成人国产一区最新在线观看| 亚洲人成77777在线视频| 成人av在线播放网站| 日本在线视频免费播放| 全区人妻精品视频| 国产成年人精品一区二区| 大型av网站在线播放| 精品久久蜜臀av无| 午夜激情av网站| 色噜噜av男人的天堂激情| 性色av乱码一区二区三区2| e午夜精品久久久久久久| 中文字幕高清在线视频| 欧美又色又爽又黄视频| 最近视频中文字幕2019在线8| 最近在线观看免费完整版| 最近最新中文字幕大全电影3| 国产精品一区二区精品视频观看| 亚洲激情在线av| 中亚洲国语对白在线视频| 国产精品久久电影中文字幕| 国产亚洲av高清不卡| 久久亚洲精品不卡| 午夜福利视频1000在线观看| 一级毛片女人18水好多| 大型黄色视频在线免费观看| 国产精品av久久久久免费| 99热6这里只有精品| 国产97色在线日韩免费| 欧美乱色亚洲激情| 国产主播在线观看一区二区| 麻豆av在线久日| www.熟女人妻精品国产| 999精品在线视频| 亚洲精品色激情综合| 毛片女人毛片| 国产1区2区3区精品| 国产精品久久久久久久电影 | 国内精品一区二区在线观看| 国产蜜桃级精品一区二区三区| 亚洲国产精品成人综合色| 精品久久蜜臀av无| 黑人巨大精品欧美一区二区mp4| 久久精品国产亚洲av香蕉五月| 岛国在线免费视频观看| 精品久久蜜臀av无| www日本在线高清视频| 亚洲国产精品999在线| 亚洲七黄色美女视频| 91麻豆av在线| 高清毛片免费观看视频网站| 免费在线观看日本一区| 嫩草影视91久久| 国产一区二区三区在线臀色熟女| 特大巨黑吊av在线直播| 亚洲人成网站在线播放欧美日韩| 久久久水蜜桃国产精品网| 亚洲欧美日韩高清专用| 在线观看舔阴道视频| 色在线成人网| 亚洲五月天丁香| 高清在线国产一区| 国产高清视频在线播放一区| 90打野战视频偷拍视频| 99热这里只有精品一区 | 不卡一级毛片| 久久久精品欧美日韩精品| 桃色一区二区三区在线观看| 国产精品99久久99久久久不卡| 婷婷六月久久综合丁香| 国产精品久久久久久人妻精品电影| 熟女少妇亚洲综合色aaa.| 婷婷精品国产亚洲av| 国产精品久久久人人做人人爽| 国模一区二区三区四区视频 | 午夜两性在线视频| videosex国产| 精品日产1卡2卡| 成人国产综合亚洲| 美女黄网站色视频| bbb黄色大片| 在线观看舔阴道视频| 美女大奶头视频| 人成视频在线观看免费观看| 人妻丰满熟妇av一区二区三区| 一进一出抽搐gif免费好疼| 亚洲国产欧美人成| 性欧美人与动物交配| 在线十欧美十亚洲十日本专区| 国产一区在线观看成人免费| 亚洲成av人片免费观看| 看片在线看免费视频| 午夜福利在线在线| 久久天躁狠狠躁夜夜2o2o| 国产aⅴ精品一区二区三区波| 日本黄色视频三级网站网址| 天堂√8在线中文| bbb黄色大片| 男插女下体视频免费在线播放| 观看免费一级毛片| 亚洲 国产 在线| 久久中文字幕一级| 欧美极品一区二区三区四区| 国产一级毛片七仙女欲春2| 亚洲精品国产精品久久久不卡| 非洲黑人性xxxx精品又粗又长| 91九色精品人成在线观看| 18禁黄网站禁片免费观看直播| 亚洲国产欧美一区二区综合| 精品久久久久久久人妻蜜臀av| 欧美又色又爽又黄视频| 老鸭窝网址在线观看| 成人18禁高潮啪啪吃奶动态图| 身体一侧抽搐| 性色av乱码一区二区三区2| 国产激情久久老熟女| 午夜两性在线视频| 国产亚洲欧美在线一区二区| 国产成人一区二区三区免费视频网站| 成年版毛片免费区| 亚洲国产欧美人成| 国产精品永久免费网站| 老汉色∧v一级毛片| 嫩草影院精品99| 精品日产1卡2卡| 亚洲中文字幕日韩| 欧美日韩中文字幕国产精品一区二区三区| 蜜桃久久精品国产亚洲av| 久热爱精品视频在线9| 国产精品一区二区三区四区久久| 狂野欧美激情性xxxx| 亚洲欧美精品综合一区二区三区| 亚洲无线在线观看| 成人18禁在线播放| 女人高潮潮喷娇喘18禁视频| 俺也久久电影网| 成人欧美大片| av欧美777| 欧美乱妇无乱码| 看黄色毛片网站| 亚洲成人中文字幕在线播放| 别揉我奶头~嗯~啊~动态视频| 亚洲成av人片在线播放无| 美女免费视频网站| 欧美人与性动交α欧美精品济南到| 91在线观看av| 免费电影在线观看免费观看| e午夜精品久久久久久久| 国语自产精品视频在线第100页| 99精品欧美一区二区三区四区| 老司机午夜十八禁免费视频| 亚洲一区中文字幕在线| av有码第一页| 欧美国产日韩亚洲一区| 一进一出抽搐gif免费好疼| 18禁美女被吸乳视频| 久久午夜综合久久蜜桃| 亚洲 国产 在线| 岛国在线免费视频观看| 啦啦啦观看免费观看视频高清| 国产男靠女视频免费网站| 亚洲国产欧美一区二区综合| 中文资源天堂在线| 十八禁网站免费在线| 国产视频内射| 亚洲国产高清在线一区二区三| 嫩草影院精品99| 国产亚洲精品一区二区www| 国产不卡一卡二| 后天国语完整版免费观看| 国产伦在线观看视频一区| 精品国产美女av久久久久小说| 日韩有码中文字幕| 亚洲欧美精品综合一区二区三区| 久久中文字幕一级| 国产精品电影一区二区三区| 99久久精品热视频| 国产区一区二久久| 色哟哟哟哟哟哟| 少妇裸体淫交视频免费看高清 | 美女免费视频网站| 三级毛片av免费| 国产黄片美女视频| av在线天堂中文字幕| www.熟女人妻精品国产| 亚洲国产精品sss在线观看| 日韩高清综合在线| 亚洲国产精品合色在线| 国产精品自产拍在线观看55亚洲| 人妻丰满熟妇av一区二区三区| 美女大奶头视频| 一本一本综合久久| 国产精品一区二区精品视频观看| 少妇裸体淫交视频免费看高清 | 久久亚洲精品不卡| 久久久国产成人精品二区| 久久精品91蜜桃| 99国产综合亚洲精品| 正在播放国产对白刺激| 日韩 欧美 亚洲 中文字幕| 久久久国产成人免费| 岛国在线观看网站| 俄罗斯特黄特色一大片| 啦啦啦观看免费观看视频高清| www日本黄色视频网| 国产精华一区二区三区| 国产精品一区二区精品视频观看| 制服丝袜大香蕉在线| 黄色a级毛片大全视频| 一级a爱片免费观看的视频| 亚洲色图 男人天堂 中文字幕| 国产免费男女视频| 亚洲国产欧洲综合997久久,| 国产久久久一区二区三区| 999久久久国产精品视频| 亚洲五月天丁香| 99热这里只有是精品50| 午夜影院日韩av| 久久午夜亚洲精品久久| 国产精品1区2区在线观看.| 一个人免费在线观看的高清视频| 怎么达到女性高潮| 成人三级黄色视频| 国产成人aa在线观看| av免费在线观看网站| 中文字幕熟女人妻在线| 国产精品电影一区二区三区| 99热只有精品国产| 国产成人啪精品午夜网站| 波多野结衣高清无吗| 男人的好看免费观看在线视频 | 在线观看免费日韩欧美大片| 老熟妇乱子伦视频在线观看| 日本免费a在线| 午夜精品一区二区三区免费看| 巨乳人妻的诱惑在线观看| 国产熟女午夜一区二区三区| 亚洲 欧美 日韩 在线 免费| 色精品久久人妻99蜜桃| 757午夜福利合集在线观看| 女生性感内裤真人,穿戴方法视频| 丰满人妻熟妇乱又伦精品不卡| 国产男靠女视频免费网站| 久久午夜亚洲精品久久| 亚洲成人免费电影在线观看| 麻豆av在线久日| 国产免费男女视频| 叶爱在线成人免费视频播放| 99国产精品99久久久久| 变态另类丝袜制服| 亚洲av成人精品一区久久| 亚洲国产看品久久| 又爽又黄无遮挡网站| 午夜福利高清视频| 成年人黄色毛片网站| 久久久久国产精品人妻aⅴ院| 色综合站精品国产| 日日爽夜夜爽网站| 长腿黑丝高跟| 国产精品一区二区免费欧美| 在线永久观看黄色视频| 亚洲欧美精品综合久久99| 成人国产一区最新在线观看| 在线观看日韩欧美| 日本黄大片高清| 色精品久久人妻99蜜桃| 免费在线观看视频国产中文字幕亚洲| or卡值多少钱| 国产精品野战在线观看| 99在线人妻在线中文字幕| 国产人伦9x9x在线观看| 在线观看一区二区三区| 99久久综合精品五月天人人| 色在线成人网| 欧美3d第一页| 色在线成人网| 欧美3d第一页| 亚洲精品一区av在线观看| 亚洲人成电影免费在线| 动漫黄色视频在线观看| 男插女下体视频免费在线播放| 免费无遮挡裸体视频| 18禁国产床啪视频网站| 国内精品久久久久久久电影| 欧美一区二区精品小视频在线| 亚洲性夜色夜夜综合| 欧美一级毛片孕妇| 一级a爱片免费观看的视频| 亚洲欧美激情综合另类| 日韩欧美国产一区二区入口| 热99re8久久精品国产| 国产一区二区三区视频了| 免费看十八禁软件| 女同久久另类99精品国产91| 精品久久久久久久末码| 国产精品一区二区精品视频观看| 69av精品久久久久久| 成人永久免费在线观看视频| 中文资源天堂在线| 国产爱豆传媒在线观看 | 我的老师免费观看完整版| 日韩高清综合在线| 全区人妻精品视频| 日韩av在线大香蕉| 在线国产一区二区在线| 午夜福利免费观看在线| 国产成人精品久久二区二区免费| 久久久久久国产a免费观看| 日本一本二区三区精品| 国产av一区二区精品久久| 成人三级做爰电影| 一区福利在线观看| 欧美另类亚洲清纯唯美| 美女免费视频网站| 久久精品国产99精品国产亚洲性色| 97碰自拍视频| 国产成人一区二区三区免费视频网站| 日韩欧美国产一区二区入口| 在线看三级毛片| 欧美一区二区精品小视频在线| 制服诱惑二区| 老汉色∧v一级毛片| 午夜福利视频1000在线观看| 亚洲中文字幕一区二区三区有码在线看 | 国产成人影院久久av| 免费无遮挡裸体视频| 两个人视频免费观看高清| 国产亚洲精品一区二区www| 天堂动漫精品| 国产99久久九九免费精品| 亚洲18禁久久av| 九色国产91popny在线| 一进一出好大好爽视频| 国内久久婷婷六月综合欲色啪| av在线播放免费不卡| 精品午夜福利视频在线观看一区| 精华霜和精华液先用哪个| 久久久国产成人精品二区| 亚洲美女黄片视频| 亚洲欧美精品综合久久99| 国产在线观看jvid| 久久精品国产99精品国产亚洲性色| 欧美日本亚洲视频在线播放| 精品久久蜜臀av无| 久久精品夜夜夜夜夜久久蜜豆 | 99国产极品粉嫩在线观看| 日本一区二区免费在线视频| 久久久久亚洲av毛片大全| bbb黄色大片| 亚洲 国产 在线| 国产麻豆成人av免费视频| 91字幕亚洲| 国产麻豆成人av免费视频| 男人舔女人的私密视频| xxxwww97欧美| 18禁黄网站禁片免费观看直播| 中出人妻视频一区二区| 亚洲成人精品中文字幕电影| 色综合欧美亚洲国产小说| 成人三级黄色视频| 校园春色视频在线观看| 在线永久观看黄色视频| 精品第一国产精品| 十八禁网站免费在线| 一级毛片女人18水好多| 九九热线精品视视频播放| 日韩欧美在线乱码| 少妇裸体淫交视频免费看高清 | 一个人免费在线观看的高清视频| 国产精品亚洲美女久久久| 免费看美女性在线毛片视频| 国产精品国产高清国产av| 亚洲aⅴ乱码一区二区在线播放 | 别揉我奶头~嗯~啊~动态视频| 欧美一区二区精品小视频在线| 国产精品美女特级片免费视频播放器 | 国产亚洲精品第一综合不卡| 欧美一级毛片孕妇| 国产精品久久久人人做人人爽| 日韩欧美国产在线观看| 深夜精品福利| 天堂av国产一区二区熟女人妻 | 怎么达到女性高潮| a级毛片a级免费在线| 韩国av一区二区三区四区| 少妇裸体淫交视频免费看高清 | 欧美性猛交╳xxx乱大交人| 欧美乱色亚洲激情| 亚洲国产欧美一区二区综合| 在线观看www视频免费| 欧美乱妇无乱码| 两个人的视频大全免费| aaaaa片日本免费| 2021天堂中文幕一二区在线观| 久久久国产成人精品二区| 啦啦啦免费观看视频1| 欧美成人免费av一区二区三区| 成人三级黄色视频| 欧美日韩亚洲国产一区二区在线观看| 无遮挡黄片免费观看| av片东京热男人的天堂| 村上凉子中文字幕在线| 欧美一级毛片孕妇| 日本 av在线| svipshipincom国产片| 日本 av在线| √禁漫天堂资源中文www| 欧美在线黄色| 久久伊人香网站| 草草在线视频免费看| 久久伊人香网站| 国产精品一及| 高潮久久久久久久久久久不卡| 天堂动漫精品| 免费一级毛片在线播放高清视频| 欧美性猛交黑人性爽| 久久久久久久久中文| or卡值多少钱| 欧美绝顶高潮抽搐喷水| 国产高清有码在线观看视频 | 18禁国产床啪视频网站| 欧美一级a爱片免费观看看 | 中文字幕高清在线视频| 国产精品av久久久久免费| 国产成+人综合+亚洲专区| 亚洲精品中文字幕一二三四区| 少妇熟女aⅴ在线视频| 久久精品国产亚洲av高清一级| 久久久久国产精品人妻aⅴ院| 欧美zozozo另类| 又紧又爽又黄一区二区| 久久午夜亚洲精品久久| 禁无遮挡网站| 亚洲电影在线观看av| 国产真实乱freesex| 欧美一区二区精品小视频在线| 女警被强在线播放| av天堂在线播放| 国产亚洲欧美98| 日韩欧美精品v在线| 91麻豆av在线| 美女黄网站色视频| 看片在线看免费视频| 久久中文字幕人妻熟女| 亚洲欧美精品综合一区二区三区| 久久香蕉激情| 国产在线观看jvid| 91大片在线观看| 国产亚洲欧美在线一区二区| 亚洲最大成人中文| 毛片女人毛片| 男女午夜视频在线观看| 亚洲国产精品久久男人天堂| 九色成人免费人妻av| 久久久久免费精品人妻一区二区| 最近最新免费中文字幕在线| 国产爱豆传媒在线观看 | 黄片小视频在线播放| 国产激情偷乱视频一区二区| 黄频高清免费视频| 夜夜看夜夜爽夜夜摸| 一进一出抽搐动态| 美女午夜性视频免费| 亚洲精品在线美女| 十八禁人妻一区二区| 国内少妇人妻偷人精品xxx网站 | 757午夜福利合集在线观看| 久久久久九九精品影院| 日韩欧美国产一区二区入口| 99热这里只有精品一区 | 午夜亚洲福利在线播放| 亚洲成人免费电影在线观看| x7x7x7水蜜桃| 久久99热这里只有精品18| 看免费av毛片| 亚洲七黄色美女视频| 日本黄色视频三级网站网址| 亚洲成a人片在线一区二区| 国产av一区二区精品久久| 亚洲精品av麻豆狂野| 搡老妇女老女人老熟妇| 久久久久久免费高清国产稀缺| 久久久久性生活片| 草草在线视频免费看| 国产91精品成人一区二区三区| 久久国产精品人妻蜜桃| 最近视频中文字幕2019在线8| 欧美精品亚洲一区二区| 免费在线观看黄色视频的| 国产真人三级小视频在线观看| xxx96com| 免费观看人在逋| 国产精品久久久久久久电影 | 1024手机看黄色片| 美女免费视频网站| 一级毛片女人18水好多| 亚洲欧美一区二区三区黑人| 色哟哟哟哟哟哟| 免费看日本二区| 99热只有精品国产| 亚洲专区字幕在线| 91国产中文字幕| 亚洲精品一区av在线观看| 法律面前人人平等表现在哪些方面| 男男h啪啪无遮挡| 国产黄色小视频在线观看| 动漫黄色视频在线观看| 国产成人欧美在线观看| 免费电影在线观看免费观看| 国产亚洲精品一区二区www| 777久久人妻少妇嫩草av网站| 在线永久观看黄色视频| 久久国产精品人妻蜜桃| 熟女电影av网| 国产精品影院久久| 国产v大片淫在线免费观看| 神马国产精品三级电影在线观看 | 成熟少妇高潮喷水视频| 少妇被粗大的猛进出69影院| 国产1区2区3区精品| 视频区欧美日本亚洲| 天天一区二区日本电影三级| 午夜福利视频1000在线观看| 日韩精品青青久久久久久| 国产69精品久久久久777片 | 在线观看午夜福利视频| 日韩精品青青久久久久久| 日韩欧美在线二视频| 九色成人免费人妻av| 国产伦在线观看视频一区| 两个人视频免费观看高清| 不卡一级毛片| 成人国语在线视频| 丰满人妻一区二区三区视频av | 国产精品亚洲av一区麻豆| av欧美777| 男女那种视频在线观看| 叶爱在线成人免费视频播放| 在线观看免费视频日本深夜| x7x7x7水蜜桃| 久久精品夜夜夜夜夜久久蜜豆 | 1024香蕉在线观看| 一级毛片高清免费大全| 久久亚洲真实| 好男人电影高清在线观看| 亚洲国产中文字幕在线视频| 天堂√8在线中文| 国产精品乱码一区二三区的特点| 亚洲成av人片免费观看| 欧美日本亚洲视频在线播放| 中文在线观看免费www的网站 | 变态另类成人亚洲欧美熟女| 亚洲人成伊人成综合网2020| 宅男免费午夜| 国产午夜精品论理片| 久久久久九九精品影院| 色在线成人网| 亚洲在线自拍视频| 亚洲七黄色美女视频| 亚洲成人久久性| 老司机福利观看| 变态另类丝袜制服| 白带黄色成豆腐渣| 黄色片一级片一级黄色片| 久久精品国产亚洲av香蕉五月| 真人做人爱边吃奶动态| 久久久久国内视频| 色在线成人网| 国产99久久九九免费精品| 亚洲国产精品成人综合色| 日本免费一区二区三区高清不卡| 亚洲国产中文字幕在线视频| 中文字幕熟女人妻在线| 中文亚洲av片在线观看爽| 女人被狂操c到高潮| 亚洲熟妇中文字幕五十中出| 757午夜福利合集在线观看| 欧美乱妇无乱码| 亚洲欧美精品综合一区二区三区| 精品国产乱码久久久久久男人| 免费看十八禁软件| 国产1区2区3区精品| 久久精品综合一区二区三区| 日本一本二区三区精品| 丰满人妻一区二区三区视频av | 国产人伦9x9x在线观看| 国产91精品成人一区二区三区| 天堂影院成人在线观看| 亚洲成人国产一区在线观看| 一本精品99久久精品77| 亚洲国产欧洲综合997久久,| 一级毛片精品| 国产精品精品国产色婷婷| 又黄又粗又硬又大视频| 69av精品久久久久久| 国产精品免费视频内射| www.www免费av| 身体一侧抽搐| 亚洲国产欧美一区二区综合| 麻豆成人av在线观看| 99riav亚洲国产免费|