• <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 ?

    Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    2014-06-01 09:42:34DefengZouYiChenYaxinHanChenLvGuanjunTu

    Defeng Zou, Yi Chen, Yaxin Han, Chen Lv, Guanjun Tu

    1 Department of Orthopedics, First Af fi liated Hospital of China Medical University, Shenyang, Liaoning Province, China

    2 Department of Orthopedics, Jinhua Central Hospital of Zhejiang University, Jinhua, Zhejiang Province, China

    Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    Defeng Zou1, Yi Chen2, Yaxin Han1, Chen Lv1, Guanjun Tu1

    1 Department of Orthopedics, First Af fi liated Hospital of China Medical University, Shenyang, Liaoning Province, China

    2 Department of Orthopedics, Jinhua Central Hospital of Zhejiang University, Jinhua, Zhejiang Province, China

    microRNAs (miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 (miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were signi fi cantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our fi ndings should facilitate the development of novel strategies for enhancing the therapeutic ef fi cacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

    nerve regeneration; microRNA-124; lentivirus; overexpression; bone marrow-derived mesenchymal stem cells; neural stem cells; spinal cord injury; neurogenesis; GeneChip; motor function; NSFC grant; neural regeneration

    Funding: This study was supported by the National Natural Science Foundation of China, No. 81070971.

    Zou DF, Chen Y, Han YX, Lv C, Tu GJ. Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells. Neural Regen Res. 2014;9(12):1241-1248.

    Introduction

    Injury to the spinal cord leads to loss of function, such as movement, sensation and autonomic control, in the regions innervated below the site of damage. Transplantation of neural stem cells to treat spinal cord injuries is currently one of the hottest research fi elds in biology (Paspala et al., 2009; Xu et al., 2012). However, neural stem cells are mainly obtained from embryos, which raise ethical and legal issues (Robertson, 1999). Bone marrow-derived mesenchymal stem cells (BMSCs) are a potentially promising source of cells for use in regenerative medicine because they are abundantly available, are easy to isolate from the patient themselves, are an autologous tissue and there is no ethical dispute over their use (Eftekharpour et al., 2007; Eftekharpour et al., 2008).

    BMSCs can be isolated and differentiated into a variety of cell lineages in vitro, including osteoblasts, myofibroblasts, chondrocytes, adipocytes and nerve cells (Jung et al., 2005; Roese-Koerner et al., 2013). Cultured BMSCs are hypoimmunogenic and capable of homing, and thus have great potential for various clinical applications (Abdallah and Kassem, 2009). However, the differentiation of BMSCs into neurons or neural stem cells remains limited. In this study, we sought to improve the differentiation ef fi ciency of transplanted BMSCs into neurons.

    Figure 1 Detection of rat miR-124 (rno-miR-124) expression in rat bone marrow-derived mesenchymal stem cells (BMSCs), cortical neurons (SDCNC) and neural stem cells (SDNSC).

    MicroRNAs (miRNAs) are 20-24-nt endogenous, evolutionarily conserved, RNA molecules that negatively regulate the translation of their target mRNAs by binding to their 3′-untranslated region (3′-UTR) (Bartel, 2004). Recent research has shown that miRNAs play essential roles in neural development and neuronal function (Bartel, 2009; Shi et al., 2010), and in the differentiation of stem cells (Krichevsky, 2007; Bak et al., 2008; Schoolmeesters et al., 2009; Liu et al., 2012; McNeill and Van Vactor, 2012; Akerblom and Jakobsson, 2013). However, the role of miRNAs in the neurogenesis of BMSCs remains unclear. For example, the brain-enriched miRNAs miR-9/9? and miR-124, which promote the assembly of neuron-specific BAF complexes (ATP-dependent chromatin remodeling complexes), are able to convert non-neuronal human dermal fibroblasts into post-mitotic neurons (Sun et al., 2013). In this study, we compared the miRNA profiles of BMSCs with cortical neurons and neural stem cells using GeneChip. We observed that miR-124 was one of the most downregulated miRNAs in BMSCs compared with cortical neurons and neural stem cells. We then overexpressed miR-124 in BMSCs using a lentiviral vector, and evaluated the effects of overexpression on the differentiation of BMSCs. We also assessed the effect of treatment with miR-124-overexpressing BMSCs in an animal model of spinal cord injury.

    Materials and Methods

    Materials

    Adult rat neural stem cells, cortical neurons from 18.5-dayold rats, and adult rat BMSCs (all from Sprague-Dawley rats) were purchased from Cyagen Biosciences Inc., Guangzhou, Guangdong Province, China. The adult male Sprague-Dawley rats used for in vivo experiments (body weight: 230-250 g) were purchased from the Chinese Medical University Laboratory Animal Center (license No. SYXK (Liao) 2008-0013). All rats were housed in a temperature and light-cycle-controlled animal laboratory and allowed free access to food and water. This study was approved by the Animal Research Committee of China Medical University, China.

    Cell culture, identi fi cation and GeneChip miRNA array

    Total RNA, containing miRNAs, was isolated from sorted cells with an miRNeasy kit (Qiagen, Frankfurt, Germany). GeneChip microarray assay was then performed by a third-party service provider (Affymetrix, CA, USA). qRTPCR was used to validate miRNA expression in the BMSCs, neural stem cells and neurons.

    Vector construction and transfection of BMSCs with miR-124

    The lentiviral vector pLVX-EN-rno-miR-124 was constructed at Yingrun Biotechnologies Inc. (Changsha, China). 293FT cells were then transiently transfected with pLVXEN-rno-miR124 (10 μg), pLP1 (6.5 μg), pLP/VSVG (3.5 μg) and pLP2 (2.5 μg) using Lipofectamine 2000 (Life Technologies, CA, USA). The virus titer was evaluated by counting the number of GFP-positive cells. We then transfected BMSCswith the pLVX-EN-rno-miR124 pseudovirion.

    Figure 2 miR-124 expression in lentiviral vector-infected BMSCs.

    Figure 4 Analysis of the effects of miR-124 on apoptosis in bone marrow-derived mesenchymal stem cells (BMSCs) following oxygen and glucose deprivation by annexin V-FITC/PI double staining.

    RT-PCR analysis of miR-124 expression in transfected BMSCs

    We performed RNA extraction using Trizol reagent (Invitrogen, Guangzhou, China). Reverse transcriptases (Life Technologies) were used to prepare complementary DNA (cDNA) according to the instructions provided by Fermentas Corporation. Expression levels were quantitatively determined with the ABI 7500 system using the SYBR GreenI dye method (TOYOBO, Shanghai, China). U6 was used as the internal reference for miRNA detection. PCR protocol: 95°C for 3 minutes; 40 cycles of 95°C for 20 seconds, 60°C for 30 seconds; and 95°C for 10 seconds (to obtain the melting curve).

    Western blot analysis

    The BMSCs were separated into three groups: (1) control (untransfected), (2) miR-124+(pLVX-EN-rno-miR124-transfected) and (3) miR-124-(pLVX-EN-rno-transfected). Western blotting was carried out using standard protocols. The cells were lysed on ice with PMSF lysis buffer (Applygen Technologies Inc, Beijing, China) for 30 minutes. Lysed cells were collected by centrifugation at 12,000 × g for 5 minutes at 4°C to obtain total protein, which was then quanti fi ed using the bicinchoninic acid (BCA) protein assay (ShineGene, Shanghai, China). A total of 50 μg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 5% to 10% gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk powder in TBST (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) and incubated overnight with mouse anti-β-III tubulin, anti-MAP-2 (1:1,000, Abgent Biotechnology, San Diego, CA, USA), anti-synaptophysin or anti-β-actin (1:3,000, Abgent Biotechnology) antibody at 4°C. After washing, the membranes were incubated with the secondary antibody, horseradish peroxidase-labeled IgG (goat anti-mouse IgG/HRP, KPL Biotechnology, Gaithersburg, MD, USA), for 1 hour and visualized with an ECL chemiluminescent reagent system (Pierce Biotechnology, Rockford, IN, USA). Gray scale densitometric scanning of the protein bands was performed with Quanti Scan software using β-actin as the control. Data were expressed as mean ± SD of the percentage ratio of the control.

    Immuno fl uorescence detection of neuronal markers

    Three groups of cells were cultured in vitro 6 days after transfection, and were fixed with 4% paraformaldehyde on coverslips and then rinsed with PBS, blocked with 10% goat serum for 1 hour at room temperature, and incubated at 4°C overnight. Sections were rinsed with PBS, and incubated with mouse anti-β-III tubulin (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-MAP-2 (1:50, Cell Signaling, Boston, MA, USA) for 24 hours at 4°C. Sections were then rinsed with PBS, followed by incubation with secondary antibodies (Dy-Light488 green fl uorescence-labeled goat anti-mouse IgG [1:250, Abcam, Cambridge, MA, USA]; Texas Red-labeled rabbit anti-mouse IgG [1:250, Merck Millipore, Billerica, MA, USA]) for 1 hour in the dark at 37°C. After rinsing in PBS, the sections were observed under a fl uorescence microscope (Olympus, Tokyo, Japan).

    Apoptosis assay

    Oxygen and glucose deprivation (OGD) was performed on cells according to Sun et al. (2013). After 12 hours, apoptosis was quanti fi ed with an annexin V-FITC/PI double staining kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Apoptosis was measured using a flow cytometer (BD Pharmingen, Franklin, NJ, USA).

    Spinal cord injury model and transplantation of BMSCs

    Sprague-Dawley rats were randomly divided into three groups: (1) spinal cord injury group (spinal cord injury, treated only with 10 μL saline, n = 20), (2) miR-124--BMSCs group (spinal cord injury followed by transplantation of 10 μL (1 × 105) miR-124--BMSCs, n = 20), and (3) miR-124+-BMSCs group (spinal cord injury followed by transplantation of 10 μL (1 × 105) miR-124+-BMSCs, n = 20). The rats were then subjected to a contusion injury of the spinal cord using a 20-g weight dropped from a height of 10 cm onto the surface of the spinal cord at T10-11exposed by laminectomy (Allen, 1911). Following this, either saline, miR-124--BMSCs or mir-124+-BMSCs were transplanted into the damaged area within 30 minutes of the spinal cord injury and were also injected intraperitoneally at a dose of 20 μL/100 g four times with an interval of 3 hours.

    Immunocytochemistry

    Animals were euthanized 7 days after injury (n = 4 for each group). A 10-mm segment of the spinal cord encompassing the injury site was then harvested. After fi xation, the tissue blocks were embedded in paraf fi n, and sectioned at 5 μm thickness. After paraffin sections were deparaffinized and rehydrated, antigen retrieval was performed in sodium citrate buffer heated to 92-98°C for 20 minutes. Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxide for 20 minutes. Non-specific binding sites were blocked by 10% normal goat serum (Zsbio, Beijing, China) for 30 minutes. Sections were incubated with primary antibody in PBS at 4°C overnight, and the following antibody was used: mouse anti-neuro fi lament 200 (NF-200, 1:100, Santa Cruz Biotechnology). After rinsing in PBS, sections were incubated with goat anti-mouse IgG for 30 minutes followed by avidin-peroxidase complex solution containing avidin-peroxidase conjugate for 30 minutes. Staining was developed by incubating in 50% 3,3-diaminobenzidine (DAB) and 3% hydrogen peroxide in 0.1 mol/L PBS. Then, the sections were dehydrated, cleared, and coverslipped. PBS instead of primary antibody was used in the negative control.

    Behavioral testing

    Locomotor activity was evaluated at 1, 4, 7, 14, 21, 28, 35 and 42 days post-injury using the Basso, Beattie and Bresnahan (BBB) score to measure locomotor ability over 4 minutes. Two independent and well-trained investigators observed movement and scored locomotor function according to the BBB scale as described previously (Caggiano et al., 2005).

    Statistical analysis

    The data were analyzed using SPSS 17.0 statistical software. Values are presented as mean ± SD. Student’s t-test was performed for statistical evaluation. Differences with a level of P < 0.05 were considered statistically signi fi cant.

    Results

    Differential expression of miRNAs in BMSCs, cortical neurons and neural stem cells

    GeneChip data revealed that compared with cortical neurons and neural stems cells, miR-124 was signi fi cantly downregulated in BMSCs (Figure 1A, C), which was confirmed by qRT-PCR (Figure 1B). Previous studies showed that miR-124 has an important role in the neurogenesis of non-neuronal cell types (Cheng et al., 2009). Therefore, we chose miR-124 for further study.

    miR-124 expression is upregulated in BMSCs after transfection with pLVX-EN-rno-miR-124

    The sequence of the lentiviral vector pLVX-EN-rno-miR-124 was confirmed by restriction enzyme digestion and DNA sequencing (Figure 1D). We observed three GFP-positive 293FT cells following addition of a 10-6dilution of the virus, indicating that there were at least three pseudovirion-transfected 293FT cells (Figure 2N). We calculated the virus titer at 1.5 × 109TU/mL. We transfected miR-124+cells with the pLVX-EN-rno-miR-124 pseudovirion and miR-124-cells with pLVX-EN-rno. Following this, the expression profiles of the three cell groups were assessed using RT-PCR. Quantitation of miR-124 was estimated based on measured Ct values. qRT-PCR revealed that miR-124 expression in miR-124+cells was significantly higher than in control or miR-124-cells (Figure 2O; ΔΔCt values 141.60 ± 8.51, 1.00 ± 0.05, 0.54 ± 0.02, respectively). This result indicated that miR-124 was upregulated after BMSCs were transfected with pLVXEN-rno-miR-124.

    Overexpression of miR-124 increases expression ofβ-III tubulin, MAP-2 and synaptophysin

    To investigate neurogenesis, we performed immuno fl uorescence for β-III tubulin and MAP-2 in the BMSCs during the earlier stages of the neural differentiation process. We observed a strong signal for β-III tubulin (a marker for neurons in the earlier stage; Figure 3A, red fl uorescence) and MAP-2 (Figure 3B, green fl uorescence) in the miR-124+neurons compared with the control or miR-124-group on the 6thday of in vitro culture. β-III tubulin and MAP-2 expression were clearly found in both the cell soma and the neurite-like structures under high magni fi cation (200 ×;Figure 3A, B) on the 6thday of differentiation. BMSCs developed dendrites and neurites, similar to neurons (Figure 3A2, A4, B2, B4).

    Overexpression of miR-124 reduces apoptosis in BMSCs following oxygen and glucose deprivation

    The effect of miR-124 on apoptosis of BMSCs following oxygen and glucose deprivation was analyzed by annexin V-FITC/PI double staining. Quanti fi cation of apoptosis was performed 6 days post-injury (n = 4/group). Early apoptosis was determined (Figure 4A-C). The rate of apoptosis in the miR-124+group was signi fi cantly lower than in the control or miR-124-group (5 ± 1% vs. 35 ± 4% or 15 ± 2%, respectively; P < 0.05,Figure 4D).

    Axonal growth assessed with NF-200 immunohistochemistry

    Six days after spinal cord injury, axonal regeneration was assessed by NF-200 immunostaining. In the miR-124+group, NF-200 immunoreactivity could be detected in a large number of cells in the area of spinal cord injury. In contrast, only a small number of NF-200-immunoreactive cells were observed in the injured area in the injury and miR-124-groups (Figure 5A-D).

    Overexpression of miR-124 promotes functional recovery after spinal cord injury

    As shown inFigure 5E, compared to the control or miR-124-group, recovery was significantly greater in the miR-124+group from day 14 after injury (P < 0.05), indicating that overexpression of miR-124 in BMSCs promotes functional recovery after spinal cord injury.

    Discussion

    miR-124 is one of the best characterized and most abundantly-expressed neuronal miRNAs (Krichevsky et al., 2003; Kim et al., 2004). Overexpression of miR-124 results in upregulation of the expression of neuronal markers, as well as morphological changes, including enhanced neurite outgrowth and complexity (Yoo et al., 2011). Some overexpression studies in vertebrates have identi fi ed miR-124 as a promoter of neuronal differentiation and an inhibitor of progenitor self-renewal (Maiorano et al., 2009; Clark et al., 2010; Liu et al., 2011; Sanuki et al., 2011; Akerblom et al., 2012; Weng and Cohen, 2012; Xia et al., 2012). However, whether miR-124 can regulate neurogenesis in BMSCs remains unknown. In this study, we succeeded in constructing a lentiviral vector for the overexpression of miR-124 in BMSCs. Overexpression of miR-124 was associated with increased expression of the proteins β-III tubulin, MAP-2 and synaptophysin a■er 6 days of in vitro culture.

    Tubulin is an important structural protein in neurons and is a marker of differentiated neurons. β-III tubulin is expressed by the neuroepithelium during embryogenesis and is widely used as a speci fi c marker of neurons (von Bohlen und Halbach, 2011). MAP-2 is a dendrite-specific protein that plays an important role in the development, formation and regeneration of the nervous system (Czikk et al., 2014). We investigated the rate of cell differentiation of transfected BMSCs into neurons based on their expression of β-III tubulin and MAP-2. In BMSCs overexpressing miR-124, the number of β-III tubulin and MAP-2-positive neurons were remarkably elevated. This result is consistent with the fi ndings of Roese-Koerner et al. (2013). However, these two markers do not demonstrate that BMSCs that have undergone neurogenesis are functionally neurons. Synaptophysin is a synaptic protein that is used as a marker of synapse formation (Czikk et al., 2014). We observed that synaptophysin expression was substantially increased in miR-124+cells compared with miR-124-or control cells. This indicates that miR-124 not only promotes neurogenesis in BMSCs, but also commits them to the development of synapses, which is essential for recovery of nerve function. After spinal cordinjury, secondary damage is triggered by multiple processes. Usually, transplanted BMSCs undergo apoptosis as a result of in fl ammatory and oxidative damage. Recently, a number of miRNAs were found to be decreased after spinal cord injury. In particular, miR-124a expression was signi fi cantly decreased 1 to 7 days after spinal cord injury (Nakanishi et al., 2010). Here, we found that BMSCs overexpressing miR-124 were relatively protected from oxygen and glucose deprivation-induced apoptosis in vitro.

    Figure 3 Neural differentiation of BMSCs on the 6thday of in vitro transfection.

    Figure 5 NF-200 immunohistochemistry in the injured rat spinal cord following miR-124-transfected bone marrow-derived mesenchymal stem cell (BMSC) transplantation.

    Although we initially showed that BMSCs could be induced to differentiate into neurons in vitro by overexpressing miR-124, we needed to confirm if they could differentiate into functional neurons in vivo. We found that overexpression of miR-124 in BMSCs not only enhances the ability of the cells to survive, but also raises the rate of differentiation of the transplanted BMSCs into neurons in the region of spinal cord injury. Our NF-200 immunohistochemistry results indicated that there were more NF-200-positive cells in the miR-124+group than in the miR-124-or control group. This result is in agreement with those of Sun et al. (2013). Our behavioral data revealed that miR-124-overexpressing BMSCs promote functional locomotor recovery after spinal cord injury.

    Previous studies demonstrated that miR-124 represses the expression of proteins with anti-neuronal activities, including repressor-element-1-silencing transcription factor (Qureshi et al., 2010; Baudet et al., 2011), small c-terminal domain phosphatase 1 (SCP1) (Visvanathan et al., 2007) and Sox9 (Sanuki et al., 2011). A study by Doeppner et al. (2013) found that miR-124 reduces expression of the target deubiquitinating enzyme Usp14, thereby increasing repressor-element-1-silencing transcription factor degradation. These observations provide insight into the molecular mechanisms underlying the differentiation of BMSCs into neurons.

    In conclusion, the transplantation of BMSCs overexpressing miR-124 may be an effective therapeutic strategy for promoting regeneration and functional recovery following spinal cord injury.

    Acknowledgments:We would like to thank the staff of the Central Laboratory of the First Affiliated Hospital of China Medical University (Shenyang, Liaoning Province, China) for their technical assistance.

    Author contributions:Zou DF performed the experiments, conducted statistical analysis, and wrote the manuscript. Chen Y and Han YX assisted in the experiments. Lv C was responsible for statistical analysis. Tu GJ directed the research and supervised manuscript writing. All authors approved the final version of the paper.

    Con fl icts of interest:None declared.

    Abdallah BM, Kassem M (2009) The use of mesenchymal (skeletal) stem cells for treatment of degenerative diseases: current status and future perspectives. J Cell Physiol 218:9-12.

    ?kerblom M, Sachdeva R, Barde I, Verp S, Gentner B, Trono D, Jakobsson J (2012) MicroRNA-124 is a subventricular zone neuronal fate determinant. J Neurosci 32:8879-8889.

    Akerblom M, Jakobsson J (2013) MicroRNAs as neuronal fate determinants. Neuroscientist 20:235-242.

    Allen A (1911) Surgery for experimental lesions of spinal cord equivalent to crush injury of fracture dislocation of spinal column: a preliminary report. JAMA 57:878-880

    Bak M, Silahtaroglu A, M?ller M, Christensen M, Rath MF, Skryabin B, Tommerup N, Kauppinen S (2008) MicroRNA expression in the adult mouse central nervous system. RNA 14:432-444.

    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-297.

    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215-233.

    Baudet ML, Zivraj KH, Abreu-Goodger C, Muldal A, Armisen J, Blenkiron C, Goldstein LD, Miska EA, Holt CE (2011) miR-124 acts through CoREST to control onset of Sema3A sensitivity in navigating retinal growth cones. Nat Neurosci 15:29-38.

    Caggiano AO, Zimber MP, Ganguly A, Blight AR, Gruskin EA (2005) Chondroitinase ABCI improves locomotion and bladder function following contusion injury of the rat spinal cord. J Neurotrauma 22: 226-239.

    Cheng LC, Pastrana E, Tavazoie M, Doetsch F (2009) MiR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci 12:399-408.

    Clark AM, Goldstein LD, Tevlin M, Tavaré S, Shaham S, Miska EA (2010) The microRNA miR-124 controls gene expression in the sensory nervous system of Caenorhabditis elegans. Nucleic Acids Res 38:3780-3793.

    Czikk MJ, Totten S, Hammond R, Richardson BS (2014) Microtubule-associated protein 2 and synaptophysin in the preterm and near-term ovine fetal brain and the effect of intermittent umbilical cord occlusion. Reprod Sci. doi:10.1177/1933719114529371

    Doeppner TR, Doehring M, Bretschneider E, Zechariah A, Kaltwasser B, Müller B, Koch JC, B?hr M, Hermann DM, Michel U (2013) MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent REST degradation. Acta Neuropathol 126:251-265.

    Eftekharpour E, Karimi-Abdolrezaee S, Wang J, El Beheiry H, Morshead C, Fehlings MG (2007) Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27: 3416-3428.

    Eftekharpour E, Karimi-Abdolrezaee S, Fehlings MG (2008) Current status of experimental cell replacement approaches to spinal cord injury. Neurosurg Focus 24:E19.

    Jung DI, Ha J, Kang BT, Kim JW, Quan FS, Lee JH, Woo EJ, Park HM (2009) A comparison of autologous and allogenic bone marrow-derived mesenchymal stem cell transplantation in canine spinal cord injury. J Neurol Sci 285:67-77.

    Kim J, Krichevsky A, Grad Y, Hayes GD, Kosik KS, Church GM, Ruvkun G (2004) Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proc Natl Acad Sci U S A 101:360-365.

    Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS (2003) A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9:1274-1281.

    Krichevsky AM (2007) MicroRNA pro fi ling: from dark matter to white matter, or identifying new players in neurobiology. Sci World J 7: 155-166.

    Liu J, Githinji J, Mclaughlin B, Wilczek K, Nolta J (2012) Role of miRNAs in neuronal differentiation from human embryonic stem cell-derived neural stem cells. Stem Cell Rev 8:1129-1137.

    Liu XS, Chopp M, Zhang RL, Tao T, Wang XL, Kassis H, Hozeska-Solgot A, Zhang L, Chen C, Zhang ZG (2011) MicroRNA pro fi ling in subventricular zone after stroke: MiR-124a regulates proliferation of neural progenitor cells through Notch signaling pathway. PLoS One 6: e23461.

    Maiorano NA, Mallamaci A (2009) Promotion of embryonic cortico-cerebral neuronogenesis by miR-124. Neural Dev 4:40.

    McNeill E, Van Vactor D (2012) MicroRNAs shape the neuronal landscape. Neuron 75:363-379.

    Nakanishi K, Nakasa T, Tanaka N, Ishikawa M, Yamada K, Yamasaki K, Kamei N, Izumi B, Adachi N, Miyaki S, Asahara H, Ochi M (2010) Responses of microRNAs 124a and 223 following spinal cord injury in mice. Spinal Cord 48:192-196.

    Paul C, Samdani AF, Betz RR, Fischer I, Neuhuber B (2009) Grafting of human bone marrow stromal cells into spinal cord injury: a comparison of delivery methods. Spine (Phila Pa 1976) 34:328-334.

    Paspala SA, Balaji AB, Nyamath P, Ahmed KS, Khan AA, Khaja MN, Narsu ML, Devi YP, Murthy TV, Habibullah CM (2009) Neural stem cells & supporting cells--the new therapeutic tools for the treatment of spinal cord injury. Indian J Med Res 130:379-391.

    Qureshi IA, Gokhan S, Mehler MF (2010) REST and CoREST are transcriptional and epigenetic regulators of seminal neural fate decisions. Cell Cycle 9:4477-4486.

    Robertson JA (1999) Ethics and policy in embryonic stem cell research. Kennedy Inst Ethics J 9:109-136.

    Roese-Koerner B, Stappert L, Koch P, Brüstle O, Borghese L (2013) Pluripotent Stem Cell-Derived Somatic Stem Cells as Tool to Study the Role of microRNAs in early human neural development. Curr Mol Med 13:707-722.

    Sanuki R, Onishi A, Koike C, Muramatsu R, Watanabe S, Muranishi Y, Irie S, Uneo S, Koyasu T, Matsui R, Chérasse Y, Urade Y, Watanabe D, Kondo M, Yamashita T, Furukawa T (2011) miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression. Nat Neurosci 14:1125-1134.

    Schoolmeesters A, Eklund T, Leake D, Vermeulen A, Smith Q, Force Aldred S, Fedorov Y (2009) Functional pro fi ling reveals critical role for miRNA in differentiation of human mesenchymal stem cells. PLoS One 4:e5605

    Shi Y, Zhao X, Hsieh J, Wichterle H, Impey S, Banerjee S, Neveu P, Kosik KS (2010) MicroRNA regulation of neural stem cells and neurogenesis. J Neurosci 30:14931-14936.

    Sun AX, Crabtree GR, Yoo AS (2013) MicroRNAs: regulators of neuronal fate. Curr Opin Cell Biol 25:215-221.

    Sun Y, Gui H, Li Q, Luo ZM, Zheng MJ, Duan JL, Liu X (2013) MicroRNA-124 protects neurons against apoptosis in cerebral ischemic stroke. CNS Neurosci Ther 19:813-819.

    Visvanathan J, Lee S, Lee B, Lee JW, Lee SK (2007) The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development. Genes Dev 21:744-749.

    von Bohlen und Halbach O (2011) Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res 345:1-19.

    Weng R, Cohen SM (2012) Drosophila miR-124 regulates neuroblast proliferation through its target anachronism. Development 139:1427-1434.

    Xia H, Cheung WK, Ng SS, Jiang X, Jiang S, Sze J, Leung GK, Lu G, Chan DT, Bian XW, Kung HF, Poon WS, Lin MC (2012) Loss of brain-enriched miR-124 microRNA enhances stem-like traits and invasiveness of glioma cells. J Biol Chem 87:9962-9971.

    Xu W, Li P, Qin K, Wang X, Jiang X (2012) MiR-124 regulates neural stem cells in the treatment of spinal cord injury. Neurosci Lett 529:12-17.

    Yoo AS, Sun AX, Li L, Shcheglovitov A, Portmann T, Li Y, Lee-Messer C, Dolmetsch RE, Tsien RW, Crabtree GR (2011) MicroRNA-mediated conversion of human fi broblasts to neurons. Nature 476:228-231.

    Copyedited by Patel B, Li CH, Song LP, Zhao M

    10.4103/1673-5374.135333

    Guanjun Tu, M.D., Department of Orthopedics, First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China,

    tu188@sina.com.

    http://www.nrronline.org/

    Accepted: 2014-05-23

    欧美日韩在线观看h| 久久久久久久国产电影| 国产黄片视频在线免费观看| 我的女老师完整版在线观看| 在线看a的网站| 十八禁网站网址无遮挡| 日韩熟女老妇一区二区性免费视频| 老司机影院毛片| 狠狠婷婷综合久久久久久88av| 永久免费av网站大全| 国产黄频视频在线观看| 欧美人与性动交α欧美精品济南到 | 免费观看av网站的网址| 国产毛片在线视频| 考比视频在线观看| 丝袜喷水一区| 精品人妻一区二区三区麻豆| 韩国高清视频一区二区三区| 中文乱码字字幕精品一区二区三区| 精品久久久久久电影网| 亚洲av欧美aⅴ国产| 欧美老熟妇乱子伦牲交| 男女无遮挡免费网站观看| 国产乱来视频区| 国精品久久久久久国模美| 欧美日韩精品成人综合77777| 国产av码专区亚洲av| 精品人妻在线不人妻| 午夜精品国产一区二区电影| 2021少妇久久久久久久久久久| 丰满迷人的少妇在线观看| 国产成人91sexporn| 永久免费av网站大全| 久久久久久久久久成人| 精品亚洲成a人片在线观看| 人妻系列 视频| 欧美激情国产日韩精品一区| 午夜激情福利司机影院| 在线观看免费高清a一片| 777米奇影视久久| 亚洲精品日韩av片在线观看| 国产精品久久久久久久电影| 久久婷婷青草| 欧美 日韩 精品 国产| 国产片特级美女逼逼视频| 免费观看性生交大片5| 一级片'在线观看视频| 国产精品秋霞免费鲁丝片| 观看av在线不卡| 最新的欧美精品一区二区| av.在线天堂| 亚洲在久久综合| 久久婷婷青草| 成人午夜精彩视频在线观看| h视频一区二区三区| 久久国产精品男人的天堂亚洲 | 亚洲精品一区蜜桃| 国产淫语在线视频| 国产成人精品在线电影| 亚洲av中文av极速乱| 一区二区av电影网| 自拍欧美九色日韩亚洲蝌蚪91| 日本wwww免费看| 免费久久久久久久精品成人欧美视频 | 亚洲经典国产精华液单| 婷婷色麻豆天堂久久| 女人精品久久久久毛片| 丰满乱子伦码专区| 日韩欧美精品免费久久| 超碰97精品在线观看| 青春草视频在线免费观看| 国产精品.久久久| 国产极品天堂在线| 五月玫瑰六月丁香| 狂野欧美激情性xxxx在线观看| 国产精品一区二区在线不卡| 国精品久久久久久国模美| 热99国产精品久久久久久7| 综合色丁香网| 久久97久久精品| 欧美日韩国产mv在线观看视频| 国产在视频线精品| 黄色视频在线播放观看不卡| 日韩亚洲欧美综合| 能在线免费看毛片的网站| 一区二区三区乱码不卡18| 能在线免费看毛片的网站| 最后的刺客免费高清国语| 亚洲综合精品二区| 美女cb高潮喷水在线观看| av电影中文网址| 一本久久精品| 2021少妇久久久久久久久久久| 中文字幕精品免费在线观看视频 | 亚洲色图综合在线观看| 91在线精品国自产拍蜜月| 亚洲精品av麻豆狂野| 一区二区三区乱码不卡18| 黄片播放在线免费| 久久精品国产亚洲网站| 日韩欧美精品免费久久| 亚洲,一卡二卡三卡| av天堂久久9| 国国产精品蜜臀av免费| 99久久中文字幕三级久久日本| 国产熟女午夜一区二区三区 | 能在线免费看毛片的网站| 免费观看性生交大片5| 亚洲精品国产色婷婷电影| 国产成人精品婷婷| 国产成人91sexporn| 视频区图区小说| 欧美精品一区二区免费开放| 视频在线观看一区二区三区| 在线看a的网站| 欧美三级亚洲精品| 人人妻人人澡人人爽人人夜夜| 亚洲国产日韩一区二区| 人人妻人人爽人人添夜夜欢视频| 国产精品国产av在线观看| av专区在线播放| 夜夜爽夜夜爽视频| 考比视频在线观看| 伦精品一区二区三区| 国产一区二区三区综合在线观看 | 伊人久久精品亚洲午夜| 欧美日本中文国产一区发布| 国产欧美亚洲国产| 欧美日韩综合久久久久久| 中文欧美无线码| av在线观看视频网站免费| 日韩不卡一区二区三区视频在线| 国产日韩一区二区三区精品不卡 | 亚洲不卡免费看| 91精品一卡2卡3卡4卡| 91精品三级在线观看| 插逼视频在线观看| 视频区图区小说| 精品少妇久久久久久888优播| 国产黄色视频一区二区在线观看| 国产亚洲av片在线观看秒播厂| 欧美+日韩+精品| 久久99一区二区三区| 成人国产麻豆网| 黄片播放在线免费| 色婷婷久久久亚洲欧美| 三上悠亚av全集在线观看| 国产精品人妻久久久久久| 国产精品一区二区在线观看99| av.在线天堂| 亚洲欧洲国产日韩| 久久ye,这里只有精品| 我要看黄色一级片免费的| 成年av动漫网址| 国产伦理片在线播放av一区| 欧美精品人与动牲交sv欧美| 我的女老师完整版在线观看| 少妇猛男粗大的猛烈进出视频| 婷婷成人精品国产| 亚洲精品一二三| 亚洲精品aⅴ在线观看| xxx大片免费视频| 我要看黄色一级片免费的| 高清在线视频一区二区三区| 成人亚洲欧美一区二区av| 国产伦精品一区二区三区视频9| 一级毛片我不卡| 精品人妻在线不人妻| 69精品国产乱码久久久| 最近手机中文字幕大全| 99国产精品免费福利视频| 亚洲国产av新网站| 天堂俺去俺来也www色官网| 成人亚洲精品一区在线观看| 久久午夜综合久久蜜桃| 亚洲av成人精品一区久久| 91午夜精品亚洲一区二区三区| av国产精品久久久久影院| tube8黄色片| 最新的欧美精品一区二区| 精品少妇内射三级| a级片在线免费高清观看视频| 国产国语露脸激情在线看| 热99国产精品久久久久久7| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 另类亚洲欧美激情| 亚洲国产av新网站| 婷婷色av中文字幕| 欧美精品人与动牲交sv欧美| 国语对白做爰xxxⅹ性视频网站| 两个人免费观看高清视频| 国产极品粉嫩免费观看在线 | 插逼视频在线观看| 91在线精品国自产拍蜜月| 免费少妇av软件| 久久99蜜桃精品久久| 午夜老司机福利剧场| 热99久久久久精品小说推荐| 最近最新中文字幕免费大全7| 秋霞在线观看毛片| 我的老师免费观看完整版| 日本爱情动作片www.在线观看| 国产一区有黄有色的免费视频| 街头女战士在线观看网站| 一级毛片我不卡| 欧美激情 高清一区二区三区| 亚洲综合色惰| 999精品在线视频| 男女免费视频国产| 啦啦啦视频在线资源免费观看| 久久精品熟女亚洲av麻豆精品| 美女主播在线视频| 少妇人妻精品综合一区二区| 国产免费福利视频在线观看| 好男人视频免费观看在线| 国产精品国产av在线观看| 精品一区二区免费观看| 嘟嘟电影网在线观看| 亚洲第一区二区三区不卡| 黄片播放在线免费| 久久毛片免费看一区二区三区| www.色视频.com| a级毛色黄片| 插逼视频在线观看| 久久 成人 亚洲| 成人午夜精彩视频在线观看| 色婷婷av一区二区三区视频| 欧美成人精品欧美一级黄| 在线看a的网站| 国产成人免费无遮挡视频| 两个人免费观看高清视频| 日本vs欧美在线观看视频| 国产亚洲一区二区精品| 国产精品 国内视频| 一区二区三区免费毛片| 欧美激情极品国产一区二区三区 | 秋霞在线观看毛片| 91久久精品电影网| 亚洲欧美清纯卡通| 国产成人精品在线电影| 久久97久久精品| 少妇的逼水好多| 国产成人免费观看mmmm| 18禁观看日本| 亚洲熟女精品中文字幕| 免费黄色在线免费观看| 人成视频在线观看免费观看| av不卡在线播放| www.av在线官网国产| 97在线视频观看| 亚洲少妇的诱惑av| 国产一区二区在线观看日韩| 久久久精品区二区三区| 婷婷色麻豆天堂久久| 高清欧美精品videossex| 成年人免费黄色播放视频| 多毛熟女@视频| 久久久久久久久久久丰满| 成人手机av| 亚洲国产色片| 亚洲国产精品一区二区三区在线| 一区二区三区乱码不卡18| 国产在线视频一区二区| 成人漫画全彩无遮挡| 夜夜爽夜夜爽视频| 97超视频在线观看视频| av在线app专区| 国产一区二区三区av在线| 国产精品一区www在线观看| 免费高清在线观看日韩| 亚洲成人手机| 成人免费观看视频高清| 一区二区三区四区激情视频| 久久久久久久久久成人| 亚洲情色 制服丝袜| 大又大粗又爽又黄少妇毛片口| 亚洲av成人精品一二三区| 热re99久久精品国产66热6| 最近的中文字幕免费完整| videosex国产| 一二三四中文在线观看免费高清| 欧美激情国产日韩精品一区| 亚洲情色 制服丝袜| 亚洲丝袜综合中文字幕| 国产亚洲欧美精品永久| 久久国产精品男人的天堂亚洲 | 性色avwww在线观看| 日韩,欧美,国产一区二区三区| 97在线视频观看| 欧美精品高潮呻吟av久久| 久久精品国产自在天天线| 天堂俺去俺来也www色官网| 成人毛片a级毛片在线播放| 一区二区三区乱码不卡18| www.av在线官网国产| 亚洲国产精品一区三区| 午夜免费男女啪啪视频观看| av一本久久久久| 国产日韩欧美在线精品| 黑人高潮一二区| 黑人巨大精品欧美一区二区蜜桃 | 啦啦啦视频在线资源免费观看| 尾随美女入室| 夫妻午夜视频| 男女无遮挡免费网站观看| 日韩大片免费观看网站| 一个人免费看片子| 一级毛片 在线播放| 内地一区二区视频在线| 国产国语露脸激情在线看| 亚洲精品456在线播放app| freevideosex欧美| 三级国产精品片| 久久久久久久久久久久大奶| 五月天丁香电影| 欧美日韩综合久久久久久| 国产免费视频播放在线视频| 精品酒店卫生间| 日本爱情动作片www.在线观看| 中国美白少妇内射xxxbb| 男女边吃奶边做爰视频| 欧美变态另类bdsm刘玥| 97在线视频观看| 在线观看人妻少妇| 国产黄频视频在线观看| 狂野欧美激情性xxxx在线观看| 欧美bdsm另类| 国产黄片视频在线免费观看| 蜜桃久久精品国产亚洲av| 成人毛片a级毛片在线播放| 中文字幕最新亚洲高清| 九色亚洲精品在线播放| 蜜桃在线观看..| 亚洲美女黄色视频免费看| 日日爽夜夜爽网站| 午夜福利视频在线观看免费| 亚洲精品自拍成人| 精品少妇内射三级| av专区在线播放| 国产欧美日韩综合在线一区二区| 日本欧美国产在线视频| 另类亚洲欧美激情| 精品久久久久久电影网| 最近的中文字幕免费完整| 女人久久www免费人成看片| 中文精品一卡2卡3卡4更新| 国产无遮挡羞羞视频在线观看| 欧美日韩一区二区视频在线观看视频在线| 在线观看国产h片| 国产亚洲av片在线观看秒播厂| 只有这里有精品99| 妹子高潮喷水视频| 免费高清在线观看视频在线观看| 欧美3d第一页| 少妇被粗大猛烈的视频| 自线自在国产av| 国产欧美日韩综合在线一区二区| 亚洲av在线观看美女高潮| 日本wwww免费看| 久久精品国产亚洲av天美| 草草在线视频免费看| 欧美成人午夜免费资源| av免费在线看不卡| 夜夜爽夜夜爽视频| 欧美国产精品一级二级三级| 日韩成人伦理影院| 黄色怎么调成土黄色| 十八禁网站网址无遮挡| 一二三四中文在线观看免费高清| 看十八女毛片水多多多| 精品久久久精品久久久| 大话2 男鬼变身卡| 午夜激情av网站| 母亲3免费完整高清在线观看 | 成年女人在线观看亚洲视频| 少妇被粗大猛烈的视频| 99久国产av精品国产电影| 欧美日本中文国产一区发布| 国产国拍精品亚洲av在线观看| 18禁观看日本| 这个男人来自地球电影免费观看 | 亚洲人成网站在线播| 久久热精品热| videossex国产| 伦精品一区二区三区| 国产成人精品婷婷| 亚洲精品乱码久久久v下载方式| 亚洲精品乱码久久久久久按摩| 午夜老司机福利剧场| 久久久久久久久久成人| 国产亚洲欧美精品永久| 日日啪夜夜爽| 欧美bdsm另类| 免费看av在线观看网站| 成人毛片a级毛片在线播放| 精品久久久精品久久久| 日本黄色日本黄色录像| 男女国产视频网站| 又黄又爽又刺激的免费视频.| 国产成人91sexporn| 亚洲精品国产av蜜桃| 这个男人来自地球电影免费观看 | av线在线观看网站| 中文欧美无线码| 狂野欧美白嫩少妇大欣赏| 中文字幕人妻丝袜制服| 日日摸夜夜添夜夜爱| 久久av网站| 97在线视频观看| 亚洲精品一区蜜桃| 超色免费av| av女优亚洲男人天堂| 国产白丝娇喘喷水9色精品| 亚洲欧洲精品一区二区精品久久久 | 嫩草影院入口| 日韩欧美一区视频在线观看| www.色视频.com| 麻豆精品久久久久久蜜桃| 美女脱内裤让男人舔精品视频| 狂野欧美激情性xxxx在线观看| 国产在线一区二区三区精| 蜜臀久久99精品久久宅男| 亚洲四区av| 亚洲av二区三区四区| 美女xxoo啪啪120秒动态图| 国产色婷婷99| 日韩不卡一区二区三区视频在线| 国产亚洲一区二区精品| 男的添女的下面高潮视频| 亚洲综合精品二区| 少妇高潮的动态图| 18禁在线播放成人免费| 两个人免费观看高清视频| 精品久久蜜臀av无| 国产在线一区二区三区精| 国产 一区精品| 韩国av在线不卡| 男的添女的下面高潮视频| 2021少妇久久久久久久久久久| 99精国产麻豆久久婷婷| 亚洲欧美清纯卡通| 久久久久久久久久久久大奶| 亚洲av综合色区一区| 日韩不卡一区二区三区视频在线| 国产日韩欧美在线精品| 久久ye,这里只有精品| 国产探花极品一区二区| 欧美日韩国产mv在线观看视频| 亚洲欧美精品自产自拍| 国产乱来视频区| 久久精品国产自在天天线| 国产亚洲精品第一综合不卡 | 热99国产精品久久久久久7| 亚洲精品自拍成人| 中文字幕免费在线视频6| 伦理电影大哥的女人| 王馨瑶露胸无遮挡在线观看| 天堂中文最新版在线下载| 男人爽女人下面视频在线观看| 这个男人来自地球电影免费观看 | 国产一区亚洲一区在线观看| kizo精华| 男男h啪啪无遮挡| 欧美xxⅹ黑人| 丰满饥渴人妻一区二区三| 免费播放大片免费观看视频在线观看| 亚洲第一av免费看| 18禁在线播放成人免费| 免费av不卡在线播放| 成年人午夜在线观看视频| 国产黄频视频在线观看| 高清在线视频一区二区三区| 免费av中文字幕在线| 亚洲在久久综合| 日韩欧美精品免费久久| 亚洲一区二区三区欧美精品| 免费黄网站久久成人精品| 亚州av有码| 国产精品国产av在线观看| 国精品久久久久久国模美| 欧美日韩一区二区视频在线观看视频在线| 亚洲精品第二区| 日韩伦理黄色片| 国产色爽女视频免费观看| 精品久久久久久久久av| 国产探花极品一区二区| 99久久精品国产国产毛片| 汤姆久久久久久久影院中文字幕| 午夜激情福利司机影院| 午夜日本视频在线| 天堂俺去俺来也www色官网| 免费人妻精品一区二区三区视频| 午夜激情福利司机影院| 久久免费观看电影| 免费日韩欧美在线观看| 国产男女超爽视频在线观看| 久久97久久精品| 成年人免费黄色播放视频| 久久99热这里只频精品6学生| 久久精品国产亚洲av涩爱| 校园人妻丝袜中文字幕| 熟女av电影| 18禁在线播放成人免费| 亚洲无线观看免费| 99九九线精品视频在线观看视频| 在线播放无遮挡| 黑人欧美特级aaaaaa片| 亚洲欧美成人精品一区二区| 激情五月婷婷亚洲| 两个人免费观看高清视频| 男女高潮啪啪啪动态图| h视频一区二区三区| 欧美另类一区| 成人午夜精彩视频在线观看| 国产视频首页在线观看| 国产精品不卡视频一区二区| 丰满迷人的少妇在线观看| 亚洲,欧美,日韩| 91在线精品国自产拍蜜月| 成年人免费黄色播放视频| 高清不卡的av网站| 欧美精品一区二区大全| 18禁动态无遮挡网站| 国产成人精品婷婷| 免费大片黄手机在线观看| 2021少妇久久久久久久久久久| 街头女战士在线观看网站| 久久久国产欧美日韩av| 成人18禁高潮啪啪吃奶动态图 | 搡老乐熟女国产| 色94色欧美一区二区| 精品人妻熟女av久视频| 国产一区二区在线观看日韩| 日韩精品免费视频一区二区三区 | 大香蕉97超碰在线| 久久99蜜桃精品久久| 国产高清三级在线| 精品少妇久久久久久888优播| 亚洲欧美一区二区三区黑人 | 国产精品国产三级国产专区5o| 色婷婷av一区二区三区视频| 久久亚洲国产成人精品v| 18禁在线无遮挡免费观看视频| 男女边摸边吃奶| 国产乱人偷精品视频| 制服人妻中文乱码| 九九在线视频观看精品| 妹子高潮喷水视频| 又粗又硬又长又爽又黄的视频| 亚洲国产日韩一区二区| 国产 精品1| 亚洲国产av影院在线观看| 亚洲精华国产精华液的使用体验| 卡戴珊不雅视频在线播放| 纵有疾风起免费观看全集完整版| 女的被弄到高潮叫床怎么办| 一级a做视频免费观看| 亚洲经典国产精华液单| 国产精品国产三级国产专区5o| 亚洲一级一片aⅴ在线观看| 欧美激情国产日韩精品一区| 老司机影院成人| 美女主播在线视频| 久热这里只有精品99| 丝袜美足系列| 男的添女的下面高潮视频| 亚洲图色成人| 成人18禁高潮啪啪吃奶动态图 | 亚洲高清免费不卡视频| 视频在线观看一区二区三区| 91成人精品电影| 精品少妇久久久久久888优播| 交换朋友夫妻互换小说| 亚洲精品乱码久久久v下载方式| 黑人高潮一二区| 18+在线观看网站| 久久久久久久久久久久大奶| 国产视频首页在线观看| 日韩在线高清观看一区二区三区| 欧美日韩视频高清一区二区三区二| 欧美日本中文国产一区发布| 国模一区二区三区四区视频| 免费观看av网站的网址| 久久久久精品性色| 视频在线观看一区二区三区| 亚洲成人一二三区av| 国产精品秋霞免费鲁丝片| 少妇精品久久久久久久| 国产高清国产精品国产三级| a级片在线免费高清观看视频| 国产成人av激情在线播放 | 精品国产一区二区三区久久久樱花| 性色avwww在线观看| a级毛片在线看网站| 超碰97精品在线观看| 亚洲国产av影院在线观看| 国产成人精品一,二区| 91久久精品国产一区二区三区| 精品少妇黑人巨大在线播放| 插阴视频在线观看视频| 免费观看av网站的网址| 美女国产高潮福利片在线看| 亚洲av欧美aⅴ国产| 91精品国产九色| 亚州av有码| 亚洲欧美成人精品一区二区| 黄片播放在线免费| 国产成人精品福利久久| 国产精品久久久久成人av| 男女边摸边吃奶| 999精品在线视频| 少妇人妻 视频| 精品久久久噜噜| 久久午夜综合久久蜜桃| 国产精品.久久久|