• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Human umbilical cord mesenchymal stem cells to treat spinal cord injury in the early chronic phase: study protocol for a prospective, multicenter, randomized, placebo-controlled, single-blinded clinical trial

    2020-01-18 06:02:46YangYangMaoPangYuYongChenLiangMingZhangHaoLiuJunTanBinLiuLiMinRong

    Yang Yang , Mao Pang , Yu-Yong Chen Liang-Ming Zhang Hao Liu, Jun Tan, Bin Liu , Li-Min Rong

    1 Department of Spine Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangdong Provincial Center for Quality Control of Minimally Invasive Spine Surgery, Guangdong Provincial Center for Engineering and Technology Research of Minimally Invasive Spine Surgery, Guangzhou, Guangdong Province, China

    2 Department of Orthopedics, West China Hospital of Sichuan University, Chengdu, Sichuan Province, China 3 Department of Orthopedics, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

    Abstract Human umbilical cord mesenchymal stem cells (hUC-MSCs) support revascularization, inhibition of inflammation, regulation of apoptosis, and promotion of the release of beneficial factors. Thus, they are regarded as a promising candidate for the treatment of intractable spinal cord injury (SCI). Clinical studies on patients with early chronic SCI (from 2 months to 1 year post-injury), which is clinically common, are rare; therefore, we will conduct a prospective, multicenter, randomized, placebo-controlled, single-blinded clinical trial at the Third Affiliated Hospital of Sun Yat-sen University, West China Hospital of Sichuan University, and Shanghai East Hospital, Tongji University School of Medicine, China. The trial plans to recruit 66 early chronic SCI patients. Eligible patients will undergo randomization at a 2:1 ratio to two arms: the observation group and the control group. Subjects in the observation group will receive four intrathecal transplantations of stem cells, with a dosage of 1 × 106/kg, at one calendar month intervals. Subjects in the control group will receive intrathecal administrations of 10 mL sterile normal saline in place of the stem cell transplantations. Clinical safety will be assessed by the analysis of adverse events and laboratory tests. The American Spinal Injury Association (ASIA) total score will be the primary efficacy endpoint, and the secondary efficacy outcomes will be the following: ASIA impairment scale, International Association of Neural Restoration-Spinal Cord Injury Functional Rating Scale, muscle tension, electromyogram, cortical motor and cortical sensory evoked potentials, residual urine volume, magnetic resonance imaging—diffusion tensor imaging, T cell subtypes in serum, neurotrophic factors and inflammatory factors in both serum and cerebrospinal fluid. All evaluations will be performed at 1, 3, 6, and 12 months following the final intrathecal administration. During the entire study procedure, all adverse events will be reported as soon as they are noted. This trial is designed to evaluate the clinical safety and efficacy of subarachnoid transplantation of hUC-MSCs to treat early chronic SCI. Moreover, it will establish whether cytotherapy can ameliorate local hostile microenvironments, promote tracking fiber regeneration, and strengthen spinal conduction ability, thus improving overall motor, sensory, and micturition/defecation function in patients with early chronic SCI. This study was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, China (approval No. [2018]-02) on March 30, 2018, and was registered with ClinicalTrials.gov (registration No. NCT03521323) on April 12, 2018. The revised trial protocol (protocol version 4.0) was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, China (approval No. [2019]-10) on February 25, 2019, and released on ClinicalTrials.gov on April 29, 2019.

    Key Words: clinical study; early chronic phase; efficacy; human umbilical cord mesenchymal stem cell; multicenter trial; prospective study; randomized controlled trial; safety; spinal cord injury; study protocol

    Introduction

    Spinal cord injury (SCI) often results in lifelong disability, muscle palsy, sensory disturbances, autonomic dysfunction, and neuropathic pain, as well as bowel and bladder incontinence, depending on the SCI severity (Eckert and Martin, 2017; Badhiwala et al., 2018; Lin and Chay, 2018, Brown and Martinez, 2019; Zhang et al., 2019). These sequelae can severely reduce quality of life for both patients and caregivers (Lynch and Cahalan, 2017; Müller et al., 2017). The pathophysiology of SCI can be divided into primary and secondary injuries, and secondary injury involves axon dislocation of damaged tracking fibers, axon demyelination caused by the loss of myelin-producing oligodendrocytes, local neuroinflammation because of tissue edema or ischemia, and parenchymal cavity or glial scar formation followed by intraspinal hemorrhage (Priest et al., 2015; Manley et al., 2017; Koda et al., 2018). To the best of our knowledge, there is no effective method that reverses the trauma, partly because of the extremely limited self-regeneration abilities of the spinal cord (Marichal et al., 2017; Donovan and Kirshblum, 2018; Kabatas et al., 2018). SCI in the secondary phase is considered to be the primary target for treatment, and a number of attempts have been made to treat this phase (Karsy and Hawryluk, 2017; Jacobs and Lovejoy, 2018). Currently, the only approved medication to treat SCI in clinic is a high dose of corticosteroid (Falavigna et al., 2018). However, there is a lack of consensus regarding its standardized application because a series of potential risks and undetermined clinical outcomes have been reported (Kube and Olby, 2008; Fehlings et al., 2017). Hence, a novel, effective treatment is urgently required for SCI.

    Human umbilical cord mesenchymal stem cells (hUCMSC) are a promising choice for SCI therapy. Their use has many benefits, including in revascularization support, control of inflammation, inhibition of cellular apoptosis, and production of multiple trophic factors, as well as the differentiation of hUC-MSCs into oligodendrocytes and neurons (Torres-Espín et al., 2013; Zhilai et al., 2016; Shende and Subedi, 2017). Moreover, additional advantages, including their lack of contamination, easy obtainability, low immunogenicity, and rapid proliferation, make them a highly suitable candidate for SCI therapy (Liu et al., 2013; Yaghoobi et al., 2016). Of the numerous possible transplantation routes, it has been demonstrated that cell engraftment and tissue sparing are significantly better after intrathecal delivery, and that the host immune response is reduced with subarachnoid infusion. It has also been reported that intrathecal administration of stem cells results in better functional recovery than other approaches of cellular delivery (Paul et al., 2009; Shin et al., 2013). Thus, minimally invasive subarachnoid administration is considered the safest and most effective approach for stem cell delivery (Cizkova et al., 2011; Krupa et al., 2018).

    SCI in the early chronic phase, although inconsistently defined, is characterized by a profound infiltration of immune cells and a peak secretion of pro-inflammatory cytokines from the injured tissues (Nutt et al., 2013; Vidal et al., 2013; Johnson et al., 2017). Because SCI patients at this stage are commonly encountered in the clinic, this study protocol describes a planned clinical trial to assess safety and efficacy of intrathecal transplantation of hUC-MSC to treat early chronic SCI.

    Subjects and Methods

    Study design

    This is the proposed protocol for a prospective, multicenter, randomized, placebo-controlled, single-blinded clinical trial, which was developed in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist (Chan et al., 2013a, b) (Figure 1, Table 1 and Additional file 1). The initiating sponsor and co-ordinator of this clinical trial is the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). The other two participants are West China Hospital of Sichuan University (Chengdu, China) and Shanghai East Hospital, Tongji University School of Medicine (Shanghai, China). For ethical reasons that more subjects benefit from any possible positive outcomes of this treatment, eligible patients will undergo randomization at a 2:1 ratio to receive subarachnoid infusion of either hUC-MSC (the observation group) or sterile normal saline (the control group). Randomization will be performed centrally according to a computer-generated number table. The use of a centrally automated allocation procedure is to ensure that randomization data are not influenced by any person. In this study, both those giving therapy and the assessors will not be intervention-blinded. Only the subjects will be treatment-blinded.

    Table 1 Standard Protocol Items: Recommendations for Interventional Trials diagram

    Figure 1 Schematic diagram of the use of human umbilical cord mesenchymal stem cells (hUC-MSC) to treat early chronic spinal cord injury.

    Subjects

    Eligibility

    The three trial centers will use the same criteria and rigidity for subject inclusion. Recruitment methodologies include advertisements in academic media as well as liaisons with general practitioners, specialist neurologists, and spinal surgeons. Inclusion criteria are as follows: (1) complete or incomplete trauma-induced SCI [American Spinal Injury Association (ASIA) A-D] at between 2 months and 1 year after the trauma (the early chronic phase) (Kalsi-Ryan et al., 2014); (2) aged between 18 and 65 years; (3) have voluntarily signed and dated an informed consent form, approved by the stem cell research ethics committee, after the nature of the clinical study has been explained and the subjects have had the opportunity to ask questions; a separate informed consent form is needed for any subject participating in this clinical study; (4) agree to be regularly followed up for 1 year after the completion of treatment. All of the aforementioned items will need to be met for any included subject.

    Exclusion

    Exclusion criteria are as follows: (1) ankylosing spondylitis, myelitis, or vascular abnormalities within the spinal cord parenchyma; (2) severe comorbidities, including but not limited to craniocerebral injury, cutaneous back infection, psychiatric disorder, or cancer; (3) pregnancy or lactation (for females); (4) predicted lifespan of less than 12 months following the end of therapy; (5) participation in any other stem cell-associated clinical trials that might affect accurate neurological evaluations in the present trial; (6) any medical condition that, in the opinion of investigators, may pose a safety risk to any subject in this study, confound safety or efficacy assessments, or interfere with study participation. If any clause of the exclusion criteria is met, the subject will be removed.

    Rejection

    Rejection criteria are as follows: (1) misdiagnosis; (2) use of any medication that may significantly impact the assessment accuracy of hUC-MSC efficacy; (3) absence of any evaluation outcome at any time point during the follow-up period. For any rejected subject, the reasons for rejection will be explained, and a case report form (CRF) containing the discontinuation date will be filed.

    Cessation

    Cessation criteria are as follows: (1) individual wishes of the subjects; (2) occurrence of any stem cell-associated serious adverse event (SAE) that may aggravate neural dysfunction, impair consciousness, or be life-threatening or lead to death in any subject; (3) detection of any major mistake in the present protocol during the implementation of this clinical trial; (4) inability to enforce the current protocol by investigators; (5) the national administration agency requires the clinical trial to be halted.

    Sample size evaluation

    A difference test was used to predict sample sizes in both the observation and control groups. The two-tailed significance level (α) was 0.05. To provide 80% power (1 - β) to detect any differences in changes in ASIA total scores, β was set as 0.2. Based on the results of the preliminary clinical trial, the estimated standard difference (S) between the two groups in ASIA total score change was 14. According to expert consensus from the spine surgery, rehabilitation, and neurology departments, a minimum improvement of 12 in the ASIA total score is believed to have clinical significance; therefore, δ was set as 12. The maximum possible dropout rate during follow-up was considered to be 20%. Hence, 66 patients will be sufficient to detect differences between the two groups.

    Intervention

    In this study, hUC-MSC is the intervention factor; thus, the 44 subjects in the observation group will receive subarachnoid infusion of hUC-MSCs, and the remaining 22 subjects in the control group will receive intrathecal injection of sterile normal saline (10 mL). The hUC-MSCs will be produced in a Good Manufacturing Practice-level laboratory. The isolation and proliferation of hUC-MSCs will be performed in several steps. After obtaining informed consent forms from the parents of healthy full-term neonates, umbilical cords will be collected and cut into segments, disinfected, and washed with sterile normal saline. Following the removal of arteries and vessels within the umbilical cord, Wharton’s jelly will be split into pieces with volumes of less than 1 cm3; these will then be cultured on the bottom of plates. Approximately 1 week later, cell clone of hUC-MSCs can be observed around the Wharton’s jelly slices. These primary hUC-MSCs will be digested by using TrypLETMExpress (Gibco) and then, single cells will be adjusted to the the concentration of 2 × 104/cm2in each plate. hUC-MSCs will be cultured in medium consisting of α-MEM (Gibco), GlutaMAXTM(Gibco), PLUSTMMSC Qualified Cell Culture Supplement (Compass Biomedical) in a humidified atmosphere with 5% CO2at 37°C. After reaching confluence of above 90%, cells will be passaged to continue proliferating. Following a series of in-depth quality control tests, including but not limited to endotoxin, exotoxin, mycoplasma, chlamydia, germ, virus, tumorigenicity, and tri-lineage differentiation tests in vitro, hUC-MSCs at passages 4-5 will be used in this clinical trial, and the transplantation dose will be 1 × 106cells/kg based on results from our preclinical studies (manuscript in preparation). For transportation, hUC-MSCs will be suspended in 10 mL of sterile normal saline and maintained in a temperature-controlled environment of approximately 4°C under sterile and dark conditions. The stem cell suspensions will be transported as rapidly as possible to ensure infusion within 8 hours of dissociation. During the intervention period of four calendar months, all subjects will receive the recommended standard of care.

    Safety indicators and primary and secondary efficacy endpoints

    The safety indicators are as follows: (1) routine tests of blood, urine, and excrement; (2) biochemistry analysis; (3) tumor indicators; (4) blood coagulation; (5) chest radiography; (6) electrocardiogram. Biological specimens will be acquired from subjects once an additional informed consent form has been obtained, and tests will be performed immediately after specimen acquisition. The ASIA total score is the primary efficacy endpoint, while the secondary efficacy outcomes are as follows: (1) ASIA impairment scale; (2) International Association of Neural Restoration-Spinal Cord Injury Functional Rating Scale; (3) muscle tension based on the Ashworth scale; (4) electromyogram results; (5) cortical motor and cortical sensory evoked potentials; (6) residual urine volume; (7) magnetic resonance imaging—diffusion tensor imaging (MRI-DTI) results; (8) T cell subtype analysis; (9) neurotrophic factors in serum and cerebrospinal fluid (e.g., brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, vascular endothelial growth factor); (10) inflammatory factors in serum and cerebrospinal fluid (e.g., tumor necrosis factor-α, transforming growth factor-β, interleukin-1β, interleukin-6). Of these secondary outcomes, the first three indicators will be assessed by evaluators according to standard scoring or grading systems, while the latter seven indicators will be tested using machines or kits.

    Study procedures

    In this clinical trial, the subarachnoid transplantation of hUC-MSCs will be performed a total of four times per patient. Before the first infusion of stem cells, safety and efficacy indicators will be acquired as baseline data. For the intrathecal transplantation process, each patient’s maximum lumbar flexion will be maintained, and cerebrospinal fluid release with a volume of approximately 10 mL will be performed. The hUC-MSC suspension will be administered manually at the L3/4, L4/5, or L5/S1 level as slowly as possible to minimize the possibility of any complications associated with intrathecal transplantation, including headache, nausea, or vomiting. One calendar month later, patients will undergo a second subarachnoid engraftment of hUC-MSC after all safety and most efficacy parameters (all but MRIDTI) have been obtained. Following this pattern, patients will receive the third and fourth rounds of stem cell transplantation, with the same collection of parameters as for the second hUC-MSC administration. After the completion of cytotherapy, the patient will be regularly followed up at four time points, scheduled at 1, 3, 6, and 12 calendar months following the final administration of hUC-MSCs. At the second follow-up, all safety and most efficacy indicators (all but MRI-DTI) will be acquired. For the other three follow-ups, all evaluation outcomes will be collected (Table 1). Any subject who has any kind of SAE, such as anaphylactic shock or pyogenic infection of the central nervous system, will be followed up immediately, and effective treatments will be initiated as rapidly as possible. Unscheduled follow-ups will then be continued until recovery of the normal condition. If any SAE occurs, the unblinding of this subject will be permissible. For mild or moderate adverse events (AE), the investigators will make a joint decision as to whether the subject receives unscheduled follow-ups or not.

    AE

    AEs refer to any unwanted symptoms or signs in a clinical investigation subject, and do not necessarily have a causal relationship with the applied intervention (Koda et al., 2018). Fever, headache, dizziness, muscle spasm, fatigue, chest distress, nausea, and vomiting are relatively common AEs associated with cytotherapy (Kakabadze et al., 2016; Vaquero et al., 2016, 2017). Of these, fever is most likely to be observed, with an approximate incidence of 30%. Once detected, non-steroidal anti-inflammatory drugs and intravenous infusions will be given. Similarly, headaches will also be treated with non-steroidal anti-inflammatory drugs. For dizziness, betahistine mesylate and flunarizine hydrochloride will be used to relieve symptoms. For the relief of muscle spasms, a muscle relaxant (baclofen) and mecobalamin will be administered. In patients with fatigue and chest distress, bed rest and intermittent oxygen inhalation will be suggested. To treat nausea and vomiting, metoclopramide or ondansetron will be injected intramuscularly. For other possible AEs with low morbidity, corresponding treatments will be recommended. If any SAE is encountered, even with extremely low incidence, it will be reported to the principal investigator of the trial, the stem cell research ethics committee, and the state supervision agency within 24 hours of its occurrence. All AEs will be coded according to the World Health Organization Adverse Reactions Terminology. At the end of the clinical trial, the intensity and relationship of any AEs with the study intervention will be identified.

    Data management

    All study documents from the three centers will be considered highly confidential and will be stored in locked filing cabinets in a room with restricted access. On a paper CRF, data will be collected and managed with a four-digit pseudonym. In the restricted study office, three independent trial coordinators will jointly check the completeness and consistency of CRFs. Then, under the supervision of independent trial inspectors, implausible or missing outcomes will be confirmed or added after consulting the investigator via the data query form. All correct data from paper CRFs that have undergone careful screening will be translated into electronic information and stored in the database. During this procedure, data will be entered twice by two subject-blinded people to allow a double check for accuracy. When data revision is required, the corrected data will be entered by independent trial coordinators under the supervision of both trial investigators and inspectors. Access to the database will be strongly restricted. The principal investigator and biostatistician will be able to log into the data set and get full access to the information only upon permission from the head of this study. Data backups and paper CRF archiving will be performed regularly by trial coordinators. If required, data transfer between centers will be encrypted, and any information capable of identifying individuals will be removed.

    Trial quality assurance

    It will be ensured that this study is of high quality and is delivered in accordance with the present trial protocol, which will not be amended unless any SAE occurs during its implementation. All research staff, including investigators, research assistants, and outcome assessors, will be trained in advance to be able to competently administer items as per the protocol. Once the clinical trial begins, an independent trial inspector will visit each study site monthly and will be responsible for reviewing the following: overall research progress and integrity, adherence to the selection criteria for all included subjects, compliance with the scheduled intervention for each participant, compliance with national regulations, and the handling of practical problems. Moreover, this trial inspector will occasionally provide suggestions to the principal investigator, who will make any final decisions about trial modifications, continuation, or termination.

    Statistical analysis

    In this study, three analysis sets will be required. For the safety analysis set, all safety indicators will be obtained from those who have undergone one or multiple stem cell transplantations following randomization. In the full analysis set, subjects will be excluded if they received no intervention or never gave any evaluation outcome. For the per protocol set, subjects should achieve at least 90% integrity of all of the following criteria: (1) underwent either stem cell or normal saline injection; (2) showed no violation of the present protocol; (3) completed the full follow-up; (4) obtained all required data. For the baseline characteristics, continuous variables will be expressed as means ± standard deviations, while categorical data will be given as frequencies. The independent t-test and Pearson chi-test will be applied to analyze continuous and categorical variables, respectively. For the safety indicators, primary endpoint, and most secondary outcomes, including blood coagulation function, routine tests (blood, urine, and excrement), biochemistry and tumor findings, ASIA total score, International Association of Neural Restoration-Spinal Cord Injury Functional Rating Scale, residual urine volume, MRI-DTI (fractional anisotropy, apparent diffusion coefficient), electromyogram (conduction speed, latency, amplitude), cortical motor and cortical sensory evoked potentials (latency, amplitude), and contents of neurotrophic and inflammatory factors, the independent t-test will be used to detect any differences between the two groups. To compare the remaining two secondary indicators (ASIA impairment scale and muscle tension classification) a nonparametric test (the Wilcoxon rank sum test) will be performed between the two groups. If required, statistical analysis between subgroups will be performed. All statistical analyses will be performed using Statistical Product and Service Solutions (Version 22.0, IBM, New York, NY, USA) by the principle biostatistician. P < 0.05 will be considered statistically significant.

    Patient and public involvement statements

    After the completion of the protocol design by investigators, subjects will be included in this clinical trial. Outcome measures will be developed based on the priority of this study, as well as the experience and preferences of investigators. When asked to assess the energy and time requirements to participate in this clinical trial, subjects are not expected to consider it a burden. Included participants will not be responsible for the official dissemination of the final results and conclusions.

    Ethical considerations

    The study ethics and trial protocol have been approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, Human Cell Clinical Research Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine, and Ethics Committee on Stem Cell Clinical Research, West China Hospital of Sichuan University (Additional file 2) and all the relevant regulatory authorities. The clinical study will be conducted in accordance with all regulations. An informed consent form (Additional file 3) that has the approval of the ethics committee will be obtained from all participants before their inclusion. This clinical trial complies with the principles of the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects, with all its amendments, as well as with the principles of Good Clinical Practice.

    Informed consent forms and privacy protection of subjects

    As far as possible, informed consent forms and associated explanatory information will be prepared in simple language. They will be clearly explained to subjects or their legal representatives by investigators before obtaining their informed consent. If trial participants are not able to sign their names, an allograph by their legal representatives will be allowed. No study procedure will be implemented prior to the acquisition of informed consent forms. During the study procedures, only the investigators and inspectors will have partial access to the personal information of subjects, and this will occur under the strict supervision of the study’s principal investigator. No investigators will reveal any data associated with this clinical trial, except upon an official request by the national administration agency. If there are any changes to the informed consent form during this clinical study, the revised informed consent form must receive another written approval from the stem cell research ethics committee before its use, and subjects must re-consent to the revised version of the informed consent form.

    Public dissemination and literature publication

    After consensus in the results and conclusions of this clinical trial are obtained, they will be submitted to the chief investigators in both the sponsor and participating hospitals, and will be reserved in paper form for at least 30 years. Even if the final results and conclusions are not desirable, despite performing the trial in strict accordance with all standard operating procedures, a paper describing this study will be written by professional writers in the sponsor institute. If investigators in the participating agencies plan to publish partial outcomes of this clinical trial at academic conferences or in journals, permission will be obtained in advance from the principal investigator in the sponsor hospital. When preparing the associated articles, the confidentiality of all subjects will be ensured.

    Discussion

    To the best of our knowledge, this will be the first systematic study to demonstrate the clinical safety and efficacy of hUCMSCs in treating early chronic SCI, which is relatively common in the clinic. Several studies have reported only preliminary results of the efficacy of hUC-MSCs in treating SCI, and these have not reported detailed safety evaluations and generally have not involved the treatment of SCI in the early chronic phase (Cheng et al., 2014; Miao et al., 2015; Zhao et al., 2017). Furthermore, these previous studies have many shortcomings, with small sample sizes, limited evaluation indicators, and the use of more invasive methodologies for performing stem cell transplantation. Hence, the therapeutic potential of hUC-MSC may not have been fully demonstrated. In comparison with previous studies, the proposed protocol of the present, well-designed clinical trial is aimed at early chronic SCI, and includes a variety of functional assessments, imaging evaluations, and humoral indicators that will overcome most of the aforementioned shortcomings of previous studies. The transplantation dose will be set at 1 × 106cells/kg in the current clinical trial, which is similar to that of other reports (Hur et al., 2016; Satti et al., 2016). Toxicity is extremely unlikely because our preclinical study has demonstrated very good safety in vivo after intrathecal administration with this dosage (manuscript in preparation).In summary, hUC-MSC holds great promise for the effective treatment of SCI, which is a serious public health problem. It is believed that the current protocol will be able to definitively elucidate the therapeutic efficacy and safety concerns of hUC-MSCs in treating early chronic SCI. If the present study successfully demonstrates the efficacy and safety of subarachnoid transplantation of hUC-MSCs, more in-depth clinical studies will be conducted to further verify the feasibility of their application in treating early chronic SCI. Moreover, the results of this study may substantially save costs by reducing the need for other expensive, but mostly ineffective therapies, and may also improve the quality of life of affected people. The current clinical study of intrathecal administration of hUC-MSCs is hoped to be a profound milestone for the treatment of early chronic SCI.

    Trial Status

    The study was registered with ClinicalTrials.gov on April 12, 2018 (registration No. NCT03521323), and the revised trial protocol (Protocol version 4.0) was released on April 29, 2019. Upon submission, this study is not yet in the process of patient recruitment. It is estimated that subject enrollment will begin on September 1, 2019, and be completed at the end of July 2020.

    Acknowledgments:We deeply thank Professor Chang-Hai Ding from Menzies Research Institute Tasmania, University of Tasmania, Australia for the help of language polishing in this article.

    Author contributions:All authors make substantial contributions to the conception and design of the present protocol. Data analysis for the current protocol: LMR, YY, BL, MP, HL, JT; manuscript drafting: YY, MP; manuscript revising: YY, MP; present protocol revising: YYC, LMZ. After all aspects of the present manuscript are appropriately investigated and checked, the manuscript in this version is sent to be published by all authors.

    Conflicts of interest:The authors declare that they have no conflicts of interest.

    Financial support:This work was supported by the National Key Research and Development Program of China, No. 2017YFA0105403 (to LMR); the Key Research and Development Program of Guangdong Province of China, No. 2019B020236002 (to LMR); The Clinical Innovation Research Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory of China, No. 2018GZR0201006 (to LMR); the National Natural Science Foundation of China, Nos. 81772349 (to BL), 31470949 (to BL); the Guangzhou Science and Technology Project of China, Nos. 201704020221 (to LMR), 201707010115 (to BL); the Natural Science Foundation of Guangdong Province of China, No. 2017A030313594 (to BL); the Medical Scientific Research Foundation of Guangdong Province of China, No. A2018547 (to MP). All the funding bodies do not participate in the design detailed of the study or collection, analysis, and interpretation of data or manuscript preparation.

    Institutional review board statement:The study was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, China (approval No. [2018]-02) on March 30, 2018, and was registered with ClinicalTrials.gov (registration No. NCT03521323) on April 12, 2018. The revised trial protocol (Protocol version 4.0) was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, China (approval No. [2019]-10) on February 25, 2019, and released on ClinicalTrials.gov on April 29, 2019.

    Declaration of subject consent:The authors certify that they will obtain all appropriate subject consent forms from the subjects. In the forms, the subjects will give their consent for their images and other clinical information to be reported in the journal. The subjects understand that their names and initials will not be published and due efforts will be made to conceal their identity.

    Reporting statement:This study followed the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidance for protocol reporting.

    Biostatistics statement:The statistical methods of this study were reviewed by the biostatistician of the The Third Affiliated Hospital of Sun Yat-sen University, China.

    Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

    Data sharing statement:The purpose of the data sharing in this study is to provide original data for any legal and reasonable use. In this clinical trial, all data from included subjects, including the unlabeled table, figure and attachment in the Results section of the article, can be obtained upon request from the readers. Other associated information, including but not limited to study protocol, statistical analyses and informed consent form, can also be obtained upon reasonable request. All these information will be supplied to readers in need through the Internet after the publication of this clinical study.

    Plagiarism check:Checked twice by iThenticate.

    Peer review:Externally peer reviewed.

    Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribu-tion-Non-Commercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

    Open peer reviewer:Mercedes Fernandez, University of Bologna, Italy.

    Additional files:

    Additional file 1:SPIRIT checklist.

    Additional file 2:Hospital ethics approval (Chinese).

    Additional file 3:Informed consent form (Chinese).

    亚洲av成人不卡在线观看播放网| 真人做人爱边吃奶动态| 97超级碰碰碰精品色视频在线观看| 在线观看免费午夜福利视频| 国产亚洲精品久久久com| 国模一区二区三区四区视频| 99久久无色码亚洲精品果冻| 性色av乱码一区二区三区2| 深爱激情五月婷婷| 亚洲av电影在线进入| 亚洲18禁久久av| 亚洲激情在线av| 久久精品国产自在天天线| 欧美在线黄色| 国产一区二区在线观看日韩 | 香蕉久久夜色| 亚洲在线自拍视频| 久久人妻av系列| 久久国产精品人妻蜜桃| 久久人妻av系列| 亚洲avbb在线观看| 老司机午夜福利在线观看视频| 中出人妻视频一区二区| 99久久综合精品五月天人人| 少妇的丰满在线观看| 精品无人区乱码1区二区| 狂野欧美白嫩少妇大欣赏| 成人av在线播放网站| 欧美日韩福利视频一区二区| 一本一本综合久久| 亚洲国产色片| 国产成人av教育| 女警被强在线播放| 成人av在线播放网站| 美女 人体艺术 gogo| 成人性生交大片免费视频hd| 久久久色成人| 国产麻豆成人av免费视频| 国产美女午夜福利| 在线免费观看不下载黄p国产 | 小说图片视频综合网站| 麻豆成人av在线观看| 成年版毛片免费区| 白带黄色成豆腐渣| 一个人看的www免费观看视频| 又紧又爽又黄一区二区| 午夜福利在线观看免费完整高清在 | 1000部很黄的大片| 亚洲一区二区三区色噜噜| 三级男女做爰猛烈吃奶摸视频| 欧美激情久久久久久爽电影| 亚洲午夜理论影院| 国产精品亚洲美女久久久| 天天一区二区日本电影三级| 欧美乱色亚洲激情| 又黄又爽又免费观看的视频| 国产精品1区2区在线观看.| 99久久九九国产精品国产免费| 亚洲最大成人中文| 少妇高潮的动态图| 特级一级黄色大片| a级一级毛片免费在线观看| 国产激情偷乱视频一区二区| 日韩大尺度精品在线看网址| 午夜视频国产福利| 9191精品国产免费久久| 国内精品久久久久精免费| 美女被艹到高潮喷水动态| 可以在线观看毛片的网站| 男女那种视频在线观看| www国产在线视频色| 欧美黄色淫秽网站| 一级黄片播放器| 欧美色视频一区免费| 最后的刺客免费高清国语| 母亲3免费完整高清在线观看| 亚洲五月婷婷丁香| 久久天躁狠狠躁夜夜2o2o| 在线观看美女被高潮喷水网站 | 精品福利观看| 国产色婷婷99| 人妻丰满熟妇av一区二区三区| 最新美女视频免费是黄的| 国内久久婷婷六月综合欲色啪| 国产精品永久免费网站| 天堂影院成人在线观看| 国内精品久久久久精免费| 国产伦精品一区二区三区四那| 国产伦精品一区二区三区四那| 亚洲人成网站在线播| 午夜精品在线福利| 老鸭窝网址在线观看| 国产亚洲精品久久久com| 别揉我奶头~嗯~啊~动态视频| 中文字幕精品亚洲无线码一区| 日本撒尿小便嘘嘘汇集6| 最新在线观看一区二区三区| 精品熟女少妇八av免费久了| 一本一本综合久久| 三级毛片av免费| 少妇人妻精品综合一区二区 | 韩国av一区二区三区四区| 成人精品一区二区免费| 美女被艹到高潮喷水动态| 波多野结衣巨乳人妻| 麻豆一二三区av精品| 亚洲一区二区三区不卡视频| 国产精品99久久久久久久久| 无限看片的www在线观看| 久久精品国产亚洲av涩爱 | 黄片大片在线免费观看| 亚洲精华国产精华精| 高清毛片免费观看视频网站| av片东京热男人的天堂| 夜夜爽天天搞| 欧美乱妇无乱码| 女人被狂操c到高潮| 丰满人妻熟妇乱又伦精品不卡| 国产在线精品亚洲第一网站| 看片在线看免费视频| 日本熟妇午夜| 国语自产精品视频在线第100页| 中文亚洲av片在线观看爽| 日韩人妻高清精品专区| 久久欧美精品欧美久久欧美| 搡老岳熟女国产| 国产精品,欧美在线| 亚洲熟妇中文字幕五十中出| a级毛片a级免费在线| 在线观看美女被高潮喷水网站 | 久久精品国产自在天天线| 久久精品国产清高在天天线| 久久香蕉国产精品| 日韩欧美免费精品| 亚洲av熟女| 亚洲国产色片| 精品午夜福利视频在线观看一区| 最新美女视频免费是黄的| 怎么达到女性高潮| 久久精品国产综合久久久| 99久久九九国产精品国产免费| 亚洲成人久久性| 国产真人三级小视频在线观看| 少妇丰满av| 深夜精品福利| 一个人免费在线观看电影| 日本一二三区视频观看| xxxwww97欧美| 亚洲精品乱码久久久v下载方式 | 亚洲,欧美精品.| 丰满乱子伦码专区| 国产精品 国内视频| 日韩有码中文字幕| 国产单亲对白刺激| 级片在线观看| 国产精品亚洲美女久久久| 一级黄色大片毛片| 亚洲 国产 在线| 午夜福利欧美成人| 国产91精品成人一区二区三区| 久久久国产精品麻豆| 久久6这里有精品| 午夜精品久久久久久毛片777| 成年免费大片在线观看| 国产av一区在线观看免费| 18+在线观看网站| 国语自产精品视频在线第100页| 国产精品综合久久久久久久免费| 亚洲精品成人久久久久久| 久久精品影院6| 精品欧美国产一区二区三| 久久久国产成人精品二区| 国产 一区 欧美 日韩| 99久久99久久久精品蜜桃| 亚洲av五月六月丁香网| 欧美日韩黄片免| 欧美+亚洲+日韩+国产| 久久亚洲精品不卡| 久久久国产精品麻豆| 欧美xxxx黑人xx丫x性爽| 三级毛片av免费| 精品99又大又爽又粗少妇毛片 | 国产精品日韩av在线免费观看| 精品免费久久久久久久清纯| 国产亚洲精品久久久com| 免费一级毛片在线播放高清视频| 丰满人妻熟妇乱又伦精品不卡| 国产色婷婷99| 久久精品国产亚洲av涩爱 | 成熟少妇高潮喷水视频| 欧洲精品卡2卡3卡4卡5卡区| 午夜视频国产福利| 成年女人看的毛片在线观看| 亚洲国产精品sss在线观看| 99在线人妻在线中文字幕| 最近最新中文字幕大全免费视频| av女优亚洲男人天堂| 亚洲男人的天堂狠狠| 精品久久久久久久人妻蜜臀av| 精品不卡国产一区二区三区| 中文字幕av成人在线电影| 91麻豆精品激情在线观看国产| 久久久国产精品麻豆| 午夜免费激情av| e午夜精品久久久久久久| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 夜夜爽天天搞| 亚洲人与动物交配视频| 日韩欧美国产在线观看| 小蜜桃在线观看免费完整版高清| 婷婷亚洲欧美| 国产精品综合久久久久久久免费| 国产精品av视频在线免费观看| 国产精品日韩av在线免费观看| 亚洲精品久久国产高清桃花| 免费看日本二区| 脱女人内裤的视频| 三级男女做爰猛烈吃奶摸视频| 99国产极品粉嫩在线观看| 十八禁网站免费在线| 两个人视频免费观看高清| 给我免费播放毛片高清在线观看| 18禁黄网站禁片午夜丰满| 国产精品 欧美亚洲| 中文字幕av在线有码专区| 99久久九九国产精品国产免费| 午夜福利高清视频| 色噜噜av男人的天堂激情| 国产亚洲欧美在线一区二区| 一区二区三区免费毛片| www国产在线视频色| 两个人看的免费小视频| 手机成人av网站| 久久99热这里只有精品18| 精品久久久久久久末码| 欧美中文综合在线视频| 精品福利观看| 岛国视频午夜一区免费看| 淫秽高清视频在线观看| www.999成人在线观看| 日韩高清综合在线| 岛国在线免费视频观看| 1024手机看黄色片| 亚洲av电影不卡..在线观看| 一级毛片女人18水好多| 欧美精品啪啪一区二区三区| 欧美日韩综合久久久久久 | 长腿黑丝高跟| 精品久久久久久久末码| 日韩有码中文字幕| 99热这里只有是精品50| 日本熟妇午夜| 久久久久久九九精品二区国产| 丰满的人妻完整版| 国产精品精品国产色婷婷| 久久性视频一级片| 99久久综合精品五月天人人| www.熟女人妻精品国产| 久久久久免费精品人妻一区二区| 每晚都被弄得嗷嗷叫到高潮| 在线观看一区二区三区| 免费大片18禁| 99热6这里只有精品| 中文字幕久久专区| 亚洲av中文字字幕乱码综合| 亚洲人成电影免费在线| 久久99热这里只有精品18| 欧美日本视频| 日本撒尿小便嘘嘘汇集6| 国产野战对白在线观看| 女同久久另类99精品国产91| 12—13女人毛片做爰片一| 黄色女人牲交| 欧美成人性av电影在线观看| www国产在线视频色| 脱女人内裤的视频| 国产爱豆传媒在线观看| 国产蜜桃级精品一区二区三区| 90打野战视频偷拍视频| 精品欧美国产一区二区三| av黄色大香蕉| 少妇熟女aⅴ在线视频| 亚洲真实伦在线观看| 国产黄a三级三级三级人| 一区二区三区免费毛片| 国产成人欧美在线观看| 亚洲av中文字字幕乱码综合| 日本成人三级电影网站| 18禁黄网站禁片免费观看直播| 亚洲av电影在线进入| 熟女人妻精品中文字幕| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 亚洲无线在线观看| 免费观看的影片在线观看| 又爽又黄无遮挡网站| 在线播放无遮挡| 日韩欧美国产一区二区入口| 久久久久久久久久黄片| 一本久久中文字幕| 内地一区二区视频在线| 欧美激情久久久久久爽电影| 中文字幕人成人乱码亚洲影| 免费观看人在逋| 国产精品亚洲av一区麻豆| 成人欧美大片| 999久久久精品免费观看国产| 美女被艹到高潮喷水动态| 香蕉久久夜色| 人人妻,人人澡人人爽秒播| 亚洲无线在线观看| 欧美日韩福利视频一区二区| 精品久久久久久久久久免费视频| 国产视频一区二区在线看| 一进一出好大好爽视频| 在线观看午夜福利视频| 国产久久久一区二区三区| 亚洲国产精品合色在线| 国产不卡一卡二| 91在线精品国自产拍蜜月 | 久久欧美精品欧美久久欧美| 久久久国产精品麻豆| 99久久精品国产亚洲精品| 又黄又粗又硬又大视频| 亚洲av成人不卡在线观看播放网| 免费观看人在逋| 欧美另类亚洲清纯唯美| 欧美+日韩+精品| 亚洲国产精品999在线| 搡老妇女老女人老熟妇| 亚洲av不卡在线观看| 国产毛片a区久久久久| 男女做爰动态图高潮gif福利片| 亚洲精品久久国产高清桃花| 欧美一级a爱片免费观看看| 精品国产超薄肉色丝袜足j| 亚洲内射少妇av| 成人性生交大片免费视频hd| 午夜日韩欧美国产| 人人妻人人澡欧美一区二区| 国产精品自产拍在线观看55亚洲| 一级黄片播放器| 亚洲国产欧美网| 国产av在哪里看| 亚洲av一区综合| 很黄的视频免费| 在线十欧美十亚洲十日本专区| 亚洲美女视频黄频| 中国美女看黄片| 真实男女啪啪啪动态图| 国产爱豆传媒在线观看| 久久久精品欧美日韩精品| 黄色片一级片一级黄色片| 免费在线观看日本一区| 999久久久精品免费观看国产| 久久香蕉国产精品| 黄色片一级片一级黄色片| netflix在线观看网站| 国产成人系列免费观看| 欧美黑人巨大hd| 老司机福利观看| 熟女电影av网| 99热只有精品国产| 国产淫片久久久久久久久 | 亚洲欧美日韩高清专用| 窝窝影院91人妻| av中文乱码字幕在线| 在线观看美女被高潮喷水网站 | 性色av乱码一区二区三区2| 亚洲国产中文字幕在线视频| 亚洲中文字幕日韩| 国产欧美日韩精品亚洲av| 亚洲成a人片在线一区二区| 老司机在亚洲福利影院| 午夜免费观看网址| av女优亚洲男人天堂| 香蕉久久夜色| 一区福利在线观看| 99riav亚洲国产免费| 99热精品在线国产| 亚洲成人中文字幕在线播放| 亚洲黑人精品在线| 国产成人av激情在线播放| 男人舔女人下体高潮全视频| 真人一进一出gif抽搐免费| 伊人久久精品亚洲午夜| 老汉色av国产亚洲站长工具| 精品日产1卡2卡| 国产伦一二天堂av在线观看| 国产精品久久久人人做人人爽| 久99久视频精品免费| 老司机深夜福利视频在线观看| 丰满的人妻完整版| 操出白浆在线播放| 伊人久久精品亚洲午夜| 久久99热这里只有精品18| 麻豆国产av国片精品| 久久精品亚洲精品国产色婷小说| 一区福利在线观看| 午夜老司机福利剧场| 人妻丰满熟妇av一区二区三区| 97人妻精品一区二区三区麻豆| 毛片女人毛片| 欧美+日韩+精品| 久久99热这里只有精品18| 女警被强在线播放| 成人国产综合亚洲| 国产av不卡久久| 免费看日本二区| 亚洲 欧美 日韩 在线 免费| a级毛片a级免费在线| 亚洲国产中文字幕在线视频| 欧美日韩黄片免| 天天添夜夜摸| 校园春色视频在线观看| 国产亚洲精品久久久久久毛片| 亚洲精品国产精品久久久不卡| 91麻豆av在线| 国产极品精品免费视频能看的| 国产精品日韩av在线免费观看| 成熟少妇高潮喷水视频| 中文亚洲av片在线观看爽| 亚洲熟妇中文字幕五十中出| 少妇的丰满在线观看| 国产精品日韩av在线免费观看| 1024手机看黄色片| 日韩欧美在线乱码| 最新中文字幕久久久久| 麻豆成人午夜福利视频| 天天一区二区日本电影三级| 日本精品一区二区三区蜜桃| 两人在一起打扑克的视频| 动漫黄色视频在线观看| 琪琪午夜伦伦电影理论片6080| 亚洲av电影不卡..在线观看| 国产一区二区三区视频了| 18禁美女被吸乳视频| 亚洲在线自拍视频| 欧美bdsm另类| 熟女人妻精品中文字幕| 亚洲av五月六月丁香网| 有码 亚洲区| 午夜福利成人在线免费观看| 成人av一区二区三区在线看| 99国产精品一区二区三区| 婷婷精品国产亚洲av在线| 免费人成视频x8x8入口观看| 日本一本二区三区精品| 日韩免费av在线播放| 两个人视频免费观看高清| 国产高清三级在线| 亚洲精品一区av在线观看| 老鸭窝网址在线观看| 淫妇啪啪啪对白视频| 亚洲精品亚洲一区二区| 成人午夜高清在线视频| 高潮久久久久久久久久久不卡| 伊人久久大香线蕉亚洲五| 夜夜爽天天搞| 可以在线观看毛片的网站| 国产一区在线观看成人免费| 国产精华一区二区三区| 搞女人的毛片| 长腿黑丝高跟| 蜜桃久久精品国产亚洲av| 大型黄色视频在线免费观看| 欧美黑人巨大hd| 久久久久久久久中文| 丰满人妻一区二区三区视频av | av黄色大香蕉| 97碰自拍视频| 免费观看的影片在线观看| 国产成+人综合+亚洲专区| 久久中文看片网| 亚洲成人久久爱视频| 国产精品1区2区在线观看.| 最近视频中文字幕2019在线8| 怎么达到女性高潮| 五月伊人婷婷丁香| 国产成人av教育| 夜夜看夜夜爽夜夜摸| 69av精品久久久久久| 窝窝影院91人妻| 国产一区二区在线观看日韩 | av专区在线播放| 成人国产一区最新在线观看| 国产亚洲av嫩草精品影院| 欧美高清成人免费视频www| 丰满的人妻完整版| 国产97色在线日韩免费| 成人国产一区最新在线观看| 日韩高清综合在线| 成人高潮视频无遮挡免费网站| 少妇的逼好多水| 欧美成人免费av一区二区三区| 身体一侧抽搐| av视频在线观看入口| 亚洲午夜理论影院| 久久香蕉国产精品| 黑人欧美特级aaaaaa片| 一a级毛片在线观看| 免费在线观看亚洲国产| 国产99白浆流出| 国产精品久久视频播放| 无限看片的www在线观看| 欧美一区二区国产精品久久精品| 国产高清videossex| www日本黄色视频网| 久久午夜亚洲精品久久| 亚洲人成网站在线播放欧美日韩| 成人午夜高清在线视频| 中文资源天堂在线| 亚洲av成人av| 色尼玛亚洲综合影院| 久久国产乱子伦精品免费另类| 99精品久久久久人妻精品| 国产精品98久久久久久宅男小说| 日本a在线网址| 亚洲午夜理论影院| 成人特级av手机在线观看| 99热精品在线国产| 精品乱码久久久久久99久播| 婷婷丁香在线五月| 亚洲国产精品999在线| e午夜精品久久久久久久| 久久精品人妻少妇| 精品一区二区三区视频在线 | 69av精品久久久久久| 熟妇人妻久久中文字幕3abv| av在线天堂中文字幕| 18+在线观看网站| 亚洲国产欧美人成| 啦啦啦观看免费观看视频高清| 在线免费观看的www视频| 亚洲天堂国产精品一区在线| 亚洲成人中文字幕在线播放| 欧美一级毛片孕妇| 美女大奶头视频| 日本黄色片子视频| 国产三级中文精品| 真人一进一出gif抽搐免费| 国产精品久久久久久久电影 | 亚洲最大成人中文| 亚洲av一区综合| 欧美+日韩+精品| 少妇裸体淫交视频免费看高清| 搡老熟女国产l中国老女人| 90打野战视频偷拍视频| 窝窝影院91人妻| 亚洲内射少妇av| 国产精品电影一区二区三区| 19禁男女啪啪无遮挡网站| 在线播放无遮挡| 99国产综合亚洲精品| 欧美日本视频| 国产精品爽爽va在线观看网站| 亚洲av中文字字幕乱码综合| 长腿黑丝高跟| 日日摸夜夜添夜夜添小说| 国产一区二区亚洲精品在线观看| 窝窝影院91人妻| 麻豆国产97在线/欧美| 国产欧美日韩精品一区二区| 变态另类丝袜制服| 国产激情欧美一区二区| 长腿黑丝高跟| 国产探花在线观看一区二区| 天堂√8在线中文| 国产精品野战在线观看| 久久久久久久午夜电影| 色综合婷婷激情| 亚洲性夜色夜夜综合| 一本综合久久免费| 制服丝袜大香蕉在线| 99国产精品一区二区三区| 亚洲欧美一区二区三区黑人| 亚洲国产精品合色在线| 免费搜索国产男女视频| 黄色成人免费大全| 男人的好看免费观看在线视频| 国产精品久久久久久久电影 | 精品国内亚洲2022精品成人| 亚洲激情在线av| 亚洲av成人精品一区久久| АⅤ资源中文在线天堂| 99久久精品一区二区三区| 噜噜噜噜噜久久久久久91| 99久久99久久久精品蜜桃| 亚洲av一区综合| 国产精品一区二区三区四区免费观看 | 日韩中文字幕欧美一区二区| tocl精华| 成人国产一区最新在线观看| 在线看三级毛片| 亚洲国产精品999在线| 色av中文字幕| 久久精品国产清高在天天线| 亚洲欧美日韩高清在线视频| 少妇的逼好多水| 久久久久免费精品人妻一区二区| 我要搜黄色片| 欧美日韩精品网址| 免费看十八禁软件| 国产一区二区三区视频了| 18禁黄网站禁片午夜丰满| 99国产极品粉嫩在线观看| 欧美色欧美亚洲另类二区| 亚洲自拍偷在线| 亚洲avbb在线观看| 欧美黄色片欧美黄色片| 国内精品久久久久精免费| 中文字幕熟女人妻在线| 日韩国内少妇激情av| 免费搜索国产男女视频| 在线天堂最新版资源| 国产美女午夜福利|