Difference between revisions of "Team:SYSU-MEDICINE/Results"

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{{SYSU-MEDICINE}}
+
 
 
<html>
 
<html>
  
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 +
<head>
 +
    <meta charset="UTF-8">
 +
    <link rel="icon" type="image/png" href="https://static.igem.org/mediawiki/2016/2/2a/T--SYSU-MEDICINE--logo_32%2A32.png" sizes="16x16">
 +
    <link rel="icon" type="image/png" href="https://static.igem.org/mediawiki/2016/2/2a/T--SYSU-MEDICINE--logo_32%2A32.png" sizes="32x32">
 +
    <!-- Latest compiled and minified CSS -->
 +
    <!--<link href='//cdn.webfont.youziku.com/webfonts/nomal/92549/46882/57f3d7e1f629d81a5077d03f.css' rel='stylesheet' type='text/css' />-->
 +
    <link rel="stylesheet" href="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.7/css/bootstrap.min.css" integrity="sha384-BVYiiSIFeK1dGmJRAkycuHAHRg32OmUcww7on3RYdg4Va+PmSTsz/K68vbdEjh4u" crossorigin="anonymous">
 +
    <!--jQuery-->
 +
    <script  src="https://code.jquery.com/jquery-3.1.1.min.js"  integrity="sha256-hVVnYaiADRTO2PzUGmuLJr8BLUSjGIZsDYGmIJLv2b8="  crossorigin="anonymous"></script>
 +
    <!-- Latest compiled and minified JavaScript -->
 +
    <script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.7/js/bootstrap.min.js" integrity="sha384-Tc5IQib027qvyjSMfHjOMaLkfuWVxZxUPnCJA7l2mCWNIpG9mGCD8wGNIcPD7Txa" crossorigin="anonymous"></script>
 +
    <style>
 +
        /*font*/
 +
        /*font*/
 +
        @font-face{font-family:"HelveticaNeueW02-45Ligh";src:url('//www.eyeem.com/css/fonts/88fcd49a-13c7-4d0c-86b1-ad1e258bd75d.eot?#iefix');src:url('//www.eyeem.com/css/fonts/88fcd49a-13c7-4d0c-86b1-ad1e258bd75d.eot?#iefix') format("eot"),url('//www.eyeem.com/css/fonts/9a2e4855-380f-477f-950e-d98e8db54eac.woff') format("woff"),url('//www.eyeem.com/css/fonts/fa82d0ee-4fbd-4cc9-bf9f-226ad1fcbae2.ttf') format("truetype"),url('//www.eyeem.com/css/fonts/48d599a6-92b5-4d43-a4ac-8959f6971853.svg#48d599a6-92b5-4d43-a4ac-8959f6971853') format("svg")}
 +
        *{
 +
            font-family:"HelveticaNeueW02-45Ligh","Arial",sans-serif;
 +
            letter-spacing: 0.4px;
 +
            line-height: 1.5em;
 +
        }
 +
        #HQ_page h1,#HQ_page h2,#HQ_page h3,#HQ_page h4,#HQ_page h5,#HQ_page p{
 +
            font-family:"HelveticaNeueW02-45Ligh","Arial",sans-serif;
 +
        }
 +
        #HQ_page p{
 +
            font-size: 20px;
 +
        }
 +
        #HQ_page td{
 +
            padding:0;
 +
            border:0;
 +
        }
 +
        #HQ_page table {
 +
            border:0;
 +
        }
 +
        /*override wiki*/
 +
        .menu_wrapper, #top_title, #sideMenu{display: none;}
 +
        #content {
 +
            width:100%;
 +
            padding:0;
 +
            margin: 16px auto;
 +
        }
 +
        #top_menu_under {
 +
            height: 0;
 +
        }
 +
        #top_menu_14 {
 +
            border-bottom: 0;
 +
        }
 +
        #globalWrapper {
 +
            font-size: 100%;
 +
            padding-bottom: 0;
 +
        }
 +
        #content {
 +
            margin-bottom: 0;
 +
        }
 +
        /*nav*/
 +
        .navbar {
 +
            border-radius: 0;
 +
            height: 50px;
 +
            margin-bottom:0;
 +
            z-index: 25;
 +
        }
 +
        .navbar-inverse .navbar-brand {
 +
            color: #fff;
 +
        }
 +
        .navbar-inverse .navbar-nav>.active>a, .navbar-inverse .navbar-nav>.active>a:focus, .navbar-inverse .navbar-nav>.active>a:hover {
 +
            background-color: rgba(0,0,0,0);
 +
            color: #999;
 +
            font-weight:600;
 +
        }
 +
        .navbar-inverse .navbar-nav>li>a {
 +
            color: #fff;
 +
            font-weight:600;
 +
        }
 +
        .navbar-inverse .navbar-nav>li>a:hover {
 +
            color: #999;
 +
            font-weight:600;
 +
        }
 +
        .navbar-nav>li{
 +
            top: -5px;
 +
        }
 +
        /*.mw-content-ltr ul, .mw-content-rtl .mw-content-ltr ul {*/
 +
        /*margin: 0 0.1;*/
 +
        /*}*/
 +
        .dropdown-menu>li>a {
 +
            font-weight: 600;
 +
        }
 +
        .third-level-list {
 +
            top: -10px;
 +
            left: 150px;
 +
        }
 +
        .jumbotron{
 +
            margin-bottom:0;
 +
            padding-top:10px;
 +
            padding-bottom:20px;
 +
        }
 +
        #jumbo-footer.jumbotron {
 +
            padding-top: 10px;
 +
            padding-bottom: 30px;
 +
            margin-bottom: 0;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container{
 +
            width: 1170px;
 +
            margin: 20px auto;
 +
            font-size: 18px;
 +
            color: #888;
 +
            /*font-weight: bold;*/
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .line{
 +
            height: 2px;
 +
            background-color: #bbb;
 +
            width: 100%;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact {
 +
            margin-top: 20px;
 +
            margin-left: 10px;
 +
            height: 140px;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .title{
 +
            font-weight: bold;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul{
 +
            list-style: none;
 +
            padding-left: 0;
 +
            margin-left: 0;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul.float-contact {
 +
            float:left;
 +
            width: 400px;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul li {
 +
            margin-top: 5px;
 +
            margin-bottom: 5px;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul li a img{
 +
            width: 30px;
 +
            height: 30px;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul li a {
 +
            color: #888;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact ul li a:hover, #jumbo-footer.jumbotron>.footer-container .contact ul li a:active, #jumbo-footer.jumbotron>.footer-container .contact ul li a:focus {
 +
            color: #888;
 +
            text-decoration: none;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .contact .float-contact-right {
 +
            float:right;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .copyright{
 +
            margin-top: 5px;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .copyright .text {
 +
            font-size: 16px;
 +
            float: left;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .copyright .icon{
 +
            float: right;
 +
        }
 +
        #jumbo-footer.jumbotron>.footer-container .copyright .icon img {
 +
            width: 25px;
 +
            height: 25px;
 +
        }
 +
        #wechatModal .modal-dialog{
 +
            width: 332px;
 +
            margin-top: 100px;
 +
            z-index: 2000;
 +
        }
 +
        /*content*/
 +
        .jumbotron .my-content{
 +
            margin: 0 auto;
 +
            width: 900px;
 +
        }
 +
        .jumbotron .my-content h2 {
 +
            text-align: center;
 +
            font-weight: bolder;
 +
            font-size: 40px;
 +
            margin-bottom:30px;
 +
        }
 +
        .jumbotron .my-content p {
 +
            line-height: 1.5em;
 +
            font-size: 20px;
 +
            color: #333;
 +
        }
 +
        .jumbotron#content{
 +
            background-color: #fff;
 +
        }
 +
        .jumbotron#content p a{
 +
            color: #333;
 +
        }
 +
        .jumbotron#content img{
 +
            width: 90%;
 +
            margin: 10px auto;
 +
            display: block;
 +
        }
 +
        .jumbotron#content span.note{
 +
            width: 80%;
 +
            font-size: 16px;
 +
            color: #999;
 +
            margin: 0 auto;
 +
            display: block;
 +
        }
 +
        /*mynav*/
 +
        .mynav{
 +
            position: fixed;
 +
            /*width:250px;*/
 +
            left: 10px;
 +
            opacity: 0.2;
 +
            top: 300px;
 +
            background-color: #f2f2f2;
 +
            border-radius: 10px;
 +
            -webkit-transition: opacity 0.2s, width 0.2s;
 +
            -moz-transition: opacity 0.2s, width 0.2s;
 +
            -ms-transition: opacity 0.2s, width 0.2s;
 +
            -o-transition: opacity 0.2s, width 0.2s;
 +
            transition: opacity 0.2s, width 0.2s;
 +
            padding-right: 20px;
 +
        }
 +
        .mynav:hover {
 +
            opacity: 1;
 +
 +
        }
 +
        .mynav ol{
 +
            list-style: none;
 +
            padding-left:20px;
 +
            margin:0;
 +
 +
        }
 +
        .mynav ol li ol{
 +
            display:none;
 +
        }
 +
        .mynav li a{
 +
            color: #333;
 +
            font-size: 18px;
 +
            width: 300px;
 +
        }
 +
        .mynav li a:hover, .mynav li a:active,.mynav li a:focus{
 +
            color:#e57d22;
 +
        }
 +
        .mynav li li a{
 +
            font-size: 16px;
 +
        }
 +
        .mynav li li li a{
 +
            font-size: 14px;
 +
        }
 +
        /*backtoTop*/
 +
        .backtoTop{
 +
            height: 50px;
 +
            width: 50px;
 +
            position: absolute; //绝对定位
 +
        padding-top: 8px;
 +
            right: 10px;
 +
            z-index: 9999;
 +
            /*避免闪烁*/
 +
            -moz-transition: all 1s ease;
 +
            -webkit-transition: all 1s ease;
 +
            -o-transition: all 1s ease;
 +
            transition: all 1s ease;
 +
            /*避免闪烁*/
 +
            /*background: blue;*/
 +
            cursor: pointer;
 +
            text-align: center;
 +
        }
 +
    </style>
 +
    <script type="text/javascript">
 +
        $(document).ready(function() {
 +
//          wechat qr code
 +
            var canHide= true;
 +
            $('#wechatModal').on('show.bs.modal', function (e) {
 +
                // do something...
 +
                $("#wechatModal").after('<div class="modal-backdrop fade in"></div>');
 +
            });
 +
            $(".modal").click(function() {
 +
//                console.log("click");
 +
                //$(this).remove();
 +
                if (canHide) {
 +
                    $(".modal-backdrop").remove();
 +
                    $('#wechatModal').modal('hide');
 +
                }
 +
            });
 +
            $(".modal-dialog").click(function() {
 +
                canHide = false;
 +
            });
 +
            $(".modal").mousemove(function() {
 +
                canHide = true;
 +
            });
 +
            $(".third-level-list").css("left", "120px");
 +
//          third level menu on nav
 +
            $(".third-level-menu").mouseenter(function() {
 +
                console.log("mouse in");
 +
                $(this).addClass("open");
 +
 +
            });
 +
            $(".third-level-menu").mouseleave(function() {
 +
                console.log("mouse in");
 +
                $(this).removeClass("open");
 +
            });
 +
            $(".dropdown").mouseenter(function() {
 +
                $(this).addClass("open");
 +
            });
 +
            $(".dropdown").mouseleave(function() {
 +
                $(this).removeClass("open");
 +
            });
 +
            //            mynav
 +
            $(".mynav li").mouseenter(function() {
 +
                $(this).children('ol').slideDown();
 +
            });
 +
            $(".mynav li").mouseleave(function() {
 +
                $(this).children('ol').slideUp();
 +
            });
 +
 +
//            backtotop
 +
            $(window).scroll( function() {              //滚动时触发
 +
                var top = $(document).scrollTop(),      //获取滚动条到顶部的垂直高度
 +
                        height = $(window).height();
 +
//                        height = 600;
 +
                console.log(height+" "+top);//获得可视浏览器的高度
 +
                if(top > 100){
 +
                    $("#backToTop").show(200, function(){
 +
                        $("#backToTop").css({
 +
                            top: height + top - 80
 +
                        })
 +
                    });
 +
                }
 +
            });
 +
            /*点击回到顶部*/
 +
            $('#backToTop').click(function(){
 +
                $('html, body').animate({
 +
                    scrollTop: 0
 +
                }, 500);
 +
            });
 +
        });
 +
 +
    </script>
 +
</head>
 +
<body>
 +
<nav class="navbar navbar-inverse">
 +
    <div class="container-fluid">
 +
        <!-- Brand and toggle get grouped for better mobile display -->
 +
        <div class="navbar-header">
 +
            <button type="button" class="navbar-toggle collapsed" data-toggle="collapse" data-target="#bs-example-navbar-collapse-1" aria-expanded="false">
 +
                <span class="sr-only">Toggle navigation</span>
 +
                <span class="icon-bar"></span>
 +
                <span class="icon-bar"></span>
 +
                <span class="icon-bar"></span>
 +
            </button>
 +
            <a class="navbar-brand" href="https://2016.igem.org/Team:SYSU-MEDICINE">
 +
                <img style="height: 40px; width: 40px; margin-top: -10px " src="https://2016.igem.org/wiki/skins/Igem/images/IGEM_white_letters.png">
 +
            </a>
 +
            <a class="navbar-brand" href="https://2016.igem.org/Team:SYSU-MEDICINE">SYSU-MEDICINE</a>
 +
        </div>
 +
 +
        <!-- Collect the nav links, forms, and other content for toggling -->
 +
        <div class="collapse navbar-collapse" id="bs-example-navbar-collapse-1">
 +
            <ul class="nav navbar-nav navbar-right">
 +
                <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE">HOME</a></li>
 +
                <li class="dropdown active">
 +
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">PROJECT<span class="caret"></span></a>
 +
                    <ul class="dropdown-menu">
 +
                        <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Description">Description</a></li>
 +
                        <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Results">Results</a></li>
 +
                        <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Protocol">Protocol</a></li>
 +
                        <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Notebook">Notebook</a></li>
 +
                        <li class="dropdown third-level-menu">
 +
                            <a href="#" class="dropdown-toggle" data-toggle="dropdown" aria-haspopup="true" aria-expanded="false">For Judgement <span class="glyphicon glyphicon-chevron-right"></span></a>
 +
                            <ul class="third-level-list dropdown-menu">
 +
                                <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Proof">Proof of Concept</a></li>
 +
                                <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Demonstrate">Demonstrate</a></li>
 +
                            </ul>
 +
                        </li>
 +
                    </ul>
 +
                </li>
 +
                <li class="dropdown">
 +
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-haspopup="true" aria-expanded="false">HUMAN PRACTICES<span class="caret"></span></a>
 +
                    <ul class="dropdown-menu">
 +
                        <li><a href="https://2016.igem.org/Team:SYSU-MEDICINE/Our_Story">Our Story</a></li>
 +
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<div class="jumbotron">
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    <div class="my-content">
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        <h1 align="center">Results</h1>
 +
    </div>
 +
</div>
 +
<div class="jumbotron" id="content">
 +
    <div class="mynav">
 +
        <ol>
 +
            <li><a href="#Results1">Major Achievements</a></li>
 +
            <li><a href="#Results2">Details</a></li>
 +
        </ol>
 +
    </div>
 +
    <div class="my-content">
 +
        <h2 id="Results1">Major achievements:</h2>
 +
        <p>
 +
            <a href="#Results1.1">1. Our modified MSCs still remained their own characteristics.</a><br/>
 +
            <a href="#Results1.2">2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.</a><br/>
 +
            <a href="#Results1.3">3. Chemotaxis of our engineered MSCs improved.</a><br/>
 +
            <a href="#Results1.4">4. Marking proteins (eGFP/dTomato) worked.</a><br/>
 +
            <a href="#Results1.5">5. Therapeutic effects of our engineered MSCs for IBD (inflammatory bowel disease) improved.</a><br/>
 +
            <a href="#Results1.6">6. Therapeutic effects of our engineered MSCs for DTH (delayed type hypersensitivity) improved.</a><br/>
 +
            <a href="#Results1.7">7. The switch element worked.</a><br/>
 +
        </p>
 +
        <h2 id="Results2">Details: </h2>
 +
        <p>
 +
            <b id="Results1.1"><big>1. Our modified MSCs still remained their own characteristics. </big></b><br/>
 +
            <br/>
 +
        </p>
 +
 +
        <p>
 +
            <img style="width:500px" src="https://static.igem.org/mediawiki/2016/1/1e/T--SYSU-MEDICINE--project-2.1.1.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.1.1<br/>
 +
            </span>
 +
        </p>
 +
 +
 +
        <p>
 +
            <img style="width:500px" src="https://static.igem.org/mediawiki/2016/e/e5/T--SYSU-MEDICINE--project-2.1.2.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.1.2<br/>
 +
            </span>
 +
        </p>
 +
 +
        </p>
 +
 +
 +
        <p>
 +
            Our modified MSCs were positive for CD105, CD90, CD29 and CD73 and negative for CD34 (Figure 2.1.1 A-E), and were able to differentiate to osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiating conditions (Figure 2.1.2 F-K). (Meet the standard of International Society for Cellular Therapy (ISCT))<br/>
 +
            <br/>
 +
            <b id="Results1.2"><big>2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.</big></b><br/>
 +
            <br/>
 +
        </p>
 +
        <table>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/9/96/T--SYSU-MEDICINE--project-2.2.1.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/8/89/T--SYSU-MEDICINE--project-2.2.2.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/d/d6/T--SYSU-MEDICINE--project-2.2.3.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.2.1<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.2.2<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.2.3<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
        </table>
 +
        <p>
 +
            <img src="https://static.igem.org/mediawiki/2016/9/97/T--SYSU-MEDICINE--project-2.2.4.tif">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.2.4<br/>
 +
            </span>
 +
            <br/>
 +
            mRNA and protein levels of CCR7, CXCR4 and CXCR5 of MSCs and our modified MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were semi-quantified and detected by qPCR and western blot, respectively. mRNA and protein levels of CCR7, CXCR4 and CXCR5 all elevated in our modified MSCs.<br/>
 +
            <br/>
 +
            <b id="Results1.3"><big>3. Chemotaxis of our engineered MSCs improved. </big></b><br/>
 +
            <br/>
 +
        </p>
 +
        <table>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/a/aa/T--SYSU-MEDICINE--project-2.3.1.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/8/8b/T--SYSU-MEDICINE--project-2.3.2.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/2/26/T--SYSU-MEDICINE--project-2.3.3.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.3.1<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.3.2<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.3.3<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
        </table>
 +
        <p>
 +
            <img src="https://static.igem.org/mediawiki/2016/0/04/T--SYSU-MEDICINE--project-2.3.4.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.3.4<br/>
 +
            </span>
 +
            <br/>
 +
            Chemotaxis of our engineered MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were examined against CCL19, CXCL12 and CXCL13, respectively. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs all improved.<br/>
 +
            <br/>
 +
            <b id="Results1.4"><big>4. Marking proteins (eGFP/dTomato) worked. </big></b><br/>
 +
            <br/>
 +
            <img style="width:800px" src="https://static.igem.org/mediawiki/2016/7/72/T--SYSU-MEDICINE--2.4.1.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.4.1<br/>
 +
            </span>
 +
            <br/>
 +
            <b>4.1</b> 2 days later, after transduction into 293FT cells, marking proteins (eGFP/dTomato) expressed.<br/>
 +
            Figure A: 293FT cells (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A- hFTH)<br/>
 +
            Figure B: 293FT cells (BBa_K1993005: EF-1α-CXCR5-IRES-eGFP)<br/>
 +
            Figure C: 293FT cells (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)<br/>
 +
            Figure D: 293FT cells (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP) <br/>
 +
            <br/><a style="width:600px">
 +
            <img style="width:600px" src="https://static.igem.org/mediawiki/2016/f/fb/T--SYSU-MEDICINE--2.4.2.png"></a>
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.4.2<br/>
 +
            </span>
 +
            <br/>
 +
            <b>4.2</b> 4 days later, after transduction into MSCs, marking proteins (eGFP/dTomato) expressed.<br/>
 +
            Figure A: MSCs (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A-hFTH)<br/>
 +
            Figure B: MSCs (BBa_K1993005: EF-1α-CXCR5-IRES-eGFP)<br/>
 +
            Figure C: MSCs (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)<br/>
 +
            Figure D: MSCs (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP) <br/>
 +
            <br/>
 +
            <img style="width:400px" src="https://static.igem.org/mediawiki/2016/6/68/T--SYSU-MEDICINE--BBa_K1071009-fig4.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.4.3<br/>
 +
            </span>
 +
            <br/>
 +
            <b>4.3</b> After intraperitoneal injection of Luciferin, MSCs engineered with GFP and Luciferase were administrated intravenously to caudal vein. Oxidized Luciferin was observed under IVIS spectrum system. As a result, mice administered with engineered MSCs （BBa_K1993008: EF-1α-Luciferase-IRES-eGFP）<br/>
 +
            <br/>
 +
            <b id="Results1.5"><big>5.Therapeutic effects of our engineered MSCs for inflammatory bowel disease(IBD) improved.</big></b><br/>
 +
            <br/>
 +
            Our MSCs were modified by BBa_K1993009 (EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP).<br/>
 +
            <br/>
 +
        </p>
 +
        <table>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/a/ae/T--SYSU-MEDICINE--project-2.5.1.1.jpg">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/e/e9/T--SYSU-MEDICINE--project-2.5.1.2.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.1.1<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.1.2<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/d/d8/T--SYSU-MEDICINE--project-2.5.1.3.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/6/6a/T--SYSU-MEDICINE--project-2.5.1.4.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.1.3<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.1.4<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
        </table>
 +
        <p>
 +
            <b>5.1</b> We established murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-colitis (IBD animal model). After injecting with MSCs<sup>CXCR4</sup>, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.<br/>
 +
            <br/>
 +
            <img  src="https://static.igem.org/mediawiki/2016/c/c9/T--SYSU-MEDICINE--project-2.5.2.jpg">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.5.2<br/>
 +
            </span>
 +
            <br/>
 +
            <b>5.2</b> Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCs<sup>CXCR4</sup> displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.<br/>
 +
            <br/>
 +
 +
        </p>
 +
        <table>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/1/11/T--SYSU-MEDICINE--project-2.5.3.1.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/9/90/T--SYSU-MEDICINE--project-2.5.3.2.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.3.1<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.3.2<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    <img src="https://static.igem.org/mediawiki/2016/3/31/T--SYSU-MEDICINE--project-2.5.3.3.png">
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <img  src="https://static.igem.org/mediawiki/2016/3/35/T--SYSU-MEDICINE--project-2.5.3.4.png">
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.3.3<br/>
 +
                    </span>
 +
                </td>
 +
                <td style="text-align: center">
 +
                    <span class="note">
 +
                        Figure 2.5.3.4<br/>
 +
                    </span>
 +
                </td>
 +
            </tr>
 +
        </table>
 +
        <p>
 +
            <b>5.3</b> Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSC<sup>CXCR4</sup> group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSC<sup>CXCR4</sup> group.<br/>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/6/63/T--SYSU-MEDICINE--project-2.5.4.1.jpg">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.5.4.1<br/>
 +
            </span>
 +
            <br/>
 +
            <b>5.4</b> Under IVIS Spectrum, MSCs<sup>CXCR4</sup> exhibited enhanced capacities for targeted migration to the bowels in inflammatory condition in vivo.<br/>
 +
            <br/>
 +
            <br/>
 +
            <b id="Results1.6"><big>6. Therapeutic effects of our engineered MSCs for delayed type hypersensitivity(DTH)) improved. </big></b><br/>
 +
            <br/>
 +
            Our MSCs were modified by BBa_K1993005 (EF-1α-CXCR5-IRES-eGFP).<br/>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/7/77/T--SYSU-MEDICINE--project-2.6.1.1.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.6.1.1<br/>
 +
            </span>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/4/42/T--SYSU-MEDICINE--project-2.6.1.2.png">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.6.1.2<br/>
 +
            </span>
 +
            <br/>
 +
            <b>6.1</b> MSCs<sup>CXCR5</sup> infusion dramatically ameliorated DTH. MSCs<sup>CXCR5</sup> displayed better treatment efficacy than DTH+MSC<sup>eGFP</sup> group, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCs<sup>CXCR5</sup> significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection.<br/>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/4/4e/T--SYSU-MEDICINE--project-2.6.2.jpg">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.6.2<br/>
 +
            </span>
 +
            <br/>
 +
            <b>6.2</b> Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of the anti-inflammatory cytokines (IL-10), elevated in DTH+MSCs<sup>CXCR5</sup> group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCs<sup>CXCR5</sup> group.<br/>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/6/6c/T--SYSU-MEDICINE--project-2.6.3.jpg">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.6.3<br/>
 +
            </span>
 +
            <br/>
 +
            <b>6.3</b> The inflamed ears were collected from each group on Day2 post-injection, and subjected to in situ immunofluorescence staining. Our results revealed that MSCs<sup>eGFP</sup> were almost undetectable, whereas MSCs<sup>CXCR5</sup> were highly accumulated in the inflamed ears of DTH mice.<br/>
 +
            <br/>
 +
 +
            <b id="Results1.7"><big>7. The switch element worked.</big></b><br/>
 +
            <br/>
 +
            <img src="https://static.igem.org/mediawiki/2016/9/9b/T--SYSU-MEDICINE--project-2.7.jpg">
 +
            <span class="note"  style="text-align: center">
 +
                Figure 2.7<br/>
 +
            </span>
 +
            <br/>
 +
            Transduction of our switch plasmid into MSCs. After administering TGF-β1 (7ng/mL), α-SMA was expressed and green fluorescence could be detected in MSCs (BBa_K1993014: pMSA-eGFP worked), which indicated our switch was running.<br/>
 +
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Latest revision as of 19:54, 19 October 2016

Results

  1. Major Achievements
  2. Details

Major achievements:

1. Our modified MSCs still remained their own characteristics.
2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.
3. Chemotaxis of our engineered MSCs improved.
4. Marking proteins (eGFP/dTomato) worked.
5. Therapeutic effects of our engineered MSCs for IBD (inflammatory bowel disease) improved.
6. Therapeutic effects of our engineered MSCs for DTH (delayed type hypersensitivity) improved.
7. The switch element worked.

Details:

1. Our modified MSCs still remained their own characteristics.

Figure 2.1.1

Figure 2.1.2

Our modified MSCs were positive for CD105, CD90, CD29 and CD73 and negative for CD34 (Figure 2.1.1 A-E), and were able to differentiate to osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiating conditions (Figure 2.1.2 F-K). (Meet the standard of International Society for Cellular Therapy (ISCT))

2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.

Figure 2.2.1
 		Figure 2.2.2
 		Figure 2.2.3

Figure 2.2.4
mRNA and protein levels of CCR7, CXCR4 and CXCR5 of MSCs and our modified MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were semi-quantified and detected by qPCR and western blot, respectively. mRNA and protein levels of CCR7, CXCR4 and CXCR5 all elevated in our modified MSCs.

3. Chemotaxis of our engineered MSCs improved.

Figure 2.3.1
 		Figure 2.3.2
 		Figure 2.3.3

Figure 2.3.4
Chemotaxis of our engineered MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were examined against CCL19, CXCL12 and CXCL13, respectively. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs all improved.

4. Marking proteins (eGFP/dTomato) worked.

Figure 2.4.1
4.1 2 days later, after transduction into 293FT cells, marking proteins (eGFP/dTomato) expressed.
Figure A: 293FT cells (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A- hFTH)
Figure B: 293FT cells (BBa_K1993005: EF-1α-CXCR5-IRES-eGFP)
Figure C: 293FT cells (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)
Figure D: 293FT cells (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP)

Figure 2.4.2
4.2 4 days later, after transduction into MSCs, marking proteins (eGFP/dTomato) expressed.
Figure A: MSCs (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A-hFTH)
Figure B: MSCs (BBa_K1993005: EF-1α-CXCR5-IRES-eGFP)
Figure C: MSCs (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)
Figure D: MSCs (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP)

Figure 2.4.3
4.3 After intraperitoneal injection of Luciferin, MSCs engineered with GFP and Luciferase were administrated intravenously to caudal vein. Oxidized Luciferin was observed under IVIS spectrum system. As a result, mice administered with engineered MSCs （BBa_K1993008: EF-1α-Luciferase-IRES-eGFP）

5.Therapeutic effects of our engineered MSCs for inflammatory bowel disease(IBD) improved.

Our MSCs were modified by BBa_K1993009 (EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP).

Figure 2.5.1.1
 		Figure 2.5.1.2
Figure 2.5.1.3
 		Figure 2.5.1.4

5.1 We established murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-colitis (IBD animal model). After injecting with MSCsCXCR4, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.

Figure 2.5.2
5.2 Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCsCXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.

Figure 2.5.3.1
 		Figure 2.5.3.2
Figure 2.5.3.3
 		Figure 2.5.3.4

5.3 Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSCCXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSCCXCR4 group.

Figure 2.5.4.1
5.4 Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in inflammatory condition in vivo.


6. Therapeutic effects of our engineered MSCs for delayed type hypersensitivity(DTH)) improved.

Our MSCs were modified by BBa_K1993005 (EF-1α-CXCR5-IRES-eGFP).

Figure 2.6.1.1
Figure 2.6.1.2
6.1 MSCsCXCR5 infusion dramatically ameliorated DTH. MSCsCXCR5 displayed better treatment efficacy than DTH+MSCeGFP group, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCsCXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection.

Figure 2.6.2
6.2 Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of the anti-inflammatory cytokines (IL-10), elevated in DTH+MSCsCXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCsCXCR5 group.

Figure 2.6.3
6.3 The inflamed ears were collected from each group on Day2 post-injection, and subjected to in situ immunofluorescence staining. Our results revealed that MSCseGFP were almost undetectable, whereas MSCsCXCR5 were highly accumulated in the inflamed ears of DTH mice.

7. The switch element worked.

Figure 2.7
Transduction of our switch plasmid into MSCs. After administering TGF-β1 (7ng/mL), α-SMA was expressed and green fluorescence could be detected in MSCs (BBa_K1993014: pMSA-eGFP worked), which indicated our switch was running.