这项工作是格兰特DK078732支持由美国国立卫生研究院（的PTF）。

1。腺病毒介导和培养大鼠胰岛<ol><li>准备加入所需数量的2毫升（载8毫米葡萄糖，10％胎牛血清，50个单位/ ml青霉素，50μg/ mL链霉素的RPMI 1640培养媒体）媒体6以及非组织文化的涂层板井。例如，一个典型的实验可能需要三口井 - 一个无病毒控制，病毒控制（例如，表达GFP的腺病毒），实验组。 </li><li>暖板放置到组织文化的孵化器，为至少30分钟至37°C。 </li><li>紧随6以及非组织文化的涂层板个别井大鼠胰岛分离18,19 100-200胰岛。第六胰岛细胞所需的胰岛素分泌和胸腺嘧啶掺入法。其余的胰岛细胞可用于RNA基因表达研究或蛋白质的免疫隔离的隔离。 </li></ol> [注:从这点出发，请遵循和生物危险材料的处理，使用，处置制度的协议。] <ol start="4"><li>轻轻地旋转以及中心板带来的小岛。 </li><li>直接吸取上盘中心的胰岛腺病毒。使用100-500多重感染（内政部，比靶细胞病毒蚀斑形成单位）。 </li><li>让胰岛休息5分钟。 </li><li>将组织文化的孵化器（37℃，5％CO 2）板块。 </li><li> 24小时后，轻轻地摇动板带来的胰岛井中心和转让使用P200的微管以及含有新鲜的媒体到一个新的胰岛。如果胰岛成为附着板，就可以轻轻抛下枪头。 </li></ol> [注:为了验证足够的传导efficienCY，控制病毒表达GFP的使用是有益的，如胰岛细胞，然后可以通过共聚焦显微镜成像核实渗透到胰岛核心的腺病毒。] <ol start="9"><li>文化的胰岛额外的24-72小时，这取决于所需的时间从优化试验研究实验。例如，诱导的增殖反应，可能需要从24-72小时或击倒感兴趣的基因，可能需要48或72小时的时间。每天新鲜媒体的胰岛细胞转移。 </li><li>对于最后24Ĥ实验，文化媒体，含1胰岛微居里甲基-3] -thymidine/ml媒体（一般为1微升胸苷/毫升媒体）。 </li></ol> [注:从这点出发，请按照下列机构的协议处理，使用，处置放射性物质。 2。胰岛素分泌试验<ol><li>准备分泌缓冲液（SAB）的10X储备溶液（1.14 M氯化钠，氯化钾47毫米，12毫米KH 2 PO 4，硫酸镁 11.6毫米）和氯化钙 100X股票解决方案（0.25M氯化钙2）。这些股票的解决方案，可提前准备的时间，并在室温下储存。 </li><li>新鲜的准备工作审计局（5 mL 10X审计局，1毫升1 M羟乙基100X 氯化钙 ，0.5毫升，0.28毫升35％的牛血清白蛋白，0.11克碳酸氢钠3，无菌水50毫升中）在50毫升50毫升锥形管和温暖至37°C的放置在37℃水浴。 </li><li>吸取10毫升到15毫升锥形管工作审计局和增加66.8微升2.5米D-葡萄糖，准备高糖（16.7毫米）审计局。 </li><li>新增44.8微升至2.5米D-葡萄糖，审计局的工作，其余40毫升，以备低血糖（2.8毫米）审计局。 </li><li>标签三个1.7毫升，每6孔板以及离心管，加入1毫升的磷酸盐缓冲液（PBS）。 </li></oL&gt; [注:由于胰岛是放射性，放射性物质的处理，使用，处置，请按照下列机构的协议。 <ol start="6"><li>每个离心管放入20个小岛。尽一切努力向每个离心管加入同等大小岛屿。例如，每管可包含5小，10中，5个大型的胰岛细胞（ 见图1）。 </li></ol> [注:胰岛可以使用解剖立体或一个标准的显微镜可视化。] <ol start="7"><li>胰岛后定居管底部的重力（〜2分钟），吸用微量的PBS及丢弃。 </li></ol> [注:作为替代，以重力沉降管，可在300 XG离心1分钟。] <ol start="8"><li>预孵化，添加400μL低谷氨酸葡萄糖审计局，放入孵化器的组织文化（37℃，5％CO 2），60分钟前孵育管（盖打开）。吸预孵化低血糖审计局和丢弃。 </li><li>基础胰岛素分泌，添加400μL的低血糖审计局，组织培养的孵化器（37℃，5％CO 2）放入管（盖打开），孵育60分钟。收集的低血糖审计局和保存胰岛素放射免疫分析法。 </li><li>为刺激胰岛素分泌，添加400μL的高血糖审计局，组织培养的孵化器（37℃，5％CO 2）放入管（盖打开），孵育60分钟。收集高血糖审计局和保存胰岛素放射免疫分析法。 </li></ol> 3。 TdR掺入实验<ol><li>加入1 ml的PBS;重力管底部定居后的胰岛细胞，吸用微量的PBS，丢弃，重复此加强一次。 </li><li>加入500μL冰冷的三氯乙酸（TCA，10％W / V），并在冰上孵育30分钟。 </li><li> 16 000 XG 3分，在4°C离心管</li><li>吸出TCA的，加0.3 N氢氧化钠80μL，孵育室温30分钟。在此期间，大力涡样品，每10分钟为5-10秒。 </li><li>添加4毫升到7毫升液体闪烁计数管计数鸡尾酒经济安全。 </li><li>加入50微升样品闪烁计数管，帽筒，简要动摇，在液体闪烁计数器计数。 </li><li>根据制造商的协议使用的二喹啉酸（BCA）法和10μL的样品测量蛋白质浓度。 </li></ol> 4。数据分析<ol><li>按照制造商的协议执行的胰岛素放射免疫分析法。 </li><li>正常化与蛋白质浓度的胰岛素分泌和TdR掺入数据entration。 </li></ol> 5。代表结果 如图2所示的实验，以评估胰岛细胞复制和大鼠胰岛β细胞功能的例子。这个例子说明，假设"＃6"强劲刺激胰岛β细胞功能不改变复制的基因的腺病毒过度。在顶部面板中，从胸腺嘧啶掺入法结果表明，越来越多的"基因＃6"的表达，增加DNA的合成，TdR掺入测定。因为大部分在大鼠胰岛细胞的β细胞，它是可能的，这增加TdR掺​​入表明增加β细胞复制。然而，实验验证，必须要牢固树立。底部面板中，从胰岛素分泌试验的结果表明，过度的"基因＃6"不改变β细胞的主要功能之一，即，我nsulin在低和高糖的分泌。与腺病毒治疗后的胰岛胰岛分离和健康质量指示倍，在低和高血糖浓度在胰岛素分泌增加。如果增加"基因＃6"受损的β细胞功能的表达，这可能会反映在高，刺激葡萄糖浓度（16.7毫米）的胰岛素分泌减少。也可以执行一个不同的血糖浓度的剂量 - 反应曲线。 <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="图1"/> 图1。协议的概述，以评估在离体大鼠胰岛细胞，腺病毒介导的基因表达改变后的胰岛细胞复制和β细胞功能。新鲜分离的大鼠胰岛腺病毒暴露24小时，然后培养96小时。在最后的24小时，胰岛素的分泌测量TdR掺入评估高，低血糖。 <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="图2"/> 图2。使用控制腺病毒和腺病毒过度一个假设的基因标记"基因＃6"的实验结果。顶部面板显示TdR掺入和底部面板的胰岛素分泌。

<ol>
	<li>Ferrannini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Cell+function+in+subjects+spanning+the+range+from+normal+glucose+tolerance+to+overt+diabetes:+a+new+analysis.">beta-Cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis.</a> J. Clin. Endocrinol. Metab. 90, 493-500 (2005).</li><li>Weyer, C., Bogardus, C., Mott, D. M., Pratley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+insulin+secretory+dysfunction+and+insulin+resistance+in+the+pathogenesis+of+type+2+diabetes+mellitus.">The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus.</a> J. Clin. Invest. 104, 787-794 (1999).</li><li>Keenan, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+insulin+production+and+pancreatic+ss-cell+turnover+after+50+years+of+diabetes:+Joslin+Medalist+Study.">Residual insulin production and pancreatic ss-cell turnover after 50 years of diabetes: Joslin Medalist Study.</a> Diabetes. 59, 2846-2853 (2010).</li><li>Bain, J. R., Schisler, J. C., Takeuchi, K., Newgard, C. B., Becker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adenovirus+vector+for+efficient+RNA+interference-mediated+suppression+of+target+genes+in+insulinoma+cells+and+pancreatic+islets+of+langerhans.">An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.</a> Diabetes. 53, 2190-2194 (2004).</li><li>Fueger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trefoil+factor+3+stimulates+human+and+rodent+pancreatic+islet+beta-cell+replication+with+retention+of+function.">Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.</a> Mol. Endocrinol. 22, 1251-1259 (2008).</li><li>Schisler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+human+and+rat+islet+beta-cell+proliferation+with+retention+of+function+by+the+homeodomain+transcription+factor+Nkx6.1.">Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.</a> Mol. Cell Biol. 28, 3465-3476 (2008).</li><li>Chan, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+uncoupling+protein+2+inhibits+glucose-stimulated+insulin+secretion+from+rat+islets.">Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.</a> Diabetes. 48, 1482-1486 (1999).</li><li>Cozar-Castellano, I., Takane, K. K., Bottino, R., Balamurugan, A. N., Stewart, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+beta-cell+proliferation+and+retinoblastoma+protein+phosphorylation+in+rat+and+human+islets+using+adenovirus-mediated+transfer+of+cyclin-dependent+kinase-4+and+cyclin+D1.">Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.</a> Diabetes. 53, 149-159 (2004).</li><li>Icyuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+infection+activates+akt1+and+induces+cell+proliferation+in+pancreatic+islets1.">Adenovirus infection activates akt1 and induces cell proliferation in pancreatic islets1.</a> Transplantation. 87, 821-824 (2009).</li><li>Kaneto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+hexosamine+pathway+leads+to+deterioration+of+pancreatic+beta-cell+function+through+the+induction+of+oxidative+stress.">Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress.</a> J. Biol. Chem. 276, 31099-31104 (2001).</li><li>Antinozzi, P. A., Berman, H. K., O'Doherty, R. M., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+with+recombinant+adenoviruses.">Metabolic engineering with recombinant adenoviruses.</a> Annu. Rev. Nutr. 19, 511-544 (1999).</li><li>Newgard, C. B., Becker, T. C., Berman, H. K., O'Doherty, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+overexpressed+hexokinases+in+liver+and+islet+cells.">Regulation of overexpressed hexokinases in liver and islet cells.</a> Biochem. Soc. Trans. 25, 118-122 (1997).</li><li>Becker, T. C., BeltrandelRio, H., Noel, R. J., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+hexokinase+I+in+isolated+islets+of+Langerhans+via+recombinant+adenovirus.+Enhancement+of+glucose+metabolism+and+insulin+secretion+at+basal+but+not+stimulatory+glucose+levels.">Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels.</a> J. Biol. Chem. 269, 21234-21238 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+to+pancreatic+islets+does+not+alter+islet+function.">Adenoviral-mediated gene transfer to pancreatic islets does not alter islet function.</a> Transplant Proc. 26, 756-757 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+to+pancreatic+islets+mediated+by+adenoviral+vectors.">Efficient gene transfer to pancreatic islets mediated by adenoviral vectors.</a> Transplantation. 59, 263-268 (1995).</li><li>Meng, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+liver+X+receptors+inhibits+pancreatic+islet+beta+cell+proliferation+through+cell+cycle+arrest.">Activation of liver X receptors inhibits pancreatic islet beta cell proliferation through cell cycle arrest.</a> Diabetologia. 52, 125-135 (2009).</li><li>Ronnebaum, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyruvate+cycling+pathway+involving+cytosolic+NADP-dependent+isocitrate+dehydrogenase+regulates+glucose-stimulated+insulin+secretion.">A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion.</a> J. Biol. Chem. , (2006).</li><li>Milburn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cells+in+obesity.+Evidence+for+induction+of+functional,+morphologic,+and+metabolic+abnormalities+by+increased+long+chain+fatty+acids.">Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.</a> J. Biol. Chem. 270, 1295-1299 (1995).</li><li>Szot, G., Koudria, P., Bluestone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Islet+Isolation.">Murine Pancreatic Islet Isolation.</a> J. Vis. Exp. (7), e255 (2007).</li></ol>

没有利益冲突的声明。

建立通路，可调制刺激的复制和增强β细胞的功能有关糖尿病的两个主要形式。因为功能的β细胞质量是依赖胰岛素​​分泌细胞的存在和功能上，评估这些因素，同时有它的优势。这个协议描述为确定一种蛋白质的过度表达或抑制是否会导致功能的β细胞质量的变化， 在体外培养 ，然后可以测试体内疗效的精简协议。 这个协议的一个限制是，胰岛是一种微型器官的多种细胞类型，包括组成，而不是仅限于α，β，δ，ε，和PP细胞。胰岛细胞复制的变化可能不会完全转化为β细胞复制的变化，因为80-90％的大鼠胰岛细胞β细胞。观测到的变化胰岛的可能性复制是由于非β细胞复制存在。因此，这个协议的逻辑和验证的下一步可能会研究使用胸苷类似物加上免疫或流式细胞仪分析5,6β细胞复制。这验证分析，也可以缓解成胰岛细胞的增殖独立非特异性TdR掺入潜在的问题。 另一个潜在的缺点在于对腺病毒转导效率。转导效率的60-70％是合理的实现，但成效的一个关键因素是腺病毒转导的时机。至关重要的是尽快传导效率最大化的腺病毒的分离胰岛文化。胰岛分离后的几个小时内，胰岛开始收缩，从而限制了腺病毒的能力，深入渗透到胰岛核心。记者构造的使用，如腺病毒表达GFP，加上共聚焦显微镜可能是有益的评估转染效率。 该协议的主要优点是:1）同一池中的小岛上的测试功能的β细胞质量的多个因素的效率和2）的要求执行协议（一个典型的大鼠胰岛分离得到400胰岛的胰岛小） 。这些优势使该协议将作为一个稳健的步伐多个基因的筛检工具。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI 1640培养媒体</td><td> Gibco公司</td><td> 11879 </td><td></td></tr><tr><td>青霉素/ </td><td> Gibco公司</td><td> 15140 </td><td></td></tr><tr><td> 6孔板</td><td> BD-猎鹰</td><td> 35-1146 </td><td>非TC治疗</td></tr><tr><td> [甲基-3H]-胸苷</td><td> Perkin Elmer公司</td><td> NET027Z001MC </td><td> 1毫居里/毫升</td></tr><tr><td>微型离心管</td><td>丹维尔</td><td> C2170 </td><td> 1.7毫升</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> 59888 </td><td></td></tr><tr><td>氯化钾</td><td>阿库罗斯福冈</td><td> 42409 </td><td></td></tr><tr><td>锦2 &lt;/ SUB&gt; PO 4 </td><td>阿库罗斯福冈</td><td> 20592 </td><td></td></tr><tr><td> 硫酸镁 </td><td>阿库罗斯福冈</td><td> 41348 </td><td></td></tr><tr><td> 氯化钙 </td><td>阿库罗斯福冈</td><td> 34961 </td><td></td></tr><tr><td>肝素钠</td><td>西格玛</td><td> H0887 </td><td> 1个M解决方案</td></tr><tr><td> 35％BSA </td><td>西格玛</td><td> A7979 </td><td></td></tr><tr><td> 碳酸氢钠 </td><td>阿库罗斯福冈</td><td> 42427 </td><td></td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G8769 </td><td></td></tr><tr><td>氯乙酸</td><td> Fisher Scientific则</td><td> SA9410-1 </td><td> 10％W / V </td></tr><tr><td>氢氧化钠</td><td>阿库罗斯福冈</td><td> 12426 </td><td></td></tr><tr><td>闪烁计数管</td><td>萨尔斯塔特</td><td> 58.536 </td><td> 7毫升，PP </td&gt; </tr><tr><td>闪烁计数管帽</td><td>萨尔斯塔特</td><td> 65.816 </td><td></td></tr><tr><td>安全的Econo计数鸡尾酒</td><td>零售物价指数</td><td> 111175 </td><td></td></tr><tr><td>胰岛素的RIA </td><td>西门子</td><td> TKIN2 </td><td></td></tr><tr><td> BCA分析套件</td><td> Thermo Scientific的</td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5415R </td><td></td></tr><tr><td>闪烁计数管架</td><td>萨尔斯塔特</td><td> 93.1431.001 </td><td></td></tr><tr><td>液体闪烁计数器</td><td> Perkin Elmer公司</td><td>三，碳水化合物2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

主要是葡萄糖稳态控制的内分泌激素的胰岛素和胰高血糖素，胰岛β和α细胞分泌，分别。功能的β细胞质量是决定解剖β细胞质量以及β细胞的能力，以应对营养负荷。一个丧失功能的β细胞质量是中心1-3糖尿病的两种主要形式。而下降功能的β细胞自身免疫性1型糖尿病发作的质量结果，在2型糖尿病，这一递减的发展都无法适当分泌胰岛素的β细胞和β细胞的破坏从干部机制。因此，努力恢复功能的β细胞质量更好的治疗和糖尿病的潜在治疗至关重要。 正在努力找出刺激的复制和增强β细胞的功能，可以利用的分子途径。理想的情况下，治疗的目标，同时提高β细胞的生长和功能。也许更重要的虽然是一种策略，刺激β细胞的生长，以确定是否损害的β细胞功能（如某些癌基因），反之亦然成本。 由系统抑制或过度在离体大鼠胰岛靶基因的表达，可以增加功能的β细胞质量4-6找出潜在的治疗靶点。腺病毒载体可有效地过度或击倒蛋白在离体大鼠胰岛4,7-15就业。在这里，我们提出了一种方法来操纵利用腺病毒转导的基因表达和评估胰岛复制，并在离体大鼠胰岛β细胞功能（ 图1）。这种方法已经被以前使用，以确定新的目标，调节β细胞复制或功能5,6,8,9,16,17。

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

该协议允许一个确定调节功能的β细胞质量的因素，找到治疗糖尿病的潜在的治疗靶点。该协议包括一个精简的方法来评估胰岛细胞复制和β细胞功能，在离体大鼠胰岛细胞与腺病毒基因表达操纵。

复制和β细胞功能评估Adenovirally转分离的啮齿动物胰岛

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

assessing-replication-beta-cell-function-adenovirally-transduced

Nanyang Technological University

Research

JoVE Journal

Medicine

13.9K 次观看.  Indiana University School of Medicine. 该协议允许一个确定调节功能的β细胞质量的因素，找到治疗糖尿病的潜在的治疗靶点。该协议包括一个精简的方法来评估胰岛细胞复制和β细胞功能，在离体大鼠胰岛细胞与腺病毒基因表达操纵。

视频: Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

科研

医学

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

评估与远藤的PAT 2000内皮血管舒张功能

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

在女小鼠害保留神经的中期尿道梗阻（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

复制和β细胞功能评估Adenovirally转分离的啮齿动物胰岛

摘要

探索更多视频

此视频中的章节

在分离的小鼠使用红桃心功能和能量学的评估³¹ P核磁共振