Difference between revisions of "NJU-China-parts.html"

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<title>project</title>
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<title>humanpractice</title>
  
  
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</style>
  
 
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#top-section {
 
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</style>
 
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<style type="text/css">
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</style>
  
  
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     <TR height="100%">
 
     <TR height="100%">
 
<TD width="27%" bgColor=#E6E8FA style="vertical-align:top">
 
<TD width="27%" bgColor=#E6E8FA style="vertical-align:top">
−
<ul id="nav" style="height:100px;line-height:100px;margin-top:20%;margin-left:10%;list-style-type:none" class="dyn">
+
</br>
−
<li><a href="https://2015.igem.org/Team:NJU-China" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Home</a></li>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Home</a></li>
−
<li><a href="https://2015.igem.org/NJU-China-background.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Background</a></li>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-background.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Background</a></li>
−
<li><a href="https://2015.igem.org/NJU-China-project.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Project</a></li>
+
<div style="line-height:250%;margin-left:10%" id="main1" onClick="document.all.child0.style.display=(document.all.child0.style.display =='none')?'':'none'" ><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Project</a></div>
−
<li><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Model</a>
+
<div id="child0" style="display:none">
−
        <ul>  
+
<ul>
−
            <li><a href="https://2015.igem.org/NJU-China-model.html#deliverymodel" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Delivery model</a></li>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/Design" style="font-weight:bold;font-family:幼圆;color:black">design</a></li>
−
            <li><a href="#" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">RNAi model</a></li>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/result.html" style="font-weight:bold;font-family:幼圆;color:black">results</a></li>
−
        </ul>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/conclusion.html" style="font-weight:bold;font-family:幼圆;color:black">conclusion</a></li>
−
</li>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-project/future work.html" style="font-weight:bold;font-family:幼圆;color:black">future work</a></li>
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-human-practice.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li>
+
</ul>
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-parts.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Parts</a></li>
+
</div>
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-team.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Team</a></li>
+
<div style="line-height:250%;margin-left:10%" id="main1" onClick="document.all.child1.style.display=(document.all.child1.style.display =='none')?'':'none'" ><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Model</a></div>
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-attribution.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Attribution</a></li>
+
<div id="child1" style="display:none">
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-colaboration.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Colaborations</a></li>
+
<ul>
−
<li style="line-height:250%"><a href="https://2015.igem.org/NJU-China-safty.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Safety</a></li>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-model.html" style="font-weight:bold;font-family:幼圆;color:black">Delivery model</a></li>
−
<li style="line-height:250%"><a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Notebook</a>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/RNAi" style="font-weight:bold;font-family:幼圆;color:black">RNAi model</a></li>
−
        <ul>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/Team:NJU-China/signaling" style="font-weight:bold;font-family:幼圆;color:black">Signaling</a></li>
−
            <li><a href="https://2015.igem.org/NJU-China-notebook.html#methods" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Methods</a></li>  
+
</ul>
−
            <li><a href="https://2015.igem.org/NJU-China-notebook.html#protocal" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Protocal</a></li>  
+
</div>
−
            <li><a href="https://2015.igem.org/NJU-China-notebook.html#notebook" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Notebook</a></li>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-human-practice.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Human Practice</a></li>
−
        </ul>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-parts.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Parts</a></li>
−
</li>
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-team.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Team</a></li>
−
</ul>  
+
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-attribution.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Attribution</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-colaboration.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Colaborations</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-safty.html" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Safety</a></li>
 +
<div style="line-height:250%;margin-left:10%" id="main2" onClick="document.all.child2.style.display=(document.all.child2.style.display =='none')?'':'none'" > <a href="#" style="font-weight:bold;font-family:幼圆;font-size:25px;color:black">Notebook </div>
 +
<div id="child2" style="display:none">
 +
<ul>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#methods" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Methods</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#protocal" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Protocal</a></li>
 +
<li style="line-height:250%;margin-left:10%"><a href="https://2015.igem.org/NJU-China-notebook.html#notebook" style="font-weight:bold;font-family:幼圆;font-size:20px;color:black">Notebook</a></li>
 +
</ul>
 +
</div>
  
  
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     <TD width="73%" bgColor=#FFFFFF style="vertical-align:top;padding-left:80px;padding-right:80px;padding-top:50px;padding-bottom:50px;word-wrap:break-word;">
 
     <TD width="73%" bgColor=#FFFFFF style="vertical-align:top;padding-left:80px;padding-right:80px;padding-top:50px;padding-bottom:50px;word-wrap:break-word;">
 
</TD>
 
</TD>
−
<h1> <font color=#FF0000>  1.Bba_K1633003 </font> </h1>
 
−
 
−
<B> MOR siRNA -1(siRNA for mouse Mu opioid receptor) </B>
 
−
 
−
<h2> INTRODUCTION </h2>
 
−
 
−
This part is an artificially designed RNA strand. It serves as an element of the Team
 
−
 
−
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
 
−
 
−
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
 
−
 
−
software accessible online. This tool can find the best siRNA sequences on target gene
 
−
 
−
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
 
−
 
−
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
 
−
 
−
the plasmid. Then we order them through a DNA synthesis company.  We got four such RNA.
 
−
 
−
MOR siRNA-1 is a backup. 
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第一张图>
 
−
 
−
Figure 1. The sequence of MOR siRNA-1
 
−
 
−
<br><br>
 
−
 
−
<h2> USAGE AND BIOLOGY </h2>
 
−
 
−
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
 
−
 
−
expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
 
−
 
−
modified exosomes into the bloodstream, exosome will specifically recognize
 
−
 
−
acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
 
−
 
−
Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
 
−
 
−
decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
 
−
 
−
function will result in the inhabitation of the secretion of GABA and the suppression of
 
−
 
−
the dopaminergic reward pathway, which ultimately have some therapeutic effects on
 
−
 
−
opioid dependence.
 
−
 
−
<br><br>
 
−
 
−
 
−
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
 
−
 
−
different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
 
−
 
−
cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
 
−
 
−
sequence with the best interfering effect was selected for further study. Not showing
 
−
 
−
the best efficiency, MOR siRNA-1 finally functions just as a backup.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第二张图>
 
−
 
−
Figure 2. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
 
−
 
−
plasmids.
 
−
 
−
<br><br>
 
−
 
−
<h1> <font color=#FF0000>  2.Bba_K1633004 </font> </h1>
 
−
 
−
<B> MOR siRNA -2(siRNA for mouse Mu opioid receptor) </B>
 
−
 
−
<h2> <B> INTRODUCTION </B> </h2>
 
−
 
−
This part is an artificially designed RNA strand. It serves as an element of the Team
 
−
 
−
NJU CHINA RNAi module. We use them as siRNA medicine to downregulate the expression of
 
−
 
−
Mu opioid receptor in brain tissue. We designed specific MOR siRNAs based on a free
 
−
 
−
software accessible online. This tool can find the best siRNA sequences on target gene
 
−
 
−
MOR to insure the maximum gene-specificity and silencing efficacy. This tool also
 
−
 
−
designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in
 
−
 
−
the plasmid. Then we order them through a DNA synthesis company.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第三张图>
 
−
 
−
Figure 3. The sequence of MOR siRNA-2.
 
−
 
−
<br><br>
 
−
 
−
 
−
<h2> <B> USAGE AND BIOLOGY </B> </h2>
 
−
 
−
We package MOR siRNA into exosomes by transfecting HEK293 cells with a plasmid
 
−
 
−
expressing MOR siRNA and then collect siRNA-encapsulated exosomes. When inject the
 
−
 
−
modified exosomes into the bloodstream, exosome will specifically recognize
 
−
 
−
acetylcholine receptors and fuse with neurons under the direction of the RVG peptide.
 
−
 
−
Once inside neurons, MOR siRNA will degrade MOR mRNA by base-pairing, resulting in sharp
 
−
 
−
decrease of MOR on neuron membrane. As a consequence, MOR reduction and disturbed
 
−
 
−
function will result in the inhabitation of the secretion of GABA and the suppression of
 
−
 
−
the dopaminergic reward pathway, which ultimately have some therapeutic effects on
 
−
 
−
opioid dependence.
 
−
 
−
<br><br>
 
−
 
−
To ensure the interference efficiency of the MOR siRNA, three siRNA sequences targeting
 
−
 
−
different sites of MOR mRNA were designed and transfected into the mouse neuroblastoma
 
−
 
−
cell line Neuro2A. Efficient knockdown of MOR in Neuro2A cells is observed, and the
 
−
 
−
sequence with the best interfering effect was selected for further study. Not showing
 
−
 
−
the best efficiency, MOR finally functions just as a backup. Showing the best
 
−
 
−
efficiency, MOR siRNA-2 is used for further study.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第四张图>
 
−
 
−
Figure 4. Relative level of MOR mRNA in Neuro2A cell after transfection of MOR siRNA-1
 
−
 
−
plasmid.
 
−
 
−
<br><br>
 
−
 
−
 
−
<h2> <B> CHARACTERIZATION </B> <h2>
 
−
 
−
` <h3> <B> 3.1 Package of MOR siRNA into exosomes </B> </h3>
 
−
 
−
The levels of MOR siRNA in isolated exosomes were assayed by a quantitative RT-PCR
 
−
 
−
assay. The MOR siRNA concentration in exosomes was calculated to be approximately 0.14
 
−
 
−
pmol/μg. The results showed that MOR siRNA can be successfully packaged into exosomes,
 
−
 
−
no matter the exosomes were modified on the outside membrane with or without RVG
 
−
 
−
peptide.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第五张图>
 
−
 
−
Figure 5. The concentration of MOR siRNA in unmodified or RVG-modified exosomes.
 
−
 
−
<br><br>
 
−
 
−
<h3> <B> 3.2 TEM photographs of exosomes carrying MOR siRNA inside and RVG on
 
−
 
−
membranes </B> </h3>
 
−
 
−
 
−
We next characterized the RVG exosomes loaded with MOR siRNA using transmission electron
 
−
 
−
microscopy (TEM). The TEM photographs showed that the exosomes presented normal
 
−
 
−
morphological characteristics after outside modification and siRNA loading, with a
 
−
 
−
diameter of approximately 90 nm and a double-layer membrane surrounded. These
 
−
 
−
characteristics indicate that the exosome properties were not affected by the
 
−
 
−
modifications.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第六张图>
 
−
 
−
Figure 6. TEM photographs of the exosomes with outside RVG modification and inside siRNA
 
−
 
−
loading.
 
−
 
−
<br><br>
 
−
 
−
<h3> <B> 3.3 RVG exosomes specifically deliver MOR siRNA into neuronal cells
 
−
 
−
</B> </h3>
 
−
 
−
Subsequently, MOR siRNA levels were assayed in recipient Neuro2A cells when incubating
 
−
 
−
with RVG exosomes loaded with MOR siRNA. The siRNAs concentrations were barely detected
 
−
 
−
in untreated control cells or in cells treated with RVG exosomes or unmodified exosomes
 
−
 
−
loaded with MOR siRNA. In contrast, a significant amount of siRNAs were detected in
 
−
 
−
Neuro2A cells after treatment with RVG exosomes loaded with MOR siRNA. As a control, MOR
 
−
 
−
siRNA was also barely detected in C2C12 cells treated with RVG-exosome loaded with MOR
 
−
 
−
siRNA. Taken together, these results clearly demonstrate that the RVG peptide
 
−
 
−
modification on the exosome membrane specifically guides exosomes to target neuronal
 
−
 
−
cells bearing the surface acetylcholine receptor, allowing for the efficient delivery of
 
−
 
−
MOR siRNA into the recipient cells.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第七张图>
 
−
 
−
Figure 7. Quantitative RT-PCR analysis of MOR siRNA concentration in Neuro2A and C2C12
 
−
 
−
cells treated with RVG exosomes (RVG exosome), unmodified exosomes loaded with MOR siRNA
 
−
 
−
(siRNA-exosome) or RVG exosomes loaded with MOR siRNA (siRNA-RVG exosome).
 
−
 
−
<br><br>
 
−
 
−
<h3> <B> RVG exosomes loaded with MOR siRNA specifically reduce MOR expression
 
−
 
−
in neuronal cells </B> </h3>
 
−
 
−
We next evaluate the effect of RVG exosome-delivered siRNA on MOR expression in vitro.
 
−
 
−
MOR expression levels were assayed in Neuro2A cells after treatment with RVG exosomes
 
−
 
−
loaded with MOR siRNA. Compared with control cells, MOR protein and mRNA levels were
 
−
 
−
dramatically reduced by RVG exosome-delivered siRNA, while no reduction in the MOR
 
−
 
−
protein and mRNA levels were observed by exosomes without the RVG peptide on their
 
−
 
−
surface. The results suggest that the RVG peptide modification on the exosome membrane
 
−
 
−
can specifically guides exosomes to target neuronal cells, allowing for the delivery of
 
−
 
−
MOR siRNA into the neuronal cells to reduce MOR expression levels.
 
−
 
−
<br><br>
 
−
 
−
<!-- 插入第八张图>
 
−
 
−
Figure 8. RVG exosome-delivered siRNA specifically enters Neuro2A cells and reduce MOR
 
−
 
−
expression. (A) Western blot analysis of MOR protein levels in untreated control Neuro2A
 
−
 
−
cells or cells treated with MOR siRNA loaded in normal exosomes or RVG exosomes. (B)
 
−
 
−
qRT-PCR analysis of MOR mRNA levels in untreated control Neuro2A cells or cells treated
 
−
 
−
with MOR siRNA loaded in normal exosomes or RVG exosomes.
 
−
 
−
<br><br> 
 
−
 
−
 
−
<h3> <B> 3.5 The effects of siRNA delivered by RVG exosomes on morphine-induced
 
−
 
−
CPP </B> </h3>
 
−
 
−
MOR and its signaling pathway are known to be involved in the dependence and relapse of
 
−
 
−
opioids such as morphine and heroin. Importantly, relapse always disrupts the process of
 
−
 
−
opioid withdrawal. Subsequently, we focus on investigating the effect of exosomal siRNA
 
−
 
−
of MOR on opioid relapse. We evaluate the consequences of MOR knockdown by exosomal
 
−
 
−
siRNA in the animals by conducting the morphine-induced conditioned place preference
 
−
 
−
(CPP) test, a mouse model for morphine wanting/liking behaviors. In the CPP paradigm,
 
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mice learned to associate the rewarding effect of morphine with a drug-paired
 
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environment. The CPP test was designed to mimick the process of relapse of morphine
 
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(Fig. 5A). Before conditioning, the mice showed a preference for visiting black chamber.
 
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Then, morphine dependence was developed when mice were place-conditioned by
 
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intraperitoneal injection with morphine in the white chamber on even-numbered days (on
 
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days 2, 4, 6, 8 and 10) and with saline in the black chamber on odd-numbered days (on
 
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days 3, 5, 7, 9 and 11). On day 12, CPP test 1 was conducted by allowing the mice to
 
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freely visit the morphine-paired white chamber or saline-paired black chambers. As
 
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expected, mice showed a significant preference in visiting the morphine-paired white
 
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chamber, suggesting the development of morphine dependence. Then, morphine treatment was
 
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removed for several days. On day 26, CPP test 2 was conducted and mice spent less time
 
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in the morphine-paired white chamber than the saline-paired black chamber, suggesting
 
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the disappearance of morphine dependence. Then, mice were intravenously injected with
 
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saline or with siRNAs loaded in normal exosome or RVG exosome once every two days for a
 
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total of four times, and CPP test 3 was performed on day 32. Mice maintained their
 
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natural preference for the black chamber, suggesting that MOR siRNA had no effect on the
 
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behavior of the mice. Finally, mice were relapsed on morphine on day 33, and CPP test 4
 
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was performed the next day. Interestingly, the mice treated with RVG exosome-delivered
 
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siRNAs maintained their natural preference for the black chamber, while the mice treated
 
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with saline or with siRNAs loaded in normal exosome show preference to morphine-paired
 
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white chamber, suggesting that the MOR siRNAs delivered by RVG exosome restrain drug
 
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addiction when the mice were re-exposed to morphine.
 
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<br><br>
 
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<!-- 插入第九张图>
 
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Figure 9. The effects of siRNA delivered by RVG exosomes on morphine-induced CPP. A flow chart depicting the experimental design is shown. The panel is represented by the value of the time mice stay in morphine-paired white chamber minus the time mice stay in saline-paired black chamber.
 
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<br><br>
 
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h3> <B> 3.6 The effects of siRNA delivered by RVG exosomes on MOR expression in vivo </B> </h3>
 
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After the CPP test, mice were sacrificed, and total RNA and protein were extracted from mouse brain to evaluate the expression levels of MOR in vivo. Both MOR protein and mRNA levels were reduced in the mice treated with RVG exosome-delivered siRNA. In contrast, siRNAs delivered by unmodified exosome could not reduce MOR mRNA and protein levels in mouse brain. Thus, these results clearly demonstrate that exosomes with RVG modification passed through the BBB and delivered MOR siRNA into the central nervous system to regulate MOR expression, while natural exosomes without the RVG modification were not capable of delivering siRNA into the central nervous system or regulating target gene expression.
 
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<br><br>
 
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<!-- 插入第十张图>
 
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Figure 10. RVG exosomes can transfer MOR siRNA through the BBB and reduce MOR expression levels in vivo. (A) Western blot analysis of MOR protein levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes. (B) qRT-PCR analysis of MOR mRNA levels in the brains of mice following injection with saline or with MOR siRNA loaded in normal exosomes or RVG exosomes.
 
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<br><br>
 
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     </TR>
 
     </TR>
 
</TABLE>
 
</TABLE>

Revision as of 01:53, 18 September 2015

humanpractice