外泌体研究小组第二次会议总结

惜名

2015年4月18日，周六，上海的下午不冷也不热，正是小聚好时节。来自中科院上海生命科学院、复旦医学院、上交医学院、上海二军大、上海大学、浙大医学院等6所院校（排名不分先后）的16名外泌体研究者，在生科院高大上的会议室里举行了“外泌体研究小组第二次聚会”。

本次聚会有6人（Johnny、倩倩、惜名、宝莲、Alice、游虾）进行了PPT交流。会议基本上可以分为两个阶段，第一阶段是以PPT演讲为中心的交流，第二阶段是自由讨论。

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PPT内容有部分重叠，不再一一赘述，下面分享几个重点讨论的问题。

1、外泌体提取哪家强？

毫无疑问，山东蓝翔排第一。

排第二的或许应该还是超离吧，这是经典大paper久经实践的好方法。至于得率，Johnny用Nano对比了超离和SBI提取后exo的总粒子数，竟然发现二者在一个数量级。这似乎是说SBI的提取效率和超离差不多？当然，也有人认为，Nano检测粒子数不能准确反应exo含量，误差太大。

应该说，不少高分文献也在用SBI，因此这个kit可靠性还是比较高的。笔者使用过101bio和SBI，个人经验是二者提取后进行下游干预实验都能得到阳性结果，未对比二者的提取效率差异。

倩倩用101bio提取exo后做了质谱分析，发现含量最高的蛋白竟然是BSA（培养基中含有的一种成分），如果倩倩的操作没问题、质谱分析也可靠的话，这无疑说明101bio提取的exo含有杂蛋白。SBI这方面的试验我们小组没人做过。当然，值得一提是的，倩倩的质谱分析竟然连exo的已知marker都没检测出来，这个质谱分析或许需要重复。

Life的使用者应翔因故未参加此次聚会。

最后总结，有人强烈认为kit提取exo杂质较多，倩倩的质谱分析或许也提供了即使是不太可靠的证据。笔者认为，kit提取exo即使含有一定量杂质，只要你的实验设计能够容忍这些杂志，或许也是能接受的，毕竟，kit更加简单，而且也经过了高分文献的验证。当然，如果条件允许，用超离更加好。

2、外泌体电镜下形态是否有标准？

经典文献的exo电镜图片以“杯托样”结构为主，也可以形象的看成“嘴唇样”、“咖啡豆样”，其鲜明的特征是有“褶皱”。Johnny用超离提取exo，电镜图片与经典文献吻合度很高。而其余人员（包括复旦、上交、浙大）基本都是用kit提取的exo，电镜下显示为一个空泡样结构，形状基本是正圆形，没有褶皱。文献认为，这种形态上的区别和提取方法有关系。超速离心会使得exo被强烈挤压，因此产生褶皱。Kit提取则没有这个现象。

3、电镜下外泌体亚结构和直径是否很重要？

经典文献的exo电镜图有时可以看到一些“亚结构”，“杯托样”exo的内部还有一些密度不均匀、明暗相间的小区域，但此次小组人员用kit提取的则没有看到这些结构。沛渊认为，这或许和电镜拍摄时电压强度有关系，电压越大，投射性越好，越可以看清楚内部结构。沛渊介绍，生科院电镜室老师建议120kv的电压。另外，这或许也和染色过程有关系，目前exo染色以磷戊酸或铀负染为主，操作还是比较简单的。Johnny介绍了一种更加复杂的染色方法，但是效果不佳。

关于直径，kit提取的exo似乎直径偏小，较大者70nm，大多数的均在20nm上下，有人还看到了更大的，直径在400-500nm。Johnny用超离提取exo，Nano结果提示粒子直径峰值在100nm，结果与文献较为符合。

关于直径，目前一般认为30-100nm的称为exo，更大的则属于MV了。游虾经过仔细查阅文献，发现30-100nm的直径有深层次原因。Exo是脂质双层结构，其分泌依赖细胞内器，脂质双层结构决定了exo不能太小，否则便无法成为一个囊状结构了，分泌过程依赖细胞内器决定其也不能太大，不能大于其来源的母体。

4、蛋白Marker跑不出来怎么办？

目前已知的exo marker达十余种之多，有些marker并非每种细胞来源的exo都会表达，因此当marker跑不出来时，可以考虑换其他marker试试。本次聚会认为，跑出来2-3个marker就OK了。譬如，本次聚会很多人都跑不出CD9，CD63和Alix似乎比较好跑，也有人推荐试试TSG101。沛渊强调，exo marker甚至有某种程度的种属特异性，因此跑marker时大家应该尽量参考你的细胞来源的exo在文献中被证实存在哪些marker，不要盲目尝试。

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第二阶段，自由讨论环节，该过程大约1.5小时。

每个人就个人经验和兴趣与他人进行了针对性探讨。这部分内容比较杂乱，有些人会讨论很基础、很技术的细节，有些人会讨论整个实验思路，因此也就没办法记录了。

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感谢Johnny安排了本次活动，借用的生科院的会议室非常专业。Johnny自费为大家购买了一些小零食，还给与会者泡了他们家乡今年的新茶，我怎么喝都比西湖龙井更加袭人。感谢提供PPT进行交流的童鞋，也感谢所有参与人员的经验分享。

本次聚会讨论的还是以基础方法为主，因为大家进展都比较缓慢。我们暂定法桐叶变黄的时节再次小聚，期待届时能有更多童鞋参加，也祝愿在这半年里大家能做出更多的结果。

P.S. 本小结帖凭记忆所写，如有谬误，请各位及时指出，我第一时间更改。

旋转的尾音

非常感谢~！
PS：我是蓝翔高级技修学院毕业的

Damon

顶一下，很好的总结。

yy8651

赞一个，非常感谢无私分享

yy8651

In human melanoma cells, by contrast, the depletion of neutral sphingomyelinases impairs
neitherMVB biogenesis (Van Niel et al. 2011) nor exosome secretion (G. Van Niel & G. Raposo,
unpublished data); instead, a CD63-dependent mechanism is required for ILV/exosome formation.
CD63 is instrumental in targeting the EBV-encoded LMP1 protein to ILVs and allowing
its subsequent release in exosomes (Verweij et al. 2011). In HEK293 cells, CD9 or CD82 (but
not CD63) overexpression was shown to induce secretion of β-catenin by exosomes, which were
still generated through a ceramide-dependent mechanism (Chairoungdua et al. 2010). In a rat
pancreatic adenocarcinoma cell line, expression of the tetraspanin Tspan8 led to modifications in
the exosome content in mRNA and transmembrane proteins (VCAM-1, α4 integrin) (Nazarenko
et al. 2010). CD81-enriched domains have been proposed recently as sorting platforms for exosomal
proteins (Perez-Hernandez et al. 2013) and may certainly account for ESCRT-independent
sorting of some cargoes and the formation of a population of ILVs: Proteins that are known to
interact with certain tetraspanins were found in exosomes via mass spectrometry, and in CD81-
deficient animals, exosomes were found to be devoid of CD81-interacting molecules, which are
normally loaded onto exosomes.

Finally, a small integral membrane protein of lysosomes and late endosomes, called SIMPLE,
was recently shown to be secreted in association with exosomes, and fibroblasts expressing its
mutant form found in Charcot-Marie-Tooth disease patients, CMT1C, secreted less CD63- and
ALIX-containing exosomes, whereas flotillin secretion was unaffected (Zhu et al. 2013). How
SIMPLE regulates exosome secretion, and whether it is through binding to TSG101 or to Nedd4
type-3 ubiquitin ligase, two proteins for which SIMPLE contains a binding domain, was not
elucidated in this study. The latter interaction is potentially relevant to exosome biogenesis and
secretion, because a transmembrane protein able to bindNedd4, Nedd-family interacting protein
1, has been shown to promote exosome secretion and targeting of cytosolic proteins, such as the
PTEN tumor suppressor, into these exosomes (Putz et al. 2012).


这段话说的是不是和4有关呢？求指点

yy8651

In human melanoma cells, by contrast, the depletion of neutral sphingomyelinases impairs
neitherMVB biogenesis (Van Niel et al. 2011) nor exosome secretion (G. Van Niel & G. Raposo,
unpublished data); instead, a CD63-dependent mechanism is required for ILV/exosome formation.
CD63 is instrumental in targeting the EBV-encoded LMP1 protein to ILVs and allowing
its subsequent release in exosomes (Verweij et al. 2011). In HEK293 cells, CD9 or CD82 (but
not CD63) overexpression was shown to induce secretion of β-catenin by exosomes, which were
still generated through a ceramide-dependent mechanism (Chairoungdua et al. 2010). In a rat
pancreatic adenocarcinoma cell line, expression of the tetraspanin Tspan8 led to modifications in
the exosome content in mRNA and transmembrane proteins (VCAM-1, α4 integrin) (Nazarenko
et al. 2010). CD81-enriched domains have been proposed recently as sorting platforms for exosomal
proteins (Perez-Hernandez et al. 2013) and may certainly account for ESCRT-independent
sorting of some cargoes and the formation of a population of ILVs: Proteins that are known to
interact with certain tetraspanins were found in exosomes via mass spectrometry, and in CD81-
deficient animals, exosomes were found to be devoid of CD81-interacting molecules, which are
normally loaded onto exosomes.
Finally, a small integral membrane protein of lysosomes and late endosomes, called SIMPLE,
was recently shown to be secreted in association with exosomes, and fibroblasts expressing its
mutant form found in Charcot-Marie-Tooth disease patients, CMT1C, secreted less CD63- and
ALIX-containing exosomes, whereas flotillin secretion was unaffected (Zhu et al. 2013). How
SIMPLE regulates exosome secretion, and whether it is through binding to TSG101 or to Nedd4
type-3 ubiquitin ligase, two proteins for which SIMPLE contains a binding domain, was not
elucidated in this study. The latter interaction is potentially relevant to exosome biogenesis and
secretion, because a transmembrane protein able to bindNedd4, Nedd-family interacting protein
1, has been shown to promote exosome secretion and targeting of cytosolic proteins, such as the
PTEN tumor suppressor, into these exosomes (Putz et al. 2012).

有没有人看过这一段话，是什么意思呢？跟楼主所说的4有没有关系呢？求指点

丁倩倩

师哥是个好秘书

itooth

蓝翔毕业哪个强？

ffp1988

好人

hereissyk

记录很详细！希望下次开会的时候大家都发文章了！我是游虾