Small extracellular vesicles encapsulating lefty1 mRNA inhibit hepatic fibrosis

Liver fibrosis is the deposition of extracellular matrix(ECM)in the liver caused by persistent chronic injury,which can lead to more serious diseases such as cirrhosis or cancer.Blocking the effect of transforming growth factorβ1(TGF-β1),one of the most important cytokines in liver fibrosis,may be one of the effective ways to inhibit liver fibrosis.As a kind of natural nano-scale vesicles,small extracellular vesicles(sEvs)have displayed excellent delivery vehicle properties.Herein,we prepared hepatic stellate cell(HSC)-derived sEvs loading left-right determination factor 1(lefty1)mRNA(sEvLs)and we wanted to verify whether they can inhibit fibrosis by blocking the TGF-β1 signaling pathway.The results showed that sEvLs had effective cell uptake and reduced activation of HSCs.Rats that were injected with CCl 4 by intraperitoneal injection for 6 weeks exhibited obvious symptoms of liver fibrosis and were treated with systemically administered sEvLs and free sEvs for 4 weeks.Rats injected with olive oil alone served as sham controls.Administration of sEvLs significantly reduced the area of fibrosis compared with free sEvs.We demonstrated that sEvLs inhibited HSCs activation and ECM production,and promote ECM degradation by downregulatingα-smooth muscle actin(α-SMA),collagen I,tissue inhibitor of metalloproteinase(TIMP)-1 and upregulating matrix metalloprotease(MMP)-1.In summary,as an endogenous delivery vehicle,sEvs could deliver mRNA to attenuate hepatic fibrosis by blocking the TGF-β/Smad signaling pathway.

lefty1基因真核表达载体构建鉴定及转染

目的:构建lefty1基因与pcDNA3.1(+)相融合的真核表达载体,并在lefty1基因的下游加上Flag标签,转染人肾小管上皮细胞株验证重组质粒活性。方法:设计并合成lefty1基因上下游引物,同时加上Flag标签序列,PCR扩增基因,并将其连接到真核表达载体pcDNA3.1(+)上,双酶切图谱分析和扩增产物测序鉴定所构建的真核表达载体。脂质体转染法将重组质粒pcDNA3.1(+)-lefty1-Flag和pcDNA3.1(+)分别转染至人肾小管上皮细胞株HK-2,qPCR和Western blot检测lefty1在mRNA和蛋白水平的表达。结果:以lefty1基因质粒模板扩增得到的片段约1 150bp,双酶切得到目的基因片段,测序的结果显示与lefty1基因序列相同。转染肾小管上皮细胞后,pcDNA3.1(+)-lefty1-Flag能表达lefty1蛋白。结论:成功构建lefty1基因真核表达载体,为一步研究lefty1基因功能奠定了基础。

Lefty1对小鼠胚胎干细胞分化过程的影响

目的:研究Lefty1在小鼠胚胎干细胞分化过程中的作用。方法:根据染色质免疫沉淀测序结果,在临近Lefty1转录起始位点以及与之相距10 kb的上游区域有TGF-β信号通路Smad2/3蛋白的四个结合区域,通过CRISPR/Cas9方法获得四个区域敲除的单克隆细胞,利用荧光实时定量PCR(qRT-PCR)检测各细胞中Lefty1的转录水平,并用TGF-β信号通路的激活剂AC和抑制剂SB分别处理敲除的细胞,检测其对TGF-β信号的响应,最后通过胚状体形成实验,检测敲除细胞系在中内胚层分化过程中的标志分子Gsc和Mixl1的表达。结果:利用CRISPR/Cas9方法成功获得不同区域敲除的单克隆细胞,与野生型E14细胞相比,四种区域敲除的细胞中Lefty1 RNA含量明显降低,并且在干细胞状态和分化状态下,敲除细胞系对TGF-β信号的响应减弱。在胚状体形成的实验中,与野生型E14细胞相比,敲除细胞系在分化过程中Lefty1表达的基础水平明显降低,中内胚层分化的标志分子Gsc和Mixl1转录水平也明显下降。结论:在胚胎干细胞中,Lefty1转录起始位点附近以及上游10 kb的这四段区域通过TGF-β信号通路对Lefty1的转录发挥调控作用,从而影响中内胚层分化过程中的标志分子Gsc和Mixl1的表达。