中国科学院昆明动物研究所机构知识库

毒品成瘾是困扰人类健康的重大社会问题，其致病机制研究是亟待解决的重大科学问题。研究表明，基因表达长期改变是成瘾长期性的重要基础。吗啡是阿片类毒品的典型代表，研究其机制将为临床治疗吗啡成瘾人群带来福音。我们前期的研究表明，吗啡能够诱导自噬发生。然而，吗啡能否诱导特异性的分子伴侣介导的自噬发生，目前还不清楚。Hsc70和Lamp2A是分子伴侣介导自噬的生物学标志物，对于吗啡成瘾动物和吗啡处理的细胞中这两个标记基因的检测，有望确认分子伴侣介导自噬是否参与吗啡成瘾。本研究中，我们通过Microarray芯片筛选，试图获得一批在吗啡成瘾的大鼠海马区表达长期改变的基因。结果显示，分子伴侣介导自噬的标记物Hsc70的mRNA表达水平显著上调。此外，Hspa1b、Stip1和Cryab这三个小分子伴侣的mRNA表达水平也升高。这个结果提示分子伴侣介导的自噬可能参与吗啡成瘾。进一步对于吗啡处理的小鼠海马、大鼠神经胶质瘤细胞C6和大鼠嗜铬瘤细胞PC12细胞进行检测Hsc70、Hspa1b、Stip和Cryab的表达变化。结果显示，分子伴侣介导的自噬的标记物HSC70和LAMP2A蛋白水平显著上调，表明吗啡诱导了分子伴侣介导的自噬发生。我们同时发现，Hspa1b基因的mRNA和蛋白表达水平在这些组织和细胞中显著上调，Stip1和Cryab基因mRNA水平也显著上调，但蛋白水平变化不显著。因此，我们选择Hspa1b基因进行深入研究，通过在细胞水平上过表达和敲除Hspa1b基因，来研究其对于吗啡诱导的分子伴侣介导的自噬的影响。我们采用目前最常用的基因编辑技术CRISPR/Cas9技术来实现Hspa1b基因的敲除。通过向导RNA识别目标DNA靶序列，Cas9蛋白联合crRNA和tracrRNA一起切割外源基因组DNA，可实现基因编辑。该过程具有高效、普适、简便、可操作性高以及精确打靶的优点。我们成功地构建和筛选了一株PC12细胞和四株C6细胞的Hspa1b敲除细胞系。其中，PC12基因敲除细胞包括Hspa1b-KO-28# (c. 207_208del)。C6基因敲除细胞系包括Hspa1b-KO-43# (c.213_214insA)、Hspa1b-KO-9# (c. 197_211del)、Hspa1b-KO-6# (c. 203_225del; c. 203_208del)和Hspa1b-KO-21# (c. 208_215del; c. 211del)。我们在这些Hspa1b敲除细胞系进行了一些功能探究。敲除Hspa1b基因后，HSC70和LAMP2A蛋白水平显著下调，表明分子伴侣介导自噬减弱。过表达Hspa1b基因后，HSC70和LAMP2A蛋白水平显著上调，结果表明分子伴侣介导自噬增强。综上所述，我们发现在吗啡成瘾大鼠和小鼠的海马组织以及细胞系中Hspa1b基因的转录及翻译水平都显著上调。同时，我们在吗啡成瘾大鼠海马和细胞系中发现分子伴侣介导的自噬发生增强。过表达Hspa1b增强分子伴侣介导的自噬和巨自噬，Hspa1b基因敲除细胞系中发现分子伴侣介导自噬减弱。这些结果表明Hspa1b参与到吗啡诱导的分子伴侣介导的自噬过程中。我们通过CRISPR/Cas9这种基因编辑技术构建的Hspa1b基因敲除细胞系，可为深入研究该基因在吗啡成瘾过程中的潜在作用提供基础。

Drug addiction is a major social problem that plagues human health, but its mechanism has not been sufficiently determined. Studies have shown that gene expression with long-term changes is an important basis for long-term drug addiction. Morphine is a typical opioids drug. The study of morphine addiction will pave the way for the clinical treatment of morphine addiction. Our previous studies showed that morphine can induce autophagy. However, it is unclear whether morphine can induce chaperone-mediated autophagy (CMA). Hsc70 and Lamp2A are biomarker genes of chaperone-mediated autophagy. We aim to study whether the chaperone-mediated autophagy is involved in morphine addiction by detecting the altered expression of Hsc70 and Lamp2A.In this study, we tried to screen a batch of genes with long-term changes in hippocampus tissues from rats with morphine addiction by RNA microarray chip analysis. The results showed that the mRNA expression level of Hsc70, which is the marker of chaperone-mediated autophagy, was significantly up-regulated in rat with morphine treatment. In addition, the mRNA levels of the three small chaperones Hspa1b, Stip1, and Cryab were also increased. These observations suggested that chaperone-mediated autophagy may be induced by morphine.We further validated the expression changes of Hsc70, Lamp2A, Hspa1b, Stip1 and Cryab in mouse hippocampus and cell lines in response to morphine treatment. The protein levels of HSC70 and LAMP2A were significantly upregulated in rat hippocampus, glioma cell C6 and pheochromocytoma PC12 cells, indicating that morphine induces chaperone-mediated autophagy. We also found that the mRNA and protein levels of the Hspa1b were significantly upregulated in these tissues and cells. The mRNA levels of Stip1 and Cryab were also significantly upregulated, but the protein levels did not change significantly. Therefore, we next characterize the function of Hspa1b on the morphine-induced chaperone-mediated autophagy by overexpressing and knocking out this gene.We used the CRISPR/Cas9 technology, which is most commonly used for making gene knockout, to establish the Hspa1b knockout cell lines. This technology employs guide RNA and Cas9 to identify and cut the target genomic DNA. This process has the advantages of high efficiency, universality, simplicity, high operability, and accurate shooting. We successfully constructed a strain of Hspa1b knockout PC12 cell line and four knockout strains of C6 cells. The PC12 knockout (KO) cell line Hspa1b-KO-28# contains a mutation which is c. 207_208del. The C6 KO cell lines Hspa1b-KO-43# (c.213_214ins), Hspa1b-KO-9# (c. 197_211del), Hspa1b-KO-6# (c. 203_225del; c. 203_208del) and Hspa1b-KO-21# (c. 208_215del; c. 211del) contain different mutations, respectively.We performed some functional assays using these Hspa1b knockout cell lines. We found that the protein levels of HSC70 and LAMP2A were significantly down-regulated in the Hspa1b knockout cell lines, indicating that morphine induces chaperone-mediated autophagy was attenuated. Overexpression of Hspa1b significantly upregulated the protein levels of HSC70 and LAMP2A, indicating that the chaperone-mediated autophagy was enhanced.In summary, we found that the mRNA and protein levels of Hspa1b were significantly up-regulated in hippocampus of rat and mice and cell lines treated with morphine. Preliminary analysis showed that the chaperone-mediated autophagy was involved in morphine addiction. Overexpression of Hspa1b enhanced the chaperone-mediated autophagy, but the chaperone-mediated autophagy was attenuated in Hspa1b knockout cell lines. Our results indicated that Hspa1b is involved in the chaperone-mediated autophagy induced by morphine. The Hspa1b knockout cell lines that were constructed by CRISPR/Cas9 gene editing technology could provide a basis for further study of Hspa1b in morphine addiction.