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Home > Learning center > NGS_RNA-Seq > ´Ù¾çÇÑ ½ÇÇè Àû¿ë »ç·Ê > scRNA-seq°ú bulk RNA-seqÀÇ ÅõÆ®·¢

scRNA-seq°ú bulk RNA-seqÀÇ ÅõÆ®·¢

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µÎ°¡Áö RNA-seq ¹æ¹ýÀ» »ç¿ëÇÑ resident memory T cellÀÇ reverse liver fibrosis¿¡ ´ëÇÑ ¿¬±¸

°£¼¶À¯Áõ (liver fibrosis)Àº ±×µ¿¾È Ä¡·á°¡ ºÒ°¡´ÉÇÏ´Ù°í »ý°¢µÇ¾î ¿Ô´Ù. ±×·¯³ª ÃÖ±Ù Nature¿¡¼­ ¹ßÇ¥ÇÑ ¿¬±¸¿¡¼­ °£¼¶À¯Áõ (liver fibrosis) Ä¡·á¸¦ À§ÇÑ »õ·Î¿î mouse ¸ðµ¨À» °³¹ßÇß´Ù (Koda et al developed and characterized a new mouse model of liver fibrosis resolution). ÀÌ ¿¬±¸¿¡¼­´Â »õ·Î¿î mouse ¸ðµ¨°ú single-cell RNA-seq (scRNA-seq), bulk mRNA-seq µÎ°¡Áö RNA-seq ¹æ¹ýÀ» »ç¿ëÇÑ ±â´É¿¬±¸ °á°ú ƯÁ¤ÇÑ Å¸ÀÔÀÇ T-cellÀÌ °£¼¶À¯Áõ (liver fibrosis)ÀÇ ÁÖ¿ä ¿øÀÎÀÎ hepatic stellate cellÀ» apoptosis ½ÃŲ´Ù´Â Á¡À» ¹àÇû´Ù.

¡á Inflammation from NAFLD and NASH leads to liver fibrosis
Non-alcoholic fatty liver disease (NAFLD)´Â Àü¼¼°è¿¡¼­ ´Ù¾çÇÑ ¸¸¼º °£ ÁúȯÀÇ ÁÖ ¿øÀÎÀ¸·Î ¾Ë·ÁÁ® ÀÖ´Ù. NAFLD´Â Áö¹æÁõ (steatosis)À̶ó ºÎ¸£´Â °£¼¼Æ÷ (hepatocyte)¿¡¼­ÀÇ °úµµÇÑ Áö¹æ ÃàÀûÀÌ Æ¯Â¡ÀÌ´Ù. ¶ÇÇÑ Áö¹æÁõ (steatosis)Àº °£ÀÇ µ¹ÃâºÎ¿¡ ¿°ÁõÀÌ ¹ß»ýÇϸé non-alcoholic steatohepatitis (NASH)·Î º°µµ·Î ºÐ·ùµÇ±âµµ ÇÑ´Ù. ÀÌ·± NAFLD¿Í NASH ȯÀÚµéÀÇ ÀϺδ °£¼¶À¯Áõ (liver fibrosis)¸¦ °Þ°í ÀÖ°í °£°æÈ­Áõ (cirrhosis) ¶Ç´Â °£¾Ï (liver carcinoma)±îÁö ¾ÇÈ­µÇ±âµµ ÇÑ´Ù.

ºÐÀÚ»ý¹°ÇÐÀÇ ¼öÁØ¿¡¼­ NAFLD¿Í NASH¿¡ ÀÇÇØ hepatocyte°¡ Áö¼ÓÀûÀ¸·Î »ç¸êÇϸé ħÀ± ¸é¿ª¼¼Æ÷ (infiltrating immune cell)¿¡ ÀÇÇØ ´Ù¾çÇÑ chemokine °ú cytokineÀÌ ¹æÃâµÇ°í ÀÌ·Î ÀÎÇØ ¿°Áõ ¹ÝÀÀÀÌ À¯¹ßµÈ´Ù. ÀÌ·¯ÇÑ chemokine °ú cytokineÀÌ °£¼¶À¯Áõ (liver fibrosis)ÀÇ ÁÖ¿ä ¿øÀÎÀÎ hepatic stellate cellÀ» È°¼ºÈ­½ÃŲ´Ù (activation of hepatic stellate cells, which once activated, are the main drivers of liver fibrosis.).

¡á A new mouse model for liver fibrosis resolution: the RES model
°£¼¶À¯Áõ (liver fibrosis)ÀÇ Ä¡·á ¸ÅÄ¿´ÏÁòÀ» ¿¬±¸Çϱâ À§ÇØ ¿ì¼± °£¼¶À¯Áõ (liver fibrosis)ÀÌ Ä¡·áµÇ´Â °ÍÀ» °üÂûÇÒ ¼ö ÀÖ´Â ¸ðµ¨ÀÌ ÇÊ¿äÇß´Ù. À̸¦ À§ÇØ ¸ÕÀú ±âÁ¸ÀÇ mouse ¸ðµ¨¿¡ Áö¼ÓÀûÀ¸·Î °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ¸ÔÀ̸¦ ÁÖ¾î NASH¸¦ À¯¹ßÇÏ°í 24ÁÖ µÚ Á¤»ó ½Ä´ÜÀ¸·Î ´Ù½Ã ÀüȯÇÏ¿´´Ù (RES ¸ðµ¨·Î ¸í½Ã). ÀÌ mouse modelÀº °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü (HFHC diet) µ¿¾È È®¿¬ÇÑ °£¼¶À¯Áõ (liver fibrosis)À» º¸¿´°í, Á¤»ó ½Ä´ÜÀ¸·Î ÀüȯÇÑÁö 8ÁÖ ÈÄ¿¡ º»·¡ÀÇ »óÅ·Πȸº¹µÇ¾ú´Ù.


±×¸² 1. Á¤»ó ½Ä´Ü (ND) / °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü (HFHC)/ °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü¿¡¼­ Á¤»ó ½Ä´ÜÀ¸·Î ÀüȯÇÑ Áö 8ÁÖ µÚ Mouse ¸ðµ¨ (RES)
Image adapted from Koda et al. 2021, Nat. Commun. under a CC BY 4.0 license.

¡á Uncovering a mechanism for reversal of liver fibrosis using both single-cell and bulk mRNA-seq
¿¬±¸ °á°ú, Á¤»ó ½Ä´Ü (ND) ¸ðµ¨º¸´Ù RES ¸ðµ¨ÀÇ mouse¿¡¼­ CD8+ T cellÀÌ ´õ ³ô°Ô À¯ÁöµÇ¾ú´Ù. µû¶ó¼­ ÀÌ·¯ÇÑ ¼¼Æ÷¼ö°¡ °£¼¶À¯Áõ (liver fibrosis)ÀÇ Ä¡·á¿¡¼­ ÁÖ¿äÇÑ ¿ªÇÒÀ» ÇÒ °ÍÀ̶ó°í °¡Á¤ÇÏ°í ÀÌÈÄ ½ÇÇèÀ» ÁøÇàÇß´Ù. À̸¦ Áõ¸íÇϱâ À§ÇØ ¿¬±¸ÀÚµéÀº scRNA-seqÀ» »ç¿ëÇØ ND (Á¤»ó ½Ä´Ü) / HFHC (°íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü) / RES (°íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü¿¡¼­ Á¤»ó ½Ä´ÜÀ¸·Î ÀüȯÇÑ Áö 8ÁÖ µÚ)ÀÇ ¼¼°¡Áö Mouse ¸ðµ¨ÀÇ CD8+ T cellÀ» 17°³ÀÇ cluster·Î ºÐ·ùÇß´Ù. ±×¸®°í ÀÌ 17°³ÀÇ cluster µéÀ» ´Ù½Ã 4°³ÀÇ CD8+ T cell ÇÏÀ§ ±×·ìÀ¸·Î ºÐ·ùÇß´Ù. ±× °á°ú RES mouse ¸ðµ¨¿¡¼­ ƯÁ¤ ÇÏÀ§ ±×·ìÀÇ CD8+ T cell (the resident memory T cells (trm cells))ÀÇ ¾çÀÌ Áõ°¡µÈ °ÍÀ» È®ÀÎÇß´Ù.


±×¸² 2. Á¤»ó ½Ä´Ü (ND) / °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü (HFHC)/ °íÁö¹æ, °íÄÝ·¹½ºÅ×·Ñ ½Ä´Ü¿¡¼­ Á¤»ó ½Ä´ÜÀ¸·Î ÀüȯÇÑ Áö 8ÁÖ µÚ Mouse ¸ðµ¨ (RES)ÀÇ °£¿¡¼­ ºÐ¸®µÈ CD8+ T cellÀÇ UMAP projection (Trm ÇÏÀ§ ±×·ì¿¡ ¼ÓÇÏ´Â clusterÀÇ ºÎºÐÀÌ Ç¥Áö µÇ¾îÀÖ´Ù.)
Image adapted Koda et al. 2021, Nat. Commun. under a CC BY 4.0 license.

ÀÌÈÄ Trm cellÀÌ °£¼¶À¯Áõ (Liver fibrosis)ÀÇ Ä¡·á¿¡ ¾î¶»°Ô ±â¿©ÇÒ ¼ö ÀÖ´ÂÁö¸¦ ´õ¿í È®ÀÎÇϱâ À§ÇØ ¿¬±¸ÀÚµéÀº bulk full-length RNA-seqÀÎ SMART-seq v4 Ultra Low Input RNA Kit for Sequencing (ÇØ´ç Á¦Ç°Àº ÇöÀç SMART-Seq¢ç mRNA·Î Á¦Ç°¸íÀÌ º¯°æµÊ)À» »ç¿ëÇß´Ù. ÀÌ·¯ÇÑ Á¢±Ù¹ýÀ» »ç¿ëÇϸé RES ¸ðµ¨, HFHC ¸ðµ¨, ND ¸ðµ¨¿¡¼­ CD8+ T cell ÇÏÀ§ ±×·ì¿¡¼­ transcriptome Â÷ÀÌÁ¡À» ºñ±³ÇÏ´Â °ÍÀÌ °¡´ÉÇÏ´Ù. ¶ÇÇÑ bulk RNA-seq µ¥ÀÌÅÍ´Â scRNA-seq µ¥ÀÌÅÍÀÇ °á°ú¸¦ º¸°­ÇÏ´Â °Í »Ó¸¸ ¾Æ´Ï¶ó RES ¸ðµ¨°ú HFHC ¸ðµ¨ÀÇ trm cell¿¡¼­ ¼¼Æ÷µ¶¼º°ú ÁÖÈ­¼ºÀÇ °ü·ÃÀÎÀÚ (cytotoxic and chemotactic mediators)ÀÇ ¹ßÇöÀÌ Áõ°¡ÇÏ´Â °ÍÀ» Ãß°¡·Î ¹ß°ßÇß´Ù. ƯÈ÷ ÀÌÁß ¸Å¿ì °­ÇÏ°Ô ¹ßÇöµÇ´Â Ccl5¿¡¼­ ¹ßÇöµÇ´Â ´Ü¹éÁúÀÇ receptor°¡ fibrosis Ä¡·á Áß¿¡´Â hepatic stellate cell¿¡¼­ ¹ßÇöµÇ´Â ¹Ý¸é, fibrosis°¡ ÁøÇàÁß¿¡´Â ¹ßÇö µÇÁö ¾Ê´Â´Ù´Â Á¡À» ¹ß°ßÇß´Ù. Transcriptome ºÐ¼® °á°ú¸¦ ¹ÙÅÁÀ¸·Î ¿¬±¸ÀÚµéÀº functional study¸¦ ÅëÇØ trm ¼¼Æ÷°¡ CCR5¿¡ ÀÇÁ¸ÀûÀ¸·Î hepatic stellate cell¿¡¼­ ¼¼Æ÷»ç¸êÀ» À¯µµÇÏ¿© °£¼¶À¯Áõ (liver fibrosis)¸¦ Ä¡·áÇÑ´Ù´Â Á¡À» º¸¿©ÁÖ¾ú´Ù.

À§ÀÇ Koda¿Í ±× µ¿·áµéÀÇ ¿¬±¸ »ç·Ê¿¡¼­ º¼ ¼ö ÀÖµí, single cell°ú bulk RNA sequencing Áß ÇϳªÀÇ Á¢±Ù¹ý¸¸ ¼±ÅÃÇÏ´Â °Í º¸´Ù µÎ°¡Áö¸¦ ¸ðµÎ ÁøÇàÇÏ´Â °ÍÀ¸·Î ¿¬±¸¿¡¼­ ÁýÁßÇØ¾ß ÇÒ cell typeÀ» ½ºÅ©¸®´× ÇÒ »Ó ¾Æ´Ï¶ó transcriptome¿¡ ´ëÇÑ º¸´Ù ½ÉµµÀÖ´Â ºÐ¼®ÀÌ °¡´ÉÇÏ´Ù. ÀÌ·¯ÇÑ µ¥ÀÌÅÍ¿¡ ±âÃÊÇÑ functional study´Â ÀáÀçÀûÀ¸·Î NAFLD, NASH¹× °£¼¶À¯Áõ (liver fibrosis)ÀÇ Ä¡·á¸¦ À§ÇÑ »ý¹°ÇÐÀû ¸ÅÄ¿´ÏÁòÀÇ ¹ß°ßÀ» À̲ø¾ú´Ù.

¡á Reference
Koda, Y. et al. CD8+ tissue-resident memory T cells promote liver fibrosis resolution by inducing apoptosis of hepatic stellate cells. Nat Commun 12, 4474 (2021).

Tsuchida, T., Friedman, S. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14, 397-411 (2017).

¡á ¿ø¹®
https://www.takarabio.com/about/bioview-blog/single-cell-analysis/two-rna-seq-approaches-reveal-resident-memory-t-cells-hold-the-power-to-reverse-liver-fibrosis