長鏈非編碼核糖核酸在硫酸吲哚酚毒性中的作用並運用外泌體作為糖尿病治療工具

The Role of LncRNAs in the Toxicity of Indoxyl Sulfate and Application of Exosomes as a Therapeutic Tool in Diabetes Mellitus

黃鈺琴(Yu-Chin Huang)

指導教授 ： 賴亮全

國立臺灣大學/醫學院/生理學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200944

摘要

心血管疾病（Cardiovascular Disease; CVD）是導致糖尿病（Diabetes Mellitus; DM）死亡的主要原因。目前已知糖尿病會導致免疫調節功能障礙，且糖尿病若合併慢性腎臟病(Chronic Kidney Disease; CKD)更會大幅增加心血管疾病(CVD)的產生與死亡。DM/CKD病人體內的內皮損傷及血液中積累的尿毒素被認為是CVD發生的機制，然而在DM/CKD病人中為何CVD會異常增加的分子機制仍然不明確。長鏈的非蛋白質編碼核糖核酸(Long non-protein-coding RNAs; lncRNAs)已被證實具有調節多種細胞功能的重要作用，因此我們設計實驗，使用高濃度葡萄糖處理的內皮細胞來模擬DM，使用尿毒素硫酸吲哚酚(Indoxyl Sulfate; IS)來模擬與CKD相關的內皮損傷，藉由高通量RNA定序結果，分析在這些不同損傷下內皮細胞有差異表達的lncRNAs。我們發現與單獨使用高糖組相比，IS處理後內皮細胞的lnc-SLC15A1-1的表達顯著增加，且通過對miR-27b、miR-297和miR-150b的海綿效應(spongy effect)可以上調趨化因子CXCL10和CXCL8的信號。此實驗結果發現新調控途徑很可能是導致DM/CKD的病人體內內皮細胞發炎的原因,進而導致CVD的發生率大幅增加，此新調控途徑在未來可能可以成為臨床應用於治療糖尿病併慢性腎臟病病人心血管疾病的新策略。但如何將實驗室結果轉化為臨床運用是一大問題。因出於安全考慮，很難運用病毒轉染的方法來調病人體內的基因表達程度。細胞外囊泡(Extra vesicles; EVs)可以從大多數各種類型細胞中釋放出來，並具有在細胞之間傳遞生物活性成分、激活信號通路和促進細胞間通訊的能力。其中外泌體(exosomes)是屬於EVs的一種分型，已在人類臨床前期研究中應用於各種器官疾病的治療，具有作為生物藥物的潛力。
外泌體，幾乎所有種類的原核生物和真核生物都會分泌外泌體，因具有在細胞間傳遞訊息的作用，因此可以多方面的影響細胞功能，在生理和病理疾病中也扮演著重要角色。研究證實間質幹細胞(Mesenchymal stem cells; MSCs) 所分泌的外泌體，具有與其親代細胞相同的再生潛力和免疫調節能力。間質幹細胞也是目前再生醫學領域中運用最廣泛的體幹細胞，由於其分化能力和旁分泌作用，在許多臨床前期疾病模型治療試驗中，表現出具有潛能的治療效果。因此，MSCs外泌體(無細胞治療方法)會具有和其親代細胞的一樣的生物學特性，現在已成功應用於治療各種疾病。為了評估應用無細胞治療方法運用於CKD病人心血管疾病治療的可能性，我們進行了一項體外研究。運用不同條件處理下所收集的富含exosomes的臍帶間質幹細胞(Umbilical Code-MSCs; UC-MSCs)的條件培養基(UC-MSCs Condition Medium; UC-MSCs-CM)去治療受IS 損害的人臍靜脈內皮細胞，結果證明具有降低生長抑制的治療作用。因此可以預期，MSCs exosomes可以在未來發展成為一類用於治療CKD心血管疾病的生物藥物。但儘管MSC exosomes具有在各種臨床環境中應用的潛力，但實際上仍有一些障礙必須克服，且出於安全考慮，MSCs exosomes作為生物療法的使用應仍須有一套定義精確的監管條例。未來，基於分子研究技術的精進和細胞再生藥物的進步，將可以加速了解複雜臨床問題的分子機制及提供新治療方法的契機。

關鍵字

糖尿病 ； 慢性腎臟病 ； 心血管疾病 ； 硫酸吲哚酚 ； 長鏈非編碼核糖核酸 ； 趨化因子 ； 間質幹細胞 ； 外泌體

並列摘要

Cardiovascular disease (CVD) is the leading cause of mortality in diabetes mellitus (DM). Immunomodulatory dysfunction is a primary feature of DM, and the emergence of chronic kidney disease (CKD) in DM abruptly increases CVD mortality compared with DM alone. Endothelial injury and the accumulation of uremic toxins in the blood of DM/CKD patients are known mechanisms for the pathogenesis of CVD. However, the molecular factors that cause this disproportional increase in CVD in the DM/CKD population are still unknown. Since long non-protein-coding RNAs (lncRNAs) play an important role in regulating multiple cellular functions, human endothelial cells treated with high glucose to mimic DM and with the uremic toxin indoxyl sulfate (IS) to mimic the endothelial injury associated with CKD was used. Differentially expressed lncRNAs in these conditions were analyzed by RNA sequencing. The lnc-SLC15A1-1 expression was significantly increased upon IS treatment in comparison with high glucose alone was discovered, and then cascaded the signal of chemokines CXCL10 and CXCL8 via sponging miR-27b, miR-297, and miR-150b. This novel pathway might be responsible for the endothelial inflammation implicated in augmenting CVD in DM/CKD and could be a therapeutic target with future clinical applications. But it is a problem to translate the results from bench to bed due to the difficulty of modulating patients’ gene expression levels via the method of virus transfection for safety concerns. Extracellular vesicles (EVs) are released from a majority of cell types and have the ability to transmit bioactive components between cells, activating signaling pathways and facilitating intercellular communication. Exosomes, one kind of EVs, have been applied to the treatment of various organ diseases in human preclinical studies and possess the potential as biomedicine.
Exosomes, almost all kinds of prokaryotes, and eukaryotes have the ability to secrete, indicating that they play important roles in multifaceted physiological and pathological functions of intercellular communication. Exosomes of mesenchymal stem cells (MSCs) are secreted by live cells and possess the same regenerative potential and immunomodulatory ability as their parental cells. Mesenchymal stem cells (MSCs) are the most widely used somatic stem cells in regenerative medicine and exhibit potential therapeutic effects in many preclinical disease models because of their differentiation ability and paracrine effects. Furthermore, MSC exosomes exhibit the biological properties of their parent cells and have been successfully applied for the treatment of various diseases. For evaluating the possibility of applying the acellular therapeutic method to treat the CVD in CKD, an in vitro study was conducted. Human umbilical vein endothelium cells injured by the IS were treated by different processes produced from the umbilical-cord mesenchymal stem cells’ (UC-MSCs) secretomes, which were rich with exosomes in conditioned medium (CM) and this was proved to exert therapeutic effects. In the future, application of the genetic modified UC-MSCs exosomes as vector to carry specific RNAs to treat injured HUVECs based on my study result was planned to try. It could be expected that MSC exosomes might be developed into a class of bio-pharmaceuticals for the treatment of CVD in CKD. Despite the potential of MSC exosomes to be applied in various clinical settings, there are some hurdles must be overcome. For safety considerations, the usage of MSC exosomes as biological therapeutics should be defined by a precise set of regulatory requirements. Together, the progress in research techniques and regeneration medicines have expedited resolving the complicated clinical problems from this point forward.

並列關鍵字

diabetes mellitus ； chronic kidney disease ； cardiovascular disease ； indoxyl sulfate ； long non-coding RNAs ； chemokines ； mesenchymal stem cell ； exosomes

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