間充質干細胞旁分泌物質誘導肝星狀細胞凋亡的體外研究
發(fā)布時間：2018-08-25 09:59
【摘要】：研究背景： 肝硬化是嚴重危害人類健康的常見病之一,臨床上尚無特效的治療手段。間充質干細胞(Mesenchymal stem cell, MSC)治療被認為是較有希望的新療法,多個國家探索性地應用MSC移植治療肝硬化,Ⅰ、Ⅱ期臨床研究結果也非常令人鼓舞。多項研究結果顯示肝硬化患者經外周靜脈或門靜脈輸注MSC后,患者的肝功能好轉,主要體現(xiàn)在膽紅素、血清白蛋白以及MELD評分的改善,但具體機制尚不十分清楚。有觀點認為可能與MSC直接分化為肝細胞或/和旁分泌途徑有關。近來的研究發(fā)現(xiàn),MSC經門靜脈移植后在受損肝臟中定植率僅2-5%,僅極少數(shù)分化為功能性肝細胞,部分MSC通過與肝細胞融合而整合到肝臟。因此,MSC通過分化為肝細胞而起到治療作用的觀點受到質疑,MSC的旁分泌功能日益受到重視。有學者通過向肝損傷小鼠體內分別輸注MSC和MSC條件培養(yǎng)液(Mesenchymal stem cell-conditioned medium, MSC-CM)發(fā)現(xiàn)兩組的治療效果無顯著差別,證明MSC的治療作用主要通過旁分泌功能而實現(xiàn)。在肝臟損傷環(huán)境中,MSC可以分泌不同水平的細胞因子及生長因子,表現(xiàn)出抗炎、促增殖等多種生物學作用,并能激活肝內的肝干細胞,促進肝細胞再生。進一步的研究發(fā)現(xiàn)MSC分泌的多種細胞因子、生長因子可以參與調節(jié)細胞的凋亡、增殖、炎癥反應等生理過程,但其對肝纖維化的影響及其機制尚不十分清楚。 肝纖維化是肝硬化發(fā)展的必經階段,而肝星狀細胞(Hepatic stellate cell,HSC)激活又是肝纖維化的中樞事件。激活的HSC過度分泌膠原蛋白,造成細胞外基質(Extracellular matrix, ECM)大量增生沉積,最終導致肝纖維化和肝硬化。而抑制HSC活化,減少活化的HSC數(shù)量能有效緩解肝纖維化的發(fā)生發(fā)展。因此,誘導和促進活化的HSC凋亡是治療肝纖維化的策略之一。 研究目的： 為進一步了解MSC的旁分泌物質對肝纖維化的作用,本實驗以活化的肝星狀細胞系Lx-2為研究對象,通過MSC與Lx-2Transwell共培養(yǎng),了解培養(yǎng)上清中MSC旁分泌物質對Lx-2的影響并探討其機制。 研究方法： 采用密度梯度離心法分離骨髓間充質干細胞(Bone marrow mesenchymal stem cell, BM-MSC),并將其與人肝星狀細胞系Lx-2Transwell共培養(yǎng)組作為實驗組,單獨培養(yǎng)的Lx-2作為對照組,研究各組中MSC旁分泌物質對Lx-2的作用。采用流式細胞術及Hoechst33342染色法分別檢測Lx-2細胞凋亡的情況,qRT-PCR和Western Blot檢測Lx-2中解耦聯(lián)蛋白2(Uncoupling protein2, UCP2)的mRNA和蛋白水平的表達量,熒光探針法檢測Lx-2細胞內及線粒體中活性氧(Reactive oxygen species, ROS)的熒光強度,脂質過氧化物試劑盒檢測細胞培養(yǎng)上清中丙二醛(Malondialdehyde, MDA)的含量。 研究結果： 1、流式細胞術和Hoechst33342染色法示：與對照組相比,實驗組Lx-2細胞凋亡增多,可見染色質固縮、顆粒狀熒光等凋亡細胞典型表現(xiàn),凋亡率是對照組的2.6倍,兩組差異具有統(tǒng)計學意義； 2、qRT-PCR和Western Blot檢測示：實驗組Lx-2中UCP2的蛋白表達量降低,對照組UCP2mRNA的表達量是實驗組的2.16倍,兩組相比P值小于0.05； 3、細胞免疫熒光和脂質過氧化物檢測示：實驗組Lx-2細胞內和線粒體中ROS水平明顯升高,細胞培養(yǎng)上清中MDA的含量是對照組的2.5倍左右,兩組相比P值小于0.05。 結論： 1、MSC旁分泌物質有誘導Lx-2細胞凋亡的作用； 2、此作用可能與MSC旁分泌物質抑制UCP2的表達,促進細胞內及線粒體ROS過量生成有關。
[Abstract]:Research background:
Liver cirrhosis is one of the common diseases that seriously endanger human health. There is no effective treatment in clinic. Mesenchymal stem cell (MSC) therapy is considered as a promising new therapy. MSC transplantation is exploratory used in many countries to treat liver cirrhosis. The results of phase I and phase II clinical studies are also very encouraging. The results showed that the liver function of patients with liver cirrhosis was improved after MSC was transfused via peripheral vein or portal vein. The improvement of bilirubin, serum albumin and MELD score was mainly manifested, but the specific mechanism was not clear. After portal vein transplantation, only 2-5% of the damaged liver cells were colonized, and only a few of them differentiated into functional hepatocytes. Some MSCs were integrated into the liver by fusing with hepatocytes. Therefore, the viewpoint that MSCs play a therapeutic role by differentiating into hepatocytes was questioned, and the paracrine function of MSCs was paid more and more attention. In vivo infusion of MSC and MSC conditioned medium (MSC-CM) showed no significant difference between the two groups, suggesting that the therapeutic effect of MSC was mainly achieved by paracrine function. Further studies have shown that MSC secretes a variety of cytokines, growth factors, which can participate in the regulation of cell apoptosis, proliferation, inflammation and other physiological processes, but the effects of MSC on liver fibrosis and its mechanism are still unclear.
Hepatic fibrosis is a necessary stage in the development of liver cirrhosis, and the activation of hepatic stellate cell (HSC) is the central event of liver fibrosis. Activated HSC oversecretes collagen, resulting in a large number of proliferation and deposition of extracellular matrix (ECM), eventually leading to liver fibrosis and cirrhosis. Activated HSC can effectively alleviate the occurrence and development of hepatic fibrosis. Therefore, inducing and promoting apoptosis of activated HSC is one of the strategies for treating hepatic fibrosis.
Research purposes:
To further understand the effect of MSC paracrine on hepatic fibrosis, the activated hepatic stellate cell line Lx-2 was co-cultured with Lx-2 Transwell to investigate the effect of MSC paracrine on Lx-2 and its mechanism.
Research methods:
Bone marrow mesenchymal stem cells (BM-MSC) were isolated by density gradient centrifugation and co-cultured with human hepatic stellate cell line Lx-2 Transwell as experimental group. Lx-2 was cultured separately as control group. The effects of MSC paracrine on Lx-2 in each group were studied. Flow cytometry and Hoechst 33342 staining were used. Apoptosis of Lx-2 cells was detected by colorimetric assay. Expression of Uncoupling protein 2 (UCP2) in Lx-2 cells and mitochondria was detected by qRT-PCR and Western Blot. Reactive oxygen species (ROS) fluorescence intensity in Lx-2 cells and mitochondria was detected by fluorescence probe. Lipid peroxide kit was used to detect fine protein. The content of Malondialdehyde (MDA) in cell culture supernatant.
Research findings:
1. Flow cytometry and Hoechst 33342 staining showed that compared with the control group, apoptosis of Lx-2 cells in the experimental group increased, and typical apoptotic cells such as chromatin pyknosis and granular fluorescence were observed. The apoptotic rate of Lx-2 cells in the experimental group was 2.6 times higher than that in the control group.
2. QRT-PCR and Western Blot assay showed that the expression of UCP2 protein in Lx-2 of experimental group was decreased, and that of UCP2 mRNA in control group was 2.16 times higher than that in experimental group, P value was less than 0.05.
3. The levels of ROS in Lx-2 cells and mitochondria of the experimental group were significantly higher than those of the control group. The content of MDA in the supernatant of Lx-2 cells was about 2.5 times higher than that of the control group. The P value of the two groups was less than 0.05.
Conclusion:
1, paracrine substances in MSC can induce apoptosis of Lx-2 cells.
2. This effect may be related to the inhibition of UCP2 expression by MSC paracrine and the promotion of ROS overproduction in cells and mitochondria.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.2
[Abstract]:Research background:
Liver cirrhosis is one of the common diseases that seriously endanger human health. There is no effective treatment in clinic. Mesenchymal stem cell (MSC) therapy is considered as a promising new therapy. MSC transplantation is exploratory used in many countries to treat liver cirrhosis. The results of phase I and phase II clinical studies are also very encouraging. The results showed that the liver function of patients with liver cirrhosis was improved after MSC was transfused via peripheral vein or portal vein. The improvement of bilirubin, serum albumin and MELD score was mainly manifested, but the specific mechanism was not clear. After portal vein transplantation, only 2-5% of the damaged liver cells were colonized, and only a few of them differentiated into functional hepatocytes. Some MSCs were integrated into the liver by fusing with hepatocytes. Therefore, the viewpoint that MSCs play a therapeutic role by differentiating into hepatocytes was questioned, and the paracrine function of MSCs was paid more and more attention. In vivo infusion of MSC and MSC conditioned medium (MSC-CM) showed no significant difference between the two groups, suggesting that the therapeutic effect of MSC was mainly achieved by paracrine function. Further studies have shown that MSC secretes a variety of cytokines, growth factors, which can participate in the regulation of cell apoptosis, proliferation, inflammation and other physiological processes, but the effects of MSC on liver fibrosis and its mechanism are still unclear.
Hepatic fibrosis is a necessary stage in the development of liver cirrhosis, and the activation of hepatic stellate cell (HSC) is the central event of liver fibrosis. Activated HSC oversecretes collagen, resulting in a large number of proliferation and deposition of extracellular matrix (ECM), eventually leading to liver fibrosis and cirrhosis. Activated HSC can effectively alleviate the occurrence and development of hepatic fibrosis. Therefore, inducing and promoting apoptosis of activated HSC is one of the strategies for treating hepatic fibrosis.
Research purposes:
To further understand the effect of MSC paracrine on hepatic fibrosis, the activated hepatic stellate cell line Lx-2 was co-cultured with Lx-2 Transwell to investigate the effect of MSC paracrine on Lx-2 and its mechanism.
Research methods:
Bone marrow mesenchymal stem cells (BM-MSC) were isolated by density gradient centrifugation and co-cultured with human hepatic stellate cell line Lx-2 Transwell as experimental group. Lx-2 was cultured separately as control group. The effects of MSC paracrine on Lx-2 in each group were studied. Flow cytometry and Hoechst 33342 staining were used. Apoptosis of Lx-2 cells was detected by colorimetric assay. Expression of Uncoupling protein 2 (UCP2) in Lx-2 cells and mitochondria was detected by qRT-PCR and Western Blot. Reactive oxygen species (ROS) fluorescence intensity in Lx-2 cells and mitochondria was detected by fluorescence probe. Lipid peroxide kit was used to detect fine protein. The content of Malondialdehyde (MDA) in cell culture supernatant.
Research findings:
1. Flow cytometry and Hoechst 33342 staining showed that compared with the control group, apoptosis of Lx-2 cells in the experimental group increased, and typical apoptotic cells such as chromatin pyknosis and granular fluorescence were observed. The apoptotic rate of Lx-2 cells in the experimental group was 2.6 times higher than that in the control group.
2. QRT-PCR and Western Blot assay showed that the expression of UCP2 protein in Lx-2 of experimental group was decreased, and that of UCP2 mRNA in control group was 2.16 times higher than that in experimental group, P value was less than 0.05.
3. The levels of ROS in Lx-2 cells and mitochondria of the experimental group were significantly higher than those of the control group. The content of MDA in the supernatant of Lx-2 cells was about 2.5 times higher than that of the control group. The P value of the two groups was less than 0.05.
Conclusion:
1, paracrine substances in MSC can induce apoptosis of Lx-2 cells.
2. This effect may be related to the inhibition of UCP2 expression by MSC paracrine and the promotion of ROS overproduction in cells and mitochondria.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.2
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