间充质干细胞旁分泌物质诱导肝星状细胞凋亡的体外研究
发布时间：2018-08-25 09:59
【摘要】：研究背景： 肝硬化是严重危害人类健康的常见病之一,临床上尚无特效的治疗手段。间充质干细胞(Mesenchymal stem cell, MSC)治疗被认为是较有希望的新疗法,多个国家探索性地应用MSC移植治疗肝硬化,Ⅰ、Ⅱ期临床研究结果也非常令人鼓舞。多项研究结果显示肝硬化患者经外周静脉或门静脉输注MSC后,患者的肝功能好转,主要体现在胆红素、血清白蛋白以及MELD评分的改善,但具体机制尚不十分清楚。有观点认为可能与MSC直接分化为肝细胞或/和旁分泌途径有关。近来的研究发现,MSC经门静脉移植后在受损肝脏中定植率仅2-5%,仅极少数分化为功能性肝细胞,部分MSC通过与肝细胞融合而整合到肝脏。因此,MSC通过分化为肝细胞而起到治疗作用的观点受到质疑,MSC的旁分泌功能日益受到重视。有学者通过向肝损伤小鼠体内分别输注MSC和MSC条件培养液(Mesenchymal stem cell-conditioned medium, MSC-CM)发现两组的治疗效果无显著差别,证明MSC的治疗作用主要通过旁分泌功能而实现。在肝脏损伤环境中,MSC可以分泌不同水平的细胞因子及生长因子,表现出抗炎、促增殖等多种生物学作用,并能激活肝内的肝干细胞,促进肝细胞再生。进一步的研究发现MSC分泌的多种细胞因子、生长因子可以参与调节细胞的凋亡、增殖、炎症反应等生理过程,但其对肝纤维化的影响及其机制尚不十分清楚。 肝纤维化是肝硬化发展的必经阶段,而肝星状细胞(Hepatic stellate cell,HSC)激活又是肝纤维化的中枢事件。激活的HSC过度分泌胶原蛋白,造成细胞外基质(Extracellular matrix, ECM)大量增生沉积,最终导致肝纤维化和肝硬化。而抑制HSC活化,减少活化的HSC数量能有效缓解肝纤维化的发生发展。因此,诱导和促进活化的HSC凋亡是治疗肝纤维化的策略之一。 研究目的： 为进一步了解MSC的旁分泌物质对肝纤维化的作用,本实验以活化的肝星状细胞系Lx-2为研究对象,通过MSC与Lx-2Transwell共培养,了解培养上清中MSC旁分泌物质对Lx-2的影响并探讨其机制。 研究方法： 采用密度梯度离心法分离骨髓间充质干细胞(Bone marrow mesenchymal stem cell, BM-MSC),并将其与人肝星状细胞系Lx-2Transwell共培养组作为实验组,单独培养的Lx-2作为对照组,研究各组中MSC旁分泌物质对Lx-2的作用。采用流式细胞术及Hoechst33342染色法分别检测Lx-2细胞凋亡的情况,qRT-PCR和Western Blot检测Lx-2中解耦联蛋白2(Uncoupling protein2, UCP2)的mRNA和蛋白水平的表达量,荧光探针法检测Lx-2细胞内及线粒体中活性氧(Reactive oxygen species, ROS)的荧光强度,脂质过氧化物试剂盒检测细胞培养上清中丙二醛(Malondialdehyde, MDA)的含量。 研究结果： 1、流式细胞术和Hoechst33342染色法示：与对照组相比,实验组Lx-2细胞凋亡增多,可见染色质固缩、颗粒状荧光等凋亡细胞典型表现,凋亡率是对照组的2.6倍,两组差异具有统计学意义； 2、qRT-PCR和Western Blot检测示：实验组Lx-2中UCP2的蛋白表达量降低,对照组UCP2mRNA的表达量是实验组的2.16倍,两组相比P值小于0.05； 3、细胞免疫荧光和脂质过氧化物检测示：实验组Lx-2细胞内和线粒体中ROS水平明显升高,细胞培养上清中MDA的含量是对照组的2.5倍左右,两组相比P值小于0.05。 结论： 1、MSC旁分泌物质有诱导Lx-2细胞凋亡的作用； 2、此作用可能与MSC旁分泌物质抑制UCP2的表达,促进细胞内及线粒体ROS过量生成有关。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2
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