間充質(zhì)干細(xì)胞旁分泌物質(zhì)誘導(dǎo)肝星狀細(xì)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(yán)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(zhì)干細(xì)胞(Mesenchymal stem cell, MSC)治療被認(rèn)為是較有希望的新療法,多個國家探索性地應(yīng)用MSC移植治療肝硬化,Ⅰ、Ⅱ期臨床研究結(jié)果也非常令人鼓舞。多項(xiàng)研究結(jié)果顯示肝硬化患者經(jīng)外周靜脈或門靜脈輸注MSC后,患者的肝功能好轉(zhuǎn),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(jī)制尚不十分清楚。有觀點(diǎn)認(rèn)為可能與MSC直接分化為肝細(xì)胞或/和旁分泌途徑有關(guān)。近來的研究發(fā)現(xiàn),MSC經(jīng)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(xì)胞,部分MSC通過與肝細(xì)胞融合而整合到肝臟。因此,MSC通過分化為肝細(xì)胞而起到治療作用的觀點(diǎn)受到質(zhì)疑,MSC的旁分泌功能日益受到重視。有學(xué)者通過向肝損傷小鼠體內(nèi)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(shí)現(xiàn)。在肝臟損傷環(huán)境中,MSC可以分泌不同水平的細(xì)胞因子及生長因子,表現(xiàn)出抗炎、促增殖等多種生物學(xué)作用,并能激活肝內(nèi)的肝干細(xì)胞,促進(jìn)肝細(xì)胞再生。進(jìn)一步的研究發(fā)現(xiàn)MSC分泌的多種細(xì)胞因子、生長因子可以參與調(diào)節(jié)細(xì)胞的凋亡、增殖、炎癥反應(yīng)等生理過程,但其對肝纖維化的影響及其機(jī)制尚不十分清楚。 肝纖維化是肝硬化發(fā)展的必經(jīng)階段,而肝星狀細(xì)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(xì)胞外基質(zhì)(Extracellular matrix, ECM)大量增生沉積,最終導(dǎo)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(dǎo)和促進(jìn)活化的HSC凋亡是治療肝纖維化的策略之一。 研究目的： 為進(jìn)一步了解MSC的旁分泌物質(zhì)對肝纖維化的作用,本實(shí)驗(yàn)以活化的肝星狀細(xì)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(zhì)對Lx-2的影響并探討其機(jī)制。 研究方法： 采用密度梯度離心法分離骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cell, BM-MSC),并將其與人肝星狀細(xì)胞系Lx-2Transwell共培養(yǎng)組作為實(shí)驗(yàn)組,單獨(dú)培養(yǎng)的Lx-2作為對照組,研究各組中MSC旁分泌物質(zhì)對Lx-2的作用。采用流式細(xì)胞術(shù)及Hoechst33342染色法分別檢測Lx-2細(xì)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(dá)量,熒光探針法檢測Lx-2細(xì)胞內(nèi)及線粒體中活性氧(Reactive oxygen species, ROS)的熒光強(qiáng)度,脂質(zhì)過氧化物試劑盒檢測細(xì)胞培養(yǎng)上清中丙二醛(Malondialdehyde, MDA)的含量。 研究結(jié)果： 1、流式細(xì)胞術(shù)和Hoechst33342染色法示：與對照組相比,實(shí)驗(yàn)組Lx-2細(xì)胞凋亡增多,可見染色質(zhì)固縮、顆粒狀熒光等凋亡細(xì)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(xué)意義； 2、qRT-PCR和Western Blot檢測示：實(shí)驗(yàn)組Lx-2中UCP2的蛋白表達(dá)量降低,對照組UCP2mRNA的表達(dá)量是實(shí)驗(yàn)組的2.16倍,兩組相比P值小于0.05； 3、細(xì)胞免疫熒光和脂質(zhì)過氧化物檢測示：實(shí)驗(yàn)組Lx-2細(xì)胞內(nèi)和線粒體中ROS水平明顯升高,細(xì)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(jié)論： 1、MSC旁分泌物質(zhì)有誘導(dǎo)Lx-2細(xì)胞凋亡的作用； 2、此作用可能與MSC旁分泌物質(zhì)抑制UCP2的表達(dá),促進(jìn)細(xì)胞內(nèi)及線粒體ROS過量生成有關(guān)。
[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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.2
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