慢病毒介导的miRNA-302c对腹膜间皮细胞EMT的干预作用及机制研究
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摘要
研究背景
慢性肾脏病(Chronic Kidney Disease, CKD)是全球关注的公共卫生问题之一,国内外发病率逐年上升。约5%的CKD患者最终将发展成终末期肾功能衰竭,需终身接受肾脏替代治疗。其中,腹膜透析(peritoneal dialysis, PD)是符合我国国情的主要肾脏替代治疗方法,但长期PD可引起腹膜纤维化,导致超滤衰竭(ultrafiltration failure, UFF),进而影响PD的长期应用,是患者退出PD的主要原因。转化生长因子β(transforming growth factor-P, TGF-P)是腹膜纤维化发病机制中的关键因素,近年来多项研究发现腹膜间皮细胞上皮-间充质转分化(epithelial mesenchymal transition, EMT)在TGF-β诱导的腹膜纤维化中起重要作用,是腹膜纤维化启动和可逆的关键环节。结缔组织生长因子(connective tissue growth factor, CTGF)具有促进细胞增殖、迁移和分化等作用,研究证实CTGF通过介导TGF-β1,或直接作用其下游信号分子,与许多组织器官纤维化的发生发展密切相关。已有研究发现,CTGF不仅在PD腹膜纤维化中起到关键作用,并且在纤维化的始动过程EMT的发生中也起到重要促进作用。
microRNAs(miRNA)属于功能性非编码RNA,通过降解目标mRNA或抑制其翻译调节基因转录后水平的表达,参与调控生长发育,细胞分化、增殖、凋亡,肿瘤等疾病的发生发展,是目前生命科学领域的研究热点。有研究证实miRNA是TGF-P1诱导EMT的重要调节因子,如miRNA-200家族和miRNA-205。但是,miRNA在腹膜纤维化领域的研究尚未见文献报道。近期,我们采用高通道miRNA芯片技术对PD病人腹透流出液腹膜间皮细胞miRNA的表达进行了图谱分析,发现miRNA的异常表达与PD患者腹膜EMT及纤维化密切相关。
随着基因治疗技术及基因载体系统的发展,对于PD患者腹膜纤维化的分子机制和干预治疗研究取得了进一步的发展,大量在PD的动物模模型中进行的基因治疗研究证实了基因治疗方法在腹膜纤维化机制研究和治疗中的可行性。慢病毒载体(lentiviral vector, LV)作为目前被广泛应用的基因载体,具有转染效率高,插入外源基因容量大,能感染分裂期和非分裂期细胞,并且不会引起明显免疫和炎症反应等优势,将其应用于腹膜纤维化的研究十分有意义。
基于以上分析,我们认为某些miRNA可参与调节腹膜间皮细胞EMT,以慢病毒载体为基因转染工具,从miRNA着手研究其在PD过程中EMT及纤维化的作用及分子机制具有重要的理论和现实意义。
第一章腹腔注射慢病毒转染小鼠腹膜间皮细胞效应的研究
目的通过小鼠腹腔注射不同滴度的携带绿色荧光蛋白(green fluorescence protein, GFP)基因的慢病毒,观察其转染小鼠腹膜间皮细胞的效应,探讨腹腔注射慢病毒转染小鼠腹膜间皮细胞最佳效应滴度。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为对照组(n=5)、LV-GFP1组(n=5)、LV-GFP2组(n=5)、LV-GFP3组(n=5)。分别一次腹腔注射1.5m10.9%生理盐水或者不同滴度的慢病毒。于第28天处死小鼠,壁层腹膜用于HE和Masson染色观察腹膜形态变化、冰冻切片观察GFP表达,脏层腹膜用于提取组织mRNA和蛋白,采用Real Time PCR和Western blot方法检测GFP mRNA和蛋白表达。
结果
1.倒置荧光显微镜观察小鼠腹膜组织冰冻切片,与对照组相比,腹腔注射慢病毒小鼠腹膜组织有GFP表达；
2.小鼠腹膜组织石蜡切片HE、Masson染色观察,与对照组相比,腹腔注射慢病毒小鼠腹膜组织形态学无明显改变；
3. Real Time PCR和western blot的方法检测,腹腔注射慢病毒小鼠腹膜组织有GFP mRNA和蛋白表达;
4.使用2×1O8GTU LV-GFP组的转染效率高于1×108GTU LV-GFP组,而与3×108GTU LV-GFP组转染效率差异不大。
结论
腹腔注射慢病毒能安全有效转染小鼠腹膜组织,2×108GTU为最佳效应滴度。
第二章miRNA-302c在腹膜透析小鼠腹膜间皮细胞EMT中的作用与机制
目的利用慢病毒载体将miRNA-302c导入PD小鼠腹膜组织,研究miRNA-302c在PD小鼠腹膜间皮细胞EMT中的作用与机制。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为空白对照组(n=5)：腹腔注射1.5ml0.9%生理盐水,每天一次x28天；PDF组(n=5)：腹腔注射1.5ml含4.25%葡萄糖的标准腹透液,每天一次x28天; LV-mmu-miRNA-302c+PDF组(n=5)：腹腔注射1.5ml含2×108GTU的LV-mmu-miRNA-302c,第三天开始腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液,每天一次x28天；LV-pGIPZ+PDF组：腹腔注射1.5ml含2×108GTU的LV-pGIPZ,第三天开始腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液,每天一次×28天。于第28天处死小鼠,壁层腹膜用于制作石蜡切片HE和Masson染色观察腹膜形态变化、冰冻切片观察GFP表达,脏层腹膜用于提取组织蛋白和mRNA,并采用Westem-blot、Real Time-PCR等方法检测miRNA-302c、 E-cadherin、α-SMA\collagenⅠ、CTGF的表达。
结果
1.小鼠膜组织石蜡切片HE、Masson染色观察,与空白对照组相比,PDF组小鼠腹膜组织可见EMT的形态变化,而LV-mmu-miR-302c+PDF组腹膜组织形态学变化介于空白对照组和PDF组之间,EMT改变较PDF组减轻；
2. TaqMan探针实时荧光定量Real Time PCR检测发现,与空白对照组相比,腹腔注射4.25%PDF的各组小鼠脏层腹膜组织miRNA-302c表达均有下调,LV-mmu-miR-302c+PDF组较PDF组miRNA-302c表达增高；
3. Real Time PCR、western blot证实每天腹腔注射4.25%PDF1.5ml,连续28天,可引起小鼠腹膜组织EMT, E-cadherin表达减少,a-SMA及collagen I表达增高。而LV-mmu-miR-302c转染上调miRNA-302c可抑制腹透小鼠腹膜组织a-SMA、collagen I表达增加,E-cadherin表达降低；与空白对照组相比,各组腹透小鼠腹膜组织CTGF表达均有上调,LV-mmu-miR-302c+PDF组较PDF组CTGF表达降低。
结论
高糖腹透液可引起小鼠腹膜间皮细胞EOT; LV-mmu-miR-302c可有效转染小佩腹膜上调miRNA-302c的表达,抑制CTGF表达,减轻腹透小鼠腹膜间皮细胞EMT。
第三章miRNA-302c调控TGF-β1诱导的腹膜间皮细胞EMT形成的机理
目的通过观察慢病毒介导的miRNA-302c对TGF-β1诱导人腹膜间皮细胞(human peritoneal mesothelial cells, HPMCs) EMT过程中CTGF表达的影响,探讨miRNA-302c调控TGF-β1诱导的HPMCsEMT形成的机制。
方法.常规培养正常HPMCs,随机分为:空白对照组:不加任何干预;TGF-β1组：用5ng/mlTGF-β1刺激48小时；LV-hsa-miR-302c+TGF-β1组：转染载有miRNA-302c的慢病毒]LV-hsa-miR-302c后,用5ng/mlTGF-β1刺激48小时；LV-pGIPZ+TGF-β1组：转染载有对照质粒pGIPZ的慢病毒LV-pGIPZ后,用5ng/mlTGF-β1刺激48小时。采用荧光显微镜观察GFP荧光蛋白表达以判断病毒转染效率。采用Westem-blot、Real Time-PCR等方法分别检测miRNA-302c、 E-cadherin、a-SMA、collagen I、CTGF等在HPMCs中的表达变化及其相互关系。
结果
1. TaqMan探针实时荧光定量Real Time PCR检测发现,与空白对照组相比,TGF-β1刺激的各组腹膜间皮细胞miRNA-302c表达均有下调,LV-hsa-miR-3O2c+TGF-β1组miRNA-302c表达较TGF-β1组增高；
2. HPMCs经5ng/ml TGF-β1刺激48小时后,细胞出现成纤维样细胞形态改变,而LV-hsa-rniR-302c+TGF-β1组细胞形态变化介于空白对照组和TGF-β1组之间,细胞EMT形态改变较TGF-β1组减轻；
3. Regtl Time PCR、western blot证实5ng/ml TGF-β1刺激48h可引起HPMCsEMT, E-cadherin表达减少,a-SMA及collagen I表达增高；而LV-bsa-miR-302c转染上调miR-302c可抑制TGF-β1诱导的α-SMA、collagen I表达增加,E-cadherin表达降低；与空白对照组相比,TGF-β1刺激的各组腹膜间皮细胞CTGF表达均有上调,LV-hsa-miR-302c+TGF-β1组CTGF表达较TGF-β1组降低。
结论
TGF-β1可诱导HPMCs EMT; LV-hsa-miR-302c转染上调HPMCs miRNA-302c表达,抑制CTGF表达,减轻TGF-β1诱导的HPMCsEMT。
Background Chronic kidney disease (CKD) is a public health problem all over the world, the morbidity is about11%, and the incidence increased year by year.5%of the CKD patients will eventually develop into the end-stage renal failure, and should have life-long renal replacement therapy. Peritoneal dialysis (PD) is an important modality of renal replacement therapy according with China catual condition. But long-term PD can lead to peritoneal fibrosis and ultrafiltration failure which ultimately limits the long term clinical application of PD and makes patients drop out of PD. Transforming growth factor-β (TGF-β) is a key factor in the mechanism of peritoneal fibrosis, recently some studies have found that epithelial mesenchymal transition (EMT) plays a important role in TGF-βinduced peritoneal fibrosis, it is the initiate and reversible process of fibrosis. Connective tissue growth factor (CTGF) can accelerate cell proliferation, migration and differentiation. Some studies have showed that CTGF acts both as an effector in the downstream cascade of fibrosis induced by TGF-β and as a pro-fibrotic factor, it is closely related to the occurrence and development of many organs fibrosis. It also has been proved that CTGF not only play a important role in PD related peritoneal fibrosis, but also facilitate EMT in the process of fibrosis.
MicroRNAs are non-coding RNAs that post-transcriptionally regulate gene expression by inducing target mRNA degradation or inhibiting its translation. They participate in regulating growth and development, cell differentiation, proliferation, apoptosis and theevelopment of tumour, nowadays become the hot topic in the field of life sciences. Some studies have proved that microRNAs can participate in regulating the development of EMT, and they are important regulators3n the process of TGF-β induced EMT such as microRNA-200family and microRNA-205.
With the development of gene therapy technology and gene delivery system, amounts of significant results have been achieved in rnderstanding the mechanism of PD related peritoneal fibrosis. Promising results from numerous studies in animal models of peritoneal dialysis have demonstrated that gene delivery method is an innovative and feasible strategy for mechanistic investigation and potential treatment of peritoneal fibrosis. Lentiviral vector (LV) has been widely used as an effective genomic vector, it has a relatively large capacity (close to10kb) and high transfection efficiency that can mediate stable gene transfer in dividing and non-dividing cells without potent immunogenic or inflammatory response. It is very meaningful to apply it in peritoneal fibrosis research.
As mentioned above, we suppose that novel microRNA may participate in regulating EMT and peritoneal fibrosis in PD patients. Using the tool of lentiviral vectors, it has important theoretical and practical significance to research the role of microRNA in EMT and peritoneal fibrosis in the process of PD.
Chapter I Effect of lentivirus infecting peritoneum in mice through intraperitoneal injection
ObjectiveTo investigate the effect of lentivirus infecting peritoneum in mcting lentivirus carring GFP (LV-GFP) with different titer.and determine the best effective titer.
Methods Male ICR mice were randomly divided into control group (n=5), LV-GFP1group (n=5), LV-GFP2group (n=5), LV-GFP3group (n=5). The mice reoeived an infusion of1.5ml0.9%N.S. or LV-GFP with different titer. The mice were sacrificed at day28. Parietal peritoneum (?) collected for-HE and Masson staining or frozen section to゜serve was morphology of peritoneum and the fluorescence of GFP. Visceral p(?)toneum were harvested to extract tissue protein and mRNA for examining the GFP expression by Real Time PCR and western blot.
Results
1. Observing through fluorescence microscope, the frozen sections of control group have no fluorescence of GFP, the frozen sections of LV-GFP1group, LV-GFP2group and LV-GFP3group have fluorescence of GFP.
2. The paraffin sections of mice peritoneal with HE、Masson staining showed that compared with the control group, LV-GFP1group, LV-GFP2group and LV-GFP3group have no apparent morphology changes.
3. Real Time PCR and western blot showed that the control group has no GFP expression, LV-GFP1group, LV-GFP2group and LV-GFP3group have GFP expression.
4. The expression of GFP in LV-GFP2group and LV-GFP3-group is stronger than LV-GFP1group, the expression of GFP between LV-GFP2group and LV-GFP3group is similar.
Conclusion The lentivirus can successfully infect the peritoneum-of mouse by intraperitoneal injection, and2×108GTU is the best effcetive tiler.
Chapter Ⅱ The effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanism
Objective To investigate the effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanism by intraperitoneal injecting LV-mmu-miR-302c to infect mice peritoneum.
Methods Male ICR mice were randomly divided into control group (n=5), PDF group (n=5), LV-mmu-miR-302c+PDF group (n=5), LV-pGIPZ+PDF group (n=5). The mice received an infusion of1.5ml0.9%N.S. or1.5ml4.25%PDF in the presence or in the absence of LV-mmu-miR-302c for28days. The mice were sacrificed at day28. Parietal peritoneum was collected for HE and Masson staining or frozen section to observe the morphology of peritoneum and the fluorescence of GFP. Visceral peritoneum were harvested to extract tissue protein and mRNA for examining the expression of miRNA-302c、E-cadherin、 α-SMA、collagen Ⅰ、CTGF by Real Time PCR and western blot.
Results
1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the intraperitoneal injection of4.25%PDF, but (?) expression of miRNA-302c was rised in the presence of LV-mmu-miR-302c.
2. The structure of mice peritoneum has changed with the intraperitoneal injection of4.25%PDF, which manifesting the loss of mesothelial cell monolayer, the thickness and fibrosis of submesothelial zone, but the presence of LV-mmu-miR-302c can ameliorate the structural change.
3. Real Time PCR and western blot showed that intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT and fibrosis with a decrease in the expression of E-cadherin and increase in the expression of a-SMA and collagen I, but the presence of LV-mmu-miR-302c can ameliorate the change. Meanwhile, compared with the control group, the expression of CTGF was rising with the intraperitoneal injection of4.25%PDF, but the presence of LV-mmu-miR-302c can ameliorate the increase of CTGF.
Conclusion Treat the mice with intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT; intraperitoneal injection of LV-mmu-miR-302c can successfully upregulated the expression of miR-302c;:miR-302c can ameliorate the EMT and fibrosis of peritoneum in mice on peritoneal dialysis through suppresssing the expression of CTGF.
ChaptcrⅢ miRNA-302c modulate TGF-β1induce human peritoneal mesothelial cells EMT and the mechanism
ObjectiveTo investigate the mechanism of miRNA-302c modulating TGF-β1-induced human peritoneal mesothelial cells EMT by infecting human peritoneal mesothelial cells with LV-hsa-miR-302c.
Methods We treated the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours in the presence or absence of LV-hsa-miR-302c. To estimate the transfection efficiency of LV-hsa-miR-302c by observing the fluorescence of GFP through fluorescence microscope. To detect the expression of miRNA-302c、 E-cadherin、α-SMA、collagen I、CTGF by Real Time PCR and western blot.
Results
1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the stimulation of TGF-β1, but the expression of miRNA-302c was rised in the presenc of LV-hsa-miR-302c.
2. The morphology of human mesothelial cells has changed from epithelial phenotype to mesenchymal phenotype with the stimulation of but the presence of LV-hsa-miR-302c can ameliorate the morphology change.
3. Real Time RCR and western blot showed that treating the human peritoneal mesotholial cells with5ng/ml TGF-β1for48hours can cause EMT and fibrosis with a decrease in e expression of E-cadherin and increase in the expression of α-SMA and collagen I, but the presence of LV-hsa-miR-302c can ameliorate the change. Mesanwhile, compared with the control group, the expression of CTGF was rising with the stimulation of TGF-β1, but the presence of LV-hsa-miR-302c can ameliorate the increase of CTGF.
Conclusion Treat the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours can cause HPMCs EMT and fibrosis; the infection of LV-hsa-miR-302c can upregulate the expression of miR-302c; miR-302c can ameliorate the TGF-β1-induced human peritoneal mesothelial cells EMT and fibrosis through suppressing the expression of CTGF.
慢性肾脏病(Chronic Kidney Disease, CKD)是全球关注的公共卫生问题之一,国内外发病率逐年上升。约5%的CKD患者最终将发展成终末期肾功能衰竭,需终身接受肾脏替代治疗。其中,腹膜透析(peritoneal dialysis, PD)是符合我国国情的主要肾脏替代治疗方法,但长期PD可引起腹膜纤维化,导致超滤衰竭(ultrafiltration failure, UFF),进而影响PD的长期应用,是患者退出PD的主要原因。转化生长因子β(transforming growth factor-P, TGF-P)是腹膜纤维化发病机制中的关键因素,近年来多项研究发现腹膜间皮细胞上皮-间充质转分化(epithelial mesenchymal transition, EMT)在TGF-β诱导的腹膜纤维化中起重要作用,是腹膜纤维化启动和可逆的关键环节。结缔组织生长因子(connective tissue growth factor, CTGF)具有促进细胞增殖、迁移和分化等作用,研究证实CTGF通过介导TGF-β1,或直接作用其下游信号分子,与许多组织器官纤维化的发生发展密切相关。已有研究发现,CTGF不仅在PD腹膜纤维化中起到关键作用,并且在纤维化的始动过程EMT的发生中也起到重要促进作用。
microRNAs(miRNA)属于功能性非编码RNA,通过降解目标mRNA或抑制其翻译调节基因转录后水平的表达,参与调控生长发育,细胞分化、增殖、凋亡,肿瘤等疾病的发生发展,是目前生命科学领域的研究热点。有研究证实miRNA是TGF-P1诱导EMT的重要调节因子,如miRNA-200家族和miRNA-205。但是,miRNA在腹膜纤维化领域的研究尚未见文献报道。近期,我们采用高通道miRNA芯片技术对PD病人腹透流出液腹膜间皮细胞miRNA的表达进行了图谱分析,发现miRNA的异常表达与PD患者腹膜EMT及纤维化密切相关。
随着基因治疗技术及基因载体系统的发展,对于PD患者腹膜纤维化的分子机制和干预治疗研究取得了进一步的发展,大量在PD的动物模模型中进行的基因治疗研究证实了基因治疗方法在腹膜纤维化机制研究和治疗中的可行性。慢病毒载体(lentiviral vector, LV)作为目前被广泛应用的基因载体,具有转染效率高,插入外源基因容量大,能感染分裂期和非分裂期细胞,并且不会引起明显免疫和炎症反应等优势,将其应用于腹膜纤维化的研究十分有意义。
基于以上分析,我们认为某些miRNA可参与调节腹膜间皮细胞EMT,以慢病毒载体为基因转染工具,从miRNA着手研究其在PD过程中EMT及纤维化的作用及分子机制具有重要的理论和现实意义。
第一章腹腔注射慢病毒转染小鼠腹膜间皮细胞效应的研究
目的通过小鼠腹腔注射不同滴度的携带绿色荧光蛋白(green fluorescence protein, GFP)基因的慢病毒,观察其转染小鼠腹膜间皮细胞的效应,探讨腹腔注射慢病毒转染小鼠腹膜间皮细胞最佳效应滴度。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为对照组(n=5)、LV-GFP1组(n=5)、LV-GFP2组(n=5)、LV-GFP3组(n=5)。分别一次腹腔注射1.5m10.9%生理盐水或者不同滴度的慢病毒。于第28天处死小鼠,壁层腹膜用于HE和Masson染色观察腹膜形态变化、冰冻切片观察GFP表达,脏层腹膜用于提取组织mRNA和蛋白,采用Real Time PCR和Western blot方法检测GFP mRNA和蛋白表达。
结果
1.倒置荧光显微镜观察小鼠腹膜组织冰冻切片,与对照组相比,腹腔注射慢病毒小鼠腹膜组织有GFP表达；
2.小鼠腹膜组织石蜡切片HE、Masson染色观察,与对照组相比,腹腔注射慢病毒小鼠腹膜组织形态学无明显改变；
3. Real Time PCR和western blot的方法检测,腹腔注射慢病毒小鼠腹膜组织有GFP mRNA和蛋白表达;
4.使用2×1O8GTU LV-GFP组的转染效率高于1×108GTU LV-GFP组,而与3×108GTU LV-GFP组转染效率差异不大。
结论
腹腔注射慢病毒能安全有效转染小鼠腹膜组织,2×108GTU为最佳效应滴度。
第二章miRNA-302c在腹膜透析小鼠腹膜间皮细胞EMT中的作用与机制
目的利用慢病毒载体将miRNA-302c导入PD小鼠腹膜组织,研究miRNA-302c在PD小鼠腹膜间皮细胞EMT中的作用与机制。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为空白对照组(n=5)：腹腔注射1.5ml0.9%生理盐水,每天一次x28天；PDF组(n=5)：腹腔注射1.5ml含4.25%葡萄糖的标准腹透液,每天一次x28天; LV-mmu-miRNA-302c+PDF组(n=5)：腹腔注射1.5ml含2×108GTU的LV-mmu-miRNA-302c,第三天开始腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液,每天一次x28天；LV-pGIPZ+PDF组：腹腔注射1.5ml含2×108GTU的LV-pGIPZ,第三天开始腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液,每天一次×28天。于第28天处死小鼠,壁层腹膜用于制作石蜡切片HE和Masson染色观察腹膜形态变化、冰冻切片观察GFP表达,脏层腹膜用于提取组织蛋白和mRNA,并采用Westem-blot、Real Time-PCR等方法检测miRNA-302c、 E-cadherin、α-SMA\collagenⅠ、CTGF的表达。
结果
1.小鼠膜组织石蜡切片HE、Masson染色观察,与空白对照组相比,PDF组小鼠腹膜组织可见EMT的形态变化,而LV-mmu-miR-302c+PDF组腹膜组织形态学变化介于空白对照组和PDF组之间,EMT改变较PDF组减轻；
2. TaqMan探针实时荧光定量Real Time PCR检测发现,与空白对照组相比,腹腔注射4.25%PDF的各组小鼠脏层腹膜组织miRNA-302c表达均有下调,LV-mmu-miR-302c+PDF组较PDF组miRNA-302c表达增高；
3. Real Time PCR、western blot证实每天腹腔注射4.25%PDF1.5ml,连续28天,可引起小鼠腹膜组织EMT, E-cadherin表达减少,a-SMA及collagen I表达增高。而LV-mmu-miR-302c转染上调miRNA-302c可抑制腹透小鼠腹膜组织a-SMA、collagen I表达增加,E-cadherin表达降低；与空白对照组相比,各组腹透小鼠腹膜组织CTGF表达均有上调,LV-mmu-miR-302c+PDF组较PDF组CTGF表达降低。
结论
高糖腹透液可引起小鼠腹膜间皮细胞EOT; LV-mmu-miR-302c可有效转染小佩腹膜上调miRNA-302c的表达,抑制CTGF表达,减轻腹透小鼠腹膜间皮细胞EMT。
第三章miRNA-302c调控TGF-β1诱导的腹膜间皮细胞EMT形成的机理
目的通过观察慢病毒介导的miRNA-302c对TGF-β1诱导人腹膜间皮细胞(human peritoneal mesothelial cells, HPMCs) EMT过程中CTGF表达的影响,探讨miRNA-302c调控TGF-β1诱导的HPMCsEMT形成的机制。
方法.常规培养正常HPMCs,随机分为:空白对照组:不加任何干预;TGF-β1组：用5ng/mlTGF-β1刺激48小时；LV-hsa-miR-302c+TGF-β1组：转染载有miRNA-302c的慢病毒]LV-hsa-miR-302c后,用5ng/mlTGF-β1刺激48小时；LV-pGIPZ+TGF-β1组：转染载有对照质粒pGIPZ的慢病毒LV-pGIPZ后,用5ng/mlTGF-β1刺激48小时。采用荧光显微镜观察GFP荧光蛋白表达以判断病毒转染效率。采用Westem-blot、Real Time-PCR等方法分别检测miRNA-302c、 E-cadherin、a-SMA、collagen I、CTGF等在HPMCs中的表达变化及其相互关系。
结果
1. TaqMan探针实时荧光定量Real Time PCR检测发现,与空白对照组相比,TGF-β1刺激的各组腹膜间皮细胞miRNA-302c表达均有下调,LV-hsa-miR-3O2c+TGF-β1组miRNA-302c表达较TGF-β1组增高；
2. HPMCs经5ng/ml TGF-β1刺激48小时后,细胞出现成纤维样细胞形态改变,而LV-hsa-rniR-302c+TGF-β1组细胞形态变化介于空白对照组和TGF-β1组之间,细胞EMT形态改变较TGF-β1组减轻；
3. Regtl Time PCR、western blot证实5ng/ml TGF-β1刺激48h可引起HPMCsEMT, E-cadherin表达减少,a-SMA及collagen I表达增高；而LV-bsa-miR-302c转染上调miR-302c可抑制TGF-β1诱导的α-SMA、collagen I表达增加,E-cadherin表达降低；与空白对照组相比,TGF-β1刺激的各组腹膜间皮细胞CTGF表达均有上调,LV-hsa-miR-302c+TGF-β1组CTGF表达较TGF-β1组降低。
结论
TGF-β1可诱导HPMCs EMT; LV-hsa-miR-302c转染上调HPMCs miRNA-302c表达,抑制CTGF表达,减轻TGF-β1诱导的HPMCsEMT。
Background Chronic kidney disease (CKD) is a public health problem all over the world, the morbidity is about11%, and the incidence increased year by year.5%of the CKD patients will eventually develop into the end-stage renal failure, and should have life-long renal replacement therapy. Peritoneal dialysis (PD) is an important modality of renal replacement therapy according with China catual condition. But long-term PD can lead to peritoneal fibrosis and ultrafiltration failure which ultimately limits the long term clinical application of PD and makes patients drop out of PD. Transforming growth factor-β (TGF-β) is a key factor in the mechanism of peritoneal fibrosis, recently some studies have found that epithelial mesenchymal transition (EMT) plays a important role in TGF-βinduced peritoneal fibrosis, it is the initiate and reversible process of fibrosis. Connective tissue growth factor (CTGF) can accelerate cell proliferation, migration and differentiation. Some studies have showed that CTGF acts both as an effector in the downstream cascade of fibrosis induced by TGF-β and as a pro-fibrotic factor, it is closely related to the occurrence and development of many organs fibrosis. It also has been proved that CTGF not only play a important role in PD related peritoneal fibrosis, but also facilitate EMT in the process of fibrosis.
MicroRNAs are non-coding RNAs that post-transcriptionally regulate gene expression by inducing target mRNA degradation or inhibiting its translation. They participate in regulating growth and development, cell differentiation, proliferation, apoptosis and theevelopment of tumour, nowadays become the hot topic in the field of life sciences. Some studies have proved that microRNAs can participate in regulating the development of EMT, and they are important regulators3n the process of TGF-β induced EMT such as microRNA-200family and microRNA-205.
With the development of gene therapy technology and gene delivery system, amounts of significant results have been achieved in rnderstanding the mechanism of PD related peritoneal fibrosis. Promising results from numerous studies in animal models of peritoneal dialysis have demonstrated that gene delivery method is an innovative and feasible strategy for mechanistic investigation and potential treatment of peritoneal fibrosis. Lentiviral vector (LV) has been widely used as an effective genomic vector, it has a relatively large capacity (close to10kb) and high transfection efficiency that can mediate stable gene transfer in dividing and non-dividing cells without potent immunogenic or inflammatory response. It is very meaningful to apply it in peritoneal fibrosis research.
As mentioned above, we suppose that novel microRNA may participate in regulating EMT and peritoneal fibrosis in PD patients. Using the tool of lentiviral vectors, it has important theoretical and practical significance to research the role of microRNA in EMT and peritoneal fibrosis in the process of PD.
Chapter I Effect of lentivirus infecting peritoneum in mice through intraperitoneal injection
ObjectiveTo investigate the effect of lentivirus infecting peritoneum in mcting lentivirus carring GFP (LV-GFP) with different titer.and determine the best effective titer.
Methods Male ICR mice were randomly divided into control group (n=5), LV-GFP1group (n=5), LV-GFP2group (n=5), LV-GFP3group (n=5). The mice reoeived an infusion of1.5ml0.9%N.S. or LV-GFP with different titer. The mice were sacrificed at day28. Parietal peritoneum (?) collected for-HE and Masson staining or frozen section to゜serve was morphology of peritoneum and the fluorescence of GFP. Visceral p(?)toneum were harvested to extract tissue protein and mRNA for examining the GFP expression by Real Time PCR and western blot.
Results
1. Observing through fluorescence microscope, the frozen sections of control group have no fluorescence of GFP, the frozen sections of LV-GFP1group, LV-GFP2group and LV-GFP3group have fluorescence of GFP.
2. The paraffin sections of mice peritoneal with HE、Masson staining showed that compared with the control group, LV-GFP1group, LV-GFP2group and LV-GFP3group have no apparent morphology changes.
3. Real Time PCR and western blot showed that the control group has no GFP expression, LV-GFP1group, LV-GFP2group and LV-GFP3group have GFP expression.
4. The expression of GFP in LV-GFP2group and LV-GFP3-group is stronger than LV-GFP1group, the expression of GFP between LV-GFP2group and LV-GFP3group is similar.
Conclusion The lentivirus can successfully infect the peritoneum-of mouse by intraperitoneal injection, and2×108GTU is the best effcetive tiler.
Chapter Ⅱ The effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanism
Objective To investigate the effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanism by intraperitoneal injecting LV-mmu-miR-302c to infect mice peritoneum.
Methods Male ICR mice were randomly divided into control group (n=5), PDF group (n=5), LV-mmu-miR-302c+PDF group (n=5), LV-pGIPZ+PDF group (n=5). The mice received an infusion of1.5ml0.9%N.S. or1.5ml4.25%PDF in the presence or in the absence of LV-mmu-miR-302c for28days. The mice were sacrificed at day28. Parietal peritoneum was collected for HE and Masson staining or frozen section to observe the morphology of peritoneum and the fluorescence of GFP. Visceral peritoneum were harvested to extract tissue protein and mRNA for examining the expression of miRNA-302c、E-cadherin、 α-SMA、collagen Ⅰ、CTGF by Real Time PCR and western blot.
Results
1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the intraperitoneal injection of4.25%PDF, but (?) expression of miRNA-302c was rised in the presence of LV-mmu-miR-302c.
2. The structure of mice peritoneum has changed with the intraperitoneal injection of4.25%PDF, which manifesting the loss of mesothelial cell monolayer, the thickness and fibrosis of submesothelial zone, but the presence of LV-mmu-miR-302c can ameliorate the structural change.
3. Real Time PCR and western blot showed that intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT and fibrosis with a decrease in the expression of E-cadherin and increase in the expression of a-SMA and collagen I, but the presence of LV-mmu-miR-302c can ameliorate the change. Meanwhile, compared with the control group, the expression of CTGF was rising with the intraperitoneal injection of4.25%PDF, but the presence of LV-mmu-miR-302c can ameliorate the increase of CTGF.
Conclusion Treat the mice with intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT; intraperitoneal injection of LV-mmu-miR-302c can successfully upregulated the expression of miR-302c;:miR-302c can ameliorate the EMT and fibrosis of peritoneum in mice on peritoneal dialysis through suppresssing the expression of CTGF.
ChaptcrⅢ miRNA-302c modulate TGF-β1induce human peritoneal mesothelial cells EMT and the mechanism
ObjectiveTo investigate the mechanism of miRNA-302c modulating TGF-β1-induced human peritoneal mesothelial cells EMT by infecting human peritoneal mesothelial cells with LV-hsa-miR-302c.
Methods We treated the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours in the presence or absence of LV-hsa-miR-302c. To estimate the transfection efficiency of LV-hsa-miR-302c by observing the fluorescence of GFP through fluorescence microscope. To detect the expression of miRNA-302c、 E-cadherin、α-SMA、collagen I、CTGF by Real Time PCR and western blot.
Results
1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the stimulation of TGF-β1, but the expression of miRNA-302c was rised in the presenc of LV-hsa-miR-302c.
2. The morphology of human mesothelial cells has changed from epithelial phenotype to mesenchymal phenotype with the stimulation of but the presence of LV-hsa-miR-302c can ameliorate the morphology change.
3. Real Time RCR and western blot showed that treating the human peritoneal mesotholial cells with5ng/ml TGF-β1for48hours can cause EMT and fibrosis with a decrease in e expression of E-cadherin and increase in the expression of α-SMA and collagen I, but the presence of LV-hsa-miR-302c can ameliorate the change. Mesanwhile, compared with the control group, the expression of CTGF was rising with the stimulation of TGF-β1, but the presence of LV-hsa-miR-302c can ameliorate the increase of CTGF.
Conclusion Treat the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours can cause HPMCs EMT and fibrosis; the infection of LV-hsa-miR-302c can upregulate the expression of miR-302c; miR-302c can ameliorate the TGF-β1-induced human peritoneal mesothelial cells EMT and fibrosis through suppressing the expression of CTGF.
引文
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