NG2蛋白多糖在LPS诱导的肾小球炎症及系膜细胞增殖中的作用

发布时间：2018-06-09 03:26

本文选题：NG2蛋白多糖 + 肾小球系膜细胞　；参考：《华中科技大学》2012年博士论文

【摘要】：第一部分NG2蛋白多糖在肾小球中的表达及定位目的：观察大鼠乳鼠肾组织中NG2蛋白多糖的表达及其在肾小球的定位,对三种肾小球固有细胞系进行检测,明确NG2在肾小球三种固有细胞中的具体表达情况。方法：选择出生1天的Wistar大鼠乳鼠,将其肾组织进行固定、包埋后制成石蜡切片,免疫组化检测观察NG2在大鼠乳鼠肾组织的分布,对肾小球中NG2的表达进行定位。以RPMI1640培养基培养条件永生化小鼠足细胞,DMEM培养基培养大鼠系膜细胞,DMEM/F12培养基培养小鼠内皮细胞。间接免疫荧光染色共聚焦显微镜下观察三种肾小球固有细胞中NG2蛋白的表达情况。以RT-PCR法检测足细胞、系膜细胞和内皮细胞中NG2mRNA的表达。结果：NG2蛋白在肾组织中有阳性表达,在肾小球中主要定位于系膜细胞及其周围系膜区。在系膜细胞中NG2蛋白呈阳性表达,NG2均匀分布在系膜细胞胞膜上,而在细胞核上无表达；NG2蛋白在足细胞和内皮细胞中均无阳性表达。RT-PCR从mRNA水平证实了在三种肾小球固有细胞中,NG2仅在系膜细胞中表达。结论：肾小球中NG2表达于间叶组织来源的肾小球系膜细胞,在系膜细胞及其周围的系膜区均有表达,而在另外两种肾小球固有细胞中无表达,提示肾小球中NG2由系膜细胞分泌,并向周围系膜基质中释放。第二部分LPS刺激后大鼠肾小球中NG2蛋白多糖和炎症因子的表达变化目的：观察脂多糖(LPS)刺激后大鼠乳鼠肾小球中NG2蛋白多糖的表达及促炎因子肿瘤坏死因子-a(TNF-aa)和白细胞介素-1p(IL-1β)的表达变化,初步探讨NG2与肾小球炎症反应之间的关系。方法：选择出生1天的Wistar大鼠乳鼠30只,分为3组：对照组、LPS组和LPS+Dex组,分别腹腔注射生理盐水、LPS和LPS+Dexamethasone。12小时后,取其一侧肾组织进行固定、包埋后制成石蜡切片,免疫组化检测NG2在肾小球中的表达变化。另一侧肾组织筛网法分离肾小球,提取肾小球mRNA和蛋白质,实时荧光定量PCR检测NG2、TNF-aa和IL-1βmRNA的表达,ELISA检测肾小球中NG2、TNF-a和IL-1β蛋白的表达。结果：NG2免疫组化结果显示,NG2蛋白在肾小球系膜细胞及周围基质中表达,与对照组相比,LPS刺激后NG2蛋白表达明显增多；LPS刺激并与Dexamethasone干预后NG2蛋白表达较LPS组减少。分离的肾小球提取RNA,实时荧光定量PCR结果显示,LPS刺激后肾小球中NG2、TNF-a和IL-1β mRNA相对表达水平均高于对照组,差异有统计学意义(P0.01)；LPS刺激并与Dexamethasone干预后肾小球中NG2、TNF-a和IL-1β mRNA相对表达水平均较LPS组降低,差异有统计学意义(P0.01)。分离的肾小球提取蛋白,ELISA结果显示,与对照组相比,LPS刺激后RMC中NG2、TNF-a和IL-1p蛋白表达水平均升高,差异有统计学意义(P0.01)；LPS刺激并与Dexamethasone干预后RMC中NG2、TNF-a和IL-1p蛋白表达水平较LPS组均降低,差异有统计学意义(P0.01)。结论：肾小球系膜细胞NG2高表达与肾小球炎症密切相关,NG2可诱导促炎因子TNF-a和IL-1p高表达,通过TNF-a和IL-1β介导肾小球炎症反应。介素-1p 第三部分NG2蛋白多糖在肾小球炎症与系膜细胞增殖中的作用目的：观察LPS刺激后NG2蛋白多糖和促炎因子TNF-a和IL-1p在大鼠系膜细胞(RMC)的表达变化,以及RMC的增殖情况；NG2-siRNA转染RMC下调NG2表达后,观察促炎因子TNF-a和IL-1p的表达变化及RMC增殖的变化,探讨NG2与肾小球炎症反应及系膜细胞增殖之间的关系。方法：(1)体外培养RMC,将细胞分为3组：对照组、LPS组和LPS+Dex组,干预8小时后,间接免疫荧光染色观察NG2蛋白的表达,实时荧光定量PCR检测RMC中NG2、TNF-a和IL-1β mRNA的表达,ElISA检测RMC培养上清液中NG2、TNF-a和IL-1p,MTT检测RMC增殖情况。(2)设计及合成NG2-siRNA,用脂质体法将其转入体外培养的RMC,24小时后荧光显微镜下观察转染标志Cy3的表达；48小时后PCR检测NG2的干扰效率。(3)将细胞分为3组：正常对照组、LPS干预组和LPS干预+转染NG2-siRNA组。NG2-siRNA转染及LPS干预8小时后,实时荧光定量PCR检测RMC中NG2、TNF-a和IL-1βmRNA的表达,ElISA检测RMC培养上清液中NG2、TNF-a和IL-1β,MTT检测RMC增殖情况。结果：(1)LPS刺激RMC8小时后免疫荧光结果显示,与对照组相比LPS组中NG2蛋白表达明显增多,且分布均匀。LPS+Dex组NG2蛋白表达较LPS组减少,但仍多于对照组。(2)实时荧光定量PCR和ELISA结果显示,LPS刺激8小时后RMC中NG2、TNF-α、IL-1β mRNA和RMC培养上清液中NG2、TNF-a、IL-1p蛋白表达水平均升高,RMC增殖效率上升,与对照组相比,差异均有统计学意义(P0.01)。Dexamethasone干预可部分逆转上述反应。(3)NG2-siRNA转染24小时后,Cy3呈强荧光表达；实时荧光定量PCR结果显示,与未转染对照组及阴性siRNA转染组相比,NG2-siRNA转染组NG2表达显著下降,达到下调NG2表达的效果。(4)LPS刺激RMC8小时后,与对照组相比,RMC中NG2、TNF-α、IL-1β mRNA和RMC培养上清液中NG2、TNF-a、IL-1β蛋白表达水平均升高, RMC增殖效率上升,而NG2-siRNA可逆转上述反应,差异有统计学意义(P0.01)。结论：体外LPS刺激RMC后可NG2表达增加,促炎因子TNF-a和IL-1β的释放增加,RMC增殖效率上升,NG2基因沉默可逆转上述反应。因此,NG2介导促炎因子TNF-a和IL-1β等的释放,且通过肾小球炎症反应的激活促进系膜细胞增殖。
[Abstract]:The first part is the expression and localization of NG2 proteoglycan in the glomerulus.
Objective: To observe the expression of NG2 proteoglycan in the renal tissue of rats and the location of the glomeruli, to detect the three kinds of glomerular innate cell lines and to clarify the specific expression of NG2 in the three kinds of glomerular mesangial cells.
Methods: the Wistar rats of 1 days of birth were selected to fix the renal tissue and be embedded into paraffin section. The distribution of NG2 in the renal tissue of rats was detected by immunohistochemistry and the expression of NG2 in the glomeruli was located. The rat mesangial cells were immortalized by the culture condition of RPMI1640 medium, and the mesangial cells were cultured in the DMEM medium. Mouse endothelial cells were cultured on DMEM/F12 medium. The expression of NG2 protein in three glomerular innate cells was observed under indirect immunofluorescence staining microscope. The expression of NG2mRNA in podocytes, mesangial cells and endothelial cells was detected by RT-PCR.
Results: the positive expression of NG2 protein in the renal tissue is mainly located in the mesangial cells and the surrounding mesangial region. In the mesangial cells, the NG2 protein is positive, and the NG2 is distributed evenly on the membrane of the mesangial cells, but no expression in the nucleus, and the NG2 protein has no positive expression of.RT-PCR from mRNA in the podocytes and endothelial cells. It is confirmed that NG2 is only expressed in mesangial cells in three types of glomerular intrinsic cells.
Conclusion: NG2 expressed in mesangial mesangial cells derived from mesangial cells and the mesangial region around mesangial cells, but not expressed in the other two kinds of glomerular innate cells, suggesting that the glomerular NG2 is secreted by mesangial cells and released into the peripheral mesangial matrix. The second part of LPS stimulates the rat glomeruli. Expression of NG2 proteoglycan and inflammatory factors
Objective: To observe the expression and promotion of NG2 proteoglycan in glomeruli of rats after lipopolysaccharide (LPS) stimulation.
Expression of inflammatory factors, tumor necrosis factor -a (TNF-aa) and interleukin -1p (IL-1 beta), were preliminarily studied.
To investigate the relationship between NG2 and glomerular inflammatory response.
Methods: 30 Wistar rats were selected for 1 days of birth. They were divided into 3 groups: control group, LPS group and LPS+Dex group. After intraperitoneal injection of saline, LPS and LPS+Dexamethasone.12 hours, the kidney tissue was fixed and embedded into paraffin section. The expression of NG2 in the glomeruli was detected by immunohistochemistry. The other side of renal tissue was screened. Glomeruli was separated by net method. Glomerular mRNA and protein were extracted. The expression of NG2, TNF-aa and IL-1 beta mRNA were detected by real-time fluorescence quantitative PCR. The expression of NG2, TNF-a and IL-1 beta in glomeruli was detected by ELISA.
Results: the results of NG2 immunohistochemical staining showed that NG2 protein was expressed in mesangial cells and surrounding matrix. Compared with the control group, the expression of NG2 protein increased significantly after LPS stimulation. LPS stimulated and NG2 protein expression decreased after Dexamethasone intervention. The isolated glomeruli extracted RNA, real-time fluorescent quantitative PCR results showed, LPS stimulation after stimulation. The relative expression level of NG2, TNF-a and IL-1 beta mRNA in the glomeruli was higher than that in the control group (P0.01). LPS stimulation and the relative expression level of TNF-a and IL-1 beta mRNA after Dexamethasone intervention were lower than that of LPS group. Compared with the control group, the expression level of NG2, TNF-a and IL-1p protein in RMC increased after LPS stimulation, and the difference was statistically significant (P0.01). LPS stimulation and NG2, TNF-a and IL-1p protein expression levels in RMC decreased after the intervention of Dexamethasone, and the difference was statistically significant.
Conclusion: the high expression of NG2 in mesangial mesangial cells is closely related to glomerular inflammation. NG2 can induce the high expression of TNF-a and IL-1p, and mediate the glomerular inflammatory reaction through TNF-a and IL-1 beta. The mediator -1p is mediated.
The third part is the role of NG2 proteoglycan in glomerulonephritis and mesangial cell proliferation.
Objective: To observe the expression of NG2 proteoglycan and proinflammatory factor TNF-a and IL-1p in rat mesangial cells (RMC) and the proliferation of RMC after LPS stimulation. After NG2-siRNA transfected with RMC, the expression of TNF-a and IL-1p and the proliferation of RMC were observed and the proliferation of glomerular inflammation and mesangial cells were observed. The relationship between them.
Methods: (1) the RMC was cultured in vitro, and the cells were divided into 3 groups: the control group, the LPS group and the LPS+Dex group. After 8 hours of intervention, the expression of NG2 protein was observed by indirect immunofluorescence staining. The expression of NG2, TNF-a and IL-1 beta mRNA in RMC was detected by real-time fluorescent quantitative PCR, and the proliferation of RMC culture supernatant was detected by ElISA detection. (2) design and NG2-siRNA was synthesized with liposome method and transferred into RMC in vitro. After 24 hours, the expression of transfection marker Cy3 was observed. The interference efficiency of NG2 was detected by PCR after 48 hours. (3) the cells were divided into 3 groups: normal control group, LPS intervention group and LPS intervention + transfected NG2-siRNA group.NG2-siRNA transfection and LPS intervention for 8 hours, real time fluorescence Quantitative PCR was used to detect the expression of NG2, TNF-a and IL-1 beta mRNA in RMC. ElISA was used to detect NG2, TNF-a and IL-1 beta in RMC culture supernatant, and the proliferation was detected by TNF-a.
Results: (1) the immunofluorescence results of LPS stimulation after RMC8 hours showed that the expression of NG2 protein in the LPS group was significantly increased compared with the control group, and the NG2 protein expression in the.LPS+Dex group was less than that in the LPS group, but it was still more than the control group. (2) the real-time fluorescence quantitative PCR and ELISA results showed that LPS stimulated 8 hours after the RMC NG2. The expression level of NG2, TNF-a, IL-1p protein in the supernatant increased and the proliferation efficiency of RMC increased. Compared with the control group, the difference was statistically significant (P0.01).Dexamethasone intervention could partly reverse the above reaction. (3) Cy3 showed strong fluorescent expression after 24 hours of NG2-siRNA transfection, and the real time fluorescence quantitative PCR results showed that the control group and negative siR were not transfected with the control group and negative siR. Compared with the NA transfection group, the expression of NG2 decreased significantly in the NG2-siRNA transfection group. (4) after LPS stimulation for RMC8 hours, the expression level of NG2, TNF- alpha, IL-1 beta and RMC culture supernatant in RMC was higher than that of the control group, and the expression level of the beta protein increased and the proliferation efficiency increased. Study meaning (P0.01).
Conclusion: after LPS stimulation of RMC in vitro, the expression of NG2 increased, the release of pro-inflammatory factor TNF-a and IL-1 beta was increased, the proliferation efficiency of RMC increased, and NG2 gene silencing could reverse the above reaction. Therefore, NG2 mediated the release of TNF-a and IL-1 beta, and promoted the proliferation of mesangial cells through the activation of glomerular inflammatory reaction.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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