第一阶段

验证高糖刺激对肾小球细胞的影响以及NADPH氧化酶的作用　

第二阶段

验证XBP1S在HG诱导的MCs细胞氧化压力和病理变化的作用

锐赛点评

该项研究的实验推进共分为两个阶段。首先对体外高糖诱导的细胞模型进行摸索，通过相关病例表型因子和氧化压力水平的检测确认体外模拟的DN的肾小球模型的科学性和可行性。第二阶段在确认模型构建成功后研究XBP1基因对高糖诱导下的MCs细胞的保护作用。整个方案流程逻辑清晰，研究目的明确，操作性合理，结合目前研究基础引入多个创新点，具有一定的临床研究的价值。

Figure 1. Effect of high glucose on the expression of XBP1S. A: diagram shows the formation of XBP1S from XBP1U mRNA, a 26 bp fragment(from 484 to 509) was deleted during splicing. B: electrophoresis of RT-PCR products indicates MCs expressed both XBP1U and XBP1S. C and D:Western blot and real time PCR result show the expression of XBP1S before and after high glucose treatment (mean 6 SEM, n = 5).E: Western blot result shows the expression of XBP1U before and after high glucose treatment for 48 h (mean 6 SEM, n = 6) *p,0.05 compared to NG; ***p,0.001 compared to NG

研究方案设计思路清晰。首先通过调整MCs状态，设计多次预实验摸索糖浓度，HG处理时间，用以确认细胞状态，最佳糖浓度和HG处理时间，确立体外模拟体内高糖应激的最佳模型。其次，通过特异性引物的巧妙设计，结合WB实验，确认了XBP1基因在高糖应激条件下的表达模式，明确后续重点关注XBP1S的研究思路。

HG处理后FN和collagen IV表达增高，而NADPH氧化酶抑制剂DPI能逆转这种HG诱导的现象。

Figure 2. Effects of high glucose treatment on collagen IV and fibonectin. Western blot results show high glucose treatment increases collagen IV (A) and fibronectin (B) levels (mean 6 SEM, n = 6). DPI reverses high glucose-induced increase in collagen IV and fibronectin expressions(C) (mean 6 SEM, n = 5). *p,0.05 compared to NG; ***p,0.001 compared to NG. #p,0.05 compared with HG.

高糖处理后MCs胞外基质蛋白的分泌明显增高，应证了DN的相关病例表型，证明HG条件下肾小球系膜细胞的体外模型的科学性，同时DPI的实验数据证明了NADPH氧化酶参与了肾小球系膜细胞在ER stress条件下的细胞病理改变，并在此过程中发挥了重要作用。

HG处理后p47表达增高，胞内ROS浓度升高。而NADPH氧化酶抑制剂DPI能逆转这种HG诱导的现象。

Figure 3. Effects of high glucose on ROS generation and p47phox expression. A and B: Observation of ROS generation by DHE fluorescent probe assay (mean 6 SEM, n = 7). C: Western blot analysis on p47phox expression (mean 6 SEM, n = 6). ***p,0.001 compared to NG; ###p,0.001compared to HG.

高糖处理后MCs胞内ROS浓度明显增高，同时p47表达增加。应证了DN的相关病例表型，证明HG条件下肾小球系膜细胞的体外模型的科学性。

XBP1 OE腺病毒感染MCs后靶基因过表达效果明显

Figure 4. Identification of XBP1S expression after transfection of Ad-GFP or Ad-XBP1S. A: Observation of the cell under fluorescent microscope after transfection of Ad-GFP or Ad-XBP1S for 24 h. B: Western blot analysis on XBP1S protein level after Ad-GFP or Ad-XBP1S transfection for 48 h.

多次预实验结果显示MCs细胞比较敏感，对腺病毒纯度要求高。同时在HG诱导的模拟病理状态下，病毒纯度和感染量以及overexpression效果的平衡则尤为重要，最终实验显示感染效果非常好，过表达效果显著，同时对细胞状态的影响较小，满足了后续研究的需求。

XBP1过表达后，HG诱导的FN和collagenIV表达上调受到抑制，提示XBP1参与了HG诱导的MCs细胞应激过程，对HG对MCs细胞的损伤有保护作用

Figure 5. Western blot analysis on ECM expressions in thecultured MCs with or without Ad-XBP1S transfection for 48 h.A: Collagen IV; B: Fibronectin (mean 6 SEM, n = 5). *p,0.05 comparedto NG; #p,0.05 compared with HG.

XBP1抑制肾小球系膜细胞ECM分泌的效果显著，证明了XBP1对HG诱导的肾小球系膜细胞损伤的保护作用。

XBP1过表达后，HG诱导的p47表达上调和ROS浓度升高受到抑制，提示XBP1参与了HG诱导的MCs细胞胞内氧化应激，对HG对MCs细胞的损伤有保护作用

Figure 6. Changes in p47 and ROS generation after transfection of Ad-GFP or Ad-XBP1S. A: Western blot analysis on p47phox levels in the cultured MCs before and after Ad-XBP1S transfection for 48 h (mean 6 SEM, n = 5). B: Observation of ROS generation by DHE fluorescent probe assay in the cultured MCs before and after Ad-XBP1S transfection for 48 h (mean 6 SEM, n = 8). *p,0.05 compared to NG; #p,0.05 compared with HG; ##p,0.01 compared to HG. C: Western blot analysis on collagen IV and fibronectin levels after transfection of Ad-XBP1S or Ad-XBP1S+xanthine and xanthine oxidase (X?XO) for 48 h in the presence of HG. **p,0.01 compared to Ad-GFP; #p,0.05 compared with Ad-XBP1S (mean 6 SEM, n = 5).

XBP1降低肾小球系膜细胞胞内ROS的水平，证明了XBP1对HG诱导的肾小球系膜细胞损伤的保护作用。

为了进一步证明XBP1基因对MCs细胞的高糖损伤压力下的保护作用，设计者引入siRNA的技术，试图通过XBP1的沉默，实现反向证明XBP1基因的保护作用。siRNA本身是一项很成熟的技术，但是MCs细胞中XBP1基因的表达属于应激表达类型，正常状态下MCs细胞中XBP1基因的本底表达比较低，不适合进行siRNA靶点的筛选。设计者通过多次优化摸索和实验创新，筛选到敲减效果比较显著的靶点，利用该靶点达到了实验预期。此为该部分实验的难点和亮点。