SAB,SAC及其分子药对配伍对HSA诱导HK-2细胞CCL2,CCL3表达量的影响

发布时间：2018-07-08 15:44

本文选题：肾小管上皮细胞 + 丹酚酸B　；参考：《遵义医学院》2016年硕士论文

【摘要】：目的:研究SAB,SAC及其分子药对配伍对HSA诱导肾小管上皮细胞过程中CCL2,CCL3表达的影响。方法:将体外培养的HK-2细胞随机分为6组:(1)正常组(2)人血清白蛋白组(简称模型组)、(3)PDGF-C抗体组(简称抗体组)(4)丹酚酸B组(5)丹酚酸C组(6)丹酚酸B,C按1:1组分配伍组(简称:配伍组)。同步化24h后,除正常组外,其余各组均给予HSA诱导。抗体组、丹酚酸B组、丹酚酸C组、配伍组(统称为治疗组)分别加入相应药物共同培养,于24h、48h、72h分组收集细胞。采用Western Blot、免疫细胞化学及免疫荧光技术检测HK-2细胞PDGF-C、PDGFR-α、CCL2和CCL3的定位及表达水平。分别采用WST-1法、DTNB法和TBA法测定SOD、GSH及MDA的含量。结果:1.PDGF-C的检测结果1)免疫细胞化学检测结果:PDGF-C阳性表达为棕黄颗粒,表达部位主要位于HK-2细胞胞质。2)WB检测结果:与模型组相比24h、48h及72h三个时间点治疗组PDGF-C相对表达量明显减少(P0.05)。2.PDGFR-α的检测结果1)免疫细胞化学检测结果:PDGFR-α阳性表达为棕黄颗粒,表达部位主要位于HK-2细胞胞质。2)WB检测结果:与模型组相比24h、48h及72h治疗组PDGFR-α相对表达量明显减少(P0.05),其中,72h时间点抗体组及配伍组PDGFR-α相对表达量较丹酚酸B、C组显著降低(P0.05)3.CCL2的检测结果1)免疫荧光化学检测结果:CCL2阳性表达为绿色荧光,表达部位主要位于HK-2细胞胞质。2)WB检测结果:与模型组相比,24h、48h及72h三个时间点各治疗组CCL2相对表达量均明显减少(P0.05),其中,72h时间点抗体组及配伍组CCL2相对表达量较丹酚酸C组显著降低(P0.05)。4.CCL3检测结果1)免疫荧光化学检测结果:CCL3阳性表达为绿色荧光,表达部位主要位于HK-2细胞胞质。2)WB检测结果:与模型组相比,24h、48h和72h三个时间点各治疗组CCL3蛋白相对表达明显减少(P0.05)。5.SOD、GSH和MDA检测结果:与模型组相比,24h、48h及72h各治疗组SOD和GSH含量明显升高(P0.05),各治疗组内比较,配伍组SOD和GSH含量均较其他治疗组明显升高(P0.05)。与模型组相比,24h、48h及72h各治疗组MDA含量明显降低(P0.05),配伍组MDA含量较其他治疗组明显降低(P0.05)。结论:丹酚酸B、C及其药对配伍对HSA诱导HK-2细胞PDGF-C、PDGFR-α、CCL2及CCL3的表达起抑制作用,可有效增加SOD、GSH的含量及减少MDA的量。推测丹酚酸B、C及其药对配伍通过干预PDGF-C/PDGFR-α信号通路,减轻炎症反应,缓解氧化应激反应等途径,从而有可能在延缓RIF的进程中发挥作用。
[Abstract]:Aim: to study the effect of SABX SAC and its molecular drugs on the expression of CCL2 and CCL3 in renal tubular epithelial cells induced by HSA. Methods: HK-2 cells cultured in vitro were randomly divided into 6 groups: (1) normal group (2) human serum albumin group (), (3) PDGF-C antibody group (4) Salvianolic acid group B (5) Salvianolic acid group C (6) Salvianolic acid BnC divided into 1:1 group (abbreviated: compatibility group). After 24 hours of synchronization, all the groups except the normal group were induced by HSA. The antibody group, Salvianolic acid group B, Salvianolic acid group C, compatibility group (collectively referred to as treatment group) were co-cultured with corresponding drugs, and the cells were collected at 24 h, 48 h and 72 h respectively. The localization and expression of CCL2 and CCL3 in HK-2 cells were detected by Western blot, immunocytochemistry and immunofluorescence techniques. The contents of GSH and MDA were determined by WST-1 DTNB method and TBA method respectively. Results: 1) Immunocytochemistry analysis showed that the positive expression of PDGF-C was brown and yellow granules. The expression site was mainly located in the cytoplasm of HK-2 cells) WB: compared with the model group, the relative expression of PDGF-C in the treatment group at 24 h and 72 h decreased significantly (P0.05). 2. The results of the detection of PDGFR- 伪 were as follows: 1) Immunocytochemistry showed that the positive expression of PDGFR- 伪 was brown granules. The expression site of PDGFR- 伪 was mainly located in the cytoplasm of HK-2 cells. Compared with the model group, the relative expression of PDGFR- 伪 in 24 h and 72 h treatment group was significantly lower than that in the control group (P0.05), and the relative expression of PDGFR- 伪 in antibody group and compatibility group at 72 h after treatment was significantly lower than that in Salvianolic acid group (P 0.05). Low (P0.05) 3. Results of CCL2 detection. 1) the positive expression of CCL2 was green fluorescence. The expression site was mainly located in the cytoplasm of HK-2 cells. The results of WB: compared with the model group, the relative expression of CCL2 at 24 h and 72 h in each treatment group was significantly decreased (P0.05), and the relative expression of CCL2 in antibody group and compatibility group was higher than that in Dan group at 72 h after treatment (P0.05). Phenolic acid group C significantly decreased (P0.05) .4.CCL3 detection results 1) Immunofluorescence staining results showed that the positive expression of CCL3 was green fluorescence. Compared with the model group, the relative expression of CCL3 protein in each treatment group at 48h and 72h was significantly decreased (P0.05). 5. The results of GSH and MDA detection: compared with the model group, SOD was detected at 24 h, 48 h and 72 h, respectively. And GSH content increased significantly (P0.05), compared with each treatment group, The contents of SOD and GSH in compatibility group were significantly higher than those in other treatment groups (P0.05). Compared with the model group, the MDA content of 24 h and 72 h treatment group decreased significantly (P0.05), and the MDA content of compatibility group was significantly lower than that of other treatment group (P0.05). Conclusion: Salvianolic acid BfC and its drugs can inhibit the expression of PDGF-CnPDGFR- 伪 CCL2 and CCL3 in HK-2 cells induced by HSA, and can effectively increase the content of SOD- GSH and decrease the amount of MDA in HK-2 cells. It is inferred that the combination of Salvianolic acid and its admixture may play an important role in delaying the process of RIF by interfering with PDGF-C / PDGFR- 伪 signaling pathway, reducing inflammation and alleviating oxidative stress.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R277.5

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